TNF-anti-TNF Immune Complexes Inhibit IL-12/IL-23 Secretion by Inflammatory Macrophages via an Fc-dependent Mechanism

TNF-anti-TNF Immune Complexes Inhibit IL-12/IL-23 Secretion by Inflammatory Macrophages via an... Abstract Background and Aims We have recently shown that the mode of action of IgG1 anti-tumour necrosis factor [TNF] antibodies in inflammatory bowel disease [IBD] requires Fcγ-receptor [FcγR] engagement on macrophages. Here we examine the effect of Fcγ-receptor signalling by anti-TNF on macrophage IL-12/IL-23 secretion. Methods Cytokine production by human inflammatory macrophages was assessed at the level of RNA and protein. TNF-anti-TNF immune complex formation was determined by size-exclusion chromatography and signalling visualized by immunofluorescence. IL-12/IL-23p40 was measured in CD14+ lamina propria cells from IBD patients. Results Infliximab and adalimumab potently suppressed IL-12/IL-23 production by inflammatory macrophages, but Fab’ fragment certolizumab did not. IL-12/IL-23 suppression depended on Syk activity and was mediated at the level of IL-12/IL-23p40 mRNA. Etanercept, a soluble TNF receptor fused to an Fc-region, did not inhibit IL-12/L-23 secretion, suggesting that the presence of an Fc-region was not sufficient. Infliximab and adalimumab formed immune complexes with soluble TNF whereas etanercept did not, suggesting that FcγR-mediated suppression of IL-12/IL-23 required the formation of immune complexes. Indeed, non-specific IgG1 immune complexes, but not uncomplexed IgG1, similarly suppressed IL-12/IL-23 secretion. Finally, infliximab significantly decreased IL-12/IL-23p40 production in myeloid cells isolated from the lamina propria of IBD patients. Conclusions TNF-anti-TNF antibody immune complexes potently inhibit IL-12/IL-23 expression by inflammatory macrophages. Our data suggest that anti-TNFs and antibodies against IL-12/IL-23 may therefore have partially overlapping modes of action in patients with IBD. Anti-TNF, IL-12/IL-23 axis, macrophage 1. Introduction In contrast to rheumatoid arthritis, the mode of action of anti-tumour necrosis factors[TNFs] in inflammatory bowel disease [IBD] is not exclusively related to therapeutic blockade of TNF.1 We have previously demonstrated that the therapeutic efficacy of anti-TNF was completely dependent on Fc-Fcγ receptor [FcγR] interaction in a preclinical model of colitis. Additionally, we have shown that IgG1 anti-TNFs polarise macrophages to a regulatory and wound healing phenotype in an Fc-dependent manner in humans and mice.2–5 Intriguingly, it has previously been shown that activation of FcγR by immune complexes can potently inhibit IL-12 secretion by macrophages.6–8 IL-12 is a dimeric cytokine which shares its p40 subunit with IL-23. Whether immune complexes also inhibit IL-23 is not known, as the IL-23-specific p19 subunit had not yet been identified at the time these experiments were performed. IL-12 and IL-23 are mainly produced by macrophages and dendritic cells and play a key role in the pathogenesis of IBD. In the inflamed intestine of patients with Crohn’s disease, monocytes polarise towards an inflammatory macrophage phenotype and produce pro-inflammatory cytokines including TNF and IL-23.9 The blockade of IL-12p40 with ustekinumab, which neutralises both IL-12 and IL-23, is currently approved as a treatment for patients with Crohn’s disease and, more recently, the selective IL-23p19 inhibitors risankizumab and brazikumab showed promising results in clinical trials in patients with Crohn’s disease.10–13 Studies in ulcerative colitis are ongoing. The advent of this novel class of antibodies poses the question whether patients with Crohn’s disease responding to anti-TNFs and IL-12/IL-23 blockade represent distinct or overlapping groups. TNF, IL-12, and IL-23 promote inflammation via distinct pathways, suggesting that therapies blocking these cytokines may establish mucosal healing via different routes and therefore work in different patient subgroups. However, given the fact that we have previously found that FcγR signalling is involved in the therapeutic effect of anti-TNFs and the ability of FcγR signalling to suppress IL-12 secretion, we aimed to examine how different anti-TNF blocking strategies affected macrophage IL-12/IL-23 secretion. 2. Methods 2.1. Anti-TNF compounds Therapeutic anti-TNF compounds used in this study are adalimumab [Humira, AbbVie], infliximab [Remicade, MSD], golimumab [Simponi, MSD], certolizumab pegol [Cimzia, UCB Pharma], and etanercept [Enbrel, Pfizer]. Infliximab Fab fragments were generated using the Pierce Fab micro Preparation Kit [Thermo Fisher, Landsmeer, The Netherlands]. 2.2. In vitro macrophage differentiation Peripheral blood mononuclear cells [PBMC] from healthy volunteers were isolated by Ficoll Paque density-gradient centrifugation. After washing, monocytes were isolated by Percoll density-gradient centrifugation.14 Inflammatory [M1] macrophages were obtained by culturing monocytes with IFNγ [50 ng/mL, PeproTech, London, UK] for 6 days in RPMI supplemented with 10% heat-inactivated fetal calf serum [FCS]. Macrophages were then washed, reseeded, and stimulated for 48 h with lipopolysaccharide [LPS] 100 ng/ml [Sigma, Aldrich, Zwijndrecht] and anti-TNF or IgG1 [Genetex, Irvine, CA]. Complexed IgG1 was obtained by heating IgG1 at 63°C for 1 h. Culture supernatants were harvested and cytokine levels were measured by enzyme-linked immunosorbent assay [ELISA] for IL-23, IL-12p40, and IL-10 [Duoset, R&D] or by Cytometric Bead Array [Human Inflammatory Cytokine Kit, BD Biosciences]. For pSYK staining, M1 macrophages were incubated with LPS and anti-TNF for 30 min, fixed using Fixation/Permeabilization Solution Kit [BD Biosciences], and stained for anti-pSYK-PE or isotype control staining [both Cell Signaling]. Syk was inhibited with 1 μM R406 [Invivogen] added 1 before the addition of LPS and anti-TNF cytokine production in this case was measured after 24 h. 2.3. Heat-killed bacteria Heat-killed E. coli 0111:B4 was purchased from Invivogen [catalog no. tlrl-hkeb2]. E. faecalis [catalog no. 29212; ATCC] was cultured in brain-heart infusion [BHI] medium. Bacteria were harvested and washed twice with phosphate-buffered saline [PBS]. Then, bacterial suspension was heated at 80°C for 30 min, washed, resuspended in PBS, and stored at –80°C. Complete killing was confirmed by a 72-h incubation at 37°C. 2.4. Preparation of LPMCs Intestinal mucosa was obtained from surgically resected specimens from patients with IBD, diagnosed on the basis of endoscopic and histological findings according to established criteria. All experiments were performed with mucosa from macroscopically inflamed specimens. All experiments were approved by the Medical Ethical Committee of the Academic Medical Center, Amsterdam. Written informed consent was obtained from all patients. Lamina propria mononuclear cells [LPMC] were isolated from intestinal specimens as follows. Briefly, dissected mucosa was incubated in calcium and magnesium-free HBSS containing 5 mM EDTA [Sigma-Aldrich] for 20 min at 37°C to remove epithelial cells. Tissues were then cut very fine and incubated in RPMI medium containing 10% FCS, 1 mg/ml collagenase D [Roche], 1 mg/ml soybean trypsin inhibitor [Sigma], and 50 µg/ml Dnase I [Roche] for 60 min at 37°C. The fraction was pelleted and resuspended in a 30% Percoll solution, then layered on 60% Percoll before centrifugation at 500g for 10 min at room temperature. Viable LPMC were recovered from the 30–60% layer interface. LPMC were then stimulated overnight with heat-killed E. coli and E. faecalis, both 1 × 108 cells/ml, in the presence of IgG1 or anti-TNF 10 µg/ml. Golgistop [BD Biosciences] was added after 1 h. The next day, LPMC were stained for LIVE/DEAD® Fixable Green Dead Cell Stain Kit [Invitrogen], CD45-AF700 [Sony], CD14-PE-Cy7 [eBioscience], HLA-DR-PE, and CD11b-BV421 [both Biolegend] and then fixed using Fixation/Permeabilization Solution Kit [BD Biosciences]. After fixation, cells were stained for IL-12p40-APC [Biolegend] and analysed by FACS Fortessa [BD Biosciences], then analysed using FlowJo software [Treestar Inc]. 2.5. High-performance size-exclusion chromatography Recombinant human TNF was obtained from Active Bioscience. Therapeutic anti-TNF compounds [60 µg/ml, diluted in PBS] were incubated for at least 1 h with 2 µg/ml recombinant human TNF. Samples were analysed by applying 200 μl to a Superdex 200 HR 10/300 column [GE Healthcare, Uppsala Sweden], which was connected to an ÄKTAexplorer HPLC system [GE Healthcare]. Elution profiles were monitored by measuring absorbance at 215 nm. The Superdex column was calibrated using the Gel Filtration Markers Kit [29–700 kDa range, Sigma Aldrich]. 2.6. Quantitative real-time polymerase chain reaction mRNA isolation was performed using the Bioline ISOLATE II RNA Mini kit [BIO-52073, Bioline] according to manufacturers’ instructions. First-strand cDNA was synthesised using Oligo-dT [Invitrogen], random hexamer primers [Promega, Madison, IL], and RiboLock RNase and RevertAid reverse transcriptase [both Thermo Scientific]. Quantitative real-time polymerase chain reaction [qRT-PCR] was performed using sensifast SYBR No-ROX Kit [GC-biotech Bio-98020] on a BioRad iCycler. Relative gene expression was calculated using the 2−delta Ct method. GAPDH was used as reference gene. 2.7. Cohort studies of patients with Crohn’s disease treated with anti-TNF Intestinal biopsy specimens were collected in patients suffering from Crohn’s disease, before anti-TNF treatment was initiated and during treatment at the Academic Medical Center in Amsterdam and at the University Hospital in Leuven. Endoscopy was performed in all patients before the start of anti-TNF therapy and during follow-up. Seven responders and eight non-responders were included, where response versus non-response was determined by the clinician’s assessment of the endoscopic evaluation of the mucosa. Response was determined before analysis of the tissue samples [patient characteristics in Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. When comparing mRNA levels on consecutive time points within one patient, we compared biopsies within one anatomical location [i.e. colon versus colon and ileum versus ileum]. The study protocols were approved by the medical ethical committees of the Academic Medical Center, Amsterdam, The Netherlands and of the University Hospital, Leuven, The Netherlands; all participants provided written informed consent. 2.8. Statistical analysis For statistical analysis, unpaired t-test or analysis of variance [ANOVA] was used, followed by Bonferroni post-test. Normalised data were analysed with Wilcoxon signed rank test or Kuskal-Wallis, followed by Dunn’s post hoc analysis. Results were considered significant when p-value < 0.05. 3. Results 3.1. Infliximab inhibits IL-12p40 and IL-23 production by inflammatory macrophages Given the pro-inflammatory state of macrophages in the intestine of Crohn’s disease patients,9 we investigated the effects of infliximab on a priori polarised inflammatory macrophages in vitro. Inflammatory macrophages were generated by culturing human monocytes with IFNγ. For clarity, we will hereafter refer to IFNγ-induced macrophages as M1 macrophages. M1 macrophages were washed and reseeded with LPS in the presence of infliximab or IgG1 isotype control. To explore the effect of infliximab on the pro-inflammatory cytokine profile of M1 macrophages, we measured IL-12p40, IL-6, IL-8, and IL-1β production. Whereas IL-6, IL-8, and IL-1β were comparable between infliximab and IgG1-treated M1 macrophages, IL-12p40 was specifically inhibited by infliximab [Figure 1A]. Further analyses revealed that the decrease in IL-12p40 was dose-dependent [Figure 1B]. As IL-23 is composed of subunits IL-12p40 and IL-23-p19, we next measured IL-23 secretion and we found that IL-23 was also dose-dependently decreased by infliximab. [Figure 1C] Figure 1. View largeDownload slide Infliximab specifically inhibits IL-12p40 and IL-23 production by inflammatory macrophages. Human monocytes were cultured with IFNγ 50 ng/ml for 6 days to generate M1 macrophages. M1 macrophages were then reseeded with infliximab or IgG1 [both 10 µg/ml] in the presence of lipopolysaccharide [LPS] 100 ng/ml. [A] Inflammatory cytokine production by M1 macrophages cultured for 48 h in six different donors. Each dot represents an individual donor: Wilcoxon signed rank test. [B/C] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: analysis of variance [ANOVA] followed by Dunnet’s Multiple comparisons test. [D] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]: Mann‑Whitney U-test. Data are representative of at least three independent experiments with different donors and bars show means; error bars indicate standard error of the mean, *p < 0.05, **p < 0.01, ***p < 0.001. Figure 1. View largeDownload slide Infliximab specifically inhibits IL-12p40 and IL-23 production by inflammatory macrophages. Human monocytes were cultured with IFNγ 50 ng/ml for 6 days to generate M1 macrophages. M1 macrophages were then reseeded with infliximab or IgG1 [both 10 µg/ml] in the presence of lipopolysaccharide [LPS] 100 ng/ml. [A] Inflammatory cytokine production by M1 macrophages cultured for 48 h in six different donors. Each dot represents an individual donor: Wilcoxon signed rank test. [B/C] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: analysis of variance [ANOVA] followed by Dunnet’s Multiple comparisons test. [D] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]: Mann‑Whitney U-test. Data are representative of at least three independent experiments with different donors and bars show means; error bars indicate standard error of the mean, *p < 0.05, **p < 0.01, ***p < 0.001. Analysis by qPCR showed that infliximab inhibited the expression of IL-12B mRNA, the gene encoding subunit IL-12p40. The expression of IL-12A and IL-23A, encoding subunits IL-12p35 and IL-23p19, respectively, was not affected by infliximab, nor was the induction of TNFα itself [Figure 1D]. Overall, these data suggest that the suppression of the IL-23 protein is mediated by inhibition of IL-12B transcription and not by IL-23p19. 3.2. Inhibition of IL12/IL-23 is Fc-dependent To investigate if the inhibition of IL-12p40 and IL-23 was Fc-dependent, we compared infliximab with certolizumab, a TNF-neutralising Fab’ fragment. Both anti-TNF compounds blocked TNF to undetectable levels [Supplementary Figure 1, available as Supplementary data at ECCO-JCC online]. Hereby, we could distinguish between Fc-dependent mechanisms and effects that should be attributed to TNF neutralisation. Again, infliximab treatment decreased IL-12p40 and IL-23. However, the production of these cytokines remained high in certolizumab-treated M1 macrophages, indicating that the inhibition of IL-12p40 and IL-23 is mediated via the Fc-region of infliximab rather than neutralisation of TNF [Figure 2A]. Similarly, infliximab Fab fragments did not inhibit IL-12p40 production, further supporting a role for the Fc-region [Supplementary Figure 2, available as Supplementary data at ECCO-JCC online]. Figure 2. View largeDownload slide Inhibition of IL12/ IL-23 is Fc-dependent. M1 macrophages were reseeded with lipopolysaccharide [LPS] 100 ng/ml and infliximab or certolizumab 10 µg/ml for 48 h unless otherwise stated. [A] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B] Normalised IL-10 production by M1 macrophages from multiple donors: Wilcoxon signed rank test. [C] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]. [D] Macrophages were fixed 30 min after the addition of lipopolysaccharide [LPS] and anti-TNF, stained with anti-pSYK-PE or isotype control, and visualised by immunofluorescence and flow cytometry. [E] Syk-inhibitor R406 was added before the addition of LPS and anti-TNF [1 µg/ml], IL-12p40 and IL-10 production was measured by ELISA after 24 h: analysis of variance [ANOVA] followed by Bonferroni post-test. Data are representative of at least three independent experiments with different donors, bars show means, error bars indicate standard error of the mean, **p < 0.01, ***p < 0.001. Figure 2. View largeDownload slide Inhibition of IL12/ IL-23 is Fc-dependent. M1 macrophages were reseeded with lipopolysaccharide [LPS] 100 ng/ml and infliximab or certolizumab 10 µg/ml for 48 h unless otherwise stated. [A] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B] Normalised IL-10 production by M1 macrophages from multiple donors: Wilcoxon signed rank test. [C] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]. [D] Macrophages were fixed 30 min after the addition of lipopolysaccharide [LPS] and anti-TNF, stained with anti-pSYK-PE or isotype control, and visualised by immunofluorescence and flow cytometry. [E] Syk-inhibitor R406 was added before the addition of LPS and anti-TNF [1 µg/ml], IL-12p40 and IL-10 production was measured by ELISA after 24 h: analysis of variance [ANOVA] followed by Bonferroni post-test. Data are representative of at least three independent experiments with different donors, bars show means, error bars indicate standard error of the mean, **p < 0.01, ***p < 0.001. Interestingly, others have shown that FcγR binding on macrophages by IgG immune complexes not only inhibits IL-12 production, but also increases IL-10 production.6–8 We found that infliximab enhanced the production of IL-10 compared with certolizumab, confirming an Fc-dependent increase in IL-10 production [Figure 2B]. However, TNF neutralisation with certolizumab reduced IL-10 production compared with cultures treated with LPS alone. Thus, IL-10 production is regulated by both TNF blockade and Fc-engagement in opposite ways, and the outcome may differ depending on the relative strengths of these signals. This might explain why we observed some variability in the effects of infliximab on IL-10 production when analysing multiple donors [Figure 2B]. Overall, IL-10 production was always higher in infliximab-treated cultures compared with certolizumab. Analysis by qPCR showed that infliximab inhibited the expression of IL-12B mRNA, whereas certolizumab did not. In line with Figure 1, expression of IL-12A and IL-23A was not inhibited by infliximab or certolizumab [Figure 2C]. We recently showed that activating FcγR are indispensable for the therapeutic efficacy of anti-TNF in experimental colitis.2 Activated FcγR have been shown to signal via spleen tyrosine kinase [Syk], and ligation of FcγR results in the phosphorylation of Syk.15 To study this further in a human setting, we measured phosphorylated Syk in human M1 macrophages. Indeed, only in infliximab-treated M1 macrophages was the presence of phosphorylated Syk detected [Figure 2D]. Furthermore, inhibition of Syk completely reversed the infliximab-mediated decrease in IL-12p40, as well as the increase in IL-10 production [Figure 2E]. 3.3. TNF-anti-TNF complex formation crucial for IL-23 inhibition Our previous data indicate an important role for FcγR engagement in the mode of action of IgG1 anti-TNFs in IBD; however, etanercept also contains an Fc-region but failed to show clinical efficacy in Crohn’s disease.16 Interestingly, when M1 macrophages were cultured with etanercept, the production of IL-12p40 and IL-23 was not inhibited [Figure 3A]. Also, IL-10 production was decreased to levels comparable to certolizumab-treated macrophages. This suggests that the mere presence of an IgG1 Fc-region is not sufficient to increase the IL-10 to IL-12/IL-23 ratio. We hypothesised that anti-TNF antibodies like infliximab and adalimumab differ from etanercept in their ability to cross-link FcγR. Antibodies like infliximab and adalimumab have two Fab’ arms that can simultaneously bind two TNF molecules. Importantly, biologically active TNF is a homotrimer and it has been shown that TNF can be bound by three infliximab molecules at once. These features will enable the formation of TNF-anti-TNF immune complexes that can cross-link FcγR. In contrast, etanercept contains only one TNF-binding domain and has been shown to bind soluble TNF in a 1:1 ratio.17 Importantly, cross-linking of FcγR by immune complexes affects downstream FcγR effector functions to a much greater extent than Fc engagement by monomeric antibodies. Figure 3. View largeDownload slide Anti-TNF immune complexes mediate alterations in IL-10 and IL-23 production. [A] M1 macrophages stimulated with lipopolysaccharide [LPS] 100 ng/ml and different anti-TNF compounds 10 μg/ml: ytokine production after 48 h: analysis of variance [ANOVA] followed by Bonferroni post=test [B] Complex formation of TNF with anti-TNF. TNF 2 µg/ml was incubated with anti-TNF 60 µg/ml and analysed on a Superdex 200 column. For each sample, a representative elution pattern is shown out of at least two measurements. Arrows indicate elution of TNF-anti-TNF complexes. [C] M1 macrophages stimulated with IgG1 or complexed IgG1 [both 10 μg/ml] in the presence of LPS 100 ng/ml. Cytokine production measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: unpaired t-test. [D] M1 macrophages stimulated with anti-TNF and complexed IgG1 where indicated in the presence of LPS 100 ng/ml. Cytokine production measured by ELISA after 48rs: ANOVA followed by Bonferroni post-test. Data are representative at least three experiments with different donors, ***p < 0.001. Figure 3. View largeDownload slide Anti-TNF immune complexes mediate alterations in IL-10 and IL-23 production. [A] M1 macrophages stimulated with lipopolysaccharide [LPS] 100 ng/ml and different anti-TNF compounds 10 μg/ml: ytokine production after 48 h: analysis of variance [ANOVA] followed by Bonferroni post=test [B] Complex formation of TNF with anti-TNF. TNF 2 µg/ml was incubated with anti-TNF 60 µg/ml and analysed on a Superdex 200 column. For each sample, a representative elution pattern is shown out of at least two measurements. Arrows indicate elution of TNF-anti-TNF complexes. [C] M1 macrophages stimulated with IgG1 or complexed IgG1 [both 10 μg/ml] in the presence of LPS 100 ng/ml. Cytokine production measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: unpaired t-test. [D] M1 macrophages stimulated with anti-TNF and complexed IgG1 where indicated in the presence of LPS 100 ng/ml. Cytokine production measured by ELISA after 48rs: ANOVA followed by Bonferroni post-test. Data are representative at least three experiments with different donors, ***p < 0.001. To confirm these differences in TNF-anti-TNF immune complex formation for multiple anti-TNF compounds, we performed size-exclusion chromatography [Figure 3B]. Infliximab, adalimumab, and golimumab formed a complex that roughly corresponds in size to a complex where three antibodies are bound to one soluble TNF molecule. Etanercept formed a complex with soluble TNF almost equal in size to unbound etanercept, indicating that one etanercept molecule was bound to one TNF molecule [Figure 3B]. In line with these data, we have previously shown that in the presence of soluble TNF, FcγR binding capacity becomes much higher for adalimumab than for etanercept.2 Thus, although etanercept has an Fc-region, it does not bind FcγR as potently as do monoclonal antibodies infliximab or adalimumab in the presence of soluble TNF, and this is reflected by the inability of etanercept to increase the IL-10 to IL-12/IL-23 ratio. Finally, to further investigate the need for immune complex formation to achieve IL-12/IL-23 inhibition, we compared non-specific monomeric IgG1 with complexed IgG1. Indeed, complexed IgG1 increased IL-10 and inhibited both IL-12p40 and IL-23 compared with monomeric IgG1, confirming their functional difference [Figure 3C]. Furthermore, when complexed IgG1 was added to certolizumab treatment [to add TNF blockade to the FcR cross-linking by the complexed IgG], the production of IL-10, IL-12p40, and IL-23 became equal to that of infliximab-treated M1 macrophages. [Figure 3D] Thus, the increase in IL-10 versus IL-12/IL23 ratio mediated by infliximab can be mimicked by a combination of certolizumab [TNF blockade] and IgG1 immune complexes [FcγR cross-linking]. 3.4. IgG1 anti-TNF inhibits IL-12/IL-23 in mucosal CD14+ cells from IBD patients In an inflamed intestine, mucosal macrophages are constantly exposed to a wide range of microbial products. To explore if our findings could be reproduced using an equivalent stimulation, we cultured M1 macrophages with heat-killed bacteria. Indeed, when M1 macrophages were cultured with a combination of heat-killed E. coli and heat-killed E. faecalis, we found that infliximab inhibits IL-12p40 in an Fc-dependent manner in this setting also [Figure 4A]. Additionally, this was also the case when M1 macrophages were cultured with either heat-killed E. coli or heat-killed E. faecalis separately [Supplementary Figure 3, available as Supplementary data at ECCO-JCC online]. Figure 4. View largeDownload slide IgG1 anti-TNF inhibits mucosal IL-12/IL-23 in inflammatory bowel disease [IBD] patients. [A] M1 macrophages were stimulated with heat-killed E.coli and E. faecalis [both 1 × 108 cells/ml] and treated with anti-TNF [10 µg/ml] for 48 h. IL-12p40 was measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B/C] Representative histograms and graph of the CD14+ lamina propria myeloid compartment stimulated with heat-killed E. coli and E. faecalis [both 1 × 108 cells/ml] overnight in the presence of IgG1, certolizumab, or infliximab [all 10 µg/ml]. IL-12p40 was measured in CD14+ macrophages by flow cytometry: ANOVA followed by Bonferroni post-test. [D] mRNA expression of IL-12B measured by real-time polymerase chain reaction [RT-PCR] in intestinal biopsy specimens from patients with Crohn’s disease treated with infliximab or adalimumab, 7]seven endoscopic responders and eight endoscopic non-responders. Each dot represents an individual patient: Mann-Whitney U-test. Data are representative of at least three experiments with different donors or different IBD patients, and bars show means, error bars indicate standard error of the mean,*p < 0.05, **p < 0.01,***p < 0.001. Figure 4. View largeDownload slide IgG1 anti-TNF inhibits mucosal IL-12/IL-23 in inflammatory bowel disease [IBD] patients. [A] M1 macrophages were stimulated with heat-killed E.coli and E. faecalis [both 1 × 108 cells/ml] and treated with anti-TNF [10 µg/ml] for 48 h. IL-12p40 was measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B/C] Representative histograms and graph of the CD14+ lamina propria myeloid compartment stimulated with heat-killed E. coli and E. faecalis [both 1 × 108 cells/ml] overnight in the presence of IgG1, certolizumab, or infliximab [all 10 µg/ml]. IL-12p40 was measured in CD14+ macrophages by flow cytometry: ANOVA followed by Bonferroni post-test. [D] mRNA expression of IL-12B measured by real-time polymerase chain reaction [RT-PCR] in intestinal biopsy specimens from patients with Crohn’s disease treated with infliximab or adalimumab, 7]seven endoscopic responders and eight endoscopic non-responders. Each dot represents an individual patient: Mann-Whitney U-test. Data are representative of at least three experiments with different donors or different IBD patients, and bars show means, error bars indicate standard error of the mean,*p < 0.05, **p < 0.01,***p < 0.001. Next, we isolated lamina propria mononuclear cells [LMPC] from IBD patients. Cells were stimulated overnight with a combination of heat-killed E. coli and E. faecalis in the presence of infliximab, certolizumab, or an IgG1 isotype control antibody. Analyses by flow cytometry revealed that infliximab inhibited IL-12p40 protein production in the CD14+ lamina propria myeloid population to levels comparable to unstimulated cultures [Figure 4B]. Furthermore, IL-12p40 was significantly decreased in infliximab-treated CD14+ lamina propria cells compared with certolizumab or IgG1-treated LPMC [Figure 4C]. Finally, mucosal mRNA expression of the gene encoding IL-12/IL-23p40 [IL-12B] was analysed in a cohort of patients with Crohn’s disease treated with infliximab or adalimumab. We included seven endoscopic responders and eight endoscopic non-responders [patient characteristics shown in Supplementary Table 1]. The expression of IL-12B decreased significantly in responders upon anti-TNF treatment, but remained stable in non-responders [Figure 4D]. 4. Discussion The mode of action of anti-TNFs in IBD is still a matter of investigation.1 Here we find that the trimeric cytokine TNF forms immune complexes with IgG1 anti-TNF monoclonal antibodies that potently inhibit IL-12/IL-23 secretion at the level of IL12B mRNA in an Fc-dependent manner. Only a few years after the introduction of infliximab, it became clear that the mode of action of anti-TNFs in IBD was less certain than anticipated, when etanercept completely failed to achieve either clinical response or remission in patients with Crohn’s disease, with patients on etanercept doing numerically worse than on placebo.16 Also, treatment with etanercept has been associated with paradoxical development of IBD in patients with rheumatic diseases.1,18 Certolizumab is significantly better than placebo for maintenance treatment but did not reach the primary endpoint for induction of clinical remission in three independent trials.19–22 In addition, there are no placebo-controlled data that support induction of mucosal healing by certolizumab. One uncontrolled endoscopic study showed that only 4% of patients with Crohn’s disease achieved mucosal healing after 10 weeks of certolizumab treatment.23 By comparison, complete mucosal healing was observed in 31% of patients on infliximab and 28% of patients on adalimumab in the same time frame.24,25 We have previously shown that one of the potential mechanisms of action that differentiates the IgG1 antibodies infliximab and adalimumab from other anti-TNF blocking strategies is dependent on the antibody Fc region. We found that IgG1 antibodies, but not certolizumab or etanercept, generate CD206-positive macrophages with regulatory and wound healing properties, in a mixed lymphocyte reaction in vitro.5 We found that responders but not non-responders to infliximab show accumulation of such CD206+ macrophages in the lamina propria in vivo.4 In a preclinical model, we used two anti-TNF antibodies with the same variable region that differed in their ability to bind FcγR. In a head-to-head comparison we found that both antibodies were equally effective in the collagen-induced model of arthritis, but that FcγR interaction was required for mucosal healing in the CD45RBhigh transfer model of colitis.3 More recently we demonstrated that mice lacking activating FcγR failed to generate CD206+ regulatory macrophages, and were completely unresponsive to anti-TNF therapy in the same model of colitis. Conversely, a hypo-fucosylated anti-TNF with increased affinity for FcγR showed increased CD206+ macrophage formation as well as improved therapeutic efficacy.2 In all our previous work, the anti-TNF induced CD206+ macrophages were likely formed from monocyte precursors. Here, we studied the effects of Fc-engagement on pro-inflammatory human macrophages with an M1-like phenotype. We found that in addition to generating CD206+ macrophages, anti-TNF monoclonal antibodies specifically inhibit IL-12/IL-23 production by M1-like macrophages. The inhibition of IL-12/IL-23 was mediated via Syk and was only achieved with anti-TNF compounds that had an Fc-region and were capable of forming immune complexes. TNF is a homotrimeric molecule containing three binding sites for anti-TNF antibodies. For antibodies like infliximab and adalimumab, this promotes the formation of TNF-anti-TNF complexes. Complexed antibodies affect FcγR effector functions to a much greater extent than do monomeric antibodies, by virtue of their ability to cross-link multiple FcγR. Although we have confirmed the primary finding of inhibition of IL-12/IL-23 in primary intestinal macrophages, it would be interesting to further evaluate the effects of immune complexes in this particular cell type as well. In vivo, we observed a decrease in IL-12B expression in patients responding to anti-TNF therapy, but not in non-responders. It should be noted that our cohort was a clinical follow-up cohort without hard criteria for response. However, a subanalysis of the ACT-1 trial, a prospective study using well-defined endoscopic criteria, showed similar results with decreased expression of IL12B in responders after 8 weeks of therapy.26 As other current targets in IBD do not have a homomultimeric structure, it seems unlikely that other monoclonal antibodies used in IBD share a similar mode of action. However, the inhibition of IL12/IL23 by TNF-anti-TNF immune complexes does pose questions around the differentiation of therapies blocking IL-12/IL-23 directly from anti-TNF monoclonal antibodies. Our data suggest that that the mode of action of anti-TNFs in IBD may be partially overlapping with ustekinumab [anti-IL12/IL23] and IL-23-specific therapies such as risankizumab. No controlled endoscopic data are currently available for ustekinumab. In a recent phase 2 study with risankuzimab, which involved mainly anti-TNF exposed patients, the delta between placebo and the top dose was only 5% in terms of mucosal healing.10 Thus, although we need to await further data, the available endoscopic data support the idea that this therapeutic class may not be as differentiated from anti-TNFs as previously hoped. Collectively, our results point out a previously unrecognised effector function of anti-TNF therapy that involves inhibition of the IL-12/IL-23 axis. These findings could contribute to our understanding about the failure of multiple biologics in therapy-refractory patients, and also suggest a new immunosuppressive mechanism for therapeutic antibodies capable of immune complex formation. Supplementary Data Supplementary data are available at ECCO-JCC online. Funding This project was supported by Health Holland, Top Sector Life Sciences and Health. Conflict of Interest GB is currently an employee of GlaxoSmithKline. GD received consulting and/or lecture fees from AbbVie, ActoGeniX, AM Pharma, Boehringer Ingelheim GmbH, Centocor, ChemoCentryx, Cosmo Technologies, Elan Pharmaceuticals, Engene, Dr Falk Pharma, Ferring, Galapagos, Giuliani SpA, Given Imaging, GlaxoSmithKline, Jansen Biologics, Merck Sharp and Dohme Corp, Millennium Pharmaceuticals, Inc. [now Takeda], Neovacs, Novonordisk, Otsuka, PDL Biopharma, Pfizer, Receptos, Salix, Setpoint, Shire Pharmaceuticals, Schering-Plough, Tillotts Pharma, UCB Pharma, Versant, and Vifor Pharma, and reports receiving research grants from Abbott Laboratories, Jansen Biologics, Given Imaging, MSD, Dr Falk Pharma, and Photopill; and speaking honoraria from Abbott Laboratories, Tillotts, Tramedico, Ferring, MSD, UCB, Norgine, and Shire. MW reports personal fees from Takeda, personal fees and non-financial support from Janssen Parmaceuticals, grants from GlaxoSmithKline, grants from Tillotts Pharma, and grants from Immunic, outside the submitted work. CB reports being on the Advisory Board Johnson & Johnson [energy devices]. Author Contributions FB, KS, PK, and TR designed, performed, and analysed experiments. CB, JB, and WB were involved in acquisition of data. GD, GB, and MW supervised study. FB, GB, and MW wrote the manuscript. References 1. Levin AD , Wildenberg ME , van den Brink GR . Mechanism of action of anti-TNF therapy in inflammatory bowel disease . J Crohns Colitis 2016 ; 10 : 989 – 97 . Google Scholar CrossRef Search ADS PubMed 2. Bloemendaal FM , Levin AD , Wildenberg ME , et al. Anti-tumor necrosis factor with a glyco-engineered Fc-region has increased efficacy in mice with colitis . Gastroenterology 2017 ; 153 : 1351 – 62.e4 . Google Scholar CrossRef Search ADS PubMed 3. McRae BL , Levin AD , Wildenberg ME , et al. Fc Receptor-mediated effector function contributes to the therapeutic response of anti-TNF monoclonal antibodies in a mouse model of inflammatory bowel disease . J Crohns Colitis 2016 ; 10 : 69 – 76 . Google Scholar CrossRef Search ADS PubMed 4. Vos AC , Wildenberg ME , Arijs I , et al. Regulatory macrophages induced by infliximab are involved in healing in vivo and in vitro . Inflamm Bowel Dis 2012 ; 18 : 401 – 8 . Google Scholar CrossRef Search ADS PubMed 5. Vos AC , Wildenberg ME , Duijvestein M , Verhaar AP , van den Brink GR , Hommes DW . Anti-tumor necrosis factor-α antibodies induce regulatory macrophages in an Fc region-dependent manner . Gastroenterology 2011 ; 140 : 221 – 30 . Google Scholar CrossRef Search ADS PubMed 6. Gallo P , Gonçalves R , Mosser DM . The influence of IgG density and macrophage Fc [gamma] receptor cross-linking on phagocytosis and IL-10 production . Immunol Lett 2010 ; 133 : 70 – 7 . Google Scholar CrossRef Search ADS PubMed 7. Sutterwala FS , Noel GJ , Clynes R , Mosser DM . Selective suppression of interleukin-12 induction after macrophage receptor ligation . J Exp Med 1997 ; 185 : 1977 – 85 . Google Scholar CrossRef Search ADS PubMed 8. Sutterwala FS , Noel GJ , Salgame P , Mosser DM . Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I . J Exp Med 1998 ; 188 : 217 – 22 . Google Scholar CrossRef Search ADS PubMed 9. Kamada N , Hisamatsu T , Okamoto S , et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis . J Clin Invest 2008 ; 118 : 2269 – 80 . Google Scholar PubMed 10. Feagan BG , Sandborn WJ , D’Haens G , et al. Induction therapy with the selective interleukin-23 inhibitor risankizumab in patients with moderate-to-severe Crohn’s disease: a randomised, double-blind, placebo-controlled phase 2 study . Lancet 2017 ; 389 : 1699 – 709 . Google Scholar CrossRef Search ADS PubMed 11. Feagan BG , Sandborn WJ , Gasink C , et al. ; UNITI–IM-UNITI Study Group . Ustekinumab as induction and maintenance therapy for Crohn’s disease . N Engl J Med 2016 ; 375 : 1946 – 60 . Google Scholar CrossRef Search ADS PubMed 12. Sandborn WJ , Gasink C , Gao LL , et al. ; CERTIFI Study Group . Ustekinumab induction and maintenance therapy in refractory Crohn’s disease . N Engl J Med 2012 ; 367 : 1519 – 28 . Google Scholar CrossRef Search ADS PubMed 13. Sands BE , Chen J , Feagan BG , et al. Efficacy and safety of MEDI2070, an antibody against interleukin 23, in patients with moderate to severe Crohn’s disease: a phase 2a study . Gastroenterology 2017 ; 153 : 77 – 86.e6 . Google Scholar CrossRef Search ADS PubMed 14. Repnik U , Knezevic M , Jeras M . Simple and cost-effective isolation of monocytes from buffy coats . J Immunol Methods 2003 ; 278 : 283 – 92 . Google Scholar CrossRef Search ADS PubMed 15. Nimmerjahn F , Ravetch JV . Fcgamma receptors as regulators of immune responses . Nat Rev Immunol 2008 ; 8 : 34 – 47 . Google Scholar CrossRef Search ADS PubMed 16. Sandborn WJ , Hanauer SB , Katz S , et al. Etanercept for active Crohn’s disease: a randomized, double-blind, placebo-controlled trial . Gastroenterology 2001 ; 121 : 1088 – 94 . Google Scholar CrossRef Search ADS PubMed 17. Scallon B , Cai A , Solowski N , et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists . J Pharmacol Exp Ther 2002 ; 301 : 418 – 26 . Google Scholar CrossRef Search ADS PubMed 18. O’Toole A , Lucci M , Korzenik J . Inflammatory bowel disease provoked by etanercept: report of 443 possible cases combined from an IBD referral center and the FDA . Dig Dis Sci 2016 ; 61 : 1772 – 4 . Google Scholar CrossRef Search ADS PubMed 19. Sandborn WJ , Feagan BG , Stoinov S , et al. ; PRECISE 1 Study Investigators . Certolizumab pegol for the treatment of Crohn’s disease . N Engl J Med 2007 ; 357 : 228 – 38 . Google Scholar CrossRef Search ADS PubMed 20. Sandborn WJ , Schreiber S , Feagan BG , et al. Certolizumab pegol for active Crohn’s disease: a placebo-controlled, randomized trial . Clin Gastroenterol Hepatol 2011 ; 9 : 670 – 6.e3 . Google Scholar CrossRef Search ADS PubMed 21. Schreiber S , Khaliq-Kareemi M , Lawrance IC , et al. ; PRECISE 2 Study Investigators . Maintenance therapy with certolizumab pegol for Crohn’s disease . N Engl J Med 2007 ; 357 : 239 – 50 . Google Scholar CrossRef Search ADS PubMed 22. Schreiber S , Rutgeerts P , Fedorak RN , et al. ; CDP870 Crohn’s Disease Study Group . A randomized, placebo-controlled trial of certolizumab pegol [CDP870] for treatment of Crohn’s disease . Gastroenterology 2005 ; 129 : 807 – 18 . Google Scholar CrossRef Search ADS PubMed 23. Hébuterne X , Lémann M , Bouhnik Y , et al. Endoscopic improvement of mucosal lesions in patients with moderate to severe ileocolonic Crohn’s disease following treatment with certolizumab pegol . Gut 2013 ; 62 : 201 – 8 . Google Scholar CrossRef Search ADS PubMed 24. Rutgeerts P , Diamond RH , Bala M , et al. Scheduled maintenance treatment with infliximab is superior to episodic treatment for the healing of mucosal ulceration associated with Crohn’s disease . Gastrointest Endosc 2006 ; 63 : 433 – 42 ; quiz 464. Google Scholar CrossRef Search ADS PubMed 25. Rutgeerts P , Van Assche G , Sandborn WJ , et al. ; EXTEND Investigators . Adalimumab induces and maintains mucosal healing in patients with Crohn’s disease: data from the EXTEND trial . Gastroenterology 2012 ; 142 : 1102 – 11.e2 . Google Scholar CrossRef Search ADS PubMed 26. Toedter G , Li K , Marano C , et al. Gene expression profiling and response signatures associated with differential responses to infliximab treatment in ulcerative colitis . Am J Gastroenterol 2011 ; 106 : 1272 – 80 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. 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 Journal of Crohn's and Colitis Oxford University Press

TNF-anti-TNF Immune Complexes Inhibit IL-12/IL-23 Secretion by Inflammatory Macrophages via an Fc-dependent Mechanism

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Oxford University Press
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Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
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1873-9946
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1876-4479
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10.1093/ecco-jcc/jjy075
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Abstract

Abstract Background and Aims We have recently shown that the mode of action of IgG1 anti-tumour necrosis factor [TNF] antibodies in inflammatory bowel disease [IBD] requires Fcγ-receptor [FcγR] engagement on macrophages. Here we examine the effect of Fcγ-receptor signalling by anti-TNF on macrophage IL-12/IL-23 secretion. Methods Cytokine production by human inflammatory macrophages was assessed at the level of RNA and protein. TNF-anti-TNF immune complex formation was determined by size-exclusion chromatography and signalling visualized by immunofluorescence. IL-12/IL-23p40 was measured in CD14+ lamina propria cells from IBD patients. Results Infliximab and adalimumab potently suppressed IL-12/IL-23 production by inflammatory macrophages, but Fab’ fragment certolizumab did not. IL-12/IL-23 suppression depended on Syk activity and was mediated at the level of IL-12/IL-23p40 mRNA. Etanercept, a soluble TNF receptor fused to an Fc-region, did not inhibit IL-12/L-23 secretion, suggesting that the presence of an Fc-region was not sufficient. Infliximab and adalimumab formed immune complexes with soluble TNF whereas etanercept did not, suggesting that FcγR-mediated suppression of IL-12/IL-23 required the formation of immune complexes. Indeed, non-specific IgG1 immune complexes, but not uncomplexed IgG1, similarly suppressed IL-12/IL-23 secretion. Finally, infliximab significantly decreased IL-12/IL-23p40 production in myeloid cells isolated from the lamina propria of IBD patients. Conclusions TNF-anti-TNF antibody immune complexes potently inhibit IL-12/IL-23 expression by inflammatory macrophages. Our data suggest that anti-TNFs and antibodies against IL-12/IL-23 may therefore have partially overlapping modes of action in patients with IBD. Anti-TNF, IL-12/IL-23 axis, macrophage 1. Introduction In contrast to rheumatoid arthritis, the mode of action of anti-tumour necrosis factors[TNFs] in inflammatory bowel disease [IBD] is not exclusively related to therapeutic blockade of TNF.1 We have previously demonstrated that the therapeutic efficacy of anti-TNF was completely dependent on Fc-Fcγ receptor [FcγR] interaction in a preclinical model of colitis. Additionally, we have shown that IgG1 anti-TNFs polarise macrophages to a regulatory and wound healing phenotype in an Fc-dependent manner in humans and mice.2–5 Intriguingly, it has previously been shown that activation of FcγR by immune complexes can potently inhibit IL-12 secretion by macrophages.6–8 IL-12 is a dimeric cytokine which shares its p40 subunit with IL-23. Whether immune complexes also inhibit IL-23 is not known, as the IL-23-specific p19 subunit had not yet been identified at the time these experiments were performed. IL-12 and IL-23 are mainly produced by macrophages and dendritic cells and play a key role in the pathogenesis of IBD. In the inflamed intestine of patients with Crohn’s disease, monocytes polarise towards an inflammatory macrophage phenotype and produce pro-inflammatory cytokines including TNF and IL-23.9 The blockade of IL-12p40 with ustekinumab, which neutralises both IL-12 and IL-23, is currently approved as a treatment for patients with Crohn’s disease and, more recently, the selective IL-23p19 inhibitors risankizumab and brazikumab showed promising results in clinical trials in patients with Crohn’s disease.10–13 Studies in ulcerative colitis are ongoing. The advent of this novel class of antibodies poses the question whether patients with Crohn’s disease responding to anti-TNFs and IL-12/IL-23 blockade represent distinct or overlapping groups. TNF, IL-12, and IL-23 promote inflammation via distinct pathways, suggesting that therapies blocking these cytokines may establish mucosal healing via different routes and therefore work in different patient subgroups. However, given the fact that we have previously found that FcγR signalling is involved in the therapeutic effect of anti-TNFs and the ability of FcγR signalling to suppress IL-12 secretion, we aimed to examine how different anti-TNF blocking strategies affected macrophage IL-12/IL-23 secretion. 2. Methods 2.1. Anti-TNF compounds Therapeutic anti-TNF compounds used in this study are adalimumab [Humira, AbbVie], infliximab [Remicade, MSD], golimumab [Simponi, MSD], certolizumab pegol [Cimzia, UCB Pharma], and etanercept [Enbrel, Pfizer]. Infliximab Fab fragments were generated using the Pierce Fab micro Preparation Kit [Thermo Fisher, Landsmeer, The Netherlands]. 2.2. In vitro macrophage differentiation Peripheral blood mononuclear cells [PBMC] from healthy volunteers were isolated by Ficoll Paque density-gradient centrifugation. After washing, monocytes were isolated by Percoll density-gradient centrifugation.14 Inflammatory [M1] macrophages were obtained by culturing monocytes with IFNγ [50 ng/mL, PeproTech, London, UK] for 6 days in RPMI supplemented with 10% heat-inactivated fetal calf serum [FCS]. Macrophages were then washed, reseeded, and stimulated for 48 h with lipopolysaccharide [LPS] 100 ng/ml [Sigma, Aldrich, Zwijndrecht] and anti-TNF or IgG1 [Genetex, Irvine, CA]. Complexed IgG1 was obtained by heating IgG1 at 63°C for 1 h. Culture supernatants were harvested and cytokine levels were measured by enzyme-linked immunosorbent assay [ELISA] for IL-23, IL-12p40, and IL-10 [Duoset, R&D] or by Cytometric Bead Array [Human Inflammatory Cytokine Kit, BD Biosciences]. For pSYK staining, M1 macrophages were incubated with LPS and anti-TNF for 30 min, fixed using Fixation/Permeabilization Solution Kit [BD Biosciences], and stained for anti-pSYK-PE or isotype control staining [both Cell Signaling]. Syk was inhibited with 1 μM R406 [Invivogen] added 1 before the addition of LPS and anti-TNF cytokine production in this case was measured after 24 h. 2.3. Heat-killed bacteria Heat-killed E. coli 0111:B4 was purchased from Invivogen [catalog no. tlrl-hkeb2]. E. faecalis [catalog no. 29212; ATCC] was cultured in brain-heart infusion [BHI] medium. Bacteria were harvested and washed twice with phosphate-buffered saline [PBS]. Then, bacterial suspension was heated at 80°C for 30 min, washed, resuspended in PBS, and stored at –80°C. Complete killing was confirmed by a 72-h incubation at 37°C. 2.4. Preparation of LPMCs Intestinal mucosa was obtained from surgically resected specimens from patients with IBD, diagnosed on the basis of endoscopic and histological findings according to established criteria. All experiments were performed with mucosa from macroscopically inflamed specimens. All experiments were approved by the Medical Ethical Committee of the Academic Medical Center, Amsterdam. Written informed consent was obtained from all patients. Lamina propria mononuclear cells [LPMC] were isolated from intestinal specimens as follows. Briefly, dissected mucosa was incubated in calcium and magnesium-free HBSS containing 5 mM EDTA [Sigma-Aldrich] for 20 min at 37°C to remove epithelial cells. Tissues were then cut very fine and incubated in RPMI medium containing 10% FCS, 1 mg/ml collagenase D [Roche], 1 mg/ml soybean trypsin inhibitor [Sigma], and 50 µg/ml Dnase I [Roche] for 60 min at 37°C. The fraction was pelleted and resuspended in a 30% Percoll solution, then layered on 60% Percoll before centrifugation at 500g for 10 min at room temperature. Viable LPMC were recovered from the 30–60% layer interface. LPMC were then stimulated overnight with heat-killed E. coli and E. faecalis, both 1 × 108 cells/ml, in the presence of IgG1 or anti-TNF 10 µg/ml. Golgistop [BD Biosciences] was added after 1 h. The next day, LPMC were stained for LIVE/DEAD® Fixable Green Dead Cell Stain Kit [Invitrogen], CD45-AF700 [Sony], CD14-PE-Cy7 [eBioscience], HLA-DR-PE, and CD11b-BV421 [both Biolegend] and then fixed using Fixation/Permeabilization Solution Kit [BD Biosciences]. After fixation, cells were stained for IL-12p40-APC [Biolegend] and analysed by FACS Fortessa [BD Biosciences], then analysed using FlowJo software [Treestar Inc]. 2.5. High-performance size-exclusion chromatography Recombinant human TNF was obtained from Active Bioscience. Therapeutic anti-TNF compounds [60 µg/ml, diluted in PBS] were incubated for at least 1 h with 2 µg/ml recombinant human TNF. Samples were analysed by applying 200 μl to a Superdex 200 HR 10/300 column [GE Healthcare, Uppsala Sweden], which was connected to an ÄKTAexplorer HPLC system [GE Healthcare]. Elution profiles were monitored by measuring absorbance at 215 nm. The Superdex column was calibrated using the Gel Filtration Markers Kit [29–700 kDa range, Sigma Aldrich]. 2.6. Quantitative real-time polymerase chain reaction mRNA isolation was performed using the Bioline ISOLATE II RNA Mini kit [BIO-52073, Bioline] according to manufacturers’ instructions. First-strand cDNA was synthesised using Oligo-dT [Invitrogen], random hexamer primers [Promega, Madison, IL], and RiboLock RNase and RevertAid reverse transcriptase [both Thermo Scientific]. Quantitative real-time polymerase chain reaction [qRT-PCR] was performed using sensifast SYBR No-ROX Kit [GC-biotech Bio-98020] on a BioRad iCycler. Relative gene expression was calculated using the 2−delta Ct method. GAPDH was used as reference gene. 2.7. Cohort studies of patients with Crohn’s disease treated with anti-TNF Intestinal biopsy specimens were collected in patients suffering from Crohn’s disease, before anti-TNF treatment was initiated and during treatment at the Academic Medical Center in Amsterdam and at the University Hospital in Leuven. Endoscopy was performed in all patients before the start of anti-TNF therapy and during follow-up. Seven responders and eight non-responders were included, where response versus non-response was determined by the clinician’s assessment of the endoscopic evaluation of the mucosa. Response was determined before analysis of the tissue samples [patient characteristics in Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. When comparing mRNA levels on consecutive time points within one patient, we compared biopsies within one anatomical location [i.e. colon versus colon and ileum versus ileum]. The study protocols were approved by the medical ethical committees of the Academic Medical Center, Amsterdam, The Netherlands and of the University Hospital, Leuven, The Netherlands; all participants provided written informed consent. 2.8. Statistical analysis For statistical analysis, unpaired t-test or analysis of variance [ANOVA] was used, followed by Bonferroni post-test. Normalised data were analysed with Wilcoxon signed rank test or Kuskal-Wallis, followed by Dunn’s post hoc analysis. Results were considered significant when p-value < 0.05. 3. Results 3.1. Infliximab inhibits IL-12p40 and IL-23 production by inflammatory macrophages Given the pro-inflammatory state of macrophages in the intestine of Crohn’s disease patients,9 we investigated the effects of infliximab on a priori polarised inflammatory macrophages in vitro. Inflammatory macrophages were generated by culturing human monocytes with IFNγ. For clarity, we will hereafter refer to IFNγ-induced macrophages as M1 macrophages. M1 macrophages were washed and reseeded with LPS in the presence of infliximab or IgG1 isotype control. To explore the effect of infliximab on the pro-inflammatory cytokine profile of M1 macrophages, we measured IL-12p40, IL-6, IL-8, and IL-1β production. Whereas IL-6, IL-8, and IL-1β were comparable between infliximab and IgG1-treated M1 macrophages, IL-12p40 was specifically inhibited by infliximab [Figure 1A]. Further analyses revealed that the decrease in IL-12p40 was dose-dependent [Figure 1B]. As IL-23 is composed of subunits IL-12p40 and IL-23-p19, we next measured IL-23 secretion and we found that IL-23 was also dose-dependently decreased by infliximab. [Figure 1C] Figure 1. View largeDownload slide Infliximab specifically inhibits IL-12p40 and IL-23 production by inflammatory macrophages. Human monocytes were cultured with IFNγ 50 ng/ml for 6 days to generate M1 macrophages. M1 macrophages were then reseeded with infliximab or IgG1 [both 10 µg/ml] in the presence of lipopolysaccharide [LPS] 100 ng/ml. [A] Inflammatory cytokine production by M1 macrophages cultured for 48 h in six different donors. Each dot represents an individual donor: Wilcoxon signed rank test. [B/C] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: analysis of variance [ANOVA] followed by Dunnet’s Multiple comparisons test. [D] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]: Mann‑Whitney U-test. Data are representative of at least three independent experiments with different donors and bars show means; error bars indicate standard error of the mean, *p < 0.05, **p < 0.01, ***p < 0.001. Figure 1. View largeDownload slide Infliximab specifically inhibits IL-12p40 and IL-23 production by inflammatory macrophages. Human monocytes were cultured with IFNγ 50 ng/ml for 6 days to generate M1 macrophages. M1 macrophages were then reseeded with infliximab or IgG1 [both 10 µg/ml] in the presence of lipopolysaccharide [LPS] 100 ng/ml. [A] Inflammatory cytokine production by M1 macrophages cultured for 48 h in six different donors. Each dot represents an individual donor: Wilcoxon signed rank test. [B/C] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: analysis of variance [ANOVA] followed by Dunnet’s Multiple comparisons test. [D] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]: Mann‑Whitney U-test. Data are representative of at least three independent experiments with different donors and bars show means; error bars indicate standard error of the mean, *p < 0.05, **p < 0.01, ***p < 0.001. Analysis by qPCR showed that infliximab inhibited the expression of IL-12B mRNA, the gene encoding subunit IL-12p40. The expression of IL-12A and IL-23A, encoding subunits IL-12p35 and IL-23p19, respectively, was not affected by infliximab, nor was the induction of TNFα itself [Figure 1D]. Overall, these data suggest that the suppression of the IL-23 protein is mediated by inhibition of IL-12B transcription and not by IL-23p19. 3.2. Inhibition of IL12/IL-23 is Fc-dependent To investigate if the inhibition of IL-12p40 and IL-23 was Fc-dependent, we compared infliximab with certolizumab, a TNF-neutralising Fab’ fragment. Both anti-TNF compounds blocked TNF to undetectable levels [Supplementary Figure 1, available as Supplementary data at ECCO-JCC online]. Hereby, we could distinguish between Fc-dependent mechanisms and effects that should be attributed to TNF neutralisation. Again, infliximab treatment decreased IL-12p40 and IL-23. However, the production of these cytokines remained high in certolizumab-treated M1 macrophages, indicating that the inhibition of IL-12p40 and IL-23 is mediated via the Fc-region of infliximab rather than neutralisation of TNF [Figure 2A]. Similarly, infliximab Fab fragments did not inhibit IL-12p40 production, further supporting a role for the Fc-region [Supplementary Figure 2, available as Supplementary data at ECCO-JCC online]. Figure 2. View largeDownload slide Inhibition of IL12/ IL-23 is Fc-dependent. M1 macrophages were reseeded with lipopolysaccharide [LPS] 100 ng/ml and infliximab or certolizumab 10 µg/ml for 48 h unless otherwise stated. [A] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B] Normalised IL-10 production by M1 macrophages from multiple donors: Wilcoxon signed rank test. [C] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]. [D] Macrophages were fixed 30 min after the addition of lipopolysaccharide [LPS] and anti-TNF, stained with anti-pSYK-PE or isotype control, and visualised by immunofluorescence and flow cytometry. [E] Syk-inhibitor R406 was added before the addition of LPS and anti-TNF [1 µg/ml], IL-12p40 and IL-10 production was measured by ELISA after 24 h: analysis of variance [ANOVA] followed by Bonferroni post-test. Data are representative of at least three independent experiments with different donors, bars show means, error bars indicate standard error of the mean, **p < 0.01, ***p < 0.001. Figure 2. View largeDownload slide Inhibition of IL12/ IL-23 is Fc-dependent. M1 macrophages were reseeded with lipopolysaccharide [LPS] 100 ng/ml and infliximab or certolizumab 10 µg/ml for 48 h unless otherwise stated. [A] IL-12p40 and IL-23 production by M1 macrophages measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B] Normalised IL-10 production by M1 macrophages from multiple donors: Wilcoxon signed rank test. [C] mRNA expression of indicated genes normalised to GAPDH expression measured by real-time polymerase chain reaction [RT-PCR]. [D] Macrophages were fixed 30 min after the addition of lipopolysaccharide [LPS] and anti-TNF, stained with anti-pSYK-PE or isotype control, and visualised by immunofluorescence and flow cytometry. [E] Syk-inhibitor R406 was added before the addition of LPS and anti-TNF [1 µg/ml], IL-12p40 and IL-10 production was measured by ELISA after 24 h: analysis of variance [ANOVA] followed by Bonferroni post-test. Data are representative of at least three independent experiments with different donors, bars show means, error bars indicate standard error of the mean, **p < 0.01, ***p < 0.001. Interestingly, others have shown that FcγR binding on macrophages by IgG immune complexes not only inhibits IL-12 production, but also increases IL-10 production.6–8 We found that infliximab enhanced the production of IL-10 compared with certolizumab, confirming an Fc-dependent increase in IL-10 production [Figure 2B]. However, TNF neutralisation with certolizumab reduced IL-10 production compared with cultures treated with LPS alone. Thus, IL-10 production is regulated by both TNF blockade and Fc-engagement in opposite ways, and the outcome may differ depending on the relative strengths of these signals. This might explain why we observed some variability in the effects of infliximab on IL-10 production when analysing multiple donors [Figure 2B]. Overall, IL-10 production was always higher in infliximab-treated cultures compared with certolizumab. Analysis by qPCR showed that infliximab inhibited the expression of IL-12B mRNA, whereas certolizumab did not. In line with Figure 1, expression of IL-12A and IL-23A was not inhibited by infliximab or certolizumab [Figure 2C]. We recently showed that activating FcγR are indispensable for the therapeutic efficacy of anti-TNF in experimental colitis.2 Activated FcγR have been shown to signal via spleen tyrosine kinase [Syk], and ligation of FcγR results in the phosphorylation of Syk.15 To study this further in a human setting, we measured phosphorylated Syk in human M1 macrophages. Indeed, only in infliximab-treated M1 macrophages was the presence of phosphorylated Syk detected [Figure 2D]. Furthermore, inhibition of Syk completely reversed the infliximab-mediated decrease in IL-12p40, as well as the increase in IL-10 production [Figure 2E]. 3.3. TNF-anti-TNF complex formation crucial for IL-23 inhibition Our previous data indicate an important role for FcγR engagement in the mode of action of IgG1 anti-TNFs in IBD; however, etanercept also contains an Fc-region but failed to show clinical efficacy in Crohn’s disease.16 Interestingly, when M1 macrophages were cultured with etanercept, the production of IL-12p40 and IL-23 was not inhibited [Figure 3A]. Also, IL-10 production was decreased to levels comparable to certolizumab-treated macrophages. This suggests that the mere presence of an IgG1 Fc-region is not sufficient to increase the IL-10 to IL-12/IL-23 ratio. We hypothesised that anti-TNF antibodies like infliximab and adalimumab differ from etanercept in their ability to cross-link FcγR. Antibodies like infliximab and adalimumab have two Fab’ arms that can simultaneously bind two TNF molecules. Importantly, biologically active TNF is a homotrimer and it has been shown that TNF can be bound by three infliximab molecules at once. These features will enable the formation of TNF-anti-TNF immune complexes that can cross-link FcγR. In contrast, etanercept contains only one TNF-binding domain and has been shown to bind soluble TNF in a 1:1 ratio.17 Importantly, cross-linking of FcγR by immune complexes affects downstream FcγR effector functions to a much greater extent than Fc engagement by monomeric antibodies. Figure 3. View largeDownload slide Anti-TNF immune complexes mediate alterations in IL-10 and IL-23 production. [A] M1 macrophages stimulated with lipopolysaccharide [LPS] 100 ng/ml and different anti-TNF compounds 10 μg/ml: ytokine production after 48 h: analysis of variance [ANOVA] followed by Bonferroni post=test [B] Complex formation of TNF with anti-TNF. TNF 2 µg/ml was incubated with anti-TNF 60 µg/ml and analysed on a Superdex 200 column. For each sample, a representative elution pattern is shown out of at least two measurements. Arrows indicate elution of TNF-anti-TNF complexes. [C] M1 macrophages stimulated with IgG1 or complexed IgG1 [both 10 μg/ml] in the presence of LPS 100 ng/ml. Cytokine production measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: unpaired t-test. [D] M1 macrophages stimulated with anti-TNF and complexed IgG1 where indicated in the presence of LPS 100 ng/ml. Cytokine production measured by ELISA after 48rs: ANOVA followed by Bonferroni post-test. Data are representative at least three experiments with different donors, ***p < 0.001. Figure 3. View largeDownload slide Anti-TNF immune complexes mediate alterations in IL-10 and IL-23 production. [A] M1 macrophages stimulated with lipopolysaccharide [LPS] 100 ng/ml and different anti-TNF compounds 10 μg/ml: ytokine production after 48 h: analysis of variance [ANOVA] followed by Bonferroni post=test [B] Complex formation of TNF with anti-TNF. TNF 2 µg/ml was incubated with anti-TNF 60 µg/ml and analysed on a Superdex 200 column. For each sample, a representative elution pattern is shown out of at least two measurements. Arrows indicate elution of TNF-anti-TNF complexes. [C] M1 macrophages stimulated with IgG1 or complexed IgG1 [both 10 μg/ml] in the presence of LPS 100 ng/ml. Cytokine production measured by enzyme-linked immunosorbent assay [ELISA] after 48 h: unpaired t-test. [D] M1 macrophages stimulated with anti-TNF and complexed IgG1 where indicated in the presence of LPS 100 ng/ml. Cytokine production measured by ELISA after 48rs: ANOVA followed by Bonferroni post-test. Data are representative at least three experiments with different donors, ***p < 0.001. To confirm these differences in TNF-anti-TNF immune complex formation for multiple anti-TNF compounds, we performed size-exclusion chromatography [Figure 3B]. Infliximab, adalimumab, and golimumab formed a complex that roughly corresponds in size to a complex where three antibodies are bound to one soluble TNF molecule. Etanercept formed a complex with soluble TNF almost equal in size to unbound etanercept, indicating that one etanercept molecule was bound to one TNF molecule [Figure 3B]. In line with these data, we have previously shown that in the presence of soluble TNF, FcγR binding capacity becomes much higher for adalimumab than for etanercept.2 Thus, although etanercept has an Fc-region, it does not bind FcγR as potently as do monoclonal antibodies infliximab or adalimumab in the presence of soluble TNF, and this is reflected by the inability of etanercept to increase the IL-10 to IL-12/IL-23 ratio. Finally, to further investigate the need for immune complex formation to achieve IL-12/IL-23 inhibition, we compared non-specific monomeric IgG1 with complexed IgG1. Indeed, complexed IgG1 increased IL-10 and inhibited both IL-12p40 and IL-23 compared with monomeric IgG1, confirming their functional difference [Figure 3C]. Furthermore, when complexed IgG1 was added to certolizumab treatment [to add TNF blockade to the FcR cross-linking by the complexed IgG], the production of IL-10, IL-12p40, and IL-23 became equal to that of infliximab-treated M1 macrophages. [Figure 3D] Thus, the increase in IL-10 versus IL-12/IL23 ratio mediated by infliximab can be mimicked by a combination of certolizumab [TNF blockade] and IgG1 immune complexes [FcγR cross-linking]. 3.4. IgG1 anti-TNF inhibits IL-12/IL-23 in mucosal CD14+ cells from IBD patients In an inflamed intestine, mucosal macrophages are constantly exposed to a wide range of microbial products. To explore if our findings could be reproduced using an equivalent stimulation, we cultured M1 macrophages with heat-killed bacteria. Indeed, when M1 macrophages were cultured with a combination of heat-killed E. coli and heat-killed E. faecalis, we found that infliximab inhibits IL-12p40 in an Fc-dependent manner in this setting also [Figure 4A]. Additionally, this was also the case when M1 macrophages were cultured with either heat-killed E. coli or heat-killed E. faecalis separately [Supplementary Figure 3, available as Supplementary data at ECCO-JCC online]. Figure 4. View largeDownload slide IgG1 anti-TNF inhibits mucosal IL-12/IL-23 in inflammatory bowel disease [IBD] patients. [A] M1 macrophages were stimulated with heat-killed E.coli and E. faecalis [both 1 × 108 cells/ml] and treated with anti-TNF [10 µg/ml] for 48 h. IL-12p40 was measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B/C] Representative histograms and graph of the CD14+ lamina propria myeloid compartment stimulated with heat-killed E. coli and E. faecalis [both 1 × 108 cells/ml] overnight in the presence of IgG1, certolizumab, or infliximab [all 10 µg/ml]. IL-12p40 was measured in CD14+ macrophages by flow cytometry: ANOVA followed by Bonferroni post-test. [D] mRNA expression of IL-12B measured by real-time polymerase chain reaction [RT-PCR] in intestinal biopsy specimens from patients with Crohn’s disease treated with infliximab or adalimumab, 7]seven endoscopic responders and eight endoscopic non-responders. Each dot represents an individual patient: Mann-Whitney U-test. Data are representative of at least three experiments with different donors or different IBD patients, and bars show means, error bars indicate standard error of the mean,*p < 0.05, **p < 0.01,***p < 0.001. Figure 4. View largeDownload slide IgG1 anti-TNF inhibits mucosal IL-12/IL-23 in inflammatory bowel disease [IBD] patients. [A] M1 macrophages were stimulated with heat-killed E.coli and E. faecalis [both 1 × 108 cells/ml] and treated with anti-TNF [10 µg/ml] for 48 h. IL-12p40 was measured by enzyme-linked immunosorbent assay [ELISA]: analysis of variance [ANOVA] followed by Bonferroni post-test. [B/C] Representative histograms and graph of the CD14+ lamina propria myeloid compartment stimulated with heat-killed E. coli and E. faecalis [both 1 × 108 cells/ml] overnight in the presence of IgG1, certolizumab, or infliximab [all 10 µg/ml]. IL-12p40 was measured in CD14+ macrophages by flow cytometry: ANOVA followed by Bonferroni post-test. [D] mRNA expression of IL-12B measured by real-time polymerase chain reaction [RT-PCR] in intestinal biopsy specimens from patients with Crohn’s disease treated with infliximab or adalimumab, 7]seven endoscopic responders and eight endoscopic non-responders. Each dot represents an individual patient: Mann-Whitney U-test. Data are representative of at least three experiments with different donors or different IBD patients, and bars show means, error bars indicate standard error of the mean,*p < 0.05, **p < 0.01,***p < 0.001. Next, we isolated lamina propria mononuclear cells [LMPC] from IBD patients. Cells were stimulated overnight with a combination of heat-killed E. coli and E. faecalis in the presence of infliximab, certolizumab, or an IgG1 isotype control antibody. Analyses by flow cytometry revealed that infliximab inhibited IL-12p40 protein production in the CD14+ lamina propria myeloid population to levels comparable to unstimulated cultures [Figure 4B]. Furthermore, IL-12p40 was significantly decreased in infliximab-treated CD14+ lamina propria cells compared with certolizumab or IgG1-treated LPMC [Figure 4C]. Finally, mucosal mRNA expression of the gene encoding IL-12/IL-23p40 [IL-12B] was analysed in a cohort of patients with Crohn’s disease treated with infliximab or adalimumab. We included seven endoscopic responders and eight endoscopic non-responders [patient characteristics shown in Supplementary Table 1]. The expression of IL-12B decreased significantly in responders upon anti-TNF treatment, but remained stable in non-responders [Figure 4D]. 4. Discussion The mode of action of anti-TNFs in IBD is still a matter of investigation.1 Here we find that the trimeric cytokine TNF forms immune complexes with IgG1 anti-TNF monoclonal antibodies that potently inhibit IL-12/IL-23 secretion at the level of IL12B mRNA in an Fc-dependent manner. Only a few years after the introduction of infliximab, it became clear that the mode of action of anti-TNFs in IBD was less certain than anticipated, when etanercept completely failed to achieve either clinical response or remission in patients with Crohn’s disease, with patients on etanercept doing numerically worse than on placebo.16 Also, treatment with etanercept has been associated with paradoxical development of IBD in patients with rheumatic diseases.1,18 Certolizumab is significantly better than placebo for maintenance treatment but did not reach the primary endpoint for induction of clinical remission in three independent trials.19–22 In addition, there are no placebo-controlled data that support induction of mucosal healing by certolizumab. One uncontrolled endoscopic study showed that only 4% of patients with Crohn’s disease achieved mucosal healing after 10 weeks of certolizumab treatment.23 By comparison, complete mucosal healing was observed in 31% of patients on infliximab and 28% of patients on adalimumab in the same time frame.24,25 We have previously shown that one of the potential mechanisms of action that differentiates the IgG1 antibodies infliximab and adalimumab from other anti-TNF blocking strategies is dependent on the antibody Fc region. We found that IgG1 antibodies, but not certolizumab or etanercept, generate CD206-positive macrophages with regulatory and wound healing properties, in a mixed lymphocyte reaction in vitro.5 We found that responders but not non-responders to infliximab show accumulation of such CD206+ macrophages in the lamina propria in vivo.4 In a preclinical model, we used two anti-TNF antibodies with the same variable region that differed in their ability to bind FcγR. In a head-to-head comparison we found that both antibodies were equally effective in the collagen-induced model of arthritis, but that FcγR interaction was required for mucosal healing in the CD45RBhigh transfer model of colitis.3 More recently we demonstrated that mice lacking activating FcγR failed to generate CD206+ regulatory macrophages, and were completely unresponsive to anti-TNF therapy in the same model of colitis. Conversely, a hypo-fucosylated anti-TNF with increased affinity for FcγR showed increased CD206+ macrophage formation as well as improved therapeutic efficacy.2 In all our previous work, the anti-TNF induced CD206+ macrophages were likely formed from monocyte precursors. Here, we studied the effects of Fc-engagement on pro-inflammatory human macrophages with an M1-like phenotype. We found that in addition to generating CD206+ macrophages, anti-TNF monoclonal antibodies specifically inhibit IL-12/IL-23 production by M1-like macrophages. The inhibition of IL-12/IL-23 was mediated via Syk and was only achieved with anti-TNF compounds that had an Fc-region and were capable of forming immune complexes. TNF is a homotrimeric molecule containing three binding sites for anti-TNF antibodies. For antibodies like infliximab and adalimumab, this promotes the formation of TNF-anti-TNF complexes. Complexed antibodies affect FcγR effector functions to a much greater extent than do monomeric antibodies, by virtue of their ability to cross-link multiple FcγR. Although we have confirmed the primary finding of inhibition of IL-12/IL-23 in primary intestinal macrophages, it would be interesting to further evaluate the effects of immune complexes in this particular cell type as well. In vivo, we observed a decrease in IL-12B expression in patients responding to anti-TNF therapy, but not in non-responders. It should be noted that our cohort was a clinical follow-up cohort without hard criteria for response. However, a subanalysis of the ACT-1 trial, a prospective study using well-defined endoscopic criteria, showed similar results with decreased expression of IL12B in responders after 8 weeks of therapy.26 As other current targets in IBD do not have a homomultimeric structure, it seems unlikely that other monoclonal antibodies used in IBD share a similar mode of action. However, the inhibition of IL12/IL23 by TNF-anti-TNF immune complexes does pose questions around the differentiation of therapies blocking IL-12/IL-23 directly from anti-TNF monoclonal antibodies. Our data suggest that that the mode of action of anti-TNFs in IBD may be partially overlapping with ustekinumab [anti-IL12/IL23] and IL-23-specific therapies such as risankizumab. No controlled endoscopic data are currently available for ustekinumab. In a recent phase 2 study with risankuzimab, which involved mainly anti-TNF exposed patients, the delta between placebo and the top dose was only 5% in terms of mucosal healing.10 Thus, although we need to await further data, the available endoscopic data support the idea that this therapeutic class may not be as differentiated from anti-TNFs as previously hoped. Collectively, our results point out a previously unrecognised effector function of anti-TNF therapy that involves inhibition of the IL-12/IL-23 axis. These findings could contribute to our understanding about the failure of multiple biologics in therapy-refractory patients, and also suggest a new immunosuppressive mechanism for therapeutic antibodies capable of immune complex formation. Supplementary Data Supplementary data are available at ECCO-JCC online. Funding This project was supported by Health Holland, Top Sector Life Sciences and Health. Conflict of Interest GB is currently an employee of GlaxoSmithKline. GD received consulting and/or lecture fees from AbbVie, ActoGeniX, AM Pharma, Boehringer Ingelheim GmbH, Centocor, ChemoCentryx, Cosmo Technologies, Elan Pharmaceuticals, Engene, Dr Falk Pharma, Ferring, Galapagos, Giuliani SpA, Given Imaging, GlaxoSmithKline, Jansen Biologics, Merck Sharp and Dohme Corp, Millennium Pharmaceuticals, Inc. [now Takeda], Neovacs, Novonordisk, Otsuka, PDL Biopharma, Pfizer, Receptos, Salix, Setpoint, Shire Pharmaceuticals, Schering-Plough, Tillotts Pharma, UCB Pharma, Versant, and Vifor Pharma, and reports receiving research grants from Abbott Laboratories, Jansen Biologics, Given Imaging, MSD, Dr Falk Pharma, and Photopill; and speaking honoraria from Abbott Laboratories, Tillotts, Tramedico, Ferring, MSD, UCB, Norgine, and Shire. MW reports personal fees from Takeda, personal fees and non-financial support from Janssen Parmaceuticals, grants from GlaxoSmithKline, grants from Tillotts Pharma, and grants from Immunic, outside the submitted work. CB reports being on the Advisory Board Johnson & Johnson [energy devices]. Author Contributions FB, KS, PK, and TR designed, performed, and analysed experiments. CB, JB, and WB were involved in acquisition of data. GD, GB, and MW supervised study. FB, GB, and MW wrote the manuscript. References 1. Levin AD , Wildenberg ME , van den Brink GR . Mechanism of action of anti-TNF therapy in inflammatory bowel disease . J Crohns Colitis 2016 ; 10 : 989 – 97 . Google Scholar CrossRef Search ADS PubMed 2. Bloemendaal FM , Levin AD , Wildenberg ME , et al. Anti-tumor necrosis factor with a glyco-engineered Fc-region has increased efficacy in mice with colitis . Gastroenterology 2017 ; 153 : 1351 – 62.e4 . Google Scholar CrossRef Search ADS PubMed 3. McRae BL , Levin AD , Wildenberg ME , et al. Fc Receptor-mediated effector function contributes to the therapeutic response of anti-TNF monoclonal antibodies in a mouse model of inflammatory bowel disease . J Crohns Colitis 2016 ; 10 : 69 – 76 . 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Sutterwala FS , Noel GJ , Salgame P , Mosser DM . Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I . J Exp Med 1998 ; 188 : 217 – 22 . Google Scholar CrossRef Search ADS PubMed 9. Kamada N , Hisamatsu T , Okamoto S , et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis . J Clin Invest 2008 ; 118 : 2269 – 80 . Google Scholar PubMed 10. Feagan BG , Sandborn WJ , D’Haens G , et al. Induction therapy with the selective interleukin-23 inhibitor risankizumab in patients with moderate-to-severe Crohn’s disease: a randomised, double-blind, placebo-controlled phase 2 study . Lancet 2017 ; 389 : 1699 – 709 . Google Scholar CrossRef Search ADS PubMed 11. Feagan BG , Sandborn WJ , Gasink C , et al. ; UNITI–IM-UNITI Study Group . Ustekinumab as induction and maintenance therapy for Crohn’s disease . N Engl J Med 2016 ; 375 : 1946 – 60 . Google Scholar CrossRef Search ADS PubMed 12. Sandborn WJ , Gasink C , Gao LL , et al. ; CERTIFI Study Group . Ustekinumab induction and maintenance therapy in refractory Crohn’s disease . N Engl J Med 2012 ; 367 : 1519 – 28 . Google Scholar CrossRef Search ADS PubMed 13. Sands BE , Chen J , Feagan BG , et al. Efficacy and safety of MEDI2070, an antibody against interleukin 23, in patients with moderate to severe Crohn’s disease: a phase 2a study . Gastroenterology 2017 ; 153 : 77 – 86.e6 . Google Scholar CrossRef Search ADS PubMed 14. Repnik U , Knezevic M , Jeras M . Simple and cost-effective isolation of monocytes from buffy coats . J Immunol Methods 2003 ; 278 : 283 – 92 . Google Scholar CrossRef Search ADS PubMed 15. Nimmerjahn F , Ravetch JV . Fcgamma receptors as regulators of immune responses . Nat Rev Immunol 2008 ; 8 : 34 – 47 . Google Scholar CrossRef Search ADS PubMed 16. Sandborn WJ , Hanauer SB , Katz S , et al. Etanercept for active Crohn’s disease: a randomized, double-blind, placebo-controlled trial . Gastroenterology 2001 ; 121 : 1088 – 94 . Google Scholar CrossRef Search ADS PubMed 17. Scallon B , Cai A , Solowski N , et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists . J Pharmacol Exp Ther 2002 ; 301 : 418 – 26 . Google Scholar CrossRef Search ADS PubMed 18. O’Toole A , Lucci M , Korzenik J . Inflammatory bowel disease provoked by etanercept: report of 443 possible cases combined from an IBD referral center and the FDA . Dig Dis Sci 2016 ; 61 : 1772 – 4 . Google Scholar CrossRef Search ADS PubMed 19. Sandborn WJ , Feagan BG , Stoinov S , et al. ; PRECISE 1 Study Investigators . Certolizumab pegol for the treatment of Crohn’s disease . N Engl J Med 2007 ; 357 : 228 – 38 . Google Scholar CrossRef Search ADS PubMed 20. Sandborn WJ , Schreiber S , Feagan BG , et al. Certolizumab pegol for active Crohn’s disease: a placebo-controlled, randomized trial . Clin Gastroenterol Hepatol 2011 ; 9 : 670 – 6.e3 . Google Scholar CrossRef Search ADS PubMed 21. Schreiber S , Khaliq-Kareemi M , Lawrance IC , et al. ; PRECISE 2 Study Investigators . Maintenance therapy with certolizumab pegol for Crohn’s disease . N Engl J Med 2007 ; 357 : 239 – 50 . Google Scholar CrossRef Search ADS PubMed 22. Schreiber S , Rutgeerts P , Fedorak RN , et al. ; CDP870 Crohn’s Disease Study Group . A randomized, placebo-controlled trial of certolizumab pegol [CDP870] for treatment of Crohn’s disease . Gastroenterology 2005 ; 129 : 807 – 18 . Google Scholar CrossRef Search ADS PubMed 23. Hébuterne X , Lémann M , Bouhnik Y , et al. Endoscopic improvement of mucosal lesions in patients with moderate to severe ileocolonic Crohn’s disease following treatment with certolizumab pegol . Gut 2013 ; 62 : 201 – 8 . Google Scholar CrossRef Search ADS PubMed 24. Rutgeerts P , Diamond RH , Bala M , et al. Scheduled maintenance treatment with infliximab is superior to episodic treatment for the healing of mucosal ulceration associated with Crohn’s disease . Gastrointest Endosc 2006 ; 63 : 433 – 42 ; quiz 464. Google Scholar CrossRef Search ADS PubMed 25. Rutgeerts P , Van Assche G , Sandborn WJ , et al. ; EXTEND Investigators . Adalimumab induces and maintains mucosal healing in patients with Crohn’s disease: data from the EXTEND trial . Gastroenterology 2012 ; 142 : 1102 – 11.e2 . Google Scholar CrossRef Search ADS PubMed 26. Toedter G , Li K , Marano C , et al. Gene expression profiling and response signatures associated with differential responses to infliximab treatment in ulcerative colitis . Am J Gastroenterol 2011 ; 106 : 1272 – 80 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. 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)

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Journal of Crohn's and ColitisOxford University Press

Published: Sep 1, 2018

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