Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease

Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate... Abstract Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-β protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer’s-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-β1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-β1-42, vascular and parenchymal amyloid-β deposits, and astrocytosis (31%, 47–80%, and 33%, respectively; P < 0.05–0.0001). ACE10 macrophages surrounded brain and retinal amyloid-β plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05–0.001). In vitro analysis of macrophage response to well-defined amyloid-β1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-β1-42 species by ACE10 macrophages. They exhibited 2–5-fold increased surface binding to amyloid-β conformers as well as substantially more effective amyloid-β1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05–0.0001), endosomal processing (P < 0.05–0.0001), and ∼80% increased extracellular degradation of amyloid-β1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer’s-related amyloid-β1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-β forms. IGF1, TNFα, TREM2, EEA1, innate immunity Introduction Neuroinflammation is a key feature of Alzheimer’s disease, a relentless, progressive, and currently untreatable neurodegenerative disease and the leading cause of senile dementia worldwide (Wyss-Coray, 2006; Gjoneska et al., 2015; Heneka et al., 2015; Alzheimer's Association, 2019). In Alzheimer’s disease, the detrimental or therapeutic contribution of peripherally derived monocytes, compared to that of brain-resident microglia, is highly debated (Simard et al., 2006; Ajami et al., 2007; Butovsky et al., 2007; El Khoury et al., 2007; Mildner et al., 2007; Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Bernstein et al., 2014b; Jay et al., 2015a, b; Koronyo et al., 2015; Zuroff et al., 2017; Cao and Zheng, 2018; Cronk et al., 2018; Da Mesquita et al., 2018). Several studies have demonstrated that monocytes can colonize diseased brain parenchyma in murine models of Alzheimer’s disease (Simard et al., 2006; Butovsky et al., 2007; Cronk et al., 2018) as well as under irradiation-free physiological conditions (Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Michaud et al., 2013; Bernstein et al., 2014b; Koronyo et al., 2015; Rentsendorj et al., 2018). In addition, heterogeneous subpopulations of brain resident microglia have been identified as having molecular signatures that are distinct from those of peripheral monocytes, particularly disease-associated microglia located proximal to the Alzheimer’s disease pathological hallmark, amyloid-β plaques (Keren-Shaul et al., 2017; Krasemann et al., 2017; Yin et al., 2017). These microglial populations play a role in the pathogenesis of chronic inflammation (Akiyama et al., 2000; Bales et al., 2000; Colton et al., 2000; McGeer and McGeer, 2002; Wyss-Coray, 2006; Prinz et al., 2011) and synaptic loss by excessive pruning (Hong et al., 2016; Johnson and Stevens, 2018), while insufficiently clearing misfolded amyloid-β (Morgan et al., 2005; Simard et al., 2006; El Khoury et al., 2007; Hickman et al., 2008; Koronyo-Hamaoui et al., 2009; Yamanaka et al., 2012; Frenkel et al., 2013; Roussos et al., 2015). In contrast, peripherally recruited monocytes and macrophages in Alzheimer’s disease model mice were shown to exhibit a neuroprotective phenotype, regulating neuroinflammation and effectively eliminating cerebral amyloid-β pathology (Butovsky et al., 2007; Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b; Koronyo et al., 2015; Rentsendorj et al., 2018). These beneficial effects provide the rationale for a comprehensive assessment of the therapeutic potential of peripheral monocyte infusion in animal models and eventually in Alzheimer’s disease patients. The pathology of Alzheimer’s disease occurs as a complex continuum, typically characterized by early accumulation and aggregation of cerebral amyloid-β (Masters et al., 2015; Jack et al., 2018) in the form of parenchymal plaques and vascular deposits (e.g. cerebral amyloid angiopathy, CAA) (Ellis et al., 1996), which extend to the neurosensory retina in patients with Alzheimer’s disease (Koronyo-Hamaoui et al., 2011; La Morgia et al., 2016; Koronyo, 2017). The 42 amino acid-long form of amyloid-β has been regarded as pathognomonic to Alzheimer’s disease, particularly its oligomeric forms, which display high synaptotoxicity, disrupt long-term potentiation, and directly induce cognitive impairments (Selkoe, 2008; Shankar et al., 2008; Hayden and Teplow, 2012). At the same time, the precise structural characterization and impact of various amyloid-β42 species on cells have been challenging to investigate because of their highly metastable nature and their existence in dynamically changing mixtures (Walsh and Selkoe, 2004; Glabe, 2005; Kirkitadze and Kowalska, 2005). To this end, methods have been developed to chemically stabilize the different assembly types of amyloid-β42 such that a well-defined population of oligomers can be studied (Bitan et al., 2003; Bitan and Teplow, 2004; Hayden and Teplow, 2012; Lopes et al., 2012) in comparison with other naturally stable structures, including prion rod-like enriched assemblies and fibrils (Walsh et al., 1997). It is widely believed that approaches to target and remove soluble amyloid-β42 oligomers from CNS tissues hold great promise for curbing neuroinflammation and contributing to Alzheimer’s disease prevention and therapy (Haass and Selkoe, 2007; Viola and Klein, 2015). In the past decade, strategies harnessing peripheral innate immune cells such as monocytes and macrophages were effective in clearing parenchymal and vascular amyloid-β in the brain and attenuating progression in mouse models of Alzheimer’s disease (Malm et al., 2005; Butovsky et al., 2007; Town et al., 2008; Boissonneault et al., 2009; Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Michaud et al., 2013; Bernstein et al., 2014b; Koronyo et al., 2015; Ardura-Fabregat et al., 2017; Rentsendorj et al., 2018). Importantly, even in the absence of whole-body irradiation or microglial ablation via pharmacological intervention, peripheral monocytes spontaneously engrafted into the brain of transgenic APPswe/PS1ΔE9 (AD+) mice and homed to amyloid-β plaque sites (Lebson et al., 2010; Koronyo et al., 2015). In particular, enhanced recruitment of cerebral amyloid-β-associated monocytes was achieved through blood enrichment with bone marrow (BM)-derived CD115+ monocyte subsets isolated from young donor mice, leading to diminished neuropathology and preserved cognitive function (Koronyo et al., 2015). To augment these beneficial immune responses further, our group studied a mouse model of Alzheimer’s disease in which the animals were genetically modified to overexpress angiotensin-converting enzyme (ACE), an amyloid-β-degrading peptidase, controlled by a c-fms promoter (Bernstein et al., 2014b). The role of ACE and ACE inhibitors in Alzheimer’s disease is complex (Kehoe et al., 2009). In humans, a recent large genome-wide association meta-analysis (GWAS) of 94 437 individuals clinically diagnosed with late-onset Alzheimer’s disease has corroborated previous studies and identified ACE as a candidate gene (Kunkle et al., 2019). In addition, GWAS-by-familial-proxy of Alzheimer’s or dementia studies have further confirmed ACE relevance to Alzheimer’s disease and identified the single nucleotide polymorphism (SNP) combination of rs4343 and rs4351 in the ACE haplotype to possess a 45-fold increased risk of developing Alzheimer’s disease (Meng et al., 2006; Marioni et al., 2018). Importantly, individuals carrying variants of the ACE gene (D/D alleles) resulting in increased plasma expression of the enzyme were associated with lower risk for Alzheimer’s disease (Kehoe et al., 1999; Lehmann et al., 2005). Lower CSF levels of ACE and higher brain ACE were also detected in Alzheimer’s disease patients (Miners et al., 2008, 2009). In our previous studies in mice, ACE was selectively expressed in myelomonocytic cells such as monocytes, macrophages, and microglia (Bernstein et al., 2014a, b). The rationale for this experiment was 2-fold: myeloid cell ACE overexpression markedly enhances innate immune responses (Shen et al., 2007; Koronyo-Hamaoui et al., 2014; Bernstein et al., 2018), and surface expression of ACE would promote amyloid-β42 cleavage and conversion into shorter, more benign amyloid-β alloforms via ACE enzymatic activity (Hemming and Selkoe, 2005; Bernstein et al., 2014b). In fact, AD+ mice overexpressing ACE in myelomonocytes displayed minimal to no Alzheimer’s disease-like phenotype (Bernstein et al., 2014b). However, in those experiments, ACE overexpression was present throughout the life of the AD+ mice in both macrophages and microglia due to an inserted genetic modification in the ACE gene. Hence, the relative therapeutic contribution of a periodic addition of peripherally derived ACE-overexpressing monocytes and macrophages, as opposed to brain-resident microglia, has yet to be investigated. Here, we examine whether exogenous administration of bone marrow-derived CD115+ myeloid cells overexpressing ACE, either by bone marrow transplantation or by peripheral blood enrichment, can also reduce the magnitude of disease. The use of bone marrow-derived myeloid cells isolated from green fluorescent protein (GFP) mice allowed unambiguous tracing of the grafted cells’ destiny in the CNS of recipient animals; this was especially critical to distinguish clearly between the contribution of microglia and macrophages to the phenotype. Moreover, our experiments showed that ACE10 monocytes and macrophages were highly potent in clearing soluble and insoluble forms of amyloid-β42, as well as in reducing neuropathology and preserving synapses and cognitive function in murine Alzheimer’s disease models. Materials and methods Mice used in the experiments The mice selected to be recipients of bone marrow transplantation or adoptive transfer of monocytes were from the double-transgenic B6.Cg-Tg (APPSWE/PS1ΔE9)85Dbo/Mmjax hemizygous (AD+) mice strain (MMRRC stock #34832-JAX|APP/PS1) and their non-transgenic littermates (as wild-type control non-Alzheimer’s disease). These animals were purchased from Jackson Laboratories and later bred and maintained at Cedars-Sinai Medical Center. For the bone marrow transplantation experiment, we used 22 AD+ recipient mice divided into three groups as follows: BMAD+::AD+ (n = 7, four females and five males), BMWT::AD+ (n = 7, two females and five males), and BMACE10::AD+ (n = 8, three females and five males). For adoptive transfer experiments we used 30 mice, all males, divided into four groups as follows: AD+ recipients of either wild-type monocytes (MoWT) (n = 8) or MoACE10 (n = 8), PBS-injected AD+ controls (n = 7), and non-transgenic naïve wild-type mice (n = 7). The ACE10/10 donor mice strain overexpresses ACE (two alleles) in the myelomonocytic cell lineage (Supplementary Fig. 1A and B) (Bernstein et al., 2014b). The Ace genotypes used in this study were wild-type (ACEWT; n = 30) and homozygous for the targeted allele (ACE10; n = 30). The GFP donor mice were B6-TgN (β-act-EGFP) osbY01 (Okabe et al., 1997; Okwan-Duodu et al., 2010), which had been backcrossed at least 10 generations to a C57BL/6 background (Jackson Laboratory; n = 15). This line has enhanced GFP (EGFP) cDNA under the control of a chicken β-actin promoter and a cytomegalovirus enhancer. These animals are also available at Jackson Laboratory [C57BL/6-Tg(CAG-EGFP)10sb/J; #003291]. The bone marrow donor mice strains (ACE10 and GFP) were crossed to produce the genotypes GFP+AD−ACEWT (n = 76 mice), GFP+AD−ACE10 (n = 85), and GFP−AD+ACEWT (n = 4 mice) used for the in vivo experiments. Bone marrow transplantation donor mice were 12–16 weeks old (n = 11, all females). Adoptive transfer donor mice were used at the age of 8–12 weeks old (n = 154, female and males at equal numbers). All mice used for these experiments were on the genetic background of B6. All experiments were conducted in accordance with Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol. Bone marrow transplantation With their heads shielded, 2-month-old recipient AD+ mice were subjected to 9.5 Gy (950 rad) irradiation for 11 min—a dose lethal to proliferating cells. The cells were immediately reconstituted by intravenous administration of 5 × 106 donor marrow cells [isolated from 2-month-old GFP-expressing donor mice (described above)]. As irradiation of the brain significantly increases infiltration of immune cells, we protected the mice brains from irradiation with an extensive lead-shielding device that we had constructed (Supplementary Fig. 1C). After 8 weeks of bone marrow repopulation, engraftment was assessed by flow cytometry analysis of peripheral blood for GFP expression. Non-invasive retinal imaging was carried out when the mice reached 7 months of age. When they were 8 months old, the recipient mice were anaesthetized and perfused with 0.9% ice-cold saline containing 0.5 mM EDTA. Brain, eyes, and blood were collected. Retinal flatmounts were immediately isolated and fixed in 2.5% paraformaldehyde (PFA) as previously described (Koronyo-Hamaoui et al., 2011). One brain hemisphere was fixed for histology in 2.5% PFA containing 30% sucrose and the other hemisphere was snap-frozen for further analysis. Monocyte adoptive transfer After the animals had been euthanized, bone marrow cells were harvested and enriched for mononuclear cells on a Ficoll density gradient as previously described (Koronyo et al., 2015). Five to six million CD115+-MoBM cells were injected intravenously into each AD+ recipient mouse, beginning when the animal was 8 months old and continuing once a month for 3 months to ensure long-term elevated levels of transfused monocytes. Animals in the control group received intravenous injections of phosphate-buffered saline (PBS). Characterization of isolated monocytes was performed by flow cytometry. After completion of behavioural testing at 11 months old, the recipient mice were perfused, as described above, to collect tissues for analysis. Non-invasive retinal imaging After deep anaesthesia had been induced in the animals, the mouse pupils were dilated with 0.5% phenylephrine hydrochloride ophthalmic solution (Bausch and Lomb) combined with 0.5% tropicamide ophthalmic solution (Mydral, Bausch and Lomb). The retinas were imaged in vivo using the Micron III microscope with predetermined light exposure time and intensity, using identical gain and shutter modes for all animals as previously described (Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012, 2017). Images and videos of the retina were repeatedly captured at several angles to visualize a larger retinal field and eliminate non-specific reflection signals. Images were exported to ImageJ 1.46r for analysis. Behavioural tests To assess locomotor activity and cognitive functions of the treated mice, we performed open field and Barnes maze tests, as previously described (Koronyo et al., 2015). In the open field test, each mouse was placed in an individual apparatus for 60 min to record ambulatory and rearing activity. In the Barnes maze test, the mice were first trained for 4 days (acquisition-training phase), three times per day for 240 s for each trial per mouse. Following a 2-day break, the mice were retested for memory retention (retention phase, Day 7) and then short-term memory (reversal phase on Days 8 and 9) under the same conditions. Latency times and incorrect entries (errors) were recorded manually and by using a video camera (Koronyo et al., 2015). Immunohistochemistry Coronal brain sections (30 μm) and retinal flatmounts were treated for 30 min in antigen-retrieval solution (Dako S1699) prior to serum-free protein blocking (Dako X0909). Tissues were then hybridized with various primary antibodies (Table 1) overnight at 4°C. Hybridization with primary antibodies was followed by incubation with fluorophore-conjugated secondary antibodies (1 h at 37°C) (Table 1). Stained tissues were mounted using ProLong® Gold with DAPI (Molecular Probes, Life Technologies). Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labelling. Table 1 List of antibodies used in the study Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 mAb = monoclonal antibody; pAb = polyclonal antibody. * 1 μl = 1 μl per 100 000 – 1 000 000 cells. Open in new tab Table 1 List of antibodies used in the study Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 mAb = monoclonal antibody; pAb = polyclonal antibody. * 1 μl = 1 μl per 100 000 – 1 000 000 cells. Open in new tab Thioflavin-S staining For amyloid burden assessment in brain parenchyma and vasculature, sections were stained with Thioflavin S (Thio-S, 1% w/v in 70% ethanol; Sigma-Aldrich) for 10 min at room temperature, following the secondary antibody step, washed three times in 70% ethanol for 1 min each, then rinsed in distilled water before mounting. Curcumin staining Curcumin (diferuloylmethane; C21H20O6; Sigma‐Aldrich) was dissolved in 0.5 M NaOH followed by immediate dilution in PBS to a final solution of 0.1 mg/ml curcumin (pH 7.9). Brain sections were stained with curcumin solution for 10 min at room temperature, and then rinsed three times with PBS for 5 min each, as previously reported (Koronyo-Hamaoui et al., 2009). Cerebral amyloid angiopathy scoring Severity scores for the degree of CAA were defined by inspection of Thio-S-labelled brain sections using a scale of 0–4 (0 = no CAA, 1 = low, 2 = medium, 3 = high and 4 = severe CAA) (Wyss-Coray et al., 2001; Rentsendorj et al., 2018). Eighteen images encompassing three to four brain sections and covering both cortex (n = 9 images) and hippocampus (n = 9) were analysed for each animal (total of n = 22 mice, n = 6–9 per experimental group). Primary culture of bone marrow-derived macrophages Bone marrow was harvested from the femora and tibiae of wild-type or ACE10 mice donors after they had been euthanized at the age of 8–12 weeks (3–5 mice/genotype), as previously described (Rentsendorj et al., 2018). Briefly, the cells were cultured for 7 days in RPMI supplemented with 10% foetal bovine serum (FBS), 100 U/ml antibiotic, 2 mM l-glutamine, and 20 ng/ml MCSF. On the sixth day, macrophages (MΦ) were plated overnight in 24-well tissue culture plates with or without glass coverslips (105 cells/well) depending on which analysis was planned [immunocytochemistry (ICC) or ELISA]. The following day, macrophages were either unstimulated (not treated with amyloid-β) or stimulated by one of the three amyloid-β42 alloforms (100 nM and 250 nM; fibril amyloid-β42, prion rod-like amyloid-β42, oligomer amyloid-β42). MΦACE10 were analysed as compared to MΦWT for cell-surface amyloid-β42 binding; for this, the plates were chilled for 10 min on ice (0°C) followed by addition of amyloid-β42 and spun at 515 g for 1 min, followed by incubation for another 10 min at 0°C (binding – 0’ time point). MΦACE10 were also analysed for receptor expression as well as uptake of amyloid-β42 forms at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake −5, −15, −30 min). After each time point, media were collected for biochemical analysis (ELISA). The cells were rinsed with culture medium and then washed with PBS before being fixed in cold methanol (−20°C) for ICC. In parallel, another subset of MΦACE10 were treated overnight (in RPMI + 0.3% BSA) with lisinopril (1 μM) the day before plating in 24-well plates and then exposed to 100 nM fibril amyloid-β42 for 0, 5, or 30 min. Primary culture of peritoneal-derived macrophages Peritoneal macrophages were collected from the peritoneum as described previously (Okwan-Duodu et al., 2010). Briefly, the macrophages were harvested from the peritoneal cavity 4 days after a 2 ml intraperitoneal injection of 3% thioglycolate. The cells were cultured at 37°C, 5% CO2 in RPMI 1640 medium enriched with 10% foetal calf serum, 50 μM 2-ME, 0.5 mM sodium pyruvate, 10 mM HEPES buffer, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mM l-glutamine. For purification of the macrophages, peritoneal exudates were allowed to adhere for 2 h, after which non-adherent cells were removed with PBS to achieve >90% purity of macrophages. Immunocytochemistry Briefly, after serum-free protein blocking (Dako X0909), cells were hybridized with various primary antibodies (Table 1) overnight at 4°C. The following day, these cells were incubated with fluorophore-conjugated secondary antibodies (1 h at 37°C). Coverslips were mounted onto plates using ProLong® Gold with DAPI (Molecular Probes, Life Technologies). Microscopy and quantification A Karl Zeiss fluorescence microscope (Imager, Z1, ApoTome and MBF equipped) was used for microscopic analysis. For immunohistochemistry (IHC), three to six comparable coronal brain sections, originally located 150 μm apart, were used for quantitative analysis. For ICC, four to six samples were analysed. Numbers, area, and intensity of amyloid-β deposits, GFAP+ and GFP+ cells, were determined automatically by using NIH ImageJ software or an MBF stereological system. The same approach was used to analyse cytokines and other markers. Image analysis To cover the hippocampal area for synaptic analysis, 15 fields per hippocampal area per brain were taken under 100× oil objective lens and analysed (as previously described in Koronyo et al., 2015). Single optical section images at 0.25 µm intervals and 3.75 µm Zeiss ApoTome high-resolution scans were performed. Synaptic immunoreactive area and co-localization of EEA1+ with 6E10+ immunoreactive area were quantified using Puncta Analyzer as previously described (Ippolito, 2010) and ImageJ (NIH) macro and batch process. To quantify co-localized IGF1+ immunoreactive in monocytes (Mo)/MΦ (Iba1+CD45high cells), images were first compiled and analysed in Adobe Photoshop (Adobe Systems Inc., Mountain View, CA, USA). The per cent of IGF1+ foci were obtained by tracing an outline around each plaque associated with inflammation site. IGF1 (red) co-localizing with CD45 (green) was quantified by measuring the fluorescence of coincident (yellow) particles. Frequency of coincidence of IGF1+ area co-localizing with Iba+CD45high cells in cortices was plotted as percentage (%) in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 4–7 AD+ mice/group). Elongation factor The long and short axes of cells were manually measured in micrometres from microscopic images using length tools in the AxioVision Rel. 4.8 software package. At least 100 cells were examined in each experiment in which individual cells were assayed by microscopy. Quantitative Meso Scale Discovery protein assay The Meso Scale Discovery (MSD) V-PLEX proinflammatory Panel I (MSD K15048D-1) consists of highly sensitive multiplex ELISAs that quantitatively measure 10 cytokines (IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO or CXCL1, TNFα) using electrochemiluminescence detection technology. Preparation of standards/calibrators and samples The highest calibrator (Calibrator 1) was prepared by adding 1 ml of Diluent 41 to the lyophilized calibrator vial according to the manufacturer’s protocol. Seven calibrator solutions were made by first transferring 100 μl of the highest calibrator into 300 μl of Diluent 41 and then making 4-fold serial dilutions five additional times. Diluent 41 was used for the zero calibrator. Sixty microlitres of each sample was mixed with 60 µl of Diluent 41 to achieve a 2-fold dilution of each sample. Plate preparation A 1× wash buffer was made by a 20-fold dilution of the stock (supplied in the kit) using deionized water. The plate was washed with 150 µl of wash buffer three times and tapped dry. Calibrators and samples (50 µl) were pipetted into the assigned wells. The plate was incubated for 2 h at room temperature on a shaker. To make 1× detection antibodies from 50× stock, 60 μl of each detection antibody for each analyte was pipetted into 2.4 ml of Diluent 45. After the 2 h incubation, the plate was washed three times with wash buffer and tapped dry. Each well received 25 μl of detection antibody solution, after which the plate was incubated at room temperature with shaking for 2 h. To read the plate on the MSD instrument (MESO Quicklex S 120), the plate was first washed with 150 μl wash buffer three times and tapped dry, then 150 µl of 2× Read Buffer (supplied in the kit) was pipetted into each well. No incubation was needed after the read buffer was added and before reading the plate. Data analysis The calculations used to establish calibration curves and determine concentration and data analysis were carried out using MSD Workbench 4.0 software. This software fits the standard curves using a four parameter logistic fit with 1/y2 weighting. Brain/plasma amyloid-β1-42 and amyloid-β1-40 levels by ELISA Fresh-frozen brain tissues were thoroughly homogenized in PBS buffer with 0.5% Triton™ X-100 (Sigma) and 1% protease inhibitor cocktail set I (Calbiochem) and then processed as previously described (Koronyo et al., 2015). Blood was immediately centrifuged after collection (730g for 10 min at 4°C) for plasma isolation. After determination of protein concentration with the aid of the Pierce BCA Protein Assay Kit (Thermo Scientific), concentrations of amyloid-β42 (Invitrogen KHB441) in the soluble and insoluble brain protein fractions and amyloid-β40 (Invitrogen KHB3481) in the plasma were analysed using a sandwich ELISA kit (Invitrogen) as per the manufacturer’s instructions. Cell viability assay Amyloid-β42 films were dissolved in 60 mM NaOH, sonicated for 1 min in an ultra-bath sonicator, and diluted into cell culture media to the desired concentration. Aliquots of 10 μl were added to individual wells of 96-well microplates. Each well contained ∼30 000 cells in a volume of 90 μl. After an incubation lasting ∼15 h, 15 μl of MTT reagent was added to each well and the cells were incubated for 3 h at 37°C. SDS DMF HCl acetic acid stop solution (100 μl) was added to each well, and the plates were incubated overnight in the dark to ensure complete solubilization. The plates were then read using a microplate reader, and the absorbance at 570 nm (formazan product) minus the absorbance at 630 nm (background) was recorded. Controls included cells treated with NaOH only and 1 μM staurosporine (to induce apoptosis and yield maximal cell death). A minimum of six replicates were done for each treatment group, and the data from three independent experiments were combined and reported as mean ± standard error of the mean (SEM). The biological activity of amyloid-β was compared using the one-way analysis of variance Tukey test. Preparation of low molecular weight amyloid-β42 Low molecular weight (LMW) 99% HPLC purified amyloid-β42 peptide was synthesized in the UCLA Biopolymer Laboratory (Bitan and Teplow, 2005) or purchased from AnaSpec. Aggregate-free amyloid-β42 (LMW amyloid-β) was prepared as follows: Microcon YM-30 filters (EMD Millipore) were washed with 200 μl distilled deionized water. Amyloid-β was dissolved in 10% (v/v) 60 mM NaOH and 90% (v/v) 10 mM phosphate buffer, pH 7.4, at a concentration of 1 mg/ml. The solution was sonicated for 1 min and then placed into the washed 30 kDa filter. Following centrifugation at 14 000g for 20 min, the filtrate, LMW amyloid-β, was collected, and its concentration determined by optical absorption spectroscopy using a molar extinction coefficient of 1280 M−1cm−1 at 276 nm. Purified peptides were stored as lyophilizates at −20°C. Preparation of amyloid-β42 fibrils For fibrillar amyloid-β42, LMW amyloid-β42 was prepared as described above, and the peptide preparation was incubated at 100 µM with orbital shaking at 37°C until fibril formation occurred, ∼2 weeks (Koronyo et al., 2015). The presence of fibrils was confirmed by electron microscopy. Preparation of PICUP oligomeric amyloid-β42 LMW amyloid-β was stabilized to ‘freeze’ oligomers using photo-induced cross-linking of unmodified proteins (PICUP) (Bitan et al., 2001, 2003; Bitan and Teplow, 2004; Bitan, 2006) to yield a distribution of ‘oligomeric amyloid-β’. Briefly, 2 mM Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate [Ru(Bpy)3] (Aldrich) and 40 mM ammonium persulphate (APS) (Sigma) were prepared in distilled deionized water. An 18-μl aliquot of 80 μM LMW amyloid-β was placed in a PCR tube, followed by 1 μl Ru(Bpy)3 and 1 μl APS. The sample was irradiated (150 W incandescent lamp) for 1 s, and the reaction was quenched immediately with 1 M dithiothreitol. Cross-linking reagents were removed by dialysis using 3.5 kDa MWCO Slide-A-Lyzer cassettes (Pierce) against 10 mM sodium phosphate, pH 7.4. More than five changes of buffer were completed, and the protein purity and concentration were confirmed using optical absorption spectroscopy. Preparation of prion rod-like amyloid-β42 LMW amyloid-β42 was prepared as described above, and the peptide preparation was diluted to 100 μM, incubated at room temperature without agitation for 9 h, and flash frozen. Transmission electron microscopy Ten microlitres of sample were spotted onto 400-mesh carbon-coated Formvar grids (Electron Microscopy Sciences) and incubated for 2 min. Each grid was then negatively stained with a 1% (v/v) solution of uranyl acetate (Ted Pella), passed through a 0.2-μm filter, which was immediately wicked off. TEM analysis was carried out using a JEOL 1200 EX at 80 kV (Fradinger et al., 2005). Flow cytometry Brain tissues After perfusion, the mouse brains were minced and digested for 30 min with 1.5 mg/ml collagenase IV (Worthington Biochemical Corp.) and DNase I (Stem Cell Technologies) at 37°C. After centrifugation, homogenization and washing, the pellet was suspended in 70% Percoll® (GE Healthcare) and then layered under a 30%/50% Percoll® step gradient. After 30 min of centrifugation at 800g at 4°C (without break), mononuclear cells at the interface of the 70% and 50% layers were collected and washed. For surface marker staining, the cells were blocked and stained (Table 1). For intracellular staining, the cells were fixed (eBioscience; #00-8222-49), permeabilized (eBioscience: #00-8333-56), and then stained (Table 1). Cells were treated with brefeldin A to stain for secreted cytokines (eBioscience: #00-4506-51). In vitro assessment of fibril amyloid-β42 phagocytosis Primary bone marrow or peritoneal-derived CD115+ macrophages (wild-type and ACE10) were exposed to preformed 1 nM HiLyte™ Fluor 647 amyloid-β1-42 (Anaspec) fibrils. At specific time points (0, 15 and 60 min), the cells were stained and analysed by flow cytometry. Data and statistical analysis Data were analysed using GraphPad Prism v. 7.01 (GraphPad) software. Normally distributed data resulting from the IHC, ELISA, and flow cytometry analyses are expressed as means ± SEMs. One-way or two-way ANOVA with Sidak’s and Bonferroni’s post-tests was used for multiple comparisons (three or more means). The threshold for statistical significance was set at 0.05. For most behavioural testing, a power of 80% was obtained with 8–10 animals per group. For stereology or other semi- or fully quantitative analysis (i.e. q-IHC, ELISA), 6–8 mice per group have sufficient power to detect significance. Two-group comparisons were analysed using a paired or unpaired Student t-test. The statistical association between two or more variables was determined using Pearson’s correlation coefficient (r) test (GraphPad Prism) with corrections for multiple analyses. Pearson’s r indicates direction and strength of the linear relationship between two variables. Results are shown as mean ± SEM. Degrees of significance between groups are represented as follows: *P < 0.05, **P < 0.01, ***P <0 .001, and ****P < 0.0001. Data availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Results Reduced pathology in AD+ mice after partial ACE10 bone marrow replacement linked to brain and retinal monocyte recruitment In our initial experiment, we tested bone marrow transplantation in APPSWE/PS1ΔE9 double-transgenic mice (AD+ mice). Two-month-old mice (n = 22) received a high dose of radiation while placed inside a head-shielding device. Immediately afterward, the animals received an intravenous injection of either AD+ bone marrow (GFP−AD+ACEWT; n = 7, three females and four males), control bone marrow (GFP+AD−ACEWT; n = 7, two females and five males), or ACE10 bone marrow (GFP+AD−ACE10; n = 8, three females and five males) (Fig. 1A and Supplementary Fig. 1A–C). Blood analysis 8 weeks post-transplantation showed that our protocol resulted in mice with chimeric bone marrow consisting of ∼30–40% of cells from donor origin. At 7 months, the two groups with GFP-labelled bone marrow were examined using non-invasive retinal fluorescence imaging (Supplementary Fig. 1D) to track infiltration of GFP monocytes to CNS tissue using a rodent retinal Micron-III microscope (Koronyo et al., 2012, 2017). AD+ chimeric mice with BMWT had inflammatory cells surrounding amyloid-β plaques, including peripheral bone marrow-derived GFP+CD45hi monocytes (Fig. 1B). Analyses demonstrated reductions in amyloid-β plaque area (6E10+), soluble brain amyloid-β1-42 levels (ELISA), GFAP+ cells, and GFAP+ area in mice that had received ACE10 bone marrow compared to AD+ control mice (47%, 31%, 33% and 33% decreases, respectively) (Fig. 1C–F). Figure 1G and H demonstrate less cortical amyloid-β plaques stained with curcumin, a method previously described (Koronyo-Hamaoui et al., 2009, 2011; Koronyo et al., 2017), and lower cortical GFAP+ astrocytosis. Interestingly, these effects were localized in the brain as no significant differences in plasma levels of amyloid-β1-40 and amyloid-β1-42 (ELISA) or their ratios were observed between the experimental groups (Supplementary Fig. 1E). Further, no significant differences were noted between females and males. Figure 1 Open in new tabDownload slide Beneficial effects of partial ACE10 bone marrow transplantation in APPSWE/PS1ΔE9 transgenic mice. (A) Schematic illustration of experimental design and timeline. At 2 months of age, three groups of double-transgenic APPSWE/PS1ΔE9 (AD+) recipient mice (n = 22) underwent an irradiation procedure, with their heads shielded by a 3-inch-thick lead block. Mice then received a lethal dose of 950 rad irradiation for 11 min (Supplementary Fig. 1A–C). Immediately after irradiation, AD+ recipient mice received an intravenous (i.v.) injection of 5 million bone marrow cells from one of three types of donor mice: GFP−AD+ACEWT, GFP+AD−ACEWT, or GFP+AD−ACE10. The resulting BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 7–8 per group) had a partial (˜30–40%) bone marrow transplantation. The two groups of GFP-labelled bone marrow chimeric AD+ mice underwent retinal imaging at 7 months of age. Tissues were harvested for analysis from all three experimental groups when the animals were 8 months of age; the left hemisphere was used for flow cytometry, the right hindbrain for histology, and the right forebrain for protein analysis. (B) Representative fluorescence micrograph of the BMWT::AD+ chimeric mouse brain showing infiltrating monocytes (GFP+/CD45hi; arrows) near a 6E10+ amyloid-β plaque site. Scale bar = 20 μm. (C) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in brain cortices (Ctx) of BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 5–7 mice/group). (D) Sandwich ELISA analysis of cerebral soluble amyloid-β42 levels in AD+ chimeric mice (n = 6–8 mice/group). (E) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte cell count in AD+ chimeric mice (n = 6–8 mice/group). (F) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte area in AD+ chimeric mice (n = 7 mice/group). (G) Representative micrographs of cortical amyloid-β plaques stained with curcumin in BMWT::AD+ versus BMACE10::AD+ chimeric mice. (H) Representative micrographs of cortical GFAP+ astrocytes in the same experimental groups. Scale bar = 50 μm (for G and H). (I) Flow cytometry analysis for cerebral infiltrating GFP+ monocytes in AD+ chimeric mice. Combinations of markers were used to identify microglia (CD11b+CD45int.-lowGFP) versus infiltrating monocyte subsets (CD11b+CD45hiLy6ChiGFP+). (J) Representative flow cytometry map analysis showing increased infiltration of CD11bhiCD45hiLy6ChiGFP+ ACE10 versus wild-type monocytes in AD+ chimeric mice. (K and L) Quantitative flow cytometry analyses of cerebral inflammatory biomarkers in chimeric AD+ mice following partial bone marrow transplantation. (K) Percentage of GFP+CD11bhiCD45hiLy6ChiF4/80low monocytes (n = 5–6 mice/group), (L) TNFα expression in infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi Mφ (n = 4–5 mice/group). (M) Fluorescent micrographs of cortical brain regions stained against IGF1 (red) combined with Iba1 (white) CD45 (green) and nuclei (blue) in BMWT::AD+ versus BMACE10::AD+ chimeric mice. Scale bars = 10 μm. Brain-infiltrating Iba1+CD45hi Mo/Mφ abundantly expressed IGF1 in BMACE10::AD+ chimeric mice (bottom). (N) Quantitative IHC of % IGF1+ area co-localized with Mo/Mφ in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 7 mice/group). (O–Q) Representative non-invasive retinal fluorescence imaging of infiltrating GFP+ bone marrow cells in living AD+ chimeric mice. (O) A control AD+ mouse that underwent bone marrow transplantation of GFP−BMAD+ (GFP−BMAD+::AD+). (P) Retinal fluorescent images from a GFP+BMWT::AD+ mouse (P’, enlarged image). (Q) Retinal fluorescent images from a GFP+BMACE10::AD+ mouse (arrows indicate GFP+ bone marrow cells). (R) Representative microscopic image obtained during the histological examination of a retina extracted from a BMACE10::AD+ mouse whose retina had been previously imaged in vivo; ex vivo staining with GFP (green), CD45 (red), 4G8 (cyan), and DAPI (blue), validating the homing of GFP+ACE10 monocytes to retinal amyloid-β plaques. Scale bar = 5 μm. Data from an individual mouse (filled circles for males and clear circles for females) as well as group mean ± SEM are shown. Fold increase and percentage decreases compared to control groups are shown in green. *P < 0.05, **P < 0.01. ns = non-significant, using one-way ANOVA and Bonferroni’s post-test for three group comparisons. For two-group comparisons, paired or unpaired two-tailed Student t-tests were used. Aβ = amyloid-β; FS = forward scatter; SS = side scatter. Figure 1 Open in new tabDownload slide Beneficial effects of partial ACE10 bone marrow transplantation in APPSWE/PS1ΔE9 transgenic mice. (A) Schematic illustration of experimental design and timeline. At 2 months of age, three groups of double-transgenic APPSWE/PS1ΔE9 (AD+) recipient mice (n = 22) underwent an irradiation procedure, with their heads shielded by a 3-inch-thick lead block. Mice then received a lethal dose of 950 rad irradiation for 11 min (Supplementary Fig. 1A–C). Immediately after irradiation, AD+ recipient mice received an intravenous (i.v.) injection of 5 million bone marrow cells from one of three types of donor mice: GFP−AD+ACEWT, GFP+AD−ACEWT, or GFP+AD−ACE10. The resulting BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 7–8 per group) had a partial (˜30–40%) bone marrow transplantation. The two groups of GFP-labelled bone marrow chimeric AD+ mice underwent retinal imaging at 7 months of age. Tissues were harvested for analysis from all three experimental groups when the animals were 8 months of age; the left hemisphere was used for flow cytometry, the right hindbrain for histology, and the right forebrain for protein analysis. (B) Representative fluorescence micrograph of the BMWT::AD+ chimeric mouse brain showing infiltrating monocytes (GFP+/CD45hi; arrows) near a 6E10+ amyloid-β plaque site. Scale bar = 20 μm. (C) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in brain cortices (Ctx) of BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 5–7 mice/group). (D) Sandwich ELISA analysis of cerebral soluble amyloid-β42 levels in AD+ chimeric mice (n = 6–8 mice/group). (E) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte cell count in AD+ chimeric mice (n = 6–8 mice/group). (F) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte area in AD+ chimeric mice (n = 7 mice/group). (G) Representative micrographs of cortical amyloid-β plaques stained with curcumin in BMWT::AD+ versus BMACE10::AD+ chimeric mice. (H) Representative micrographs of cortical GFAP+ astrocytes in the same experimental groups. Scale bar = 50 μm (for G and H). (I) Flow cytometry analysis for cerebral infiltrating GFP+ monocytes in AD+ chimeric mice. Combinations of markers were used to identify microglia (CD11b+CD45int.-lowGFP) versus infiltrating monocyte subsets (CD11b+CD45hiLy6ChiGFP+). (J) Representative flow cytometry map analysis showing increased infiltration of CD11bhiCD45hiLy6ChiGFP+ ACE10 versus wild-type monocytes in AD+ chimeric mice. (K and L) Quantitative flow cytometry analyses of cerebral inflammatory biomarkers in chimeric AD+ mice following partial bone marrow transplantation. (K) Percentage of GFP+CD11bhiCD45hiLy6ChiF4/80low monocytes (n = 5–6 mice/group), (L) TNFα expression in infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi Mφ (n = 4–5 mice/group). (M) Fluorescent micrographs of cortical brain regions stained against IGF1 (red) combined with Iba1 (white) CD45 (green) and nuclei (blue) in BMWT::AD+ versus BMACE10::AD+ chimeric mice. Scale bars = 10 μm. Brain-infiltrating Iba1+CD45hi Mo/Mφ abundantly expressed IGF1 in BMACE10::AD+ chimeric mice (bottom). (N) Quantitative IHC of % IGF1+ area co-localized with Mo/Mφ in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 7 mice/group). (O–Q) Representative non-invasive retinal fluorescence imaging of infiltrating GFP+ bone marrow cells in living AD+ chimeric mice. (O) A control AD+ mouse that underwent bone marrow transplantation of GFP−BMAD+ (GFP−BMAD+::AD+). (P) Retinal fluorescent images from a GFP+BMWT::AD+ mouse (P’, enlarged image). (Q) Retinal fluorescent images from a GFP+BMACE10::AD+ mouse (arrows indicate GFP+ bone marrow cells). (R) Representative microscopic image obtained during the histological examination of a retina extracted from a BMACE10::AD+ mouse whose retina had been previously imaged in vivo; ex vivo staining with GFP (green), CD45 (red), 4G8 (cyan), and DAPI (blue), validating the homing of GFP+ACE10 monocytes to retinal amyloid-β plaques. Scale bar = 5 μm. Data from an individual mouse (filled circles for males and clear circles for females) as well as group mean ± SEM are shown. Fold increase and percentage decreases compared to control groups are shown in green. *P < 0.05, **P < 0.01. ns = non-significant, using one-way ANOVA and Bonferroni’s post-test for three group comparisons. For two-group comparisons, paired or unpaired two-tailed Student t-tests were used. Aβ = amyloid-β; FS = forward scatter; SS = side scatter. To characterize the types of infiltrating inflammatory cells within AD+ mouse brains, flow cytometry was used to quantitate brain inflammatory populations as indicated in Fig. 1I–L and Supplementary Fig. 1F–I. Side scatter and forward scatter, along with biomarkers of haematopoietic myelomonocytic cells such as CD11b, CD45, Ly6C, and F4/80, were identified and quantified in the brain within the GFP-negative (endogenous cells) versus GFP-positive (bone marrow-derived donor cells) populations (Fig. 1I and J). Furthermore, analyses of the expression of the three cytokines TNFα, TGFβ, and IL10 within these inflammatory cells in the brain were included. These analyses showed the presence of both GFP+ donor monocytes in the brains of AD+ animals, with a nearly 3-fold increase in ACE10 compared to ACEWT monocytes (CD11b+CD45hiLy6Cint/hi; Fig. 1K; P = 0.0101), while the microglia populations (GFP−CD11b+CD45int/lowLy6Clow) were unchanged (data not shown). Given the importance of TNFα in the induction of pro-inflammatory macrophage polarization and the pathogenesis of Alzheimer’s disease, we examined TNFα expression by both GFP−CD11b+CD45low-int microglia and brain-infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi macrophages (Fig. 1L and Supplementary Fig. 1F and G). With regards to microglia, there was no difference in TNFα in any of the groups of mice (Supplementary Fig. 1F). In contrast, there was a significant and substantial 62% decrease in TNFα expression in ACE10 macrophages in those AD+ animals that had received GFP+ACE10 bone marrow rather than GFP+ACEWT bone marrow (P < 0.05; Fig. 1L). Further, our flow cytometry analysis into the chimeric AD+ACE10 mouse brains allowed us to compare TNFα in cerebral macrophages from endogenous, non-GFP (wild-type for ACE expression) versus donor marrow, GFP+ (with overexpression of ACE) sources in individual mice (Supplementary Fig. 1G). We found that those macrophages overexpressing ACE produced 58% less cerebral TNFα (P < 0.05). No significant differences were detected in TGFβ and IL10 levels in cerebral macrophages of chimeric AD+ mice (Supplementary Fig. 1H and I). To investigate cerebral ACE10 macrophage phenotype further, we assessed insulin-like growth factor 1 (IGF1), associated with anti-inflammatory and neuro-regenerative functions (Gasparini and Xu, 2003; Butovsky et al., 2006; Koronyo-Hamaoui et al., 2009; Han et al., 2016; Bianchi et al., 2017; Spadaro et al., 2017; Vannella and Wynn, 2017) in the brains of chimeric AD+ACE10 mice compared to control mice (Fig. 1M and N and Supplementary Fig. 1J–O). Our immunohistochemistry data demonstrate a consistent 2.3-fold increase in cerebral IGF1 expression, and further, a substantial 2.5-fold increase surrounding Thio-S+ amyloid-β plaques in chimeric AD+ACE10 mice (Supplementary Fig. 1K and L; P < 0.05 and 0.0001). Moreover, cerebral IGF1 was predominantly expressed by Iba1+CD45high brain-infiltrating monocytes and macrophages, with a significant 3.2-fold elevation of IGF1 expression in brain macrophages in chimeric AD+ACE10 mice (Fig. 1M and N and Supplementary Fig. 1M and N; P < 0.01). A tight correlation was observed between cerebral IGF1 immunoreactive area and co-localized IGF1 within macrophages (Supplementary Fig. 1 O; Pearson’s r = 0.92, P < 0.0001), suggesting that infiltrating monocyte/macrophages are the main source of IGF1 in these chimeric AD+ mice. Thus, there appears to be a fundamental difference between wild-type and ACE10 macrophages in their responses to an identical inflammatory environment induced by Alzheimer’s disease in the brain. Figure 1 O–Q shows non-invasive retinal images from live AD+ mice that had received bone marrow transplantation with either non-GFP AD+ACEWT (Fig. 1 O), GFP+-AD−ACEWT (Fig. 1P and P’), or GFP+-AD−ACE10 (Fig. 1Q). Numerous macrophages within the retina were observed (Fig. 1P and Q). Supplementary Videos 1 and 2 demonstrate the ability to track individual GFP+ immune cells derived from wild-type and ACE10 bone marrow, respectively, in the live retina of an Alzheimer’s disease model mouse. Further, detailed histological examination of ex vivo retinal immunohistochemistry imaging documents homing of ACE10 macrophages to amyloid-β plaque (Fig. 1R). This was seen far more consistently in those animals that had received bone marrow from ACE10 donors than in animals that had received bone marrow from an ACEWT origin (Fig. 1P and Q). Synaptic and cognitive preservation following peripheral blood enrichment of CD115+ ACE10 monocytes Another more translational strategy that we used to assess the relative contribution and potential therapeutic effect of ACE10 monocytes involved peripheral blood enrichment with CD115+ monocytes. CD115 is the product of the Csf1r gene; monocytes that express this marker are often termed inflammatory monocytes and are recruited to inflamed sites to mediate tissue repair (Valero et al., 2017). Previous work from our laboratory has established that adoptively transferred CD115+ wild-type monocytes in AD+ mice directly ameliorate amyloid-β neuropathology and lead to reduced neuroinflammation and preservation of cognitive function (Koronyo et al., 2015). Our current findings demonstrate that ACE10 bone marrow can further reduce the neuropathology in AD+ mice more than ACEWT bone marrow; hence, the next step was to test the effects of blood enrichment with CD115+ ACE10 monocytes (Fig. 2). The experimental procedure and timeline for adoptive transfer studies can be found in Fig. 2A–D (n = 23 AD+ experimental mice and n = 7 non-transgenic naïve wild-type littermates; all males). Controls for this experiment were recipient AD+ mice that had received monthly intravenous injections of PBS (vehicle). At 11 months, open field and Barnes maze behavioural tests were performed (Fig. 2E–K and Supplementary Fig. 2A) as previously described (Bernstein et al., 2014b; Koronyo et al., 2015). There were no differences in locomotor activity, neither rearing nor ambulatory movements, between any of the groups (Fig. 2E and F). Substantial differences in performance in the Barnes cognitive test, both in incorrect entries and latency times, were recorded during the training phase of the study and during the long-term memory retention and reversal phases (Fig. 2G–K and Supplementary Fig. 2A–C). Adoptive transfer of CD115+ cells enhanced overall cognitive functions, with ACE10 monocytes consistently showing a trend of extended improvement. Figure 2 Open in new tabDownload slide Cognitive preservation and restricted pathology following adoptive transfer of a CD115+ ACE10 monocyte subset in AD+ mice transgenic mice. (A–C) Schematic representation of experimental procedure and treatment groups. (A) CD115+ monocytes (MoBM) were isolated from the bone marrow of GFP+ donor mice and enriched by MACS microbeads and an anti-CD115 antibody column sorting procedure. (B) MoBM were then intravenously (i.v.) injected into the tail vein of AD+ recipient mice (n = 8 mice/group, all males). (C) Additional control groups included naïve wild-type (WT) mice and AD+ mice injected with PBS (n = 7 mice/group, all males). (D) Schematic timeline of the in vivo preclinical experiment. Pre-symptomatic AD+ mice exhibiting neuropathology at 8 months of age received monthly injections of 5–6 million GFP+MoWT or GFP+MoACE10 or PBS for 3 months (immunization regimen indicated by green arrows). At 11 months, mice underwent behavioural tests followed by tissue collection and analysis when 12 months of age. (E and F) Open field test in all AD+ treatment and naïve wild-type groups measuring: (E) ambulatory and (F) rearing activity. (G–K) Cognitive functions assessed by the Barnes maze test in both monocyte-treated groups as compared to the control PBS-injected group and naïve cognitively normal wild-type group (n = 6–8 mice/group). Incorrect entries (errors) for the following: (G) acquisition-training phase (Days 1–4), (H) memory retention (Day 7), (I) reversal phase (Days 8 or 9), and (J and K) memory test at Day 9. (J) Errors and (K) escape latency (s). (L) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in the hippocampus (HC), cingulate cortex (CC), and total brain in MoWT- and MoACE10-treated versus PBS-injected AD+ mice (n = 7–8 mice/group). (M) CAA score assessed as vascular Thio-S+ in AD+ mice (n = 6–8 mice/group). Data from an individual mouse as well as group mean ± SEM are shown. (N) Quantitative IHC analysis of hippocampal pre-(VGluT1+) and postsynaptic (PSD95+) areas in MoACE10-treated mice compared to PBS-injected control AD+ and naïve wild-type mice (n = 6–8 mice/group). Data from individual mice, lower and upper quartiles (as lower and upper horizontal lines in box), median (midline within box), and minimum/maximum values (whiskers), are shown. Percentage increase and decrease compared to control groups are shown in green. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Significance between two-groups by unpaired two-tailed Student t-test. IR = immunoreactive. Figure 2 Open in new tabDownload slide Cognitive preservation and restricted pathology following adoptive transfer of a CD115+ ACE10 monocyte subset in AD+ mice transgenic mice. (A–C) Schematic representation of experimental procedure and treatment groups. (A) CD115+ monocytes (MoBM) were isolated from the bone marrow of GFP+ donor mice and enriched by MACS microbeads and an anti-CD115 antibody column sorting procedure. (B) MoBM were then intravenously (i.v.) injected into the tail vein of AD+ recipient mice (n = 8 mice/group, all males). (C) Additional control groups included naïve wild-type (WT) mice and AD+ mice injected with PBS (n = 7 mice/group, all males). (D) Schematic timeline of the in vivo preclinical experiment. Pre-symptomatic AD+ mice exhibiting neuropathology at 8 months of age received monthly injections of 5–6 million GFP+MoWT or GFP+MoACE10 or PBS for 3 months (immunization regimen indicated by green arrows). At 11 months, mice underwent behavioural tests followed by tissue collection and analysis when 12 months of age. (E and F) Open field test in all AD+ treatment and naïve wild-type groups measuring: (E) ambulatory and (F) rearing activity. (G–K) Cognitive functions assessed by the Barnes maze test in both monocyte-treated groups as compared to the control PBS-injected group and naïve cognitively normal wild-type group (n = 6–8 mice/group). Incorrect entries (errors) for the following: (G) acquisition-training phase (Days 1–4), (H) memory retention (Day 7), (I) reversal phase (Days 8 or 9), and (J and K) memory test at Day 9. (J) Errors and (K) escape latency (s). (L) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in the hippocampus (HC), cingulate cortex (CC), and total brain in MoWT- and MoACE10-treated versus PBS-injected AD+ mice (n = 7–8 mice/group). (M) CAA score assessed as vascular Thio-S+ in AD+ mice (n = 6–8 mice/group). Data from an individual mouse as well as group mean ± SEM are shown. (N) Quantitative IHC analysis of hippocampal pre-(VGluT1+) and postsynaptic (PSD95+) areas in MoACE10-treated mice compared to PBS-injected control AD+ and naïve wild-type mice (n = 6–8 mice/group). Data from individual mice, lower and upper quartiles (as lower and upper horizontal lines in box), median (midline within box), and minimum/maximum values (whiskers), are shown. Percentage increase and decrease compared to control groups are shown in green. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Significance between two-groups by unpaired two-tailed Student t-test. IR = immunoreactive. In the training phase, AD+ mice that had received CD115+ monocytes with either ACEWT or ACE10 showed a pattern of maze learning that was essentially identical to that of wild-type mice (Fig. 2G and Supplementary Fig. 2A). In contrast, AD+ mice that had received PBS displayed impaired training compared to wild-type mice. In the long-term memory retention test (Day 7), during the reversal phase (Day 8), and in the spatial memory analysis at Day 9, we observed significant differences between mice receiving CD115+ ACE10 cells and those receiving PBS controls. MoACE10-treated mice displayed a pattern of learning equivalent to that of wild-type mice, which was also superior to recipients of the MoWT cells (Fig. 2H–K and Supplementary Fig. 2B and C). Immunohistochemical analysis of 6E10+ plaque area in the hippocampus, cingulate cortex, and total brain shows that AD+ mice that had received an infusion of CD115+ (predominantly ACE10) monocytes had fewer hippocampal and total brain plaques than AD+ mice that had received only PBS vehicle (P < 0.05, Fig. 2L and Supplementary Fig. 2D–F). Analysis of vascular Thio-S+ amyloid-β burden calculated as CAA score (Rentsendorj et al., 2018) revealed significant 56% and 82% reductions in CAA scores in AD+ mice recipients of both BMWT and BMACE10, respectively, compared to the control group (Fig. 2M and Supplementary Fig. 2I–M; P < 0.0001). A significant reduction in CAA scores was further noted in recipients of BMACE10 versus BMWT in hippocampal and cortical regions (Fig. 2M and Supplementary Fig. 2L and M; P < 0.05). Analysis of synaptic biomarkers indicated a significant preservation of both pre- (VGluT1) and post- (PSD-95) synapses by blood enrichment with CD115+ ACE10 monocytes, promoting synaptic protection (Fig. 2N and Supplementary Fig. 2G and H; P < 0.05–P < 0.001). Enhanced binding and uptake of pathological amyloid-β42 conformers and surface expression of scavenger receptors in ACE10 macrophages Next, we characterized ACE-overexpressing macrophages for their ability to recognize, bind, internalize, and process defined structures of amyloid-β associated with Alzheimer’s disease neuropathology. To this end, we performed an experiment in which bone marrow cells were harvested from wild-type and ACE10 mice and then cultured for 6 days with MCSF to generate mature macrophages (Fig. 3A). On Day 7, these cells were mixed with well-defined and characterized populations of amyloid-β1-42 fibrils, amyloid-β1-42 prion rod-like structures, or cross-linked-stabilized soluble amyloid-β1-42 oligomers (Fig. 3A and B). The identity of amyloid-β products was confirmed by SDS-PAGE and TEM (Fig. 3B and C). Another control group for this experiment included untreated cells, which were not exposed to amyloid-β. Figure 3D shows surface binding of fibril amyloid-β42 to a class A type 1 scavenger receptor, Scara1, on the cell membrane of ACE10 macrophages (0 min time point); macrophages were immunostained for anti-amyloid-β (6E10), Scara1 (CD204), and nuclei (DAPI; Fig. 3D). Figure 3 Open in new tabDownload slide Enhanced surface recognition and uptake of diverse amyloid-β42 conformers by ACE10 macrophages. (A) Design and timeline of in vitro. Bone marrow was isolated from young wild-type (WT) and ACE10 mice (8–12 weeks old) and cultured for 6 days in MCSF-enriched media, generating primary macrophage cultures (MφWT and MφACE10, respectively). On Day 7, Mφ were either unstimulated (Un) or stimulated by one of the three amyloid-β forms: fibrils (fAβ42), prion rod-like structures (pAβ42), or cross-linked oligomers (oAβ42). MφACE10 were analysed and compared to control MφWT for cell surface amyloid-β42 binding (pre-chilled on ice; binding – 0 min) and receptor expression as well as uptake of amyloid-β42 species at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake: 5, 15, 30 min). Cells and media were isolated and further analysed using various methods including ICC, flow cytometry (FC), and quantitative protein analysis. (B) Forms of non-cross-linked (non-XL) and cross-linked (oAβ) amyloid-β42. Oligomers as large as an octamer are observed. (C) Negative stain TEM micrographs of amyloid-β42 assemblies. Left: Fibrillar amyloid-β (fAβ42) displayed long fibrils of ˜10 nm diameter with a twisted morphology typically comprising two smaller filaments. Middle: Prion rod-like amyloid-β42 (pAβ42) displayed short (<300 nm) straight rods comprising multiple filaments (blue arrows). Narrower (˜5 nm) rod-like structures appeared to comprise twisted double filaments (black arrows). The assembly indicated by the red arrow is shown in the inset at higher magnification. Right: Cross-linked oligomers (oAβ42) displayed mainly quasi-spherical assemblies (white arrows) with diameters of 5–8 nm. The assembly indicated by the red arrow is shown in the inset at higher magnification. A rare rod-like structure (yellow asterisk) was also observed. Scale bars = 100 nm or 50 nm (insets). (D) Representative fluorescence micrograph of MφACE10 prestimulated with fibril amyloid-β42 on ice (at the 0-min time point) and immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Abundance of co-surface labelling of Scara1+ and fibril amyloid-β42 (yellow spots) on the MφACE10 cell body and processes was detected, indicating large amounts of fibril amyloid-β42 bound to surface Scara1. (E) Quantitative ICC analysis of a 6E10+ immunoreactive (IR) area for cell surface binding of the three amyloid-β42 species (250 nM each) at the 0-min time point in MφACE10 versus MφWT. (F) Quantitative ICC analysis of surface Scara1 expression in ACE10 versus wild-type Mφ that were either untreated or in the presence of different amyloid-β42 forms at the 0-min time point. (G) Quantitative ICC analysis of TREM2 expression in ACE10 versus wild-type Mφ following prestimulation by the different forms of amyloid-β42 (250 nM each). (H) Representative fluorescence micrograph of MφWT and MφACE10 cells that were incubated for 5 min with 250 nM fibril amyloid-β42 and later immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Amyloid-β42 fibrils are seen in intracellular vesicles within these phagocytic cells (white arrow). (I) Panel of representative fluorescence micrographs of MφWT and MφACE10 cells following a 15-min incubation with three different amyloid-β42 forms immunolabelled for scavenger receptor type B CD36 (purple), human amyloid-β (6E10; green), and nuclei (blue). (I’) Separated single channel (cy2) for intracellular amyloid-β42 uptake. (J) Quantitative ICC analysis of CD36 expression (μm2/cell number) in ACE10 versus wild-type Mφ that were either untreated or exposed to different amyloid-β42 forms (250 nM) for 15 min. (K) Quantitative ICC analysis of 6E10+ immune-reactive area (μm2/cell number) for intracellular uptake of the three amyloid-β42 species (250 nM) for 15 min by MφACE10 versus MφWT. (L and M) Quantitative ICC analysis of (L) CD36 expression and (M) amyloid-β uptake by untreated versus ACE-inhibitor pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA) MφACE10, following exposure to 100 nM fibril amyloid-β42 for either 0, 5 or 30 min. (N–Q) Individual correlation analysis of the following: (N and O) surface fibril amyloid-β42 and Scara1 immunoreactive area at the binding time point in (N) MφWT and (O) MφACE10 (n =15 wells); (P and Q) surface fibril/oligomer amyloid-β42 and CD36 expression in MφACE10 following a 15-min exposure to either fibril amyloid-β42 or oligomer amyloid-β42. (R) Representative peak analysis in percentage mean fluorescence intensity (MFI) of MφACE10 uptake of HiLyte Fluor 647-labelled fibril amyloid-β42 at the 0-, 5-and 15-min time points. (S) Representative flow cytometry images of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI at the 15-min time point by F4/80+-MφWT versus -MφACE10. (T and U) Quantitative flow cytometry analysis of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI uptake at the 0-, 5- and 15-min time points by (T) MφWT or MφACE10 and by (U) untreated and lisinopril pretreated MφACE10. The group mean ± SEM as well as the fold reduction or increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s or Sidak’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Figure 3 Open in new tabDownload slide Enhanced surface recognition and uptake of diverse amyloid-β42 conformers by ACE10 macrophages. (A) Design and timeline of in vitro. Bone marrow was isolated from young wild-type (WT) and ACE10 mice (8–12 weeks old) and cultured for 6 days in MCSF-enriched media, generating primary macrophage cultures (MφWT and MφACE10, respectively). On Day 7, Mφ were either unstimulated (Un) or stimulated by one of the three amyloid-β forms: fibrils (fAβ42), prion rod-like structures (pAβ42), or cross-linked oligomers (oAβ42). MφACE10 were analysed and compared to control MφWT for cell surface amyloid-β42 binding (pre-chilled on ice; binding – 0 min) and receptor expression as well as uptake of amyloid-β42 species at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake: 5, 15, 30 min). Cells and media were isolated and further analysed using various methods including ICC, flow cytometry (FC), and quantitative protein analysis. (B) Forms of non-cross-linked (non-XL) and cross-linked (oAβ) amyloid-β42. Oligomers as large as an octamer are observed. (C) Negative stain TEM micrographs of amyloid-β42 assemblies. Left: Fibrillar amyloid-β (fAβ42) displayed long fibrils of ˜10 nm diameter with a twisted morphology typically comprising two smaller filaments. Middle: Prion rod-like amyloid-β42 (pAβ42) displayed short (<300 nm) straight rods comprising multiple filaments (blue arrows). Narrower (˜5 nm) rod-like structures appeared to comprise twisted double filaments (black arrows). The assembly indicated by the red arrow is shown in the inset at higher magnification. Right: Cross-linked oligomers (oAβ42) displayed mainly quasi-spherical assemblies (white arrows) with diameters of 5–8 nm. The assembly indicated by the red arrow is shown in the inset at higher magnification. A rare rod-like structure (yellow asterisk) was also observed. Scale bars = 100 nm or 50 nm (insets). (D) Representative fluorescence micrograph of MφACE10 prestimulated with fibril amyloid-β42 on ice (at the 0-min time point) and immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Abundance of co-surface labelling of Scara1+ and fibril amyloid-β42 (yellow spots) on the MφACE10 cell body and processes was detected, indicating large amounts of fibril amyloid-β42 bound to surface Scara1. (E) Quantitative ICC analysis of a 6E10+ immunoreactive (IR) area for cell surface binding of the three amyloid-β42 species (250 nM each) at the 0-min time point in MφACE10 versus MφWT. (F) Quantitative ICC analysis of surface Scara1 expression in ACE10 versus wild-type Mφ that were either untreated or in the presence of different amyloid-β42 forms at the 0-min time point. (G) Quantitative ICC analysis of TREM2 expression in ACE10 versus wild-type Mφ following prestimulation by the different forms of amyloid-β42 (250 nM each). (H) Representative fluorescence micrograph of MφWT and MφACE10 cells that were incubated for 5 min with 250 nM fibril amyloid-β42 and later immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Amyloid-β42 fibrils are seen in intracellular vesicles within these phagocytic cells (white arrow). (I) Panel of representative fluorescence micrographs of MφWT and MφACE10 cells following a 15-min incubation with three different amyloid-β42 forms immunolabelled for scavenger receptor type B CD36 (purple), human amyloid-β (6E10; green), and nuclei (blue). (I’) Separated single channel (cy2) for intracellular amyloid-β42 uptake. (J) Quantitative ICC analysis of CD36 expression (μm2/cell number) in ACE10 versus wild-type Mφ that were either untreated or exposed to different amyloid-β42 forms (250 nM) for 15 min. (K) Quantitative ICC analysis of 6E10+ immune-reactive area (μm2/cell number) for intracellular uptake of the three amyloid-β42 species (250 nM) for 15 min by MφACE10 versus MφWT. (L and M) Quantitative ICC analysis of (L) CD36 expression and (M) amyloid-β uptake by untreated versus ACE-inhibitor pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA) MφACE10, following exposure to 100 nM fibril amyloid-β42 for either 0, 5 or 30 min. (N–Q) Individual correlation analysis of the following: (N and O) surface fibril amyloid-β42 and Scara1 immunoreactive area at the binding time point in (N) MφWT and (O) MφACE10 (n =15 wells); (P and Q) surface fibril/oligomer amyloid-β42 and CD36 expression in MφACE10 following a 15-min exposure to either fibril amyloid-β42 or oligomer amyloid-β42. (R) Representative peak analysis in percentage mean fluorescence intensity (MFI) of MφACE10 uptake of HiLyte Fluor 647-labelled fibril amyloid-β42 at the 0-, 5-and 15-min time points. (S) Representative flow cytometry images of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI at the 15-min time point by F4/80+-MφWT versus -MφACE10. (T and U) Quantitative flow cytometry analysis of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI uptake at the 0-, 5- and 15-min time points by (T) MφWT or MφACE10 and by (U) untreated and lisinopril pretreated MφACE10. The group mean ± SEM as well as the fold reduction or increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s or Sidak’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Quantitative ICC analysis indicates substantial 4.9-fold and 2-fold increases of surface fibril amyloid-β42 and oligomer amyloid-β42 binding, respectively, in ACE10 macrophages as compared with wild-type macrophages (Fig. 3E; P = 0.001 and P = 0.02, respectively). Membrane scavenger receptors such as Scara1, class B scavenger receptor CD36, triggering receptor expressed on myeloid cells 2 (TREM2), and CD163 have been implicated in macrophage activation and increased phagocytosis of amyloid-β in Alzheimer’s disease (Hickman et al., 2008; Yamanaka et al., 2012; Frenkel et al., 2013; Pey et al., 2014; Kim et al., 2017). We observed significant increases in baseline Scara1, TREM2, CD163, and CD36 surface expression in untreated ACE10 versus wild-type macrophages (Fig. 3F, Supplementary Fig. 3A and B and Supplementary Table 1; P < 0.01–0.0001). These receptors have been associated with Alzheimer’s disease and specifically facilitate amyloid-β uptake by microglia and/or macrophages but have never been studied in ACE10 macrophages or in response to defined and stabilized populations of amyloid-β42 oligomers. We also found that surface expression of TREM2 and Scara1 was increased in ACE10 macrophages at the 0-min time point and 5 min after exposure to fibril amyloid-β42 (Fig. 3F–H and Supplementary Fig. 3C; P < 0.05–0.0001) but not in response to amyloid-β42 prion rod-like structures or soluble oligomers (Fig. 3F and G). As early as 5 min after contact and phagocytosis of fibril amyloid-β42, intracellular amyloid-β42 staining was visible in the cell processes and soma of wild-type macrophages, and more prominently in ACE10 macrophages (Fig. 3H). Interestingly, baseline MHC-II expression was lower by 49% in the ACE10 macrophages (Supplementary Table 1; P < 0.01). A summary of key in vitro findings comparing ACE10 and wild-type macrophages can be found in Supplementary Table 1. To assess the macrophages’ ability to uptake amyloid-β42 species at later time points (15, 30, and 60 min), we conducted a series of experiments presented in Fig. 3I–U, Supplementary Table 1, and Supplementary Fig. 3. Figure 3I displays the major differences detected in macrophage uptake of different amyloid-β42 conformers after 15 min of exposure, with fibrils being the most efficiently phagocytosed and soluble oligomers the least engulfed by these cells (Supplementary Fig. 3D–F). Quantitative ICC analysis shows that surface CD36 expression by ACE10 macrophages was substantially and consistently increased as compared to wild-type macrophages (Fig. 3J; P < 0.0001), along with increased intracellular amyloid-β42 uptake 15 min after exposure to all three forms of amyloid-β (Fig. 3K; P < 0.05–0.0001). Moreover, the expression of CD36 as well as the uptake of fibril amyloid-β42 at 5 and 30 min were found to be dependent on ACE catalytic activity, since overnight treatment with the ACE inhibitor lisinopril significantly decreased both (Fig. 3L and M). Interestingly, in ACE10 macrophages treated with lisinopril, CD36 expression was decreased and fibril amyloid-β42 uptake increased over time during a 30-min culture period, while remaining substantially lower compared to their values in the enzymatically uninhibited ACE10 macrophages (Fig. 3L and M). Supplementary Table 1 summarizes the main findings on amyloid-β surface binding and uptake as well as the expression of scavenger receptors, the immune profile, and the morphology of wild-type versus ACE10 macrophages. Analysis of discrete wild-type and ACE10 macrophage populations to determine potential associations between amyloid-β42 binding/uptake and surface expression of the scavenger receptors Scara1 and CD36, using Pearson’s r correlations, revealed strong and direct relationships in both cell genotypes (Fig. 3N and O; r = 0.73, P = 0.0022 for wild-type; r = 0.71, P = 0.003 for ACE10, for Scara1). Further, there were direct correlations between amyloid-β42 fibril and amyloid-β42 oligomer uptake after a 15-min incubation and the expression level of CD36 in ACE10 macrophages (Fig. 3P and Q; r = 0.83 and P = 0.001 for fibril amyloid-β42; r = 0.42, P = 0.038 for oligomer amyloid-β42). The strong correlation between CD36 expression and amyloid-β uptake was also true for prion rod-like amyloid-β42 uptake after 15 min in both wild-type and ACE10 macrophages (Supplementary Fig. 3K and L; r = 0.83 and 0.76, respectively, and P < 0.0001). Extended correlations are shown in Supplementary Fig. 3G–L. Using quantitative flow cytometry analysis, we validated increased binding and uptake of fluorescently labelled HiLyte 647-fibril amyloid-β42 in ACE10 versus wild-type macrophages, from both bone marrow and peritoneal sources, as well as reversal effects following inhibition of ACE catalytic domains (Fig. 3R–U and Supplementary Fig. 3M–P). Flow cytometry-based quantitative analyses confirmed the aforementioned ICC analysis and demonstrated that the rate of amyloid-β42 fibril phagocytosis by ACE10 macrophages is significantly faster than that by wild-type macrophages in the first 5, 15 and 60 min of uptake assays (Fig. 3T and Supplementary Fig. 3M–P). Interestingly, the ACE inhibitor lisinopril also had effects on wild-type macrophages (peritoneal), significantly reducing phagocytosis of amyloid-β42 fibrils by >50% (Supplementary Fig. 3P), suggesting a role for endogenous ACE in macrophage-mediated fibril amyloid-β42 phagocytosis. Superior endosomal-lysosomal and extracellular degradation of amyloid-β42 conformers by ACE10 macrophages We next studied the capacity of ACE10 macrophages for intra- and extracellular processing and degradation of the three types of amyloid-β and the intracellular localization of these amyloid-β42 species (Fig. 4). We conducted a series of studies using bone marrow-derived ACE10 and wild-type macrophages (Fig. 3A–C). Initial studies of wild-type macrophages showed that when cells were exposed to fibril amyloid-β42, the autophagy marker Beclin-1 relocated to the cell nucleus (Fig. 4A). No difference in Beclin-1 expression was detected in ACE10 versus wild-type macrophages (data not shown). In contrast to Beclin-1 expression, fibril amyloid-β42 appears to co-localize within early endosomes (EEA1) and lysosomal associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2) within lysosomal vacuoles (Fig. 4B). Further analysis of amyloid-β intake after a 15-min exposure to fibril amyloid-β42, prion amyloid-β42, and oligomer amyloid-β42 showed co-localization of the amyloid-β peptides within early endosomes (Fig. 4C–F). The uptake of amyloid-β42 conformers and co-localization in the EEA1+ endosomes was more apparent in ACE10macrophages than in wild-type cells. This was observed for both the fibrillar and the soluble oligomeric forms of amyloid-β42, although it was more intense for the fibrillar form (Fig. 4C–F). A systematic evaluation of the uptake of fibril, prion, and oligomer amyloid-β42 species by ACE10 versus wild-type macrophages was undertaken. Evaluation of the amyloid-β immunoreactive area within EEA1+ endosomes (Fig. 4G) revealed that ACE10 cells consistently had greater amounts of amyloid-β in the endosomal-lysosomal pathway, presumably fated for intracellular degradation. The larger increases in the ACE10 macrophages were most pronounced for the soluble amyloid-β oligomers, as compared to prion rod-like structures and fibrils, as they took up far more soluble amyloid than that observed in wild-type cells (Fig. 4G and H; 21- to 40-fold increases, P < 0.001–0.05). Figure 4 Open in new tabDownload slide Intra- and extracellular degradation of pathological amyloid-β42 species by ACE10 macrophages. (A) Representative fluorescence micrograph of unstimulated (top) and fibril amyloid-β42 stimulated (bottom) MφWT immunolabelled for human amyloid-β (6E10; green), autophagy marker Beclin-1 (red), and nuclei (blue). Merged channel panels show that in response to fibril amyloid-β42, Beclin-1 protein moves to the nuclei (white arrows). (B) Representative fluorescence micrograph panel of fibril amyloid-β42 (fAβ42) stimulated MφWT immunolabelled for human amyloid-β (6E10; green), endosomal markers (EEA1, Lamp-1, Lamp-2, red), and nuclei (blue). (C and D) Representative fluorescence micrograph of MφWT stimulated with the different fibril , prion rod-like and oligomer amyloid-β42 forms for 15 min and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple), and nuclei (blue). White arrows indicate co-localizing of intracellular amyloid-β42 within EEA1+ vesicles. (D) Higher magnification image of co-localized fibril amyloid-β42 within EEA1+ endosomes in MφWT (yellow spots). (E and F) Representative fluorescence micrograph of MφACE10 exposed to oligomer amyloid-β42 (100 nM for 15 min) and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple) and nuclei (blue). White arrows indicate 6E10+ and EEA1+ co-localization. (F) Higher magnification image of co-localized oligomer amyloid-β42 within EEA1+ endosomes in MφACE10 (white arrows). (G and H) Quantitative analysis of co-localized 6E10+ and EEA1+ immunoreactive area in unstimulated and fibril, prion rod-like and oligomer amyloid-β42-stimulated MφWT or MφACE10: (G) 6E10-IR area per macrophage and (H) count of 6E10+/EEA1+ vesicles using puncta count per macrophage. (I) Quantitative normalized analysis of percentage reduction of extracellular fibril amyloid-β42 in media of MφWT versus MφACE10 over 18, 20 and 24 h. (J) Quantitative ELISA analysis of total intra- and extracellular amyloid-β42 levels in the media and cell fraction of MφACE10, following fibril amyloid-β42 for 30 min and either untreated, lisinopril pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA), or pre-placed in ice bath. The group mean ± SEM as well as fold increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Figure 4 Open in new tabDownload slide Intra- and extracellular degradation of pathological amyloid-β42 species by ACE10 macrophages. (A) Representative fluorescence micrograph of unstimulated (top) and fibril amyloid-β42 stimulated (bottom) MφWT immunolabelled for human amyloid-β (6E10; green), autophagy marker Beclin-1 (red), and nuclei (blue). Merged channel panels show that in response to fibril amyloid-β42, Beclin-1 protein moves to the nuclei (white arrows). (B) Representative fluorescence micrograph panel of fibril amyloid-β42 (fAβ42) stimulated MφWT immunolabelled for human amyloid-β (6E10; green), endosomal markers (EEA1, Lamp-1, Lamp-2, red), and nuclei (blue). (C and D) Representative fluorescence micrograph of MφWT stimulated with the different fibril , prion rod-like and oligomer amyloid-β42 forms for 15 min and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple), and nuclei (blue). White arrows indicate co-localizing of intracellular amyloid-β42 within EEA1+ vesicles. (D) Higher magnification image of co-localized fibril amyloid-β42 within EEA1+ endosomes in MφWT (yellow spots). (E and F) Representative fluorescence micrograph of MφACE10 exposed to oligomer amyloid-β42 (100 nM for 15 min) and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple) and nuclei (blue). White arrows indicate 6E10+ and EEA1+ co-localization. (F) Higher magnification image of co-localized oligomer amyloid-β42 within EEA1+ endosomes in MφACE10 (white arrows). (G and H) Quantitative analysis of co-localized 6E10+ and EEA1+ immunoreactive area in unstimulated and fibril, prion rod-like and oligomer amyloid-β42-stimulated MφWT or MφACE10: (G) 6E10-IR area per macrophage and (H) count of 6E10+/EEA1+ vesicles using puncta count per macrophage. (I) Quantitative normalized analysis of percentage reduction of extracellular fibril amyloid-β42 in media of MφWT versus MφACE10 over 18, 20 and 24 h. (J) Quantitative ELISA analysis of total intra- and extracellular amyloid-β42 levels in the media and cell fraction of MφACE10, following fibril amyloid-β42 for 30 min and either untreated, lisinopril pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA), or pre-placed in ice bath. The group mean ± SEM as well as fold increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. To elucidate the relative abilities of wild-type and ACE10 macrophages to degrade amyloid-β in vitro, cells from each genotype were cultured with fibril amyloid-β42 and monitored for 18, 20 and 24 h (Fig. 4I and Supplementary Fig. 4). At the end of this period, ELISA was performed to measure extracellular amyloid-β42 levels in the supernatant. There was a substantial reduction (81%) in extracellular amyloid-β42 concentration in the media of ACE10 macrophages compared to wild-type cell media (Fig. 4I; P < 0.001). We further performed an experiment in which ACE10 macrophages were allowed to take up and degrade fibril amyloid-β42 over 30 min in the presence or absence of lisinopril (Fig. 4J). A subset of ACE10 macrophages was treated under the same conditions in an ice bath (0°C) as a control. Amyloid-β42 concentrations in the combined media and cell fraction of macrophages were analysed by performing ELISA. Again, these data suggest that clearance of fibril amyloid-β42 is dependent on ACE catalytic activity, as the group of ACE10 macrophages treated with lisinopril had significantly higher amyloid-β42 concentrations than cells in which ACE was catalytically active (Fig. 4J; P < 0.0001). Pro-healing immune profile of ACE10 macrophages exposed to Alzheimer’s disease-related amyloid-β forms To understand the biology of macrophages in response to amyloid-β forms, both wild-type and ACE10 bone marrow-derived macrophages were subjected to amyloid-β42 fibrils and oligomers, and their morphologies and immune phenotypes were assessed (Fig. 5 and Supplementary Fig. 5). As seen in Fig. 5A and C, ACE10, as opposed to wild-type macrophages, express ∼30% less iNOS and 80% more arginase-1 prior to amyloid-β42 fibril exposure (P < 0.05–0.01; Supplementary Fig. 5A and B). These differences became less apparent for arginase-1 after 5 min exposure to amyloid-β42 peptides (Fig. 5C). The 30% reduction of iNOS expression in ACE10 macrophages, compared to wild-type macrophages, was sustained even after 1-h incubation with soluble amyloid-β42 oligomers (Supplementary Fig. 5A). Further, considerable increases in expression of the amyloid-β-degrading enzyme MMP9 were observed in ACE10 macrophages, both when unstimulated and after exposure to amyloid-β42 fibrils over time (Fig. 5D and E; 1.6–2.5-fold increase, P < 0.001–0.0001). Noticeably, there was a clear difference in the physical form of the cells (Fig. 5F). The ACE10 macrophages were significantly more elongated and the percentage of elongated cells was higher than that in wild-type cells in the unstimulated state (Fig. 5G; 2.2- and 4.4-fold increase, respectively, P < 0.001). Exposing these cells to fibril amyloid-β42 for either 5 or 30 min resulted in an increased ACE10-macrophage aspect ratio (elongation factor), determined as the quotient of maximum cell process length over cell body width (Fig. 5H; 2.0- and 1.7-fold increase, respectively, P < 0.0001). Hence, the cell processes of wild-type macrophages were consistently shorter than the processes of ACE-enhanced macrophages over time (Fig. 5F–H). This type of elongated macrophage morphology was previously associated with an anti-inflammatory, pro-healing phenotype (McWhorter et al., 2013; Rentsendorj et al., 2018). Figure 5 Open in new tabDownload slide ACE overexpression in macrophages promotes a pro-healing, alternatively activated immune profile. (A) Fluorescence micrographs of inducible nitric oxide synthase (iNOS, red), scavenger receptor CD36 expression (cyan), and nuclei (blue) in MφACE10 versus MφWT (merged image on right). (B–D) Quantitative analysis showing (B) iNOS, (C) arginase-1, and (D) MMP9 in the immunoreactive area of unstimulated (Un) and fibril amyloid-β42-stimulated MφACE10 and MφWT at the 0- and 5-min time points. (E) Representative fluorescence micrographs of MφWT and MφACE10 immunolabelled for MMP-9 (red) and nuclei (blue). (F) Representative bright-field images showing a morphological difference in the cell process length of MφACE10 versus MφWT with measurements of elongation (red lines). (G) Quantitative analysis of elongation factor [ratio between total cell length and soma width (left) and percentage cell elongation (right) in unstimulated MφWT versus MφACE10]. (H) Elongation factor in MφWT versus MφACE10 stimulated with fibril amyloid-β42 for 0, 5, and 30 min. (I–L) Quantitative Meso Scale Discovery (MSD) protein assay of unstimulated (−) and stimulated with fibril amyloid-β42 [100 nM for 24 h (+)] MφWT versus MφACE10 for the following cytokines: (I) TNFα, (J) IL6, (K) CXCL1, and (L) IL1β. (M) Quantitative analysis of a cell viability MTT assay in MφWT versus MφACE10after 24 h of exposure to 1 μM fibril amyloid-β42 (n = 3 mice/genotype group, n = 6 wells/group; one-tailed Student t-test). Data from individual mouse, group mean ± SEM as well as fold increase and percentage decrease and increase between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Figure 5 Open in new tabDownload slide ACE overexpression in macrophages promotes a pro-healing, alternatively activated immune profile. (A) Fluorescence micrographs of inducible nitric oxide synthase (iNOS, red), scavenger receptor CD36 expression (cyan), and nuclei (blue) in MφACE10 versus MφWT (merged image on right). (B–D) Quantitative analysis showing (B) iNOS, (C) arginase-1, and (D) MMP9 in the immunoreactive area of unstimulated (Un) and fibril amyloid-β42-stimulated MφACE10 and MφWT at the 0- and 5-min time points. (E) Representative fluorescence micrographs of MφWT and MφACE10 immunolabelled for MMP-9 (red) and nuclei (blue). (F) Representative bright-field images showing a morphological difference in the cell process length of MφACE10 versus MφWT with measurements of elongation (red lines). (G) Quantitative analysis of elongation factor [ratio between total cell length and soma width (left) and percentage cell elongation (right) in unstimulated MφWT versus MφACE10]. (H) Elongation factor in MφWT versus MφACE10 stimulated with fibril amyloid-β42 for 0, 5, and 30 min. (I–L) Quantitative Meso Scale Discovery (MSD) protein assay of unstimulated (−) and stimulated with fibril amyloid-β42 [100 nM for 24 h (+)] MφWT versus MφACE10 for the following cytokines: (I) TNFα, (J) IL6, (K) CXCL1, and (L) IL1β. (M) Quantitative analysis of a cell viability MTT assay in MφWT versus MφACE10after 24 h of exposure to 1 μM fibril amyloid-β42 (n = 3 mice/genotype group, n = 6 wells/group; one-tailed Student t-test). Data from individual mouse, group mean ± SEM as well as fold increase and percentage decrease and increase between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Along this line, the immune profiles of ACE10 and wild-type macrophages were further analysed using a highly sensitive MSD multiplex inflammatory assay. The results are in good accordance with the results obtained in vivo (Fig. 1M and N) and show that stimulation of both wild-type and ACE10 macrophages with fibril amyloid-β42 for 24 h increased expression of TNFα (Fig. 5I; P < 0.0001). However, the magnitude of TNFα increase was significantly lower in the ACE10 macrophages (Fig. 5I; P < 0.001). While IL-6, CXCL1, and IL-1β concentrations were also elevated in both wild-type and ACE10 macrophages in response to fibril amyloid-β42 stimulation, the degree of increase was more reserved in the ACE10 macrophages than in the wild-type cells (Fig. 5J–L and Supplementary Fig. 5C–F). Therefore, this assay showed that the expression of pro-inflammatory cytokines is generally reduced in ACE10 relative to wild-type macrophages when equivalently stimulated with amyloid-β. Finally, we examined the viability of wild-type versus ACE10macrophages following a 24-h exposure to higher concentration of fibril amyloid-β42 (1 µM). In this assay, on average 50% of ACE10 macrophages appeared to survive, compared to only ∼30% of the wild-type macrophages (Fig. 5M; P < 0.05). Discussion Peripheral monocyte migration into the CNS, the role of ACE, and their possible contribution in resisting Alzheimer’s-associated neuropathology and cognitive decline are subjects of intense investigation and controversy (Ajami et al., 2007; Butovsky et al., 2007; El Khoury et al., 2007; Mildner et al., 2007; Lebson et al., 2010; Bernstein et al., 2014b; Koronyo et al., 2015). In this study, we addressed these fundamental questions and demonstrated: (i) in a partial bone marrow transplantation paradigm with head shielding (protecting the brain from irradiation), there was a measurable migration of peripheral monocytes into the retina and brain parenchyma of AD+ mice. While low in number, these cells critically localized around amyloid-β plaques and reduced cerebral amyloid-β burden and inflammation; (ii) engrafted ACE10 monocytes in the brains of AD+ mice are found at about three times higher numbers than wild-type cells and are more effective in restricting neuropathology; (iii) compared to wild-type macrophages, ACE10 cells in AD+ mouse brains express ∼60% lower levels of TNFα and 3.2-fold higher IGF1, associated with restricting neurodegeneration and cognitive deficits; (iv) non-invasive retinal imaging demonstrates the feasibility of tracking the movements of GFP+-monocytes into the retina of AD+ mice in real time; (v) adoptive transfer of CD115+ ACE10 monocytes into the peripheral blood of symptomatic AD+ mice, an irradiation-free translational approach, is highly effective in reducing vascular amyloidosis and preserving synapses and cognitive function; (vi) ACE10 macrophages exhibit strikingly unique immunological profiles, with enhanced surface receptors and augmented ability to bind, internalize, degrade, and destroy fibrillar, prion rod-like, and soluble oligomeric amyloid-β42 species; and (vii) the elongated cell morphology and molecular signature of ACE10 macrophages suggest an anti-inflammatory, pro-healing, and highly resilient phenotype that may help fight against Alzheimer’s disease. Although several studies have demonstrated the ability of human blood mononuclear cells, monocytes and macrophages, to phagocytose fibrillar amyloid-β and remove plaques from the brains of Alzheimer’s disease model mice (Malm et al., 2005; Simard et al., 2006; Butovsky et al., 2007; Fiala et al., 2007; Lai and McLaurin, 2012; Koronyo et al., 2015), their direct involvement in the clearance of non-fibrillar forms of amyloid-β and possible neuroprotection in Alzheimer’s disease is still highly debated (Fiala et al., 2005; Mildner et al., 2007). Moreover, previous studies did not measure the degree of clearance of defined and stabilized amyloid-β42 structures associated with Alzheimer’s disease neuropathology such as prion rod-like assemblies or soluble oligomers. While amyloid-β plaque burden in the brain appears very limited in predicting cognitive decline (Jansen et al., 2018) and may even serve as a mediator of an effective innate immune response against foreign pathogens (Soscia et al., 2010), soluble amyloid-β42 oligomers and prion rod-like structures have been shown to be highly neurotoxic and more elusive to phagocytic immune cells (Selkoe, 2008; Shankar et al., 2008). Because of the meta-stability of these amyloid-β42 assemblies, structure-activity determinations with respect to macrophage responses have been problematic. Here, using structurally stabilized oligomers and well-characterized prion rod-like and fibrillar amyloid-β42, we were able to accomplish these determinations with respect to binding efficacy, internalization, and degradation by wild-type and ACE10 macrophages. Our studies show that in the wild-type cells, oligomers of amyloid-β42 are found in very low amounts in endosomes and lysosomes, the cellular ubiquitin/proteasome machinery that degrades misfolded proteins (Majumdar et al., 2008). Yet, ACE overexpression and enzymatic function significantly shifted macrophage behaviour and their ability to also target amyloid-β42 oligomers to endosomes (up to 40 times higher as compared to those found in wild-type macrophages; Fig. 4C–H). As a result of the subcellular localization of amyloid-β42 in ACE10 macrophages and the increased levels of amyloid-β-degrading enzymes (i.e. ACE, MMP9), the net concentrations of amyloid-β42 remaining after a 24-h incubation with ACE10 versus wild-type macrophages were as much as 80% lower. The unique morphological and immunological profiles of ACE10 macrophages in response to amyloid-β42 were assessed using various techniques. One mechanism that could explain this superior performance is increased surface expression of scavenger receptors, including Scara1, TREM2, CD163, and especially CD36, all of which have been previously shown to facilitate amyloid-β internalization and clearance by myelomonocytic cells (Koronyo et al., 2015; Rentsendorj et al., 2018). In particular, Scara1 expression was tightly associated with the extent of phagocytosis of amyloid-β42 fibrils, but less so for prion rod-like structures and soluble oligomers. In the ACE10 macrophages, CD36 expression was drastically elevated (increased 6- to 36-fold) and tightly associated with increased uptake of all three types of amyloid-β assemblies. In contrast, the wild-type macrophages displayed no association between surface CD36 expression and uptake of amyloid-β42 fibrils and oligomers, though a strong correlation was observed with rod-like structures. The reason for this phenomenon is currently unknown. Future studies are needed to better understand the molecular immune mechanisms at play to remove pathogenic forms of amyloid-β. Further in vitro techniques supported the enhanced molecular characteristics of the ACE10 macrophages, before and after exposure to amyloid-β42 conformers. All assay findings consistently suggested that these bone marrow-derived cells exhibit an anti-inflammatory (i.e. low TNFα and iNOS, high IGF1, arginase-1, and MMP9) and a pro-healing/protective (i.e. elongated cell morphology) phenotype. These effects were dependent on ACE enzymatic activity and were eliminated when treating the cells with ACE inhibitors. In contrast, previous studies demonstrated that peritoneal-derived ACE10 macrophages, under conditions of acute foreign invasion or cancer models, enhanced their effective immune reactivity in the periphery to resolve disease while exhibiting a highly pro-inflammatory profile (Bernstein et al., 2018). These diverse phenotypes of the ACE10 cells may suggest their ability to promptly and effectively respond to insults in a tissue- and disease-specific manner. This variation in response could also be due to the different stages of healing for which these cells are recruited (i.e. chronic inflammation versus acute injury); nonetheless, their remedial capacity is strongly evident. The enhanced immune response of ACE10 macrophages to acute infection or tumour has repeatedly been shown to not be dependent on the production of angiotensin II or any angiotensin peptide by ACE (reviewed in Bernstein et al., 2018). While this was not formally tested in our study, we believe it most likely that the unique phenotype of the ACE10 macrophages here are probably also due to other mechanisms besides angiotensin II. We further note a recent study documenting that ACE cleaves dozens, if not hundreds, of different peptide substrates (Semis et al., 2019). Our in vivo studies assessed the exclusive contribution of peripherally derived ACE10 monocytes and macrophages in attenuating neuropathology and cognitive deficits in murine models of Alzheimer’s disease. Mounting evidence suggests a protective role for innate immune cells in the CNS (El Khoury et al., 2007; Koronyo-Hamaoui et al., 2009; Shechter et al., 2009; Lebson et al., 2010; Bernstein et al., 2014b; Koronyo et al., 2015), though some studies have failed to show migration of these cells under neurodegenerative conditions (Mildner et al., 2007), or showed monocyte migration to CNS without effects on behaviour and cerebral amyloid-β levels (Prokop et al., 2015; Varvel et al., 2015; Cronk et al., 2018). Our group previously demonstrated that peripherally derived GFP-labelled monocytes are naturally recruited to brains of Alzheimer's disease model mice (Butovsky et al., 2006, 2007; Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b) and that they directly reduce neuropathology and preserve cognition (Koronyo et al., 2015). This irradiation- and pharmacologically-free approach substantially enriched the CD115+ bone marrow-derived inflammatory monocyte subset in the peripheral blood of AD+ mice, resulting in spontaneous recruitment of these cells to the CNS, and led to diminished soluble and insoluble amyloid-β40,42 levels and astrocytosis as well as increased post-synaptic density and preservation of learning and memory (Koronyo et al., 2015; Rentsendorj et al., 2018). When targeting ACE overexpression in macrophages, our earlier studies reported major therapeutic effects in protecting against melanoma and microbial infections (Bernstein et al., 2014a, 2018). Our current investigation also strongly suggests neuroprotective effects of ACE10 macrophages in Alzheimer’s disease mouse models. In accordance, administration of the ACE inhibitor captopril to Alzheimer’s disease mice was reported to increase cerebral amyloid-β deposition (Zou et al., 2007). Here, ACE-overexpressing macrophages were consistently potent in clearing amyloid-β42 conformers—even the most elusive non-fibrillar forms—and successfully migrated into the mouse brain and retina with an enhanced ability to inhibit pathogenesis. While it appears that ACE overexpression in both the resident microglia and peripheral myelomonocytes work synergistically to restrict Alzheimer’s disease-associated pathology and improve cognition in animal models (Bernstein et al., 2014b; Koronyo-Hamaoui et al., 2014), the extent of the effect by peripheral ACE10 macrophages alone was nonetheless substantial, especially with regards to cerebral vascular amyloidosis. Because of growing reports on the key role of vascular abnormalities in Alzheimer’s-related cognitive decline (Ellis et al., 1996; Wyss-Coray et al., 2001; Herzig et al., 2006; Rentsendorj et al., 2018; Nation et al., 2019), the beneficial effects of ACE10 macrophages on reducing vascular amyloidosis (CAA severity score) and GFAP+ astroglial reactivation may indicate protection to the neurovascular unit and could explain synaptic and cognitive preservation seen in these mice. Similarly, previous studies in murine Alzheimer’s disease models showed that blood infusion with either wild-type or neprilysin-enriched monocytes mitigated neuropathology and cognitive deficits (Lebson et al., 2010; Koronyo et al., 2015) and monocyte depletion exacerbated amyloid-β-plaque pathology (Butovsky et al., 2007; El Khoury et al., 2007). Specifically, we have shown by two in vivo approaches that ACE-overexpressing monocytes are more effective in ameliorating Alzheimer’s disease-like pathology and preserving cognition in symptomatic murine models of Alzheimer’s disease. Our data showed that even partial bone marrow replacement with ACE10 cells (after partial-body irradiation, which avoids the skull area to prevent damage to the blood–brain barrier) leads to a substantial 3-fold increase of ACE10 monocytes in the brain, significantly decreasing cerebral amyloid-β plaques, amyloid-β’s soluble forms, and astrocytosis. Increased in vivo recruitment of ACE10 macrophages could be, at least in part, attributed to enhanced secretion of MMP9, an enzyme known to facilitate transmigration of immune cells through the blood–brain barrier (Chaturvedi and Kaczmarek, 2014). These brain-engrafted cells also exhibited ∼60% lower TNFα levels and 3.2-fold higher IGF1, adding a major advantage of this therapeutic approach. TNFα is a key pro-inflammatory cytokine that in excess can induce oxidative stress related to the release of iNOS and reactive oxygen species, which are tightly associated with neurotoxicity and functional decline. Moreover, IGF1 is a brain regenerative, neurogenesis-promoting neurotrophic factor with recently discovered anti-inflammatory functions (Gasparini and Xu, 2003). Cerebral IGF1 in murine models of Alzheimer’s disease was shown to be elevated following immunomodulation interventions involving increased brain-infiltrating monocytes/macrophages and cognitive preservation (Butovsky et al., 2006; Koronyo-Hamaoui et al., 2009), and another report has demonstrated its direct involvement in reducing amyloid-β plaque burden (Carro et al., 2002). Such substantial changes in TNFα and IGF1 levels in a chronically inflamed brain by ACE10 macrophages could, if translated into humans, potentially alter the course of Alzheimer’s disease and critically preserve synapses and cognition. This hypothesis is supported by our observations from the adoptive transfer studies in which enriching the peripheral blood of symptomatic AD+ mice with CD115+-MoACE10 cells resulted in improved cognitive function (Barnes maze test) and pre- and post-synaptic preservation. In comparison, our previous studies in murine models of Alzheimer’s disease involving life-long replacement of CNS-resident microglia and peripheral monocyte and macrophage populations with the ACE-overexpressing cells resulted in an almost complete elimination of cerebral amyloid-β burden, astrocytosis, and full preservation of cognitive functions, similar to the levels seen in the wild-type mice (Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b, 2018; Rentsendorj et al., 2018). As briefly mentioned above, here partial bone marrow replacement or short-term blood enrichment with ACE10 macrophages (once a month for 3 months) in animals that already have Alzheimer’s disease-like symptoms limited neuropathology by 30–40% (especially vascular amyloidosis), yet remarkably restored cognitive functions to levels indistinguishable from those found in cognitively normal wild-type mice. Conclusion and translational implications Despite considerable progress in understanding the aetiology of Alzheimer’s disease, an effective treatment remains elusive. The therapeutic potential evidenced in our studies favours selective increase of ACE (Al Mulhim et al., 2019), a peptidase capable of destroying amyloidogenic peptides and enhancing neuroprotective phenotype, in peripheral inflammatory cells that can migrate into the brain and powerfully support tissue-healing processes and cognition. For the first time, we demonstrate a superior ability of ACE10 macrophages to eradicate amyloid-β assemblies, particularly Alzheimer’s-associated non-fibrillar, oligomeric amyloid-β structures. The infiltration of these cells to the CNS was further validated through live non-invasive imaging of the retina, providing a feasible method to track immune cell migration during disease progression. Moreover, this study reveals the beneficial roles of CD115+ bone marrow-derived monocyte subsets and ACE in resisting Alzheimer’s pathology, especially important in light of large-scale human genetic studies identifying ACE and innate immune genes as prominently related to Alzheimer’s disease (Kehoe et al., 1999, 2003, 2004; Narain et al., 2000; Elkins et al., 2004; Kolsch et al., 2005; Lehmann et al., 2005; Meng et al., 2006; Jochemsen et al., 2014; de Oliveira et al., 2018; Marioni et al., 2018; Kunkle et al., 2019). Further, the marked reduction of Alzheimer’s-associated pathology with targeted ACE expression in inflammatory cells raises a clinically imperative question related to the outcome of life-long use of ACE inhibitors in the large number of patients with heart-related conditions (including high blood pressure), or commonly prescribed anti-inflammatory drugs (i.e. NSAIDs). For example, our results and other published data suggest caution in the use of ACE inhibitors, perhaps prioritizing those that do not penetrate the brain, such as benazepril, enalapril or quinapril (Kehoe et al., 2013). We envision the reported approach to be applicable to humans via isolation of monocytes from bone marrow or circulating blood, manipulation in vitro to overexpress ACE, followed by reinfusion of the modified cells to the patient. Future direction will focus on developing the practical and clinical protocols for each of these steps. It could involve the use of genetically modified stem cells or viral vectors to modify the isolated immune cells. In conclusion, this study may alter the vision of Alzheimer’s disease in human health in two ways: increased attention to potential neuropathological consequences of ACE inhibitors and anti-inflammatory drugs, and discovery of a novel Alzheimer’s disease immunotherapy to deliver ACE and therapeutic monocytes to the site of Alzheimer’s disease pathological manifestation. Acknowledgments The authors thank Xiao Z. Shen for his assistance with brain flow cytometry analysis, Ellen Bernstein for help with ACE10 transgenic mice, and Dr Mitra Mastali from Dr Jennifer Van Eyk’s Protein Quantification Core Laboratory at the Clinical Biosystems Research Institute, Cedars-Sinai Medical Center for assistance with the MSD-ELISA data. We also thank Mia Oviatt and Jo Ann Eliason for help with editing and Samuel Fuchs for his illustration of Supplementary Fig. 1C and help with thumbnail. The authors dedicate the manuscript to the memory of Natalie Radom Bernstein, Lillian Jones Black, and Salomon Moni Hamaoui, who died of Alzheimer’s disease. Funding Research reported in this publication was supported by the National Institute on Aging of the National Institutes of Health under Awards Number R01 AG055865 (M.K.H.) and R01 AG042195 (D.B.T.), A2013328S00 BrightFocus Foundation Award (M.K.H.), The Coins for Alzheimer’s Research Trust – CART Fund (M.K.H.), The Saban Foundation and The Marciano Foundation (M.K.H.). Competing interests The authors report no competing interests. Supplementary material Supplementary material is available at Brain online. References Ajami B , Bennett JL , Krieger C , Tetzlaff W , Rossi FM . Local self-renewal can sustain CNS microglia maintenance and function throughout adult life . Nat Neurosci 2007 ; 10 : 1538 – 43 . Google Scholar Crossref Search ADS PubMed WorldCat Akiyama H , Barger S , Barnum S , Bradt B , Bauer J , Cole GM , et al. 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Google Scholar Crossref Search ADS PubMed WorldCat Abbreviations Abbreviations AD+ = APPSWE/PS1ΔE9 double transgenic BM = bone marrow CAA = cerebral amyloid angiopathy ICC = immunocytochemistry IHC = immunohistochemistry MΦ = macrophages MSD = Meso Scale Discovery Scara1 = scavenger receptor A WT = wild-type Author notes These Yosef Koronyo and Altan Rentsendorj authors contributed equally to this work. © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Brain Oxford University Press

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Oxford University Press
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© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-β protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer’s-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-β1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-β1-42, vascular and parenchymal amyloid-β deposits, and astrocytosis (31%, 47–80%, and 33%, respectively; P < 0.05–0.0001). ACE10 macrophages surrounded brain and retinal amyloid-β plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05–0.001). In vitro analysis of macrophage response to well-defined amyloid-β1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-β1-42 species by ACE10 macrophages. They exhibited 2–5-fold increased surface binding to amyloid-β conformers as well as substantially more effective amyloid-β1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05–0.0001), endosomal processing (P < 0.05–0.0001), and ∼80% increased extracellular degradation of amyloid-β1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer’s-related amyloid-β1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-β forms. IGF1, TNFα, TREM2, EEA1, innate immunity Introduction Neuroinflammation is a key feature of Alzheimer’s disease, a relentless, progressive, and currently untreatable neurodegenerative disease and the leading cause of senile dementia worldwide (Wyss-Coray, 2006; Gjoneska et al., 2015; Heneka et al., 2015; Alzheimer's Association, 2019). In Alzheimer’s disease, the detrimental or therapeutic contribution of peripherally derived monocytes, compared to that of brain-resident microglia, is highly debated (Simard et al., 2006; Ajami et al., 2007; Butovsky et al., 2007; El Khoury et al., 2007; Mildner et al., 2007; Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Bernstein et al., 2014b; Jay et al., 2015a, b; Koronyo et al., 2015; Zuroff et al., 2017; Cao and Zheng, 2018; Cronk et al., 2018; Da Mesquita et al., 2018). Several studies have demonstrated that monocytes can colonize diseased brain parenchyma in murine models of Alzheimer’s disease (Simard et al., 2006; Butovsky et al., 2007; Cronk et al., 2018) as well as under irradiation-free physiological conditions (Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Michaud et al., 2013; Bernstein et al., 2014b; Koronyo et al., 2015; Rentsendorj et al., 2018). In addition, heterogeneous subpopulations of brain resident microglia have been identified as having molecular signatures that are distinct from those of peripheral monocytes, particularly disease-associated microglia located proximal to the Alzheimer’s disease pathological hallmark, amyloid-β plaques (Keren-Shaul et al., 2017; Krasemann et al., 2017; Yin et al., 2017). These microglial populations play a role in the pathogenesis of chronic inflammation (Akiyama et al., 2000; Bales et al., 2000; Colton et al., 2000; McGeer and McGeer, 2002; Wyss-Coray, 2006; Prinz et al., 2011) and synaptic loss by excessive pruning (Hong et al., 2016; Johnson and Stevens, 2018), while insufficiently clearing misfolded amyloid-β (Morgan et al., 2005; Simard et al., 2006; El Khoury et al., 2007; Hickman et al., 2008; Koronyo-Hamaoui et al., 2009; Yamanaka et al., 2012; Frenkel et al., 2013; Roussos et al., 2015). In contrast, peripherally recruited monocytes and macrophages in Alzheimer’s disease model mice were shown to exhibit a neuroprotective phenotype, regulating neuroinflammation and effectively eliminating cerebral amyloid-β pathology (Butovsky et al., 2007; Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b; Koronyo et al., 2015; Rentsendorj et al., 2018). These beneficial effects provide the rationale for a comprehensive assessment of the therapeutic potential of peripheral monocyte infusion in animal models and eventually in Alzheimer’s disease patients. The pathology of Alzheimer’s disease occurs as a complex continuum, typically characterized by early accumulation and aggregation of cerebral amyloid-β (Masters et al., 2015; Jack et al., 2018) in the form of parenchymal plaques and vascular deposits (e.g. cerebral amyloid angiopathy, CAA) (Ellis et al., 1996), which extend to the neurosensory retina in patients with Alzheimer’s disease (Koronyo-Hamaoui et al., 2011; La Morgia et al., 2016; Koronyo, 2017). The 42 amino acid-long form of amyloid-β has been regarded as pathognomonic to Alzheimer’s disease, particularly its oligomeric forms, which display high synaptotoxicity, disrupt long-term potentiation, and directly induce cognitive impairments (Selkoe, 2008; Shankar et al., 2008; Hayden and Teplow, 2012). At the same time, the precise structural characterization and impact of various amyloid-β42 species on cells have been challenging to investigate because of their highly metastable nature and their existence in dynamically changing mixtures (Walsh and Selkoe, 2004; Glabe, 2005; Kirkitadze and Kowalska, 2005). To this end, methods have been developed to chemically stabilize the different assembly types of amyloid-β42 such that a well-defined population of oligomers can be studied (Bitan et al., 2003; Bitan and Teplow, 2004; Hayden and Teplow, 2012; Lopes et al., 2012) in comparison with other naturally stable structures, including prion rod-like enriched assemblies and fibrils (Walsh et al., 1997). It is widely believed that approaches to target and remove soluble amyloid-β42 oligomers from CNS tissues hold great promise for curbing neuroinflammation and contributing to Alzheimer’s disease prevention and therapy (Haass and Selkoe, 2007; Viola and Klein, 2015). In the past decade, strategies harnessing peripheral innate immune cells such as monocytes and macrophages were effective in clearing parenchymal and vascular amyloid-β in the brain and attenuating progression in mouse models of Alzheimer’s disease (Malm et al., 2005; Butovsky et al., 2007; Town et al., 2008; Boissonneault et al., 2009; Koronyo-Hamaoui et al., 2009; Lebson et al., 2010; Michaud et al., 2013; Bernstein et al., 2014b; Koronyo et al., 2015; Ardura-Fabregat et al., 2017; Rentsendorj et al., 2018). Importantly, even in the absence of whole-body irradiation or microglial ablation via pharmacological intervention, peripheral monocytes spontaneously engrafted into the brain of transgenic APPswe/PS1ΔE9 (AD+) mice and homed to amyloid-β plaque sites (Lebson et al., 2010; Koronyo et al., 2015). In particular, enhanced recruitment of cerebral amyloid-β-associated monocytes was achieved through blood enrichment with bone marrow (BM)-derived CD115+ monocyte subsets isolated from young donor mice, leading to diminished neuropathology and preserved cognitive function (Koronyo et al., 2015). To augment these beneficial immune responses further, our group studied a mouse model of Alzheimer’s disease in which the animals were genetically modified to overexpress angiotensin-converting enzyme (ACE), an amyloid-β-degrading peptidase, controlled by a c-fms promoter (Bernstein et al., 2014b). The role of ACE and ACE inhibitors in Alzheimer’s disease is complex (Kehoe et al., 2009). In humans, a recent large genome-wide association meta-analysis (GWAS) of 94 437 individuals clinically diagnosed with late-onset Alzheimer’s disease has corroborated previous studies and identified ACE as a candidate gene (Kunkle et al., 2019). In addition, GWAS-by-familial-proxy of Alzheimer’s or dementia studies have further confirmed ACE relevance to Alzheimer’s disease and identified the single nucleotide polymorphism (SNP) combination of rs4343 and rs4351 in the ACE haplotype to possess a 45-fold increased risk of developing Alzheimer’s disease (Meng et al., 2006; Marioni et al., 2018). Importantly, individuals carrying variants of the ACE gene (D/D alleles) resulting in increased plasma expression of the enzyme were associated with lower risk for Alzheimer’s disease (Kehoe et al., 1999; Lehmann et al., 2005). Lower CSF levels of ACE and higher brain ACE were also detected in Alzheimer’s disease patients (Miners et al., 2008, 2009). In our previous studies in mice, ACE was selectively expressed in myelomonocytic cells such as monocytes, macrophages, and microglia (Bernstein et al., 2014a, b). The rationale for this experiment was 2-fold: myeloid cell ACE overexpression markedly enhances innate immune responses (Shen et al., 2007; Koronyo-Hamaoui et al., 2014; Bernstein et al., 2018), and surface expression of ACE would promote amyloid-β42 cleavage and conversion into shorter, more benign amyloid-β alloforms via ACE enzymatic activity (Hemming and Selkoe, 2005; Bernstein et al., 2014b). In fact, AD+ mice overexpressing ACE in myelomonocytes displayed minimal to no Alzheimer’s disease-like phenotype (Bernstein et al., 2014b). However, in those experiments, ACE overexpression was present throughout the life of the AD+ mice in both macrophages and microglia due to an inserted genetic modification in the ACE gene. Hence, the relative therapeutic contribution of a periodic addition of peripherally derived ACE-overexpressing monocytes and macrophages, as opposed to brain-resident microglia, has yet to be investigated. Here, we examine whether exogenous administration of bone marrow-derived CD115+ myeloid cells overexpressing ACE, either by bone marrow transplantation or by peripheral blood enrichment, can also reduce the magnitude of disease. The use of bone marrow-derived myeloid cells isolated from green fluorescent protein (GFP) mice allowed unambiguous tracing of the grafted cells’ destiny in the CNS of recipient animals; this was especially critical to distinguish clearly between the contribution of microglia and macrophages to the phenotype. Moreover, our experiments showed that ACE10 monocytes and macrophages were highly potent in clearing soluble and insoluble forms of amyloid-β42, as well as in reducing neuropathology and preserving synapses and cognitive function in murine Alzheimer’s disease models. Materials and methods Mice used in the experiments The mice selected to be recipients of bone marrow transplantation or adoptive transfer of monocytes were from the double-transgenic B6.Cg-Tg (APPSWE/PS1ΔE9)85Dbo/Mmjax hemizygous (AD+) mice strain (MMRRC stock #34832-JAX|APP/PS1) and their non-transgenic littermates (as wild-type control non-Alzheimer’s disease). These animals were purchased from Jackson Laboratories and later bred and maintained at Cedars-Sinai Medical Center. For the bone marrow transplantation experiment, we used 22 AD+ recipient mice divided into three groups as follows: BMAD+::AD+ (n = 7, four females and five males), BMWT::AD+ (n = 7, two females and five males), and BMACE10::AD+ (n = 8, three females and five males). For adoptive transfer experiments we used 30 mice, all males, divided into four groups as follows: AD+ recipients of either wild-type monocytes (MoWT) (n = 8) or MoACE10 (n = 8), PBS-injected AD+ controls (n = 7), and non-transgenic naïve wild-type mice (n = 7). The ACE10/10 donor mice strain overexpresses ACE (two alleles) in the myelomonocytic cell lineage (Supplementary Fig. 1A and B) (Bernstein et al., 2014b). The Ace genotypes used in this study were wild-type (ACEWT; n = 30) and homozygous for the targeted allele (ACE10; n = 30). The GFP donor mice were B6-TgN (β-act-EGFP) osbY01 (Okabe et al., 1997; Okwan-Duodu et al., 2010), which had been backcrossed at least 10 generations to a C57BL/6 background (Jackson Laboratory; n = 15). This line has enhanced GFP (EGFP) cDNA under the control of a chicken β-actin promoter and a cytomegalovirus enhancer. These animals are also available at Jackson Laboratory [C57BL/6-Tg(CAG-EGFP)10sb/J; #003291]. The bone marrow donor mice strains (ACE10 and GFP) were crossed to produce the genotypes GFP+AD−ACEWT (n = 76 mice), GFP+AD−ACE10 (n = 85), and GFP−AD+ACEWT (n = 4 mice) used for the in vivo experiments. Bone marrow transplantation donor mice were 12–16 weeks old (n = 11, all females). Adoptive transfer donor mice were used at the age of 8–12 weeks old (n = 154, female and males at equal numbers). All mice used for these experiments were on the genetic background of B6. All experiments were conducted in accordance with Cedars-Sinai Medical Center Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol. Bone marrow transplantation With their heads shielded, 2-month-old recipient AD+ mice were subjected to 9.5 Gy (950 rad) irradiation for 11 min—a dose lethal to proliferating cells. The cells were immediately reconstituted by intravenous administration of 5 × 106 donor marrow cells [isolated from 2-month-old GFP-expressing donor mice (described above)]. As irradiation of the brain significantly increases infiltration of immune cells, we protected the mice brains from irradiation with an extensive lead-shielding device that we had constructed (Supplementary Fig. 1C). After 8 weeks of bone marrow repopulation, engraftment was assessed by flow cytometry analysis of peripheral blood for GFP expression. Non-invasive retinal imaging was carried out when the mice reached 7 months of age. When they were 8 months old, the recipient mice were anaesthetized and perfused with 0.9% ice-cold saline containing 0.5 mM EDTA. Brain, eyes, and blood were collected. Retinal flatmounts were immediately isolated and fixed in 2.5% paraformaldehyde (PFA) as previously described (Koronyo-Hamaoui et al., 2011). One brain hemisphere was fixed for histology in 2.5% PFA containing 30% sucrose and the other hemisphere was snap-frozen for further analysis. Monocyte adoptive transfer After the animals had been euthanized, bone marrow cells were harvested and enriched for mononuclear cells on a Ficoll density gradient as previously described (Koronyo et al., 2015). Five to six million CD115+-MoBM cells were injected intravenously into each AD+ recipient mouse, beginning when the animal was 8 months old and continuing once a month for 3 months to ensure long-term elevated levels of transfused monocytes. Animals in the control group received intravenous injections of phosphate-buffered saline (PBS). Characterization of isolated monocytes was performed by flow cytometry. After completion of behavioural testing at 11 months old, the recipient mice were perfused, as described above, to collect tissues for analysis. Non-invasive retinal imaging After deep anaesthesia had been induced in the animals, the mouse pupils were dilated with 0.5% phenylephrine hydrochloride ophthalmic solution (Bausch and Lomb) combined with 0.5% tropicamide ophthalmic solution (Mydral, Bausch and Lomb). The retinas were imaged in vivo using the Micron III microscope with predetermined light exposure time and intensity, using identical gain and shutter modes for all animals as previously described (Koronyo-Hamaoui et al., 2011; Koronyo et al., 2012, 2017). Images and videos of the retina were repeatedly captured at several angles to visualize a larger retinal field and eliminate non-specific reflection signals. Images were exported to ImageJ 1.46r for analysis. Behavioural tests To assess locomotor activity and cognitive functions of the treated mice, we performed open field and Barnes maze tests, as previously described (Koronyo et al., 2015). In the open field test, each mouse was placed in an individual apparatus for 60 min to record ambulatory and rearing activity. In the Barnes maze test, the mice were first trained for 4 days (acquisition-training phase), three times per day for 240 s for each trial per mouse. Following a 2-day break, the mice were retested for memory retention (retention phase, Day 7) and then short-term memory (reversal phase on Days 8 and 9) under the same conditions. Latency times and incorrect entries (errors) were recorded manually and by using a video camera (Koronyo et al., 2015). Immunohistochemistry Coronal brain sections (30 μm) and retinal flatmounts were treated for 30 min in antigen-retrieval solution (Dako S1699) prior to serum-free protein blocking (Dako X0909). Tissues were then hybridized with various primary antibodies (Table 1) overnight at 4°C. Hybridization with primary antibodies was followed by incubation with fluorophore-conjugated secondary antibodies (1 h at 37°C) (Table 1). Stained tissues were mounted using ProLong® Gold with DAPI (Molecular Probes, Life Technologies). Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labelling. Table 1 List of antibodies used in the study Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 mAb = monoclonal antibody; pAb = polyclonal antibody. * 1 μl = 1 μl per 100 000 – 1 000 000 cells. Open in new tab Table 1 List of antibodies used in the study Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 Antigen Source species Dilution Commercial source Catalogue # IHC, ICC Primary antibodies Arginase1 pAb Rabbit 1:100 Santa Cruz sc-20150 Beclin1 pAb Rabbit 1:100 Abcam ab16998 CD163 pAb Rabbit 1:100 Biorbyt Orb13303 CD204 mAb Scavenger R Type I/II (SCARA-1) Rat 1:100 AbD Serotec MCA1322 CD36 mAb clone MF3 Rat 1:200 Abcam ab80080 CD45 mAb clone 30-F11 Rat 1:25 BD Pharmingen #550539 EEA1 pAb Rabbit 1:100 Millipore 07-1820 GFAP mAb Rat 1:100 Invitrogen 13-0300 GFAP pAb Rabbit 1:100 Sigma-Aldrich G926 GFP pAb Rabbit 1:500 MBL #598 Human amyloid-β residues 17–24, mAb clone 4G8 Mouse 1:100 Covance/BioLegend 800711 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:100 Covance/BioLegend 803003 Iba1 pAb Rabbit 1:200 Wako Chemicals USA #019-19741 IGF1 pAb Goat 1:30 R&D systems AF791 iNOS mAb Rabbit 1:400 Cell Signaling 13120 Lamp1 mAb Rat 1:500 Abcam ab25245 Lamp2 mAb, clone M3/84 Rat 1:500 Millipore MABC24 MMP9 Goat 1:100 R&D systems AF909 PSD95 mAb Rabbit 1:600 Abcam ab76115 TREM2 pAb Goat 1:100 Abcam ab95470 VGluT1 pAb Guinea pig 1:6000 Millipore AB5905 Secondary antibodies Cy2 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy3 (anti-mouse, rat, rabbit, goat, guinea pig IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Cy5 (anti-mouse, rat, rabbit, goat IgG) Donkey 1:200 Jackson ImmunoResearch Laboratories Flow cytometry CD11b mAb clone M1/70 (PE/Cy7) Rat 1 μl* eBioscience/ThermoFisher 25-0112-81 CD45 mAb clone 30-F11 (PE) Rat 1 μl* eBioscience/ThermoFisher 12-0451-81 F4/80 mAb clone BM8 (FITC) Rat 1 μl* eBioscience/ThermoFisher 11-4801-81 Human amyloid-β residues 1–16, mAb clone 6E10 Mouse 1:500 Covance/BioLegend 803003 IL10 mAb clone JES5-16E3 (PerCP-Cy5.5) Rat 1 μl* BioLegend 505028 Ly-6C mAb clone HK1.4 (eFluor450) Rat 1 μl* eBioscience/ThermoFisher 48-5932-80 TGFβ mAb clone TW7-16B4 (PerCP-Cy5.5) Mouse 1 μl* BioLegend 141410 TNFα mAb clone MP6-XT22 (APC) Rat 1 μl* BioLegend 506308 mAb = monoclonal antibody; pAb = polyclonal antibody. * 1 μl = 1 μl per 100 000 – 1 000 000 cells. Open in new tab Thioflavin-S staining For amyloid burden assessment in brain parenchyma and vasculature, sections were stained with Thioflavin S (Thio-S, 1% w/v in 70% ethanol; Sigma-Aldrich) for 10 min at room temperature, following the secondary antibody step, washed three times in 70% ethanol for 1 min each, then rinsed in distilled water before mounting. Curcumin staining Curcumin (diferuloylmethane; C21H20O6; Sigma‐Aldrich) was dissolved in 0.5 M NaOH followed by immediate dilution in PBS to a final solution of 0.1 mg/ml curcumin (pH 7.9). Brain sections were stained with curcumin solution for 10 min at room temperature, and then rinsed three times with PBS for 5 min each, as previously reported (Koronyo-Hamaoui et al., 2009). Cerebral amyloid angiopathy scoring Severity scores for the degree of CAA were defined by inspection of Thio-S-labelled brain sections using a scale of 0–4 (0 = no CAA, 1 = low, 2 = medium, 3 = high and 4 = severe CAA) (Wyss-Coray et al., 2001; Rentsendorj et al., 2018). Eighteen images encompassing three to four brain sections and covering both cortex (n = 9 images) and hippocampus (n = 9) were analysed for each animal (total of n = 22 mice, n = 6–9 per experimental group). Primary culture of bone marrow-derived macrophages Bone marrow was harvested from the femora and tibiae of wild-type or ACE10 mice donors after they had been euthanized at the age of 8–12 weeks (3–5 mice/genotype), as previously described (Rentsendorj et al., 2018). Briefly, the cells were cultured for 7 days in RPMI supplemented with 10% foetal bovine serum (FBS), 100 U/ml antibiotic, 2 mM l-glutamine, and 20 ng/ml MCSF. On the sixth day, macrophages (MΦ) were plated overnight in 24-well tissue culture plates with or without glass coverslips (105 cells/well) depending on which analysis was planned [immunocytochemistry (ICC) or ELISA]. The following day, macrophages were either unstimulated (not treated with amyloid-β) or stimulated by one of the three amyloid-β42 alloforms (100 nM and 250 nM; fibril amyloid-β42, prion rod-like amyloid-β42, oligomer amyloid-β42). MΦACE10 were analysed as compared to MΦWT for cell-surface amyloid-β42 binding; for this, the plates were chilled for 10 min on ice (0°C) followed by addition of amyloid-β42 and spun at 515 g for 1 min, followed by incubation for another 10 min at 0°C (binding – 0’ time point). MΦACE10 were also analysed for receptor expression as well as uptake of amyloid-β42 forms at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake −5, −15, −30 min). After each time point, media were collected for biochemical analysis (ELISA). The cells were rinsed with culture medium and then washed with PBS before being fixed in cold methanol (−20°C) for ICC. In parallel, another subset of MΦACE10 were treated overnight (in RPMI + 0.3% BSA) with lisinopril (1 μM) the day before plating in 24-well plates and then exposed to 100 nM fibril amyloid-β42 for 0, 5, or 30 min. Primary culture of peritoneal-derived macrophages Peritoneal macrophages were collected from the peritoneum as described previously (Okwan-Duodu et al., 2010). Briefly, the macrophages were harvested from the peritoneal cavity 4 days after a 2 ml intraperitoneal injection of 3% thioglycolate. The cells were cultured at 37°C, 5% CO2 in RPMI 1640 medium enriched with 10% foetal calf serum, 50 μM 2-ME, 0.5 mM sodium pyruvate, 10 mM HEPES buffer, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mM l-glutamine. For purification of the macrophages, peritoneal exudates were allowed to adhere for 2 h, after which non-adherent cells were removed with PBS to achieve >90% purity of macrophages. Immunocytochemistry Briefly, after serum-free protein blocking (Dako X0909), cells were hybridized with various primary antibodies (Table 1) overnight at 4°C. The following day, these cells were incubated with fluorophore-conjugated secondary antibodies (1 h at 37°C). Coverslips were mounted onto plates using ProLong® Gold with DAPI (Molecular Probes, Life Technologies). Microscopy and quantification A Karl Zeiss fluorescence microscope (Imager, Z1, ApoTome and MBF equipped) was used for microscopic analysis. For immunohistochemistry (IHC), three to six comparable coronal brain sections, originally located 150 μm apart, were used for quantitative analysis. For ICC, four to six samples were analysed. Numbers, area, and intensity of amyloid-β deposits, GFAP+ and GFP+ cells, were determined automatically by using NIH ImageJ software or an MBF stereological system. The same approach was used to analyse cytokines and other markers. Image analysis To cover the hippocampal area for synaptic analysis, 15 fields per hippocampal area per brain were taken under 100× oil objective lens and analysed (as previously described in Koronyo et al., 2015). Single optical section images at 0.25 µm intervals and 3.75 µm Zeiss ApoTome high-resolution scans were performed. Synaptic immunoreactive area and co-localization of EEA1+ with 6E10+ immunoreactive area were quantified using Puncta Analyzer as previously described (Ippolito, 2010) and ImageJ (NIH) macro and batch process. To quantify co-localized IGF1+ immunoreactive in monocytes (Mo)/MΦ (Iba1+CD45high cells), images were first compiled and analysed in Adobe Photoshop (Adobe Systems Inc., Mountain View, CA, USA). The per cent of IGF1+ foci were obtained by tracing an outline around each plaque associated with inflammation site. IGF1 (red) co-localizing with CD45 (green) was quantified by measuring the fluorescence of coincident (yellow) particles. Frequency of coincidence of IGF1+ area co-localizing with Iba+CD45high cells in cortices was plotted as percentage (%) in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 4–7 AD+ mice/group). Elongation factor The long and short axes of cells were manually measured in micrometres from microscopic images using length tools in the AxioVision Rel. 4.8 software package. At least 100 cells were examined in each experiment in which individual cells were assayed by microscopy. Quantitative Meso Scale Discovery protein assay The Meso Scale Discovery (MSD) V-PLEX proinflammatory Panel I (MSD K15048D-1) consists of highly sensitive multiplex ELISAs that quantitatively measure 10 cytokines (IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO or CXCL1, TNFα) using electrochemiluminescence detection technology. Preparation of standards/calibrators and samples The highest calibrator (Calibrator 1) was prepared by adding 1 ml of Diluent 41 to the lyophilized calibrator vial according to the manufacturer’s protocol. Seven calibrator solutions were made by first transferring 100 μl of the highest calibrator into 300 μl of Diluent 41 and then making 4-fold serial dilutions five additional times. Diluent 41 was used for the zero calibrator. Sixty microlitres of each sample was mixed with 60 µl of Diluent 41 to achieve a 2-fold dilution of each sample. Plate preparation A 1× wash buffer was made by a 20-fold dilution of the stock (supplied in the kit) using deionized water. The plate was washed with 150 µl of wash buffer three times and tapped dry. Calibrators and samples (50 µl) were pipetted into the assigned wells. The plate was incubated for 2 h at room temperature on a shaker. To make 1× detection antibodies from 50× stock, 60 μl of each detection antibody for each analyte was pipetted into 2.4 ml of Diluent 45. After the 2 h incubation, the plate was washed three times with wash buffer and tapped dry. Each well received 25 μl of detection antibody solution, after which the plate was incubated at room temperature with shaking for 2 h. To read the plate on the MSD instrument (MESO Quicklex S 120), the plate was first washed with 150 μl wash buffer three times and tapped dry, then 150 µl of 2× Read Buffer (supplied in the kit) was pipetted into each well. No incubation was needed after the read buffer was added and before reading the plate. Data analysis The calculations used to establish calibration curves and determine concentration and data analysis were carried out using MSD Workbench 4.0 software. This software fits the standard curves using a four parameter logistic fit with 1/y2 weighting. Brain/plasma amyloid-β1-42 and amyloid-β1-40 levels by ELISA Fresh-frozen brain tissues were thoroughly homogenized in PBS buffer with 0.5% Triton™ X-100 (Sigma) and 1% protease inhibitor cocktail set I (Calbiochem) and then processed as previously described (Koronyo et al., 2015). Blood was immediately centrifuged after collection (730g for 10 min at 4°C) for plasma isolation. After determination of protein concentration with the aid of the Pierce BCA Protein Assay Kit (Thermo Scientific), concentrations of amyloid-β42 (Invitrogen KHB441) in the soluble and insoluble brain protein fractions and amyloid-β40 (Invitrogen KHB3481) in the plasma were analysed using a sandwich ELISA kit (Invitrogen) as per the manufacturer’s instructions. Cell viability assay Amyloid-β42 films were dissolved in 60 mM NaOH, sonicated for 1 min in an ultra-bath sonicator, and diluted into cell culture media to the desired concentration. Aliquots of 10 μl were added to individual wells of 96-well microplates. Each well contained ∼30 000 cells in a volume of 90 μl. After an incubation lasting ∼15 h, 15 μl of MTT reagent was added to each well and the cells were incubated for 3 h at 37°C. SDS DMF HCl acetic acid stop solution (100 μl) was added to each well, and the plates were incubated overnight in the dark to ensure complete solubilization. The plates were then read using a microplate reader, and the absorbance at 570 nm (formazan product) minus the absorbance at 630 nm (background) was recorded. Controls included cells treated with NaOH only and 1 μM staurosporine (to induce apoptosis and yield maximal cell death). A minimum of six replicates were done for each treatment group, and the data from three independent experiments were combined and reported as mean ± standard error of the mean (SEM). The biological activity of amyloid-β was compared using the one-way analysis of variance Tukey test. Preparation of low molecular weight amyloid-β42 Low molecular weight (LMW) 99% HPLC purified amyloid-β42 peptide was synthesized in the UCLA Biopolymer Laboratory (Bitan and Teplow, 2005) or purchased from AnaSpec. Aggregate-free amyloid-β42 (LMW amyloid-β) was prepared as follows: Microcon YM-30 filters (EMD Millipore) were washed with 200 μl distilled deionized water. Amyloid-β was dissolved in 10% (v/v) 60 mM NaOH and 90% (v/v) 10 mM phosphate buffer, pH 7.4, at a concentration of 1 mg/ml. The solution was sonicated for 1 min and then placed into the washed 30 kDa filter. Following centrifugation at 14 000g for 20 min, the filtrate, LMW amyloid-β, was collected, and its concentration determined by optical absorption spectroscopy using a molar extinction coefficient of 1280 M−1cm−1 at 276 nm. Purified peptides were stored as lyophilizates at −20°C. Preparation of amyloid-β42 fibrils For fibrillar amyloid-β42, LMW amyloid-β42 was prepared as described above, and the peptide preparation was incubated at 100 µM with orbital shaking at 37°C until fibril formation occurred, ∼2 weeks (Koronyo et al., 2015). The presence of fibrils was confirmed by electron microscopy. Preparation of PICUP oligomeric amyloid-β42 LMW amyloid-β was stabilized to ‘freeze’ oligomers using photo-induced cross-linking of unmodified proteins (PICUP) (Bitan et al., 2001, 2003; Bitan and Teplow, 2004; Bitan, 2006) to yield a distribution of ‘oligomeric amyloid-β’. Briefly, 2 mM Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate [Ru(Bpy)3] (Aldrich) and 40 mM ammonium persulphate (APS) (Sigma) were prepared in distilled deionized water. An 18-μl aliquot of 80 μM LMW amyloid-β was placed in a PCR tube, followed by 1 μl Ru(Bpy)3 and 1 μl APS. The sample was irradiated (150 W incandescent lamp) for 1 s, and the reaction was quenched immediately with 1 M dithiothreitol. Cross-linking reagents were removed by dialysis using 3.5 kDa MWCO Slide-A-Lyzer cassettes (Pierce) against 10 mM sodium phosphate, pH 7.4. More than five changes of buffer were completed, and the protein purity and concentration were confirmed using optical absorption spectroscopy. Preparation of prion rod-like amyloid-β42 LMW amyloid-β42 was prepared as described above, and the peptide preparation was diluted to 100 μM, incubated at room temperature without agitation for 9 h, and flash frozen. Transmission electron microscopy Ten microlitres of sample were spotted onto 400-mesh carbon-coated Formvar grids (Electron Microscopy Sciences) and incubated for 2 min. Each grid was then negatively stained with a 1% (v/v) solution of uranyl acetate (Ted Pella), passed through a 0.2-μm filter, which was immediately wicked off. TEM analysis was carried out using a JEOL 1200 EX at 80 kV (Fradinger et al., 2005). Flow cytometry Brain tissues After perfusion, the mouse brains were minced and digested for 30 min with 1.5 mg/ml collagenase IV (Worthington Biochemical Corp.) and DNase I (Stem Cell Technologies) at 37°C. After centrifugation, homogenization and washing, the pellet was suspended in 70% Percoll® (GE Healthcare) and then layered under a 30%/50% Percoll® step gradient. After 30 min of centrifugation at 800g at 4°C (without break), mononuclear cells at the interface of the 70% and 50% layers were collected and washed. For surface marker staining, the cells were blocked and stained (Table 1). For intracellular staining, the cells were fixed (eBioscience; #00-8222-49), permeabilized (eBioscience: #00-8333-56), and then stained (Table 1). Cells were treated with brefeldin A to stain for secreted cytokines (eBioscience: #00-4506-51). In vitro assessment of fibril amyloid-β42 phagocytosis Primary bone marrow or peritoneal-derived CD115+ macrophages (wild-type and ACE10) were exposed to preformed 1 nM HiLyte™ Fluor 647 amyloid-β1-42 (Anaspec) fibrils. At specific time points (0, 15 and 60 min), the cells were stained and analysed by flow cytometry. Data and statistical analysis Data were analysed using GraphPad Prism v. 7.01 (GraphPad) software. Normally distributed data resulting from the IHC, ELISA, and flow cytometry analyses are expressed as means ± SEMs. One-way or two-way ANOVA with Sidak’s and Bonferroni’s post-tests was used for multiple comparisons (three or more means). The threshold for statistical significance was set at 0.05. For most behavioural testing, a power of 80% was obtained with 8–10 animals per group. For stereology or other semi- or fully quantitative analysis (i.e. q-IHC, ELISA), 6–8 mice per group have sufficient power to detect significance. Two-group comparisons were analysed using a paired or unpaired Student t-test. The statistical association between two or more variables was determined using Pearson’s correlation coefficient (r) test (GraphPad Prism) with corrections for multiple analyses. Pearson’s r indicates direction and strength of the linear relationship between two variables. Results are shown as mean ± SEM. Degrees of significance between groups are represented as follows: *P < 0.05, **P < 0.01, ***P <0 .001, and ****P < 0.0001. Data availability The data that support the findings of this study are available from the corresponding author, upon reasonable request. Results Reduced pathology in AD+ mice after partial ACE10 bone marrow replacement linked to brain and retinal monocyte recruitment In our initial experiment, we tested bone marrow transplantation in APPSWE/PS1ΔE9 double-transgenic mice (AD+ mice). Two-month-old mice (n = 22) received a high dose of radiation while placed inside a head-shielding device. Immediately afterward, the animals received an intravenous injection of either AD+ bone marrow (GFP−AD+ACEWT; n = 7, three females and four males), control bone marrow (GFP+AD−ACEWT; n = 7, two females and five males), or ACE10 bone marrow (GFP+AD−ACE10; n = 8, three females and five males) (Fig. 1A and Supplementary Fig. 1A–C). Blood analysis 8 weeks post-transplantation showed that our protocol resulted in mice with chimeric bone marrow consisting of ∼30–40% of cells from donor origin. At 7 months, the two groups with GFP-labelled bone marrow were examined using non-invasive retinal fluorescence imaging (Supplementary Fig. 1D) to track infiltration of GFP monocytes to CNS tissue using a rodent retinal Micron-III microscope (Koronyo et al., 2012, 2017). AD+ chimeric mice with BMWT had inflammatory cells surrounding amyloid-β plaques, including peripheral bone marrow-derived GFP+CD45hi monocytes (Fig. 1B). Analyses demonstrated reductions in amyloid-β plaque area (6E10+), soluble brain amyloid-β1-42 levels (ELISA), GFAP+ cells, and GFAP+ area in mice that had received ACE10 bone marrow compared to AD+ control mice (47%, 31%, 33% and 33% decreases, respectively) (Fig. 1C–F). Figure 1G and H demonstrate less cortical amyloid-β plaques stained with curcumin, a method previously described (Koronyo-Hamaoui et al., 2009, 2011; Koronyo et al., 2017), and lower cortical GFAP+ astrocytosis. Interestingly, these effects were localized in the brain as no significant differences in plasma levels of amyloid-β1-40 and amyloid-β1-42 (ELISA) or their ratios were observed between the experimental groups (Supplementary Fig. 1E). Further, no significant differences were noted between females and males. Figure 1 Open in new tabDownload slide Beneficial effects of partial ACE10 bone marrow transplantation in APPSWE/PS1ΔE9 transgenic mice. (A) Schematic illustration of experimental design and timeline. At 2 months of age, three groups of double-transgenic APPSWE/PS1ΔE9 (AD+) recipient mice (n = 22) underwent an irradiation procedure, with their heads shielded by a 3-inch-thick lead block. Mice then received a lethal dose of 950 rad irradiation for 11 min (Supplementary Fig. 1A–C). Immediately after irradiation, AD+ recipient mice received an intravenous (i.v.) injection of 5 million bone marrow cells from one of three types of donor mice: GFP−AD+ACEWT, GFP+AD−ACEWT, or GFP+AD−ACE10. The resulting BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 7–8 per group) had a partial (˜30–40%) bone marrow transplantation. The two groups of GFP-labelled bone marrow chimeric AD+ mice underwent retinal imaging at 7 months of age. Tissues were harvested for analysis from all three experimental groups when the animals were 8 months of age; the left hemisphere was used for flow cytometry, the right hindbrain for histology, and the right forebrain for protein analysis. (B) Representative fluorescence micrograph of the BMWT::AD+ chimeric mouse brain showing infiltrating monocytes (GFP+/CD45hi; arrows) near a 6E10+ amyloid-β plaque site. Scale bar = 20 μm. (C) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in brain cortices (Ctx) of BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 5–7 mice/group). (D) Sandwich ELISA analysis of cerebral soluble amyloid-β42 levels in AD+ chimeric mice (n = 6–8 mice/group). (E) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte cell count in AD+ chimeric mice (n = 6–8 mice/group). (F) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte area in AD+ chimeric mice (n = 7 mice/group). (G) Representative micrographs of cortical amyloid-β plaques stained with curcumin in BMWT::AD+ versus BMACE10::AD+ chimeric mice. (H) Representative micrographs of cortical GFAP+ astrocytes in the same experimental groups. Scale bar = 50 μm (for G and H). (I) Flow cytometry analysis for cerebral infiltrating GFP+ monocytes in AD+ chimeric mice. Combinations of markers were used to identify microglia (CD11b+CD45int.-lowGFP) versus infiltrating monocyte subsets (CD11b+CD45hiLy6ChiGFP+). (J) Representative flow cytometry map analysis showing increased infiltration of CD11bhiCD45hiLy6ChiGFP+ ACE10 versus wild-type monocytes in AD+ chimeric mice. (K and L) Quantitative flow cytometry analyses of cerebral inflammatory biomarkers in chimeric AD+ mice following partial bone marrow transplantation. (K) Percentage of GFP+CD11bhiCD45hiLy6ChiF4/80low monocytes (n = 5–6 mice/group), (L) TNFα expression in infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi Mφ (n = 4–5 mice/group). (M) Fluorescent micrographs of cortical brain regions stained against IGF1 (red) combined with Iba1 (white) CD45 (green) and nuclei (blue) in BMWT::AD+ versus BMACE10::AD+ chimeric mice. Scale bars = 10 μm. Brain-infiltrating Iba1+CD45hi Mo/Mφ abundantly expressed IGF1 in BMACE10::AD+ chimeric mice (bottom). (N) Quantitative IHC of % IGF1+ area co-localized with Mo/Mφ in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 7 mice/group). (O–Q) Representative non-invasive retinal fluorescence imaging of infiltrating GFP+ bone marrow cells in living AD+ chimeric mice. (O) A control AD+ mouse that underwent bone marrow transplantation of GFP−BMAD+ (GFP−BMAD+::AD+). (P) Retinal fluorescent images from a GFP+BMWT::AD+ mouse (P’, enlarged image). (Q) Retinal fluorescent images from a GFP+BMACE10::AD+ mouse (arrows indicate GFP+ bone marrow cells). (R) Representative microscopic image obtained during the histological examination of a retina extracted from a BMACE10::AD+ mouse whose retina had been previously imaged in vivo; ex vivo staining with GFP (green), CD45 (red), 4G8 (cyan), and DAPI (blue), validating the homing of GFP+ACE10 monocytes to retinal amyloid-β plaques. Scale bar = 5 μm. Data from an individual mouse (filled circles for males and clear circles for females) as well as group mean ± SEM are shown. Fold increase and percentage decreases compared to control groups are shown in green. *P < 0.05, **P < 0.01. ns = non-significant, using one-way ANOVA and Bonferroni’s post-test for three group comparisons. For two-group comparisons, paired or unpaired two-tailed Student t-tests were used. Aβ = amyloid-β; FS = forward scatter; SS = side scatter. Figure 1 Open in new tabDownload slide Beneficial effects of partial ACE10 bone marrow transplantation in APPSWE/PS1ΔE9 transgenic mice. (A) Schematic illustration of experimental design and timeline. At 2 months of age, three groups of double-transgenic APPSWE/PS1ΔE9 (AD+) recipient mice (n = 22) underwent an irradiation procedure, with their heads shielded by a 3-inch-thick lead block. Mice then received a lethal dose of 950 rad irradiation for 11 min (Supplementary Fig. 1A–C). Immediately after irradiation, AD+ recipient mice received an intravenous (i.v.) injection of 5 million bone marrow cells from one of three types of donor mice: GFP−AD+ACEWT, GFP+AD−ACEWT, or GFP+AD−ACE10. The resulting BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 7–8 per group) had a partial (˜30–40%) bone marrow transplantation. The two groups of GFP-labelled bone marrow chimeric AD+ mice underwent retinal imaging at 7 months of age. Tissues were harvested for analysis from all three experimental groups when the animals were 8 months of age; the left hemisphere was used for flow cytometry, the right hindbrain for histology, and the right forebrain for protein analysis. (B) Representative fluorescence micrograph of the BMWT::AD+ chimeric mouse brain showing infiltrating monocytes (GFP+/CD45hi; arrows) near a 6E10+ amyloid-β plaque site. Scale bar = 20 μm. (C) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in brain cortices (Ctx) of BMWT::AD+, BMACE10::AD+, and BMAD+::AD+ chimeric mice (n = 5–7 mice/group). (D) Sandwich ELISA analysis of cerebral soluble amyloid-β42 levels in AD+ chimeric mice (n = 6–8 mice/group). (E) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte cell count in AD+ chimeric mice (n = 6–8 mice/group). (F) Quantitative IHC analysis of the cortical GFAP+ reactive astrocyte area in AD+ chimeric mice (n = 7 mice/group). (G) Representative micrographs of cortical amyloid-β plaques stained with curcumin in BMWT::AD+ versus BMACE10::AD+ chimeric mice. (H) Representative micrographs of cortical GFAP+ astrocytes in the same experimental groups. Scale bar = 50 μm (for G and H). (I) Flow cytometry analysis for cerebral infiltrating GFP+ monocytes in AD+ chimeric mice. Combinations of markers were used to identify microglia (CD11b+CD45int.-lowGFP) versus infiltrating monocyte subsets (CD11b+CD45hiLy6ChiGFP+). (J) Representative flow cytometry map analysis showing increased infiltration of CD11bhiCD45hiLy6ChiGFP+ ACE10 versus wild-type monocytes in AD+ chimeric mice. (K and L) Quantitative flow cytometry analyses of cerebral inflammatory biomarkers in chimeric AD+ mice following partial bone marrow transplantation. (K) Percentage of GFP+CD11bhiCD45hiLy6ChiF4/80low monocytes (n = 5–6 mice/group), (L) TNFα expression in infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi Mφ (n = 4–5 mice/group). (M) Fluorescent micrographs of cortical brain regions stained against IGF1 (red) combined with Iba1 (white) CD45 (green) and nuclei (blue) in BMWT::AD+ versus BMACE10::AD+ chimeric mice. Scale bars = 10 μm. Brain-infiltrating Iba1+CD45hi Mo/Mφ abundantly expressed IGF1 in BMACE10::AD+ chimeric mice (bottom). (N) Quantitative IHC of % IGF1+ area co-localized with Mo/Mφ in BMWT::AD+ versus BMACE10::AD+ chimeric mice (n = 7 mice/group). (O–Q) Representative non-invasive retinal fluorescence imaging of infiltrating GFP+ bone marrow cells in living AD+ chimeric mice. (O) A control AD+ mouse that underwent bone marrow transplantation of GFP−BMAD+ (GFP−BMAD+::AD+). (P) Retinal fluorescent images from a GFP+BMWT::AD+ mouse (P’, enlarged image). (Q) Retinal fluorescent images from a GFP+BMACE10::AD+ mouse (arrows indicate GFP+ bone marrow cells). (R) Representative microscopic image obtained during the histological examination of a retina extracted from a BMACE10::AD+ mouse whose retina had been previously imaged in vivo; ex vivo staining with GFP (green), CD45 (red), 4G8 (cyan), and DAPI (blue), validating the homing of GFP+ACE10 monocytes to retinal amyloid-β plaques. Scale bar = 5 μm. Data from an individual mouse (filled circles for males and clear circles for females) as well as group mean ± SEM are shown. Fold increase and percentage decreases compared to control groups are shown in green. *P < 0.05, **P < 0.01. ns = non-significant, using one-way ANOVA and Bonferroni’s post-test for three group comparisons. For two-group comparisons, paired or unpaired two-tailed Student t-tests were used. Aβ = amyloid-β; FS = forward scatter; SS = side scatter. To characterize the types of infiltrating inflammatory cells within AD+ mouse brains, flow cytometry was used to quantitate brain inflammatory populations as indicated in Fig. 1I–L and Supplementary Fig. 1F–I. Side scatter and forward scatter, along with biomarkers of haematopoietic myelomonocytic cells such as CD11b, CD45, Ly6C, and F4/80, were identified and quantified in the brain within the GFP-negative (endogenous cells) versus GFP-positive (bone marrow-derived donor cells) populations (Fig. 1I and J). Furthermore, analyses of the expression of the three cytokines TNFα, TGFβ, and IL10 within these inflammatory cells in the brain were included. These analyses showed the presence of both GFP+ donor monocytes in the brains of AD+ animals, with a nearly 3-fold increase in ACE10 compared to ACEWT monocytes (CD11b+CD45hiLy6Cint/hi; Fig. 1K; P = 0.0101), while the microglia populations (GFP−CD11b+CD45int/lowLy6Clow) were unchanged (data not shown). Given the importance of TNFα in the induction of pro-inflammatory macrophage polarization and the pathogenesis of Alzheimer’s disease, we examined TNFα expression by both GFP−CD11b+CD45low-int microglia and brain-infiltrating GFP+CD11b+CD45hiLy6Cint-hiF4/80hi macrophages (Fig. 1L and Supplementary Fig. 1F and G). With regards to microglia, there was no difference in TNFα in any of the groups of mice (Supplementary Fig. 1F). In contrast, there was a significant and substantial 62% decrease in TNFα expression in ACE10 macrophages in those AD+ animals that had received GFP+ACE10 bone marrow rather than GFP+ACEWT bone marrow (P < 0.05; Fig. 1L). Further, our flow cytometry analysis into the chimeric AD+ACE10 mouse brains allowed us to compare TNFα in cerebral macrophages from endogenous, non-GFP (wild-type for ACE expression) versus donor marrow, GFP+ (with overexpression of ACE) sources in individual mice (Supplementary Fig. 1G). We found that those macrophages overexpressing ACE produced 58% less cerebral TNFα (P < 0.05). No significant differences were detected in TGFβ and IL10 levels in cerebral macrophages of chimeric AD+ mice (Supplementary Fig. 1H and I). To investigate cerebral ACE10 macrophage phenotype further, we assessed insulin-like growth factor 1 (IGF1), associated with anti-inflammatory and neuro-regenerative functions (Gasparini and Xu, 2003; Butovsky et al., 2006; Koronyo-Hamaoui et al., 2009; Han et al., 2016; Bianchi et al., 2017; Spadaro et al., 2017; Vannella and Wynn, 2017) in the brains of chimeric AD+ACE10 mice compared to control mice (Fig. 1M and N and Supplementary Fig. 1J–O). Our immunohistochemistry data demonstrate a consistent 2.3-fold increase in cerebral IGF1 expression, and further, a substantial 2.5-fold increase surrounding Thio-S+ amyloid-β plaques in chimeric AD+ACE10 mice (Supplementary Fig. 1K and L; P < 0.05 and 0.0001). Moreover, cerebral IGF1 was predominantly expressed by Iba1+CD45high brain-infiltrating monocytes and macrophages, with a significant 3.2-fold elevation of IGF1 expression in brain macrophages in chimeric AD+ACE10 mice (Fig. 1M and N and Supplementary Fig. 1M and N; P < 0.01). A tight correlation was observed between cerebral IGF1 immunoreactive area and co-localized IGF1 within macrophages (Supplementary Fig. 1 O; Pearson’s r = 0.92, P < 0.0001), suggesting that infiltrating monocyte/macrophages are the main source of IGF1 in these chimeric AD+ mice. Thus, there appears to be a fundamental difference between wild-type and ACE10 macrophages in their responses to an identical inflammatory environment induced by Alzheimer’s disease in the brain. Figure 1 O–Q shows non-invasive retinal images from live AD+ mice that had received bone marrow transplantation with either non-GFP AD+ACEWT (Fig. 1 O), GFP+-AD−ACEWT (Fig. 1P and P’), or GFP+-AD−ACE10 (Fig. 1Q). Numerous macrophages within the retina were observed (Fig. 1P and Q). Supplementary Videos 1 and 2 demonstrate the ability to track individual GFP+ immune cells derived from wild-type and ACE10 bone marrow, respectively, in the live retina of an Alzheimer’s disease model mouse. Further, detailed histological examination of ex vivo retinal immunohistochemistry imaging documents homing of ACE10 macrophages to amyloid-β plaque (Fig. 1R). This was seen far more consistently in those animals that had received bone marrow from ACE10 donors than in animals that had received bone marrow from an ACEWT origin (Fig. 1P and Q). Synaptic and cognitive preservation following peripheral blood enrichment of CD115+ ACE10 monocytes Another more translational strategy that we used to assess the relative contribution and potential therapeutic effect of ACE10 monocytes involved peripheral blood enrichment with CD115+ monocytes. CD115 is the product of the Csf1r gene; monocytes that express this marker are often termed inflammatory monocytes and are recruited to inflamed sites to mediate tissue repair (Valero et al., 2017). Previous work from our laboratory has established that adoptively transferred CD115+ wild-type monocytes in AD+ mice directly ameliorate amyloid-β neuropathology and lead to reduced neuroinflammation and preservation of cognitive function (Koronyo et al., 2015). Our current findings demonstrate that ACE10 bone marrow can further reduce the neuropathology in AD+ mice more than ACEWT bone marrow; hence, the next step was to test the effects of blood enrichment with CD115+ ACE10 monocytes (Fig. 2). The experimental procedure and timeline for adoptive transfer studies can be found in Fig. 2A–D (n = 23 AD+ experimental mice and n = 7 non-transgenic naïve wild-type littermates; all males). Controls for this experiment were recipient AD+ mice that had received monthly intravenous injections of PBS (vehicle). At 11 months, open field and Barnes maze behavioural tests were performed (Fig. 2E–K and Supplementary Fig. 2A) as previously described (Bernstein et al., 2014b; Koronyo et al., 2015). There were no differences in locomotor activity, neither rearing nor ambulatory movements, between any of the groups (Fig. 2E and F). Substantial differences in performance in the Barnes cognitive test, both in incorrect entries and latency times, were recorded during the training phase of the study and during the long-term memory retention and reversal phases (Fig. 2G–K and Supplementary Fig. 2A–C). Adoptive transfer of CD115+ cells enhanced overall cognitive functions, with ACE10 monocytes consistently showing a trend of extended improvement. Figure 2 Open in new tabDownload slide Cognitive preservation and restricted pathology following adoptive transfer of a CD115+ ACE10 monocyte subset in AD+ mice transgenic mice. (A–C) Schematic representation of experimental procedure and treatment groups. (A) CD115+ monocytes (MoBM) were isolated from the bone marrow of GFP+ donor mice and enriched by MACS microbeads and an anti-CD115 antibody column sorting procedure. (B) MoBM were then intravenously (i.v.) injected into the tail vein of AD+ recipient mice (n = 8 mice/group, all males). (C) Additional control groups included naïve wild-type (WT) mice and AD+ mice injected with PBS (n = 7 mice/group, all males). (D) Schematic timeline of the in vivo preclinical experiment. Pre-symptomatic AD+ mice exhibiting neuropathology at 8 months of age received monthly injections of 5–6 million GFP+MoWT or GFP+MoACE10 or PBS for 3 months (immunization regimen indicated by green arrows). At 11 months, mice underwent behavioural tests followed by tissue collection and analysis when 12 months of age. (E and F) Open field test in all AD+ treatment and naïve wild-type groups measuring: (E) ambulatory and (F) rearing activity. (G–K) Cognitive functions assessed by the Barnes maze test in both monocyte-treated groups as compared to the control PBS-injected group and naïve cognitively normal wild-type group (n = 6–8 mice/group). Incorrect entries (errors) for the following: (G) acquisition-training phase (Days 1–4), (H) memory retention (Day 7), (I) reversal phase (Days 8 or 9), and (J and K) memory test at Day 9. (J) Errors and (K) escape latency (s). (L) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in the hippocampus (HC), cingulate cortex (CC), and total brain in MoWT- and MoACE10-treated versus PBS-injected AD+ mice (n = 7–8 mice/group). (M) CAA score assessed as vascular Thio-S+ in AD+ mice (n = 6–8 mice/group). Data from an individual mouse as well as group mean ± SEM are shown. (N) Quantitative IHC analysis of hippocampal pre-(VGluT1+) and postsynaptic (PSD95+) areas in MoACE10-treated mice compared to PBS-injected control AD+ and naïve wild-type mice (n = 6–8 mice/group). Data from individual mice, lower and upper quartiles (as lower and upper horizontal lines in box), median (midline within box), and minimum/maximum values (whiskers), are shown. Percentage increase and decrease compared to control groups are shown in green. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Significance between two-groups by unpaired two-tailed Student t-test. IR = immunoreactive. Figure 2 Open in new tabDownload slide Cognitive preservation and restricted pathology following adoptive transfer of a CD115+ ACE10 monocyte subset in AD+ mice transgenic mice. (A–C) Schematic representation of experimental procedure and treatment groups. (A) CD115+ monocytes (MoBM) were isolated from the bone marrow of GFP+ donor mice and enriched by MACS microbeads and an anti-CD115 antibody column sorting procedure. (B) MoBM were then intravenously (i.v.) injected into the tail vein of AD+ recipient mice (n = 8 mice/group, all males). (C) Additional control groups included naïve wild-type (WT) mice and AD+ mice injected with PBS (n = 7 mice/group, all males). (D) Schematic timeline of the in vivo preclinical experiment. Pre-symptomatic AD+ mice exhibiting neuropathology at 8 months of age received monthly injections of 5–6 million GFP+MoWT or GFP+MoACE10 or PBS for 3 months (immunization regimen indicated by green arrows). At 11 months, mice underwent behavioural tests followed by tissue collection and analysis when 12 months of age. (E and F) Open field test in all AD+ treatment and naïve wild-type groups measuring: (E) ambulatory and (F) rearing activity. (G–K) Cognitive functions assessed by the Barnes maze test in both monocyte-treated groups as compared to the control PBS-injected group and naïve cognitively normal wild-type group (n = 6–8 mice/group). Incorrect entries (errors) for the following: (G) acquisition-training phase (Days 1–4), (H) memory retention (Day 7), (I) reversal phase (Days 8 or 9), and (J and K) memory test at Day 9. (J) Errors and (K) escape latency (s). (L) Quantitative IHC analysis of 6E10+ amyloid-β plaque areas in the hippocampus (HC), cingulate cortex (CC), and total brain in MoWT- and MoACE10-treated versus PBS-injected AD+ mice (n = 7–8 mice/group). (M) CAA score assessed as vascular Thio-S+ in AD+ mice (n = 6–8 mice/group). Data from an individual mouse as well as group mean ± SEM are shown. (N) Quantitative IHC analysis of hippocampal pre-(VGluT1+) and postsynaptic (PSD95+) areas in MoACE10-treated mice compared to PBS-injected control AD+ and naïve wild-type mice (n = 6–8 mice/group). Data from individual mice, lower and upper quartiles (as lower and upper horizontal lines in box), median (midline within box), and minimum/maximum values (whiskers), are shown. Percentage increase and decrease compared to control groups are shown in green. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Significance between two-groups by unpaired two-tailed Student t-test. IR = immunoreactive. In the training phase, AD+ mice that had received CD115+ monocytes with either ACEWT or ACE10 showed a pattern of maze learning that was essentially identical to that of wild-type mice (Fig. 2G and Supplementary Fig. 2A). In contrast, AD+ mice that had received PBS displayed impaired training compared to wild-type mice. In the long-term memory retention test (Day 7), during the reversal phase (Day 8), and in the spatial memory analysis at Day 9, we observed significant differences between mice receiving CD115+ ACE10 cells and those receiving PBS controls. MoACE10-treated mice displayed a pattern of learning equivalent to that of wild-type mice, which was also superior to recipients of the MoWT cells (Fig. 2H–K and Supplementary Fig. 2B and C). Immunohistochemical analysis of 6E10+ plaque area in the hippocampus, cingulate cortex, and total brain shows that AD+ mice that had received an infusion of CD115+ (predominantly ACE10) monocytes had fewer hippocampal and total brain plaques than AD+ mice that had received only PBS vehicle (P < 0.05, Fig. 2L and Supplementary Fig. 2D–F). Analysis of vascular Thio-S+ amyloid-β burden calculated as CAA score (Rentsendorj et al., 2018) revealed significant 56% and 82% reductions in CAA scores in AD+ mice recipients of both BMWT and BMACE10, respectively, compared to the control group (Fig. 2M and Supplementary Fig. 2I–M; P < 0.0001). A significant reduction in CAA scores was further noted in recipients of BMACE10 versus BMWT in hippocampal and cortical regions (Fig. 2M and Supplementary Fig. 2L and M; P < 0.05). Analysis of synaptic biomarkers indicated a significant preservation of both pre- (VGluT1) and post- (PSD-95) synapses by blood enrichment with CD115+ ACE10 monocytes, promoting synaptic protection (Fig. 2N and Supplementary Fig. 2G and H; P < 0.05–P < 0.001). Enhanced binding and uptake of pathological amyloid-β42 conformers and surface expression of scavenger receptors in ACE10 macrophages Next, we characterized ACE-overexpressing macrophages for their ability to recognize, bind, internalize, and process defined structures of amyloid-β associated with Alzheimer’s disease neuropathology. To this end, we performed an experiment in which bone marrow cells were harvested from wild-type and ACE10 mice and then cultured for 6 days with MCSF to generate mature macrophages (Fig. 3A). On Day 7, these cells were mixed with well-defined and characterized populations of amyloid-β1-42 fibrils, amyloid-β1-42 prion rod-like structures, or cross-linked-stabilized soluble amyloid-β1-42 oligomers (Fig. 3A and B). The identity of amyloid-β products was confirmed by SDS-PAGE and TEM (Fig. 3B and C). Another control group for this experiment included untreated cells, which were not exposed to amyloid-β. Figure 3D shows surface binding of fibril amyloid-β42 to a class A type 1 scavenger receptor, Scara1, on the cell membrane of ACE10 macrophages (0 min time point); macrophages were immunostained for anti-amyloid-β (6E10), Scara1 (CD204), and nuclei (DAPI; Fig. 3D). Figure 3 Open in new tabDownload slide Enhanced surface recognition and uptake of diverse amyloid-β42 conformers by ACE10 macrophages. (A) Design and timeline of in vitro. Bone marrow was isolated from young wild-type (WT) and ACE10 mice (8–12 weeks old) and cultured for 6 days in MCSF-enriched media, generating primary macrophage cultures (MφWT and MφACE10, respectively). On Day 7, Mφ were either unstimulated (Un) or stimulated by one of the three amyloid-β forms: fibrils (fAβ42), prion rod-like structures (pAβ42), or cross-linked oligomers (oAβ42). MφACE10 were analysed and compared to control MφWT for cell surface amyloid-β42 binding (pre-chilled on ice; binding – 0 min) and receptor expression as well as uptake of amyloid-β42 species at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake: 5, 15, 30 min). Cells and media were isolated and further analysed using various methods including ICC, flow cytometry (FC), and quantitative protein analysis. (B) Forms of non-cross-linked (non-XL) and cross-linked (oAβ) amyloid-β42. Oligomers as large as an octamer are observed. (C) Negative stain TEM micrographs of amyloid-β42 assemblies. Left: Fibrillar amyloid-β (fAβ42) displayed long fibrils of ˜10 nm diameter with a twisted morphology typically comprising two smaller filaments. Middle: Prion rod-like amyloid-β42 (pAβ42) displayed short (<300 nm) straight rods comprising multiple filaments (blue arrows). Narrower (˜5 nm) rod-like structures appeared to comprise twisted double filaments (black arrows). The assembly indicated by the red arrow is shown in the inset at higher magnification. Right: Cross-linked oligomers (oAβ42) displayed mainly quasi-spherical assemblies (white arrows) with diameters of 5–8 nm. The assembly indicated by the red arrow is shown in the inset at higher magnification. A rare rod-like structure (yellow asterisk) was also observed. Scale bars = 100 nm or 50 nm (insets). (D) Representative fluorescence micrograph of MφACE10 prestimulated with fibril amyloid-β42 on ice (at the 0-min time point) and immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Abundance of co-surface labelling of Scara1+ and fibril amyloid-β42 (yellow spots) on the MφACE10 cell body and processes was detected, indicating large amounts of fibril amyloid-β42 bound to surface Scara1. (E) Quantitative ICC analysis of a 6E10+ immunoreactive (IR) area for cell surface binding of the three amyloid-β42 species (250 nM each) at the 0-min time point in MφACE10 versus MφWT. (F) Quantitative ICC analysis of surface Scara1 expression in ACE10 versus wild-type Mφ that were either untreated or in the presence of different amyloid-β42 forms at the 0-min time point. (G) Quantitative ICC analysis of TREM2 expression in ACE10 versus wild-type Mφ following prestimulation by the different forms of amyloid-β42 (250 nM each). (H) Representative fluorescence micrograph of MφWT and MφACE10 cells that were incubated for 5 min with 250 nM fibril amyloid-β42 and later immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Amyloid-β42 fibrils are seen in intracellular vesicles within these phagocytic cells (white arrow). (I) Panel of representative fluorescence micrographs of MφWT and MφACE10 cells following a 15-min incubation with three different amyloid-β42 forms immunolabelled for scavenger receptor type B CD36 (purple), human amyloid-β (6E10; green), and nuclei (blue). (I’) Separated single channel (cy2) for intracellular amyloid-β42 uptake. (J) Quantitative ICC analysis of CD36 expression (μm2/cell number) in ACE10 versus wild-type Mφ that were either untreated or exposed to different amyloid-β42 forms (250 nM) for 15 min. (K) Quantitative ICC analysis of 6E10+ immune-reactive area (μm2/cell number) for intracellular uptake of the three amyloid-β42 species (250 nM) for 15 min by MφACE10 versus MφWT. (L and M) Quantitative ICC analysis of (L) CD36 expression and (M) amyloid-β uptake by untreated versus ACE-inhibitor pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA) MφACE10, following exposure to 100 nM fibril amyloid-β42 for either 0, 5 or 30 min. (N–Q) Individual correlation analysis of the following: (N and O) surface fibril amyloid-β42 and Scara1 immunoreactive area at the binding time point in (N) MφWT and (O) MφACE10 (n =15 wells); (P and Q) surface fibril/oligomer amyloid-β42 and CD36 expression in MφACE10 following a 15-min exposure to either fibril amyloid-β42 or oligomer amyloid-β42. (R) Representative peak analysis in percentage mean fluorescence intensity (MFI) of MφACE10 uptake of HiLyte Fluor 647-labelled fibril amyloid-β42 at the 0-, 5-and 15-min time points. (S) Representative flow cytometry images of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI at the 15-min time point by F4/80+-MφWT versus -MφACE10. (T and U) Quantitative flow cytometry analysis of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI uptake at the 0-, 5- and 15-min time points by (T) MφWT or MφACE10 and by (U) untreated and lisinopril pretreated MφACE10. The group mean ± SEM as well as the fold reduction or increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s or Sidak’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Figure 3 Open in new tabDownload slide Enhanced surface recognition and uptake of diverse amyloid-β42 conformers by ACE10 macrophages. (A) Design and timeline of in vitro. Bone marrow was isolated from young wild-type (WT) and ACE10 mice (8–12 weeks old) and cultured for 6 days in MCSF-enriched media, generating primary macrophage cultures (MφWT and MφACE10, respectively). On Day 7, Mφ were either unstimulated (Un) or stimulated by one of the three amyloid-β forms: fibrils (fAβ42), prion rod-like structures (pAβ42), or cross-linked oligomers (oAβ42). MφACE10 were analysed and compared to control MφWT for cell surface amyloid-β42 binding (pre-chilled on ice; binding – 0 min) and receptor expression as well as uptake of amyloid-β42 species at incremental time points from 5 min up to 60 min (incubated at 37°C; uptake: 5, 15, 30 min). Cells and media were isolated and further analysed using various methods including ICC, flow cytometry (FC), and quantitative protein analysis. (B) Forms of non-cross-linked (non-XL) and cross-linked (oAβ) amyloid-β42. Oligomers as large as an octamer are observed. (C) Negative stain TEM micrographs of amyloid-β42 assemblies. Left: Fibrillar amyloid-β (fAβ42) displayed long fibrils of ˜10 nm diameter with a twisted morphology typically comprising two smaller filaments. Middle: Prion rod-like amyloid-β42 (pAβ42) displayed short (<300 nm) straight rods comprising multiple filaments (blue arrows). Narrower (˜5 nm) rod-like structures appeared to comprise twisted double filaments (black arrows). The assembly indicated by the red arrow is shown in the inset at higher magnification. Right: Cross-linked oligomers (oAβ42) displayed mainly quasi-spherical assemblies (white arrows) with diameters of 5–8 nm. The assembly indicated by the red arrow is shown in the inset at higher magnification. A rare rod-like structure (yellow asterisk) was also observed. Scale bars = 100 nm or 50 nm (insets). (D) Representative fluorescence micrograph of MφACE10 prestimulated with fibril amyloid-β42 on ice (at the 0-min time point) and immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Abundance of co-surface labelling of Scara1+ and fibril amyloid-β42 (yellow spots) on the MφACE10 cell body and processes was detected, indicating large amounts of fibril amyloid-β42 bound to surface Scara1. (E) Quantitative ICC analysis of a 6E10+ immunoreactive (IR) area for cell surface binding of the three amyloid-β42 species (250 nM each) at the 0-min time point in MφACE10 versus MφWT. (F) Quantitative ICC analysis of surface Scara1 expression in ACE10 versus wild-type Mφ that were either untreated or in the presence of different amyloid-β42 forms at the 0-min time point. (G) Quantitative ICC analysis of TREM2 expression in ACE10 versus wild-type Mφ following prestimulation by the different forms of amyloid-β42 (250 nM each). (H) Representative fluorescence micrograph of MφWT and MφACE10 cells that were incubated for 5 min with 250 nM fibril amyloid-β42 and later immunolabelled for Scara1 (red), human amyloid-β (6E10; green), and nuclei (blue). Amyloid-β42 fibrils are seen in intracellular vesicles within these phagocytic cells (white arrow). (I) Panel of representative fluorescence micrographs of MφWT and MφACE10 cells following a 15-min incubation with three different amyloid-β42 forms immunolabelled for scavenger receptor type B CD36 (purple), human amyloid-β (6E10; green), and nuclei (blue). (I’) Separated single channel (cy2) for intracellular amyloid-β42 uptake. (J) Quantitative ICC analysis of CD36 expression (μm2/cell number) in ACE10 versus wild-type Mφ that were either untreated or exposed to different amyloid-β42 forms (250 nM) for 15 min. (K) Quantitative ICC analysis of 6E10+ immune-reactive area (μm2/cell number) for intracellular uptake of the three amyloid-β42 species (250 nM) for 15 min by MφACE10 versus MφWT. (L and M) Quantitative ICC analysis of (L) CD36 expression and (M) amyloid-β uptake by untreated versus ACE-inhibitor pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA) MφACE10, following exposure to 100 nM fibril amyloid-β42 for either 0, 5 or 30 min. (N–Q) Individual correlation analysis of the following: (N and O) surface fibril amyloid-β42 and Scara1 immunoreactive area at the binding time point in (N) MφWT and (O) MφACE10 (n =15 wells); (P and Q) surface fibril/oligomer amyloid-β42 and CD36 expression in MφACE10 following a 15-min exposure to either fibril amyloid-β42 or oligomer amyloid-β42. (R) Representative peak analysis in percentage mean fluorescence intensity (MFI) of MφACE10 uptake of HiLyte Fluor 647-labelled fibril amyloid-β42 at the 0-, 5-and 15-min time points. (S) Representative flow cytometry images of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI at the 15-min time point by F4/80+-MφWT versus -MφACE10. (T and U) Quantitative flow cytometry analysis of HiLyte Fluor 647-labelled fibril amyloid-β42 MFI uptake at the 0-, 5- and 15-min time points by (T) MφWT or MφACE10 and by (U) untreated and lisinopril pretreated MφACE10. The group mean ± SEM as well as the fold reduction or increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s or Sidak’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Quantitative ICC analysis indicates substantial 4.9-fold and 2-fold increases of surface fibril amyloid-β42 and oligomer amyloid-β42 binding, respectively, in ACE10 macrophages as compared with wild-type macrophages (Fig. 3E; P = 0.001 and P = 0.02, respectively). Membrane scavenger receptors such as Scara1, class B scavenger receptor CD36, triggering receptor expressed on myeloid cells 2 (TREM2), and CD163 have been implicated in macrophage activation and increased phagocytosis of amyloid-β in Alzheimer’s disease (Hickman et al., 2008; Yamanaka et al., 2012; Frenkel et al., 2013; Pey et al., 2014; Kim et al., 2017). We observed significant increases in baseline Scara1, TREM2, CD163, and CD36 surface expression in untreated ACE10 versus wild-type macrophages (Fig. 3F, Supplementary Fig. 3A and B and Supplementary Table 1; P < 0.01–0.0001). These receptors have been associated with Alzheimer’s disease and specifically facilitate amyloid-β uptake by microglia and/or macrophages but have never been studied in ACE10 macrophages or in response to defined and stabilized populations of amyloid-β42 oligomers. We also found that surface expression of TREM2 and Scara1 was increased in ACE10 macrophages at the 0-min time point and 5 min after exposure to fibril amyloid-β42 (Fig. 3F–H and Supplementary Fig. 3C; P < 0.05–0.0001) but not in response to amyloid-β42 prion rod-like structures or soluble oligomers (Fig. 3F and G). As early as 5 min after contact and phagocytosis of fibril amyloid-β42, intracellular amyloid-β42 staining was visible in the cell processes and soma of wild-type macrophages, and more prominently in ACE10 macrophages (Fig. 3H). Interestingly, baseline MHC-II expression was lower by 49% in the ACE10 macrophages (Supplementary Table 1; P < 0.01). A summary of key in vitro findings comparing ACE10 and wild-type macrophages can be found in Supplementary Table 1. To assess the macrophages’ ability to uptake amyloid-β42 species at later time points (15, 30, and 60 min), we conducted a series of experiments presented in Fig. 3I–U, Supplementary Table 1, and Supplementary Fig. 3. Figure 3I displays the major differences detected in macrophage uptake of different amyloid-β42 conformers after 15 min of exposure, with fibrils being the most efficiently phagocytosed and soluble oligomers the least engulfed by these cells (Supplementary Fig. 3D–F). Quantitative ICC analysis shows that surface CD36 expression by ACE10 macrophages was substantially and consistently increased as compared to wild-type macrophages (Fig. 3J; P < 0.0001), along with increased intracellular amyloid-β42 uptake 15 min after exposure to all three forms of amyloid-β (Fig. 3K; P < 0.05–0.0001). Moreover, the expression of CD36 as well as the uptake of fibril amyloid-β42 at 5 and 30 min were found to be dependent on ACE catalytic activity, since overnight treatment with the ACE inhibitor lisinopril significantly decreased both (Fig. 3L and M). Interestingly, in ACE10 macrophages treated with lisinopril, CD36 expression was decreased and fibril amyloid-β42 uptake increased over time during a 30-min culture period, while remaining substantially lower compared to their values in the enzymatically uninhibited ACE10 macrophages (Fig. 3L and M). Supplementary Table 1 summarizes the main findings on amyloid-β surface binding and uptake as well as the expression of scavenger receptors, the immune profile, and the morphology of wild-type versus ACE10 macrophages. Analysis of discrete wild-type and ACE10 macrophage populations to determine potential associations between amyloid-β42 binding/uptake and surface expression of the scavenger receptors Scara1 and CD36, using Pearson’s r correlations, revealed strong and direct relationships in both cell genotypes (Fig. 3N and O; r = 0.73, P = 0.0022 for wild-type; r = 0.71, P = 0.003 for ACE10, for Scara1). Further, there were direct correlations between amyloid-β42 fibril and amyloid-β42 oligomer uptake after a 15-min incubation and the expression level of CD36 in ACE10 macrophages (Fig. 3P and Q; r = 0.83 and P = 0.001 for fibril amyloid-β42; r = 0.42, P = 0.038 for oligomer amyloid-β42). The strong correlation between CD36 expression and amyloid-β uptake was also true for prion rod-like amyloid-β42 uptake after 15 min in both wild-type and ACE10 macrophages (Supplementary Fig. 3K and L; r = 0.83 and 0.76, respectively, and P < 0.0001). Extended correlations are shown in Supplementary Fig. 3G–L. Using quantitative flow cytometry analysis, we validated increased binding and uptake of fluorescently labelled HiLyte 647-fibril amyloid-β42 in ACE10 versus wild-type macrophages, from both bone marrow and peritoneal sources, as well as reversal effects following inhibition of ACE catalytic domains (Fig. 3R–U and Supplementary Fig. 3M–P). Flow cytometry-based quantitative analyses confirmed the aforementioned ICC analysis and demonstrated that the rate of amyloid-β42 fibril phagocytosis by ACE10 macrophages is significantly faster than that by wild-type macrophages in the first 5, 15 and 60 min of uptake assays (Fig. 3T and Supplementary Fig. 3M–P). Interestingly, the ACE inhibitor lisinopril also had effects on wild-type macrophages (peritoneal), significantly reducing phagocytosis of amyloid-β42 fibrils by >50% (Supplementary Fig. 3P), suggesting a role for endogenous ACE in macrophage-mediated fibril amyloid-β42 phagocytosis. Superior endosomal-lysosomal and extracellular degradation of amyloid-β42 conformers by ACE10 macrophages We next studied the capacity of ACE10 macrophages for intra- and extracellular processing and degradation of the three types of amyloid-β and the intracellular localization of these amyloid-β42 species (Fig. 4). We conducted a series of studies using bone marrow-derived ACE10 and wild-type macrophages (Fig. 3A–C). Initial studies of wild-type macrophages showed that when cells were exposed to fibril amyloid-β42, the autophagy marker Beclin-1 relocated to the cell nucleus (Fig. 4A). No difference in Beclin-1 expression was detected in ACE10 versus wild-type macrophages (data not shown). In contrast to Beclin-1 expression, fibril amyloid-β42 appears to co-localize within early endosomes (EEA1) and lysosomal associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2) within lysosomal vacuoles (Fig. 4B). Further analysis of amyloid-β intake after a 15-min exposure to fibril amyloid-β42, prion amyloid-β42, and oligomer amyloid-β42 showed co-localization of the amyloid-β peptides within early endosomes (Fig. 4C–F). The uptake of amyloid-β42 conformers and co-localization in the EEA1+ endosomes was more apparent in ACE10macrophages than in wild-type cells. This was observed for both the fibrillar and the soluble oligomeric forms of amyloid-β42, although it was more intense for the fibrillar form (Fig. 4C–F). A systematic evaluation of the uptake of fibril, prion, and oligomer amyloid-β42 species by ACE10 versus wild-type macrophages was undertaken. Evaluation of the amyloid-β immunoreactive area within EEA1+ endosomes (Fig. 4G) revealed that ACE10 cells consistently had greater amounts of amyloid-β in the endosomal-lysosomal pathway, presumably fated for intracellular degradation. The larger increases in the ACE10 macrophages were most pronounced for the soluble amyloid-β oligomers, as compared to prion rod-like structures and fibrils, as they took up far more soluble amyloid than that observed in wild-type cells (Fig. 4G and H; 21- to 40-fold increases, P < 0.001–0.05). Figure 4 Open in new tabDownload slide Intra- and extracellular degradation of pathological amyloid-β42 species by ACE10 macrophages. (A) Representative fluorescence micrograph of unstimulated (top) and fibril amyloid-β42 stimulated (bottom) MφWT immunolabelled for human amyloid-β (6E10; green), autophagy marker Beclin-1 (red), and nuclei (blue). Merged channel panels show that in response to fibril amyloid-β42, Beclin-1 protein moves to the nuclei (white arrows). (B) Representative fluorescence micrograph panel of fibril amyloid-β42 (fAβ42) stimulated MφWT immunolabelled for human amyloid-β (6E10; green), endosomal markers (EEA1, Lamp-1, Lamp-2, red), and nuclei (blue). (C and D) Representative fluorescence micrograph of MφWT stimulated with the different fibril , prion rod-like and oligomer amyloid-β42 forms for 15 min and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple), and nuclei (blue). White arrows indicate co-localizing of intracellular amyloid-β42 within EEA1+ vesicles. (D) Higher magnification image of co-localized fibril amyloid-β42 within EEA1+ endosomes in MφWT (yellow spots). (E and F) Representative fluorescence micrograph of MφACE10 exposed to oligomer amyloid-β42 (100 nM for 15 min) and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple) and nuclei (blue). White arrows indicate 6E10+ and EEA1+ co-localization. (F) Higher magnification image of co-localized oligomer amyloid-β42 within EEA1+ endosomes in MφACE10 (white arrows). (G and H) Quantitative analysis of co-localized 6E10+ and EEA1+ immunoreactive area in unstimulated and fibril, prion rod-like and oligomer amyloid-β42-stimulated MφWT or MφACE10: (G) 6E10-IR area per macrophage and (H) count of 6E10+/EEA1+ vesicles using puncta count per macrophage. (I) Quantitative normalized analysis of percentage reduction of extracellular fibril amyloid-β42 in media of MφWT versus MφACE10 over 18, 20 and 24 h. (J) Quantitative ELISA analysis of total intra- and extracellular amyloid-β42 levels in the media and cell fraction of MφACE10, following fibril amyloid-β42 for 30 min and either untreated, lisinopril pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA), or pre-placed in ice bath. The group mean ± SEM as well as fold increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. Figure 4 Open in new tabDownload slide Intra- and extracellular degradation of pathological amyloid-β42 species by ACE10 macrophages. (A) Representative fluorescence micrograph of unstimulated (top) and fibril amyloid-β42 stimulated (bottom) MφWT immunolabelled for human amyloid-β (6E10; green), autophagy marker Beclin-1 (red), and nuclei (blue). Merged channel panels show that in response to fibril amyloid-β42, Beclin-1 protein moves to the nuclei (white arrows). (B) Representative fluorescence micrograph panel of fibril amyloid-β42 (fAβ42) stimulated MφWT immunolabelled for human amyloid-β (6E10; green), endosomal markers (EEA1, Lamp-1, Lamp-2, red), and nuclei (blue). (C and D) Representative fluorescence micrograph of MφWT stimulated with the different fibril , prion rod-like and oligomer amyloid-β42 forms for 15 min and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple), and nuclei (blue). White arrows indicate co-localizing of intracellular amyloid-β42 within EEA1+ vesicles. (D) Higher magnification image of co-localized fibril amyloid-β42 within EEA1+ endosomes in MφWT (yellow spots). (E and F) Representative fluorescence micrograph of MφACE10 exposed to oligomer amyloid-β42 (100 nM for 15 min) and immunolabelled for human amyloid-β (6E10; green), endosomal marker EEA1 (red), surface scavenger receptor CD36 (purple) and nuclei (blue). White arrows indicate 6E10+ and EEA1+ co-localization. (F) Higher magnification image of co-localized oligomer amyloid-β42 within EEA1+ endosomes in MφACE10 (white arrows). (G and H) Quantitative analysis of co-localized 6E10+ and EEA1+ immunoreactive area in unstimulated and fibril, prion rod-like and oligomer amyloid-β42-stimulated MφWT or MφACE10: (G) 6E10-IR area per macrophage and (H) count of 6E10+/EEA1+ vesicles using puncta count per macrophage. (I) Quantitative normalized analysis of percentage reduction of extracellular fibril amyloid-β42 in media of MφWT versus MφACE10 over 18, 20 and 24 h. (J) Quantitative ELISA analysis of total intra- and extracellular amyloid-β42 levels in the media and cell fraction of MφACE10, following fibril amyloid-β42 for 30 min and either untreated, lisinopril pretreated (1 μM lisinopril overnight in RPMI media + 0.3% BSA), or pre-placed in ice bath. The group mean ± SEM as well as fold increase and percentage decrease between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test. To elucidate the relative abilities of wild-type and ACE10 macrophages to degrade amyloid-β in vitro, cells from each genotype were cultured with fibril amyloid-β42 and monitored for 18, 20 and 24 h (Fig. 4I and Supplementary Fig. 4). At the end of this period, ELISA was performed to measure extracellular amyloid-β42 levels in the supernatant. There was a substantial reduction (81%) in extracellular amyloid-β42 concentration in the media of ACE10 macrophages compared to wild-type cell media (Fig. 4I; P < 0.001). We further performed an experiment in which ACE10 macrophages were allowed to take up and degrade fibril amyloid-β42 over 30 min in the presence or absence of lisinopril (Fig. 4J). A subset of ACE10 macrophages was treated under the same conditions in an ice bath (0°C) as a control. Amyloid-β42 concentrations in the combined media and cell fraction of macrophages were analysed by performing ELISA. Again, these data suggest that clearance of fibril amyloid-β42 is dependent on ACE catalytic activity, as the group of ACE10 macrophages treated with lisinopril had significantly higher amyloid-β42 concentrations than cells in which ACE was catalytically active (Fig. 4J; P < 0.0001). Pro-healing immune profile of ACE10 macrophages exposed to Alzheimer’s disease-related amyloid-β forms To understand the biology of macrophages in response to amyloid-β forms, both wild-type and ACE10 bone marrow-derived macrophages were subjected to amyloid-β42 fibrils and oligomers, and their morphologies and immune phenotypes were assessed (Fig. 5 and Supplementary Fig. 5). As seen in Fig. 5A and C, ACE10, as opposed to wild-type macrophages, express ∼30% less iNOS and 80% more arginase-1 prior to amyloid-β42 fibril exposure (P < 0.05–0.01; Supplementary Fig. 5A and B). These differences became less apparent for arginase-1 after 5 min exposure to amyloid-β42 peptides (Fig. 5C). The 30% reduction of iNOS expression in ACE10 macrophages, compared to wild-type macrophages, was sustained even after 1-h incubation with soluble amyloid-β42 oligomers (Supplementary Fig. 5A). Further, considerable increases in expression of the amyloid-β-degrading enzyme MMP9 were observed in ACE10 macrophages, both when unstimulated and after exposure to amyloid-β42 fibrils over time (Fig. 5D and E; 1.6–2.5-fold increase, P < 0.001–0.0001). Noticeably, there was a clear difference in the physical form of the cells (Fig. 5F). The ACE10 macrophages were significantly more elongated and the percentage of elongated cells was higher than that in wild-type cells in the unstimulated state (Fig. 5G; 2.2- and 4.4-fold increase, respectively, P < 0.001). Exposing these cells to fibril amyloid-β42 for either 5 or 30 min resulted in an increased ACE10-macrophage aspect ratio (elongation factor), determined as the quotient of maximum cell process length over cell body width (Fig. 5H; 2.0- and 1.7-fold increase, respectively, P < 0.0001). Hence, the cell processes of wild-type macrophages were consistently shorter than the processes of ACE-enhanced macrophages over time (Fig. 5F–H). This type of elongated macrophage morphology was previously associated with an anti-inflammatory, pro-healing phenotype (McWhorter et al., 2013; Rentsendorj et al., 2018). Figure 5 Open in new tabDownload slide ACE overexpression in macrophages promotes a pro-healing, alternatively activated immune profile. (A) Fluorescence micrographs of inducible nitric oxide synthase (iNOS, red), scavenger receptor CD36 expression (cyan), and nuclei (blue) in MφACE10 versus MφWT (merged image on right). (B–D) Quantitative analysis showing (B) iNOS, (C) arginase-1, and (D) MMP9 in the immunoreactive area of unstimulated (Un) and fibril amyloid-β42-stimulated MφACE10 and MφWT at the 0- and 5-min time points. (E) Representative fluorescence micrographs of MφWT and MφACE10 immunolabelled for MMP-9 (red) and nuclei (blue). (F) Representative bright-field images showing a morphological difference in the cell process length of MφACE10 versus MφWT with measurements of elongation (red lines). (G) Quantitative analysis of elongation factor [ratio between total cell length and soma width (left) and percentage cell elongation (right) in unstimulated MφWT versus MφACE10]. (H) Elongation factor in MφWT versus MφACE10 stimulated with fibril amyloid-β42 for 0, 5, and 30 min. (I–L) Quantitative Meso Scale Discovery (MSD) protein assay of unstimulated (−) and stimulated with fibril amyloid-β42 [100 nM for 24 h (+)] MφWT versus MφACE10 for the following cytokines: (I) TNFα, (J) IL6, (K) CXCL1, and (L) IL1β. (M) Quantitative analysis of a cell viability MTT assay in MφWT versus MφACE10after 24 h of exposure to 1 μM fibril amyloid-β42 (n = 3 mice/genotype group, n = 6 wells/group; one-tailed Student t-test). Data from individual mouse, group mean ± SEM as well as fold increase and percentage decrease and increase between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Figure 5 Open in new tabDownload slide ACE overexpression in macrophages promotes a pro-healing, alternatively activated immune profile. (A) Fluorescence micrographs of inducible nitric oxide synthase (iNOS, red), scavenger receptor CD36 expression (cyan), and nuclei (blue) in MφACE10 versus MφWT (merged image on right). (B–D) Quantitative analysis showing (B) iNOS, (C) arginase-1, and (D) MMP9 in the immunoreactive area of unstimulated (Un) and fibril amyloid-β42-stimulated MφACE10 and MφWT at the 0- and 5-min time points. (E) Representative fluorescence micrographs of MφWT and MφACE10 immunolabelled for MMP-9 (red) and nuclei (blue). (F) Representative bright-field images showing a morphological difference in the cell process length of MφACE10 versus MφWT with measurements of elongation (red lines). (G) Quantitative analysis of elongation factor [ratio between total cell length and soma width (left) and percentage cell elongation (right) in unstimulated MφWT versus MφACE10]. (H) Elongation factor in MφWT versus MφACE10 stimulated with fibril amyloid-β42 for 0, 5, and 30 min. (I–L) Quantitative Meso Scale Discovery (MSD) protein assay of unstimulated (−) and stimulated with fibril amyloid-β42 [100 nM for 24 h (+)] MφWT versus MφACE10 for the following cytokines: (I) TNFα, (J) IL6, (K) CXCL1, and (L) IL1β. (M) Quantitative analysis of a cell viability MTT assay in MφWT versus MφACE10after 24 h of exposure to 1 μM fibril amyloid-β42 (n = 3 mice/genotype group, n = 6 wells/group; one-tailed Student t-test). Data from individual mouse, group mean ± SEM as well as fold increase and percentage decrease and increase between groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = non-significant, by two-way or one-way ANOVA and Bonferroni’s post-test, while asterisks in parentheses signify a two-group comparison using the unpaired two-tailed Student t-test. Along this line, the immune profiles of ACE10 and wild-type macrophages were further analysed using a highly sensitive MSD multiplex inflammatory assay. The results are in good accordance with the results obtained in vivo (Fig. 1M and N) and show that stimulation of both wild-type and ACE10 macrophages with fibril amyloid-β42 for 24 h increased expression of TNFα (Fig. 5I; P < 0.0001). However, the magnitude of TNFα increase was significantly lower in the ACE10 macrophages (Fig. 5I; P < 0.001). While IL-6, CXCL1, and IL-1β concentrations were also elevated in both wild-type and ACE10 macrophages in response to fibril amyloid-β42 stimulation, the degree of increase was more reserved in the ACE10 macrophages than in the wild-type cells (Fig. 5J–L and Supplementary Fig. 5C–F). Therefore, this assay showed that the expression of pro-inflammatory cytokines is generally reduced in ACE10 relative to wild-type macrophages when equivalently stimulated with amyloid-β. Finally, we examined the viability of wild-type versus ACE10macrophages following a 24-h exposure to higher concentration of fibril amyloid-β42 (1 µM). In this assay, on average 50% of ACE10 macrophages appeared to survive, compared to only ∼30% of the wild-type macrophages (Fig. 5M; P < 0.05). Discussion Peripheral monocyte migration into the CNS, the role of ACE, and their possible contribution in resisting Alzheimer’s-associated neuropathology and cognitive decline are subjects of intense investigation and controversy (Ajami et al., 2007; Butovsky et al., 2007; El Khoury et al., 2007; Mildner et al., 2007; Lebson et al., 2010; Bernstein et al., 2014b; Koronyo et al., 2015). In this study, we addressed these fundamental questions and demonstrated: (i) in a partial bone marrow transplantation paradigm with head shielding (protecting the brain from irradiation), there was a measurable migration of peripheral monocytes into the retina and brain parenchyma of AD+ mice. While low in number, these cells critically localized around amyloid-β plaques and reduced cerebral amyloid-β burden and inflammation; (ii) engrafted ACE10 monocytes in the brains of AD+ mice are found at about three times higher numbers than wild-type cells and are more effective in restricting neuropathology; (iii) compared to wild-type macrophages, ACE10 cells in AD+ mouse brains express ∼60% lower levels of TNFα and 3.2-fold higher IGF1, associated with restricting neurodegeneration and cognitive deficits; (iv) non-invasive retinal imaging demonstrates the feasibility of tracking the movements of GFP+-monocytes into the retina of AD+ mice in real time; (v) adoptive transfer of CD115+ ACE10 monocytes into the peripheral blood of symptomatic AD+ mice, an irradiation-free translational approach, is highly effective in reducing vascular amyloidosis and preserving synapses and cognitive function; (vi) ACE10 macrophages exhibit strikingly unique immunological profiles, with enhanced surface receptors and augmented ability to bind, internalize, degrade, and destroy fibrillar, prion rod-like, and soluble oligomeric amyloid-β42 species; and (vii) the elongated cell morphology and molecular signature of ACE10 macrophages suggest an anti-inflammatory, pro-healing, and highly resilient phenotype that may help fight against Alzheimer’s disease. Although several studies have demonstrated the ability of human blood mononuclear cells, monocytes and macrophages, to phagocytose fibrillar amyloid-β and remove plaques from the brains of Alzheimer’s disease model mice (Malm et al., 2005; Simard et al., 2006; Butovsky et al., 2007; Fiala et al., 2007; Lai and McLaurin, 2012; Koronyo et al., 2015), their direct involvement in the clearance of non-fibrillar forms of amyloid-β and possible neuroprotection in Alzheimer’s disease is still highly debated (Fiala et al., 2005; Mildner et al., 2007). Moreover, previous studies did not measure the degree of clearance of defined and stabilized amyloid-β42 structures associated with Alzheimer’s disease neuropathology such as prion rod-like assemblies or soluble oligomers. While amyloid-β plaque burden in the brain appears very limited in predicting cognitive decline (Jansen et al., 2018) and may even serve as a mediator of an effective innate immune response against foreign pathogens (Soscia et al., 2010), soluble amyloid-β42 oligomers and prion rod-like structures have been shown to be highly neurotoxic and more elusive to phagocytic immune cells (Selkoe, 2008; Shankar et al., 2008). Because of the meta-stability of these amyloid-β42 assemblies, structure-activity determinations with respect to macrophage responses have been problematic. Here, using structurally stabilized oligomers and well-characterized prion rod-like and fibrillar amyloid-β42, we were able to accomplish these determinations with respect to binding efficacy, internalization, and degradation by wild-type and ACE10 macrophages. Our studies show that in the wild-type cells, oligomers of amyloid-β42 are found in very low amounts in endosomes and lysosomes, the cellular ubiquitin/proteasome machinery that degrades misfolded proteins (Majumdar et al., 2008). Yet, ACE overexpression and enzymatic function significantly shifted macrophage behaviour and their ability to also target amyloid-β42 oligomers to endosomes (up to 40 times higher as compared to those found in wild-type macrophages; Fig. 4C–H). As a result of the subcellular localization of amyloid-β42 in ACE10 macrophages and the increased levels of amyloid-β-degrading enzymes (i.e. ACE, MMP9), the net concentrations of amyloid-β42 remaining after a 24-h incubation with ACE10 versus wild-type macrophages were as much as 80% lower. The unique morphological and immunological profiles of ACE10 macrophages in response to amyloid-β42 were assessed using various techniques. One mechanism that could explain this superior performance is increased surface expression of scavenger receptors, including Scara1, TREM2, CD163, and especially CD36, all of which have been previously shown to facilitate amyloid-β internalization and clearance by myelomonocytic cells (Koronyo et al., 2015; Rentsendorj et al., 2018). In particular, Scara1 expression was tightly associated with the extent of phagocytosis of amyloid-β42 fibrils, but less so for prion rod-like structures and soluble oligomers. In the ACE10 macrophages, CD36 expression was drastically elevated (increased 6- to 36-fold) and tightly associated with increased uptake of all three types of amyloid-β assemblies. In contrast, the wild-type macrophages displayed no association between surface CD36 expression and uptake of amyloid-β42 fibrils and oligomers, though a strong correlation was observed with rod-like structures. The reason for this phenomenon is currently unknown. Future studies are needed to better understand the molecular immune mechanisms at play to remove pathogenic forms of amyloid-β. Further in vitro techniques supported the enhanced molecular characteristics of the ACE10 macrophages, before and after exposure to amyloid-β42 conformers. All assay findings consistently suggested that these bone marrow-derived cells exhibit an anti-inflammatory (i.e. low TNFα and iNOS, high IGF1, arginase-1, and MMP9) and a pro-healing/protective (i.e. elongated cell morphology) phenotype. These effects were dependent on ACE enzymatic activity and were eliminated when treating the cells with ACE inhibitors. In contrast, previous studies demonstrated that peritoneal-derived ACE10 macrophages, under conditions of acute foreign invasion or cancer models, enhanced their effective immune reactivity in the periphery to resolve disease while exhibiting a highly pro-inflammatory profile (Bernstein et al., 2018). These diverse phenotypes of the ACE10 cells may suggest their ability to promptly and effectively respond to insults in a tissue- and disease-specific manner. This variation in response could also be due to the different stages of healing for which these cells are recruited (i.e. chronic inflammation versus acute injury); nonetheless, their remedial capacity is strongly evident. The enhanced immune response of ACE10 macrophages to acute infection or tumour has repeatedly been shown to not be dependent on the production of angiotensin II or any angiotensin peptide by ACE (reviewed in Bernstein et al., 2018). While this was not formally tested in our study, we believe it most likely that the unique phenotype of the ACE10 macrophages here are probably also due to other mechanisms besides angiotensin II. We further note a recent study documenting that ACE cleaves dozens, if not hundreds, of different peptide substrates (Semis et al., 2019). Our in vivo studies assessed the exclusive contribution of peripherally derived ACE10 monocytes and macrophages in attenuating neuropathology and cognitive deficits in murine models of Alzheimer’s disease. Mounting evidence suggests a protective role for innate immune cells in the CNS (El Khoury et al., 2007; Koronyo-Hamaoui et al., 2009; Shechter et al., 2009; Lebson et al., 2010; Bernstein et al., 2014b; Koronyo et al., 2015), though some studies have failed to show migration of these cells under neurodegenerative conditions (Mildner et al., 2007), or showed monocyte migration to CNS without effects on behaviour and cerebral amyloid-β levels (Prokop et al., 2015; Varvel et al., 2015; Cronk et al., 2018). Our group previously demonstrated that peripherally derived GFP-labelled monocytes are naturally recruited to brains of Alzheimer's disease model mice (Butovsky et al., 2006, 2007; Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b) and that they directly reduce neuropathology and preserve cognition (Koronyo et al., 2015). This irradiation- and pharmacologically-free approach substantially enriched the CD115+ bone marrow-derived inflammatory monocyte subset in the peripheral blood of AD+ mice, resulting in spontaneous recruitment of these cells to the CNS, and led to diminished soluble and insoluble amyloid-β40,42 levels and astrocytosis as well as increased post-synaptic density and preservation of learning and memory (Koronyo et al., 2015; Rentsendorj et al., 2018). When targeting ACE overexpression in macrophages, our earlier studies reported major therapeutic effects in protecting against melanoma and microbial infections (Bernstein et al., 2014a, 2018). Our current investigation also strongly suggests neuroprotective effects of ACE10 macrophages in Alzheimer’s disease mouse models. In accordance, administration of the ACE inhibitor captopril to Alzheimer’s disease mice was reported to increase cerebral amyloid-β deposition (Zou et al., 2007). Here, ACE-overexpressing macrophages were consistently potent in clearing amyloid-β42 conformers—even the most elusive non-fibrillar forms—and successfully migrated into the mouse brain and retina with an enhanced ability to inhibit pathogenesis. While it appears that ACE overexpression in both the resident microglia and peripheral myelomonocytes work synergistically to restrict Alzheimer’s disease-associated pathology and improve cognition in animal models (Bernstein et al., 2014b; Koronyo-Hamaoui et al., 2014), the extent of the effect by peripheral ACE10 macrophages alone was nonetheless substantial, especially with regards to cerebral vascular amyloidosis. Because of growing reports on the key role of vascular abnormalities in Alzheimer’s-related cognitive decline (Ellis et al., 1996; Wyss-Coray et al., 2001; Herzig et al., 2006; Rentsendorj et al., 2018; Nation et al., 2019), the beneficial effects of ACE10 macrophages on reducing vascular amyloidosis (CAA severity score) and GFAP+ astroglial reactivation may indicate protection to the neurovascular unit and could explain synaptic and cognitive preservation seen in these mice. Similarly, previous studies in murine Alzheimer’s disease models showed that blood infusion with either wild-type or neprilysin-enriched monocytes mitigated neuropathology and cognitive deficits (Lebson et al., 2010; Koronyo et al., 2015) and monocyte depletion exacerbated amyloid-β-plaque pathology (Butovsky et al., 2007; El Khoury et al., 2007). Specifically, we have shown by two in vivo approaches that ACE-overexpressing monocytes are more effective in ameliorating Alzheimer’s disease-like pathology and preserving cognition in symptomatic murine models of Alzheimer’s disease. Our data showed that even partial bone marrow replacement with ACE10 cells (after partial-body irradiation, which avoids the skull area to prevent damage to the blood–brain barrier) leads to a substantial 3-fold increase of ACE10 monocytes in the brain, significantly decreasing cerebral amyloid-β plaques, amyloid-β’s soluble forms, and astrocytosis. Increased in vivo recruitment of ACE10 macrophages could be, at least in part, attributed to enhanced secretion of MMP9, an enzyme known to facilitate transmigration of immune cells through the blood–brain barrier (Chaturvedi and Kaczmarek, 2014). These brain-engrafted cells also exhibited ∼60% lower TNFα levels and 3.2-fold higher IGF1, adding a major advantage of this therapeutic approach. TNFα is a key pro-inflammatory cytokine that in excess can induce oxidative stress related to the release of iNOS and reactive oxygen species, which are tightly associated with neurotoxicity and functional decline. Moreover, IGF1 is a brain regenerative, neurogenesis-promoting neurotrophic factor with recently discovered anti-inflammatory functions (Gasparini and Xu, 2003). Cerebral IGF1 in murine models of Alzheimer’s disease was shown to be elevated following immunomodulation interventions involving increased brain-infiltrating monocytes/macrophages and cognitive preservation (Butovsky et al., 2006; Koronyo-Hamaoui et al., 2009), and another report has demonstrated its direct involvement in reducing amyloid-β plaque burden (Carro et al., 2002). Such substantial changes in TNFα and IGF1 levels in a chronically inflamed brain by ACE10 macrophages could, if translated into humans, potentially alter the course of Alzheimer’s disease and critically preserve synapses and cognition. This hypothesis is supported by our observations from the adoptive transfer studies in which enriching the peripheral blood of symptomatic AD+ mice with CD115+-MoACE10 cells resulted in improved cognitive function (Barnes maze test) and pre- and post-synaptic preservation. In comparison, our previous studies in murine models of Alzheimer’s disease involving life-long replacement of CNS-resident microglia and peripheral monocyte and macrophage populations with the ACE-overexpressing cells resulted in an almost complete elimination of cerebral amyloid-β burden, astrocytosis, and full preservation of cognitive functions, similar to the levels seen in the wild-type mice (Koronyo-Hamaoui et al., 2009; Bernstein et al., 2014b, 2018; Rentsendorj et al., 2018). As briefly mentioned above, here partial bone marrow replacement or short-term blood enrichment with ACE10 macrophages (once a month for 3 months) in animals that already have Alzheimer’s disease-like symptoms limited neuropathology by 30–40% (especially vascular amyloidosis), yet remarkably restored cognitive functions to levels indistinguishable from those found in cognitively normal wild-type mice. Conclusion and translational implications Despite considerable progress in understanding the aetiology of Alzheimer’s disease, an effective treatment remains elusive. The therapeutic potential evidenced in our studies favours selective increase of ACE (Al Mulhim et al., 2019), a peptidase capable of destroying amyloidogenic peptides and enhancing neuroprotective phenotype, in peripheral inflammatory cells that can migrate into the brain and powerfully support tissue-healing processes and cognition. For the first time, we demonstrate a superior ability of ACE10 macrophages to eradicate amyloid-β assemblies, particularly Alzheimer’s-associated non-fibrillar, oligomeric amyloid-β structures. The infiltration of these cells to the CNS was further validated through live non-invasive imaging of the retina, providing a feasible method to track immune cell migration during disease progression. Moreover, this study reveals the beneficial roles of CD115+ bone marrow-derived monocyte subsets and ACE in resisting Alzheimer’s pathology, especially important in light of large-scale human genetic studies identifying ACE and innate immune genes as prominently related to Alzheimer’s disease (Kehoe et al., 1999, 2003, 2004; Narain et al., 2000; Elkins et al., 2004; Kolsch et al., 2005; Lehmann et al., 2005; Meng et al., 2006; Jochemsen et al., 2014; de Oliveira et al., 2018; Marioni et al., 2018; Kunkle et al., 2019). Further, the marked reduction of Alzheimer’s-associated pathology with targeted ACE expression in inflammatory cells raises a clinically imperative question related to the outcome of life-long use of ACE inhibitors in the large number of patients with heart-related conditions (including high blood pressure), or commonly prescribed anti-inflammatory drugs (i.e. NSAIDs). For example, our results and other published data suggest caution in the use of ACE inhibitors, perhaps prioritizing those that do not penetrate the brain, such as benazepril, enalapril or quinapril (Kehoe et al., 2013). We envision the reported approach to be applicable to humans via isolation of monocytes from bone marrow or circulating blood, manipulation in vitro to overexpress ACE, followed by reinfusion of the modified cells to the patient. Future direction will focus on developing the practical and clinical protocols for each of these steps. It could involve the use of genetically modified stem cells or viral vectors to modify the isolated immune cells. In conclusion, this study may alter the vision of Alzheimer’s disease in human health in two ways: increased attention to potential neuropathological consequences of ACE inhibitors and anti-inflammatory drugs, and discovery of a novel Alzheimer’s disease immunotherapy to deliver ACE and therapeutic monocytes to the site of Alzheimer’s disease pathological manifestation. Acknowledgments The authors thank Xiao Z. Shen for his assistance with brain flow cytometry analysis, Ellen Bernstein for help with ACE10 transgenic mice, and Dr Mitra Mastali from Dr Jennifer Van Eyk’s Protein Quantification Core Laboratory at the Clinical Biosystems Research Institute, Cedars-Sinai Medical Center for assistance with the MSD-ELISA data. We also thank Mia Oviatt and Jo Ann Eliason for help with editing and Samuel Fuchs for his illustration of Supplementary Fig. 1C and help with thumbnail. The authors dedicate the manuscript to the memory of Natalie Radom Bernstein, Lillian Jones Black, and Salomon Moni Hamaoui, who died of Alzheimer’s disease. Funding Research reported in this publication was supported by the National Institute on Aging of the National Institutes of Health under Awards Number R01 AG055865 (M.K.H.) and R01 AG042195 (D.B.T.), A2013328S00 BrightFocus Foundation Award (M.K.H.), The Coins for Alzheimer’s Research Trust – CART Fund (M.K.H.), The Saban Foundation and The Marciano Foundation (M.K.H.). Competing interests The authors report no competing interests. Supplementary material Supplementary material is available at Brain online. References Ajami B , Bennett JL , Krieger C , Tetzlaff W , Rossi FM . Local self-renewal can sustain CNS microglia maintenance and function throughout adult life . Nat Neurosci 2007 ; 10 : 1538 – 43 . Google Scholar Crossref Search ADS PubMed WorldCat Akiyama H , Barger S , Barnum S , Bradt B , Bauer J , Cole GM , et al. 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Google Scholar Crossref Search ADS PubMed WorldCat Abbreviations Abbreviations AD+ = APPSWE/PS1ΔE9 double transgenic BM = bone marrow CAA = cerebral amyloid angiopathy ICC = immunocytochemistry IHC = immunohistochemistry MΦ = macrophages MSD = Meso Scale Discovery Scara1 = scavenger receptor A WT = wild-type Author notes These Yosef Koronyo and Altan Rentsendorj authors contributed equally to this work. © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

BrainOxford University Press

Published: Jun 10, 9

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