Background: Several inflammatory lung diseases display abundant presence of hyaluronic acid (HA) bound to heavy chains (HC) of serum protein inter-alpha-inhibitor (IαI) in the extracellular matrix. The HC-HA modification is critical to neutrophil sequestration in liver sinusoids and to survival during experimental lipopolysaccharide (LPS)- induced sepsis. Therefore, the covalent HC-HA binding, which is exclusively mediated by tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6), may play an important role in the onset or the resolution of lung inflammation in acute lung injury (ALI) induced by respiratory infection. Methods: Reversible ALI was induced by a single intratracheal instillation of LPS or Pseudomonas aeruginosa in mice and outcomes were studied for up to six days. We measured in the lung or the bronchoalveolar fluid HC-HA formation, HA immunostaining localization and roughness, HA fragment abundance, and markers of lung inflammation and lung injury. We also assessed TSG-6 secretion by TNFα- or LPS-stimulated human alveolar macrophages, lung fibroblast Wi38, and bronchial epithelial BEAS-2B cells. Results: Extensive HC-modification of lung HA, localized predominantly in the peri-broncho-vascular extracellular matrix, was notable early during the onset of inflammation and was markedly decreased during its resolution. Whereas human alveolar macrophages secreted functional TSG-6 following both TNFα and LPS stimulation, fibroblasts and bronchial epithelial cells responded to only TNFα. Compared to wild type, TSG-6-KO mice, which lacked HC-modified HA, exhibited modest increases in inflammatory cells in the lung, but no significant differences in markers of lung inflammation or injury, including histopathological lung injury scores. Conclusions: Respiratory infection induces rapid HC modification of HA followed by fragmentation and clearance, with kinetics that parallel the onset and resolution phase of ALI, respectively. Alveolar macrophages may be an important source of pulmonary TSG-6 required for HA remodeling. The formation of HC-modified HA had a minor role in the onset, severity, or resolution of experimental reversible ALI induced by respiratory infection with gram-negative bacteria. Keywords: Extracellular matrix, Hyaluronic acid, Inter-alpha-inhibitor, Serum-derived hyaluronan-associated protein, TNFα stimulated gene 6, Lung inflammation, Lipopolysaccharide, Pseudomonas aeruginosa * Correspondence: firstname.lastname@example.org Department of Medicine, National Jewish Health, 1400 Jackson Street, Molly Blank Building, J203, Denver, CO 80206, USA Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ni et al. Respiratory Research (2018) 19:107 Page 2 of 17 Background Burlington, MA, USA) or gram negative PA bacteria (2 × The extracellular matrix actively participates in inflammatory 10 CFU, colony forming unit, PA01 strain) was instilled signaling, tissue remodeling, and repair of various tissues. A directly into the tracheas of 10–12 week old mice using a better understanding of how components of the extracellular 22-gauge oral gavage needle (7920, Cadence Science, matrix participate in acute lung injury (ALI) and repair may Cranston, RI, USA) with its distal 0.5 cm end bent 40° to provide new therapeutic targets for clinical conditions such facilitate tracheal insertion. PA was provided by Dr. Ken- as acute respiratory distress syndrome. Hyaluronic acid neth Malcolm (National Jewish Health) and originally ob- (HA) is an unsulfated glycosaminoglycan (extracellular tained from Pseudomonas Genetic Stock Center (East matrix polysaccharide with repeating disaccharide unit) that Carolina University) . PA was grown in Luria-Bertani can be covalently modified with the heavy chains (HC) of broth (LB), and PA in the exponential phase of growth serum protein inter-alpha-inhibitor (IαI) during inflamma- was centrifuged and resuspended in 50 μLPBS forinstilla- tion [1–5], in a reaction exclusively mediated by tumor ne- tion. CFU was confirmed by plating dilutions of PA on LB crosis factor α (TNFα)-stimulated gene-6 (TSG-6) protein agar. Mice weight was assessed every 24 h, for up to 6 days (Additional file 1). Building on reports that TSG-6 mediated post-instillation. formation of HC-modified HA is critical to neutrophil se- questration in liver sinusoids [6, 7] and an important pro- Methods tective mediator of survival in lipopolysaccharide (LPS) Lung HC-HA models of sepsis [6, 8–10], we investigated the role of HC-HA formation in lung tissue was measured as de- HC-HA in ALI induced by respiratory infections, modeled scribed previously  with minor modifications. Briefly, by LPS or bacteria. equal mass (50 mg) of flash frozen mouse lung tissue HC-modified HA is prominently featured in the extracel- was homogenized in PBS for 3 min using lular matrix of chronic lung diseases such as pulmonary ar- Mini-Beadbeater-16 (Biospec, Bartlesville, OK, USA) and terial hypertension , asthma , cystic fibrosis , treated with 1 U of Streptomyces hyaluronidase (389,561, and idiopathic pulmonary fibrosis , that share lung in- MilliporeSigma) or PBS control for 45 min at 4 °C with flammation in their pathogenesis, suggesting that HC-HA mechanical agitation. Samples were then centrifuged formation is important in either promoting the onset or (13,000 g, 5 min, 4 °C) and supernatants were then incu- delaying the resolution of lung inflammation. We therefore bated (37 °C; 30 min) with mechanical agitation. The used self-resolving models of ALI that were induced by samples were then combined with Laemmli Buffer, sepa- intratracheal instillation of LPS or gram negative bacteria rated by SDS-PAGE (Stain-free Criterion TGX 7.5% gels, Pseudomonas aeruginosa (PA) to study the kinetics and role Biorad, Hercucles, CA, USA) and transferred to of HC-modified HA in acute lung inflammation. In Immobilon-P PVDF membrane (MilliporeSigma) using addition to using TSG-6-knockout (KO) mice to address TransBlot Semi-Dry (Biorad). The western blot was thefunctionalroleofHC-HA formationinALI,we de- probed using rabbit-anti-hIαI antibody (A0301, DAKO, scribe that LPS and PA induce extensive HC-HA formation Agilent, Santa Clara, CA, USA), which has been vali- during the initial phase of injury, followed by fragmentation dated for detecting mouse IαI and HC-HA formation in and clearance of HC-modified HA. injured mouse lung . ChemiDoc MP (Biorad) was used to image the Stain-free gels for total protein. Densi- Materials and reagents tometry was performed using Image Studio Lite (Licor, All materials and reagents used were from ThermoFisher Lincoln, NE, USA). (Waltham, MA, USA) unless otherwise specified. Reagents from Gibco (ThermoFisher) were used for cell culture. Messenger RNA (mRNA) studies Total ribonucleic acid (RNA) was extracted from cul- Mice tured cells using RNeasy Mini Plus (Qiagen, German- All animal experiments in this paper were approved by town, MD, USA) and from whole lung using Trizol the Institutional Animal Care and Use Committee Plus RNA Purification Kit with on-column deoxyribo- (IACUC) at National Jewish Health. TSG-6-KO mice nuclease (DNAse) digest performed using PureLink (BALB/c background) were originally generated by Dr. DNase. Mouse lung was homogenized in Trizol using Katalin Mikecz . Studies were conducted using sex- Mini-Beadbeater-16 (Biospec); 1000–2000 ng of total and age matched TSG-6-KO mice and wild type (WT) extracted RNA was used to synthesize complementary and heterozygous (HT) littermate controls. DNA (cDNA) (High-Capacity cDNA Reverse Tran- scription). Real-time quantitative polymerase chain re- Murine acute lung injury models action (qPCR) was performed on the StepOnePlus Escherichia coli (E. coli) LPS (20 μg LPS in 50 μL phos- System using Taqman Universal PCR Master Mix and phate buffered saline (PBS), L2880, MilliporeSigma, Taqman probes: hTSG-6 (Hs01113602_m1), msTNFα Ni et al. Respiratory Research (2018) 19:107 Page 3 of 17 (Mm00443258_m1), msTSG-6 (Mm00493736_m1) [18, 19], Histologic ALI scoring msHAS1 (Mm03048195_m1), msHAS2 (Mm00515089_ Unlavaged mice lungs were perfused as described above. m1), msHAS3 (Mm00515092_m1), msHYAL1 (Mm004 The left lung was inflated at 20 cm H O with 0.25% agarose 76206_m1), msHYAL2 (Mm01230688_g1), msTMEM2 in 10% formalin and immersion fixed overnight in 10% for- (Mm00459599_m1), and msCEMIP (Mm00472921_m1). malin following current guidelines . The fixed lung was Relative mRNA expression was calculated using the double placed in a molding box, encased in agarose, and 3 mm delta comparative (ΔΔCt) method and 18 s RNA endogen- thick transverse pieces (apex to base) of the lung were ous control (Taqman Hs99999901_s1). sliced to ensure adequate sampling of the entire lung for histological scoring. The lung pieces were paraffin embed- HA histology ded together, sliced at 3 μm thick, and deparafinized and Mice were euthanized by isoflurane overdose, bilateral rehydrated as described above. The slides were stained with thoracotomy, and perfusion of the lungs via the right ven- Harris Hematoxylin (2 min), Clarifier 1 (1 min), Bluing re- tricle using 10 mL of blood bank saline. LPS injured lungs agent (1 min), Eosin Y (30 s), dehydrated, and mounted. 4– were inflated with a PBS equilibrated solution containing 5 fields (400X total magnification) of each transversely 4% paraformaldehyde (PFA) (15,710, Electron Microscopy sliced lung piece (4–5 pieces total) were scored by a path- Sciences, Hatfield, PA, USA) and 0.33% low melting point ologist using the scoring system published by American agarose. Inflated lungs were immersion fixed overnight Thoracic Society , which assigns weighted scores for (24 h) in 4% PFA at 4 °C with gentle rocking and then se- five parameters of ALI injury and provides a final averaged quentially incubated for 1 h in PBS and 4 h in PBS con- scorebetween0(no injury)and 1(most severe). taining 25% sucrose and 25% optimal cutting temperature (OCT) compound. The lungs were then embedded in Cell culture OCT compound and frozen using dry ice. Ten μm sec- Primary human alveolar macrophages (hAM) were ob- tions were cut using a cryostat and allowed to air dry be- tained by bronchoalveolar lavage of de-identified fore washing in PBS to remove OCT compound. non-diseased human explanted lungs and enriched by PA injured lungs were inflated with 10% neutral buff- 2 h attachment to tissue culture treated plastic in Ros- ered formalin containing 0.25% low melting point agar- well Park Memorial Institute (RPMI) media with 1% ose and then immersion fixed in 10% neutral buffered penicillin/streptomycin. Non-adherent cells were re- formalin overnight at room temperature before paraffin moved by PBS wash. Indicated treatments were per- embedding and sectioning (3 μm). Paraffin embedded formed by incubating in RPMI media with 2% fetal tissue sections were then mounted on slides and proc- bovine serum (FBS, HyClone, GE Healthcare, Marlbor- essed as follows: mounted tissues were deparafinized ough, MA, USA) and 1X penicillin-streptomycin and ei- and rehydrated using successive incubations in xylene ther vehicle (0.1% bovine serum albumin in PBS), 20 ng/ (3 × 5 min), 100% ethanol (2 × 5 min), 95% ethanol mL tumor necrosis factor α (TNFα, R&D, Minneapolis, (2 × 5 min) and equilibration in PBS followed by MN, USA), or 50 ng/mL ultrapure E. coli LPS (LPS-EK, water. The tissue was then placed in pressure cooker InvivoGen, San Diego, CA, USA) for either 6 h or 24 h. containing citric acid based antigen unmasking solu- Peripheral Blood Mononuclear Cell Derived Macro- tion (Vector Labs, Burlingame, CA, USA) and phages (PBDM) were enriched by negative selection from microwaved. whole blood using DynaBeads Untouched Human Mono- Staining of lung sections was performed as follows: tis- cyte Kit and by attachment to tissue culture plastic. Treat- sue sections were blocked using PBS solution containing ment with macrophage colony stimulating factor (20 ng/ 3% bovine serum albumin (BSA, MilliporeSigma) and mL MCSF, R&D) over six days was used to differentiate 0.1% Triton X-100 (MilliporeSigma). Biotinylated hyalur- PBMC into macrophage-like cells. Macrophage differenti- onan binding protein (50 μg/100 μl stock, 385,911, Milli- ation was performed in RPMI under serum free condi- poreSigma), rabbit anti-human HC2 (NBP2–31750, tions with supplemental 1X non-essential amino acids, Novus, Littleton, CO, USA), and rat anti-mouse CD68 1mMsodium pyruvate, 2mMglutamine,and 1X (FA-11, Biolegend, San Diego, CA) were added at 1:100 penicillin-streptomycin for days 1–3 and with additional and incubated overnight at 4 °C. Streptavidin Alexa 10% FBS for days 4–6. For experiments, cells were incu- Flour 488 (S-11223) was used at 1:1000. Cy3 donkey bated in RPMI media with 2% FBS and treated with indi- anti-rabbit (711–165-152, Jackson ImmunoResearch, cated stimuli for 24 h. West Grove, PA, USA) and Cy5 donkey anti-rat (712– BEAS-2B transformed human lung bronchial epithelial 175-153, Jackson ImmunoResearch) were used at 1:2000. cells were cultured submerged in Dulbecco’s Modified Tissue was mounted using ProLong Gold AntiFade with Eagle Medium (DMEM), high glucose (4500 mg/L) DAPI and imaged using laser scanning confocal micro- media with 10% FBS and 1% penicillin/streptomycin. For scope 700 confocal (Zeiss, Jena, Germany). experiments, cells were washed once with PBS and then Ni et al. Respiratory Research (2018) 19:107 Page 4 of 17 incubated in basal DMEM media with 2% FBS and indi- TSG-6 (2104-TS-050, R&D) in the absence and presence of cated stimuli for 24 h. FBS (2%) was used for standard curve (Additional file 2), Wi38 primary human fetal lung fibroblasts were cul- sincewefound that thepresence ofFBS loweredthe magni- tured using Minimum Essential Media (MEM) media tude of HRP substrate color development. This phenomenon with 10% FBS and 1% penicillin/streptomycin. Cells were maybedueto TSG-6forming TSG-6-HC covalent inter- used between passages 8–12 for experiments, during mediate in the presence of IαI present in the serum and may which they were incubated with the indicated stimuli in explain why efforts to directly measure TSG-6 in human basal MEM media with 2% FBS. serum have been particularly challenging . Human adipose stromal/progenitor cells (ASC) isola- tion, expansion, and characterization have been previously Time-course of macrophage expression of TSG-6 and genes described [21–23]. Briefly, ASC were obtained by liposuc- implicated in HA breakdown in LPS-challenged mice tion from three human donors (two abdominal and one Expression of mouse TSG-6 (msTSG-6),also known as flank lipoaspirate) and then digested using collagenase I TNFα-induced-protein 6 (TNFAIP6), as well as msH (Worthington, Lakewood, NJ, USA) under mechanical agi- YAL1–2, msTMEM2,and msCEMIP was identified using tation for 2 h at 37 °C and centrifuged at 300 g for 8 min to Ensembl gene annotation data in a previously published obtain a pellet containing the stromal vascular fraction. data set. The detailed methods, pathway analysis of the This fraction was filtered using 250 μm Nitex filters (Sefar RNA-sequencing (RNA-seq) data, and National Center for America, Buffalo, NY, USA), and red blood cells were lysed Biotechnology Information (NCBI) deposition have been using ammonium chloride potassium lysis buffer (154 mM described here . Briefly, RNA-seq analysis was per- NH Cl, 10 mM KHCO , and 0.1 mM ethylenediaminetet- formed on bone-marrow-derived, recruited and resident 4 3 raacetic acid (EDTA)). Cells were then cultured using macrophages isolated from bronchoalveolar lavage of Endothelial Cell Growth Medium (EGM2-MV) media intratracheal LPS treated mice (C57BL/6, 10–12 week old; (Lonza, Allendale, NJ). Cells were used for experiments be- 0, 3, 6, 9, and 12 dpi). tween passages 4–6. To stimulate TSG-6 secretion , ASC were washed with PBS to remove residual FBS and HA fragmentation assessment in whole lung then incubated in basal Endothelial Basal Medium-2 HA fragmentation in lung tissue was assessed using a (EBM2) media (Lonza) with 20 ng/mL TNFα (R&D) for protocol generously provided by Cleveland Clinic Program 24 h. Demographic information of the ASC donors have of Excellence in Glycoscience. Briefly, dedicated (non-la- been described previously . vaged) lungs were perfused with 10 mL blood buffered sa- line and flash frozen. Proteinase K (1 mg/mL) resuspended Human TSG-6 (hTSG-6) western blot in 100 mM ammonium acetate (pH 7.0) with 0.01% sodium Conditioned media was centrifuged to remove detached dodecyl sulfate was used to lyse 50 mg of tissue (24 h; 60 ° cells (5 min, 600 g) and then mixed with Laemmli buffer. C). 100% ethanol was added to precipitate glycoaminigly- Proteins were separated by sodium dodecyl sulfate poly- cans and samples were washed using 75% ethanol. Samples acrylamide gel electrophoresis (SDS-PAGE) using Stain-Free were resuspended in 100 mM ammonium acetate, and Criterion TGX 4–20% gradient gels (Biorad), transferred, 100 °C heat was used to inactivate Proteinase K. Overnight and imaged similarly as HC-HA blots. The blot was probed benzonase treatment (MilliporeSigma) was used to degrade using goat-anti-hTSG-6 antibody (AF2104, R&D). nucleic acids, and 100 °C heat was used to inactivate benzo- nase. 100 and 75% ethanol was then used to precipitate and hTSG-6 ELISA wash the samples before resuspending in 100 mM ammo- Conditioned media was collected and centrifuged (600 g, nium acetate. Samples were equally divided and paired, 5min)and humanTSG-6 (hTSG-6) wasmeasuredasprevi- having a half of the sample left untreated, and half treated ously described  using a highly sensitive sandwich ELISA with 0.2 turbidity reducing units (TRU) of Streptomyces hy- developed using commercially available antibodies and aluronidase (Seikagaku, amsbio, Cambridge, MA). All sam- validated by TSG-6 small interfering RNA (siRNA) in human ples were lyophilized and resuspended in formamide MSC andASC. Briefly, Nunc MaxiSorp 96-well (MilliporeSigma) for loading on 1% agarose gel (SeaKem plates were coated with rat anti-hTSG-6 antibody (A38.1.20; HGT Agarose, Lonza). Gels were stained overnight in Santa Cruz) diluted in 0.2 M sodium bicarbonate buffer. De- Stains-All (1.25 mg/200 mL in 30% ethanol), equilibrated in tection was performed using biotinylated goat anti-hTSG-6 water, destained using light, and imaged using Cy5 695/55 antibody (BAF2104, R&D), Streptavidin-HRP (R&D), HRP epi-fluorescence filter on ChemiDoc MP . Select-HA of Substrate (R&D) and quenched using 1 M H SO .Todeter- predetermined sizes (2500, 1000, 500, and 250 kDa HA) 2 4 mine the extent of TSG-6 secretion relative to cell number, and Select-HA HiLadder (Hyalose, Oklahoma City, Okla- viable cell numbers were assessed by trypan blue exclusion homa, USA)wereusedtosizeHA fragments.Densitometry and counted by hemocytometer. Recombinant human of the distribution of HA staining was performed using Ni et al. Respiratory Research (2018) 19:107 Page 5 of 17 ImageJ as described before . It has been described pre- ELISA viously that agarose gel electrophoresis method is optimally Albumin-, receptor for advanced glycation end products suited for resolving high and medium molecular weight (RAGE)-, and HA ELISAs were performed on the com- HA (> 200 kDa) and that chromatography and polyacryl- bined supernatant obtained from pelleting the first three amide gel electrophoresis can provide better resolution and BALF aliquots (total 2.6 mL volume). Manufacturer’sproto- quantification of low molecular weight HA [30, 31]. cols were followed, using mouse albumin ELISA quan- titation set (Bethyl Labs, Montgomery, TX); RAGE Duoset ELISA (R&D); HA Duoset ELISA (R&D), HA staining characterization using sample dilutions of 1:3000; 1:6; and 1:4 (ctl Confocal Z-stacks were de-identified for the experimental group) or 1:12 (LPS group), respectively. Capture group, and HA staining was blindly scored. Briefly, three to antibody coating and HRP detection were performed five representative 320 × 320 μm images of the left lung as described for TSG-6 ELISA. were taken from each mouse. From each image, HA stain- ing in the peri-broncho-vascular interstitium was sampled Statistics by taking five 9.4 × 9.4 μm representative subselection snap- Statistical significance was calculated using ANOVA shots. A max intensity Z projection was then prepared and Tukey’s multiple comparison test in Prism using the Fiji distribution of ImageJ  and roughness was (Graphpad, La Jolla, CA). Data points from individual calculated by determining the surface area ofthe plot- mice or independent experiments were plotted unless ted intensity of HA staining using the SurfCharJ plugin otherwise specified. Results were considered signifi- . The roughness was normalized by dividing it by the cant at P <0.05. average staining intensity of the snapshot. A mean normal- ized roughness was then determined for each mouse. Results Induction and clearance of HC-modified HA during ALI Bronchoalveolar lavage fluid (BALF) collection and flow To investigate the kinetics of HC-HA formation during re- cytometry spiratory infection-induced ALI, we delivered LPS or Tracheotomy was used to visualize the trachea and insert an Pseudomonas aeruginosa (PA)tothe lungsofadult mice 18-gauge angiocatheter (4075, JELCO-W, Smiths-Medical, andthenprobedfor HC-HAinwhole lung homogenates. Minneapolis, MN). BALF was obtained by five serial instilla- We first assessed HC-HA formation in lung tissue from tions (1 × 1 mL and 4 × 0.9 mL) of PBS containing 2 mM mice exposed to LPS or PBS control, by ex vivo treating the EDTA (a return of 4 mL of total BALF was consistently ob- saline perfused and homogenized lung tissue with hyal- tained with this protocol). For the total CD45 count, ali- uronidase, which releases any HC linked to HA, followed quots of the five lavages were combined, blocked with by western blot detection of released HC. We observed ex- CD16/CD32 (clone 93, eBioscience, ThermoFisher), stained tensive HC-HA formation in LPS-exposed lungs at one-day with CD45 (30-F11, BD, Franklin Lakes, NJ, USA), and post instillation compared to contemporaneous PBS con- mixed with 123count eBeads (eBioscience). Cells used for trols in both wild type (Fig. 1a)and TSG-6 heterozygous total cell counts were stained and fixed without any centrifu- mice (Additional file 3: Figure A). However, at 4 days gation to avoid variability introduced by pelleting and aspir- post-LPS challenge, there was minimal HC-modified HA in ating. Using the absolute concentration of the counting the lungs of wild type (Fig. 1a)or TSG-6 heterozygous mice beads added and the ratio of total CD45 events to total bead (Additional file 3: Figure A). We next assessed HC-HA for- events, the concentration of CD45 cells was determined mation using a clinically relevant model of gram negative and then multiplied by 4 mL to obtain total CD45 counts. PA-bacteria-induced ALI. The extensive HC-HA formation For BALF cellular differentials, the five lavages were com- noted in lungs 2 days post intratracheal instillation of PA bined and centrifuged, blocked with CD16/CD32 was followed by minimal HC-modified HA at 4 days post (eBioscience), and stained with CD45 (30-F11, BD), Ly6G PA instillation in wild type (Fig. 1a)or TSG-6 heterozygous (1A8, Biolegend), CD64 (X54–5/7.1, BD), CD11c (N418, mice (Additional file 3: Figure B). To confirm that TSG-6 eBioscience), F4/80 (BM8, eBioscience), CD11b (M1/70, exclusively mediates HC covalent modification, we mea- eBioscience), Siglec-F (E50–2440, BD), CD4 (RM4–5, Biole- sured HC-HA in TSG-6-KO mice and noted no HC-HA at gend), and CD8a (53–6.7, Biolegend). Flow wash buffer con- one-day post LPS instillation compared to wild type and sisting of PBS with 9% FBS and 0.5 mM EDTA was used to heterozygous littermates (Fig. 1c). resuspend and wash cells. Flow data, which included mini- Having shown HC-HA formation, we studied if lung mum of 20,000 (PBS group) and 100,000 (LPS group) levels of the eponymous TSG-6 inducer TNFα or levels CD45 leukocyte events for each sample, was collected using of TSG-6 mRNA paralleled those of HC-modified HA in LSR II cytometer (BD) and analyzed using Flowjo (Ashland, ALI induced by LPS. Both TNFα and TSG-6 were rapidly Oregon, USA). induced 1 day post LPS instillation with expression Ni et al. Respiratory Research (2018) 19:107 Page 6 of 17 WT a c LPS 1 dpi HC-modified HA PBS LPS 1 dpi LPS 4 dpi Mice WT HT KO WT HT KO (TSG-6) **** **** Mice #1 #2 #1 #2 #1 #2 HAse - - - - - - + + + + + + HAse - - - - - - + + + + + + 10 HC 75kDa HC 75kDa Total Total Protein 0 Protein PBS 1 dpi 4 dpi LPS Lung msTNF Lung msTSG-6 WT b HC-modified HA d e PBS PA 2 dpi PA 4 dpi **** **** *** 6 ** **** **** #1 #2 #1 #2 #1 #2 Mice 15 HAse - - - - - - + + + + + + HC 75kDa 5 50 Total 0 PBS 1 dpi 4 dpi PBS 1 dpi 4 dpi Protein PBS 2 dpi 4 dpi LPS LPS PA HA HC DAPI Merge Br Br Br Br Br Fig. 1 HC-HA formation after LPS or PA injury. a-c. Abundance of heavy chain (HC)-linked HA in lung lysates detected by western blot using IαI antibody (recognizing HC) on lungs before (−) and after (+) hyaluronidase (HAse), which releases HC linked to HA. Each lane represents an individual mouse lung exposed to intratracheally instilled LPS (20 μg; a)or Pseudomonas aeruginosa (PA, 2*10 CFU; b) or control PBS for the indicated time, noted as days post instillation (dpi). Lung HC abundance was expressed relative to that of total protein, measured by densitometry (a-b). c. Exclusive role of TSG-6 in forming HC-HA was confirmed using wild type (WT), heterozygous (HT), and knockout (KO) for TSG-6. d-e. msTNFα and msTSG-6 expression levels measured by qPCR in whole lung following LPS. Data in a-b and d-e analyzed by ANOVA with Tukey’s multiple comparisons; **P < 0.01, ***P < 0.001, ****P < .0001. f. Immunofluorescence images of HA and HC localization in formalin-fixed, paraffin-embedded lung sections from control (0 dpi) and PA-injured (1 and 2 dpi) mice, using antibodies against HA binding protein (red) or HC2 (green), and staining for nuclei with DAPI (Blue). Staining control provided in the last row, using secondary antibody only. Note HC-HA co-localization in the peri-broncho (Br)-vascular (V) interstitium (white arrow); scale bar 50 μm. levels returning to baseline by day 4 (Fig. 1d-e). To uninjured lungs, but was abundant in the determine the localization of HC-HA formation, lung peri-broncho-vascular interstitium following 1- and sections from PA-injured lungs were stained for HA 2 days post PA instillation (Fig. 1f). These results and HC. HC-modified HA determined by suggest rapid formation and clearance of HC-HA dur- co-localization of HA and HC staining was absent in ing respiratory infection-induced ALI. PA 2 dpi (2nd Ab only) PA 2 dpi PA 1 dpi PA 0 dpi HC relative abundance HC relative abundance (vs. total protein) (vs. total protein) msTNF mRNA (fold vs. PBS) msTSG-6 mRNA (fold vs. PBS) Ni et al. Respiratory Research (2018) 19:107 Page 7 of 17 TSG-6 production by lung resident cells presence of serum IαI-containing FBS (Fig. 2a-b). Both To determine which lung cells produce the TSG-6 that is LPS and TNFα, the standard and eponymous inducer required for forming HC-HA, we investigated TSG-6 pro- of TSG-6 production, induced TSG-6 secretion in duction by cultured lung macrophages, bronchoepithelial hAM (Fig. 2b) at levels consistent with the magnitude cells, and fibroblasts. Cells were stimulated with LPS since of mRNA induction (Fig. 2c). previous work identified it as potent stimulator of TSG-6 When we next compared different lung cell types to adi- RNA induction and secretion by myeloid cells [35, 36]. As pose stromal/progenitor cell, which are known to potently a first step, primary alveolar macrophage (hAM) and per- secrete TSG-6 in response to TNFα, we noticed signifi- ipheral blood mononuclear cell derived macrophage cantly different patterns of TSG-6 secretion in response to (hPBDM) were stimulated with vehicle or LPS, then TNFα or LPS (Fig. 2d). While hAM secreted TSG-6 more TSG-6 secretion and functionality was assessed in condi- robustly in response to LPS than TNFα, adipose stromal/ tioned media by western blot in the presence of serum progenitor cell had a potent response to TNFα, but similar containing IαI (the source of HC) or in the absence of levels of TSG-6 secretion in response to LPS as the hAM. serum (as a negative control). As anticipated, both hAM In contrast, lung fibroblast and bronchoepithelial cells and hPBDM secreted TSG-6 (35 kDa) only after stimula- only responded to TNFα, and bronchoepithelial cells only tion with LPS (Fig. 2a). Notably, secreted TSG-6 formed showed comparatively minor levels of TSG-6 secretion in covalent TSG-6-HC intermediates (130 kDa) only in the response to TNFα. hTSG-6 secretion hTSG-6 secretion hAM hPBDM hAM LPS -+-+ + veh TNFLPS kDa kDa TSG-6-HC1&2 TSG-6-HC1&2 TSG-6 37 TSG-6 2% 0% FBS hAM hTSG-6 *** * * 06 24 06 24 TNF (h) LPS (h) hTSG-6 secretion ** ** ** **** *** ** *** ** 0 0 hAM Wi38 hASC BEAS-2B Fig. 2 TSG-6 induction by TNFα or LPS stimulation of lung cells. a. Presence of TSG-6 in conditioned media of cultured human peripheral blood mononuclear cell-derived macrophages (hPBDM) and in human alveolar macrophages (hAM) following LPS stimulation (50 ng/mL, 24 h) detected by western blotting with TSG-6 antibody. Note that TSG-6 forms covalent TSG-6-HC intermediates only in the presence of 2% FBS (which contains serum inter-alpha-inhibitor that provides HC1 and HC2). b-c. TSG-6 secreted protein in supernatants (b; 2% FBS) and mRNA expression (c) of hAM stimulated with TNFα (20 ng/mL, 24 h or indicated time) or LPS (50 ng/mL, 24 h or indicated time), or vehicle (veh) assessed by western blot and qPCR, respectively. d. Levels of TSG-6 protein secreted in supernatant of hAM, human lung fibroblasts Wi38, human adipose stromal/progenitor cells (hASC), and human bronchoepithelial cells BEAS-2B stimulated with TNFα or LPS, measured by ELISA. In c-d, each data point represents an independent experiment; data analyzed with ANOVA and Tukey’s multiple comparisons. *P <0.05, **P < 0.01, ***P < 0.001, ****P <.0001 veh TNF LPS veh TNF LPS veh TNF LPS veh TNF LPS hTSG-6 hTSG-6 mRNA (ng/mL/10 cells) (fold vs. 0 h) Ni et al. Respiratory Research (2018) 19:107 Page 8 of 17 To validate that AM produce TSG-6 in vivo and to de- Assessment of HA remodeling during ALI termine the relative difference in TSG-6 induction in Since HC-modified HA was present during early inflam- bone-marrow-derived, recruited vs. resident AM, we mation, but absent at later time points (i.e. day 4), we evaluated our RNA-seq database of resident and re- sought to determine whether there was breakdown of high cruited alveolar macrophages isolated from bronchoalve- molecular weight (HMW) HA. Accordingly, the abun- olar lavage of LPS treated mice (previously published in dance of HA fragments of various molecular weights in ). We noted similar early induction of TSG-6 mRNA lung tissue were measured in LPS- or PBS exposed mice. that was highest at day 3 following LPS and noted gener- When compared to control conditions, shortly following ally a more robust induction in recruited- compared to LPS injury (days 1 and 2), the abundance of HMW HA resident AM (Additional file 4). fragments (1000–2500 kDa) decreased, while the - Hyaluronidase + Hyaluronidase LPS LPS PBS 1 dpi 2 dpi PBS 1 dpi 2 dpi 2500K 1510K 1000K 1090K 966K 572K 500K 495K 250K HA fragmentation PBS LPS 1 dpi LPS 2 dpi 1.0 0.8 0.6 0.4 0.2 0.0 2500 1000 500 250 Select HA Ladder (kDa) HMW HA (1000-2500 kDa) HA (250-500 kDa) 50 ** ** ** 0 0 PBS 1 dpi 2 dpi PBS 1 dpi 2 dpi LPS LPS Fig. 3 Effect of LPS on lung HA molecular weight distribution. a. Detection of HA by Stains-All staining of agarose gel-resolved glycosaminoglycans extracted from lung tissue of mice following intratracheal instillation of vehicle (PBS) control or LPS for the indicated time. Extracted samples were examined prior to (−) and following hyaluronidase treatment (+). Select-HA consisting of 2500, 1000, 500, and 250 kDa HA and Select-HA HiLadder consisting of 1510, 1090, 966, 572, and 495 kDa HA were used to determine HA molecular weight. b. Distribution of HA abundance (mean +/− SEM) by molecular weight size, determined by Select-HA: PBS (n =3), LPS 1 dpi (n =4), LPS 2 dpi (n =3). c. Levels of HA ranging from 1000 to 2500 kDa (HMW) or 250–500 kDa levels were determined by integrating the area of HA abundance over the specified molecular weight ranges for each individual mice. Each data point represents an individual mouse lung. n =3–4 mice per group. Data analyzed with ANOVA and Tukey’smultiple comparisons, *P < 0.05, **P < 0.01. HMW, high molecular weight; SEM, standard error of the mean; AU, arbitrary unit HA abundance HA abundance (AU) (AU) HA abundance (AU) Ni et al. Respiratory Research (2018) 19:107 Page 9 of 17 abundance of medium molecular weight HA fragments and 6 (Additional file 5: Figure B). Similar to findings in (250–500 kDa) increased (Fig. 3a-c). Of note, the increased whole lung, there was no significant difference between appearance of medium molecular weight HA fragments in TSG-6-KO and control littermates in HA levels in the whole lungs following LPS exposure was similar in bronchoalveolar lavage (Additional file 5:FigureB). TSG-6-KO mice compared to littermate heterozygous con- To further quantify and localize HA remodeling, we trol mice (Additional file 5: Figure A), suggesting that these adapted a previously described method , to detect HA fragments are generated independently of HC-modified immunofluorescence on paraformaldehyde-fixed frozen HA. In parallel with these changes in whole lungs, we mea- lung sections assessed with confocal microscopy. Com- sured the total HA content in bronchoalveolar lavage, using pared to the lamellar pattern of HA staining seen in the ELISA (which detects HA fragments of all sizes with a peri-broncho-vascular regions of PBS-exposed control minimum limit of detection between 6 KDa and 15 kDa mice, mouse lungs exposed to LPS exhibited a more ). These data showed increased HA levels at day 1 granular and rough HA staining (Fig. 4a,Additionalfile 6). following LPS which returned towards baseline at days 4 To quantify this change in the pattern of HA staining, HA CD68 DAPI HA Surface Plot Br z (AU) Br 9µm 9µm z (AU) Br Br 9µm 9µm HA CD68 DAPI Merge Br Br b c HA staining PBS 0.0005 0.0004 0.0003 0.0002 0 0.0001 0.0000 LPS 4 dpi PBS LPS 4 dpi 10 20 30 40 50 µm Fig. 4 Roughness of peribronchial HA following LPS. a. Identification of HA staining in peri-broncho-vascular interstitial areas bordered by blood vessels (V) and bronchi (Br) in paraformaldehyde fixed, frozen sections of lungs instilled with LPS (4 dpi) or control (PBS) and immunostained with HA-binding protein (green), antibody against CD68 (macrophage marker, white), and the nuclear stain DAPI (blue). A negative control for staining, using secondary antibody only is shown in the bottom row. Surface plots of the maximum intensity of staining (Z-projection) spanning 9.4 × 9.4 μm areas were generated, shown to the right, measured in arbitrary units (AU); Scale bar 50 μm. b. Representative line profile of HA staining in the max intensity Z-projection (50 × 50 μm). c. The normalized surface roughness of HA staining intensity was determined for both PBS and LPS (4 dpi) treated lungs by dividing the surface area of the intensity plots by the average staining intensity (n = 3 mice per group); ANOVA with Tukey’s multiple comparisons; *P < 0.05) LPS 4 dpi (2nd Ab only) LPS 4 dpi PBS HA Intensity (AU) Normalized surface roughness (R ) S Ni et al. Respiratory Research (2018) 19:107 Page 10 of 17 Lung msHAS1 Lung msHAS2 Lung msHAS3 8 2.5 *** ** 3 **** **** **** **** 2.0 1.5 1.0 0.5 0 0.0 PBS 1 dpi 4 dpi PBS 1 dpi 4 dpi PBS 1 dpi 4 dpi LPS LPS LPS b c Lung msHYAL1 Lung msHYAL2 Lung msTMEM2 Lung msCEMIP **** **** 1.5 *** 8 1.5 **** 1.5 *** **** **** **** ** **** 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0 0.0 0 PBS 1 dpi 4 dpi 6 dpi PBS 1 dpi 4 dpi PBS 1 dpi 4 dpi 6 dpi PBS 1 dpi 4 dpi LPS LPS LPS LPS 60 LPS 0dpi 3dpi 6dpi 9dpi 12 dpi p=0.16 p=0.061 Resident AM Recruited AM Resident AM Recruited AM Resident AM Recruited AM HYAL1 HYAL2 TMEM2 LPS ** * * 0dpi 3dpi 6dpi 9dpi 12 dpi Resident AM Recruited AM CD44 Fig. 5 Effect of LPS on genes implicated in HA synthesis and breakdown in whole lung and alveolar macrophage. a-c. Expression of HA synthases (HAS1–3, a), hyaluronidases (HYAL1–2, b), transmembrane protein 2 (TMEM2) and cell migration-inducing and HA-binding CEMIP (c) was assessed by qPCR in lungs of mice instilled intratracheally with vehicle (PBS) or LPS for the indicated duration. Each data point represents an individual mouse. d-e. Expression of genes implicated in HA breakdown HYAL1, HYAL2, TMEM2, and CD44 in resident and recruited mouse alveolar macrophages isolated from bronchoalveolar lavage of mice treated with LPS (20 μg intratracheal; 0, 3, 6, 9, and 12 dpi). Expression level was assessed by RNA-seq and shown as transcripts per million (TPM). Mean (n = 3 independently pooled samples per time point, 4–7 mice for each pool) and SD plotted. NA, not applicable; SD, standard deviation. Data analyzed with ANOVA and Tukey’s multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < .0001 which is thought to represent HA remodeling driven by exhibit RNA expression in the lung [39–41], TMEM2 HA fragmentation and synthesis, we constructed intensity (transmembrane protein 2) expression was also persistently surface plots of HA staining and calculated their surface downregulated; however, KIAA1199/CEMIP (cell migration roughness (Fig. 4b-c). HA staining from LPS-exposed lung inducing and hyaluronan binding protein) [40, 41]was sections had significantly greater surface roughness than significantly increased at 1 day following LPS exposure, control sections (Fig. 4c). returningtobaselinebyday4 (Fig. 5c). These results suggested HC-modification-independent Since alveolar macrophages can bind and degrade HA HA fragmentation during ALI. Since HA synthases and hy- , we additionally analyzed hyaluronidase expression aluronidases are major determinants of HA turnover, we in alveolar macrophage isolated from bronchoalveolar lav- measured their transcript abundance in lungs exposed to age using the RNA-seq database described above (Fig. 5d). LPS. All three HA synthases (HAS1–3) were upregulated in In contrast to the whole lung, we noted a stable expres- mouse lungs at 1 day following LPS exposure, returning to sion of msHYAL1–2 and early induction of msTMEM2 baselinebyday 4(Fig. 5a). In contrast, the expression levels that was highest at day 3 in both resident and recruited al- of key somatic tissue hyaluronidases (HYAL1 and HYAL2) veolar macrophages. Compared to msHYAL2 and were persistently downregulated in murine lungs following msTMEM2, msHYAL1 was expressed minimally and LPS injury (for up to 6 days after instillation) (Fig. 5b). Of msCEMIP was not detected (one transcript per million the two proteins recently implicated in HA breakdown that threshold cut-off). Since HYAL2’s ability to degrade HA msHYAL1 mRNA TPM msHAS1 mRNA (fold vs. PBS) TPM (fold vs. PBS) NA NA msHAS2 mRNA msHYAL2 mRNA (fold vs. PBS) (fold vs. PBS) NA msHAS3 mRNA (fold vs. PBS) msTMEM2 mRNA (fold vs. PBS) NA msCEMIP mRNA (fold vs. PBS) Ni et al. Respiratory Research (2018) 19:107 Page 11 of 17 TSG-6 mice + BALF CD45 total count a b p=0.013 het **** TSG-6 p=0.096 null WT 1500 HT -5 KO -10 -15 1 dpi PBS 4 dpi 6 dpi (LPS) LPS (dpi) + + cd BALF Ly6G total count BALF CD11b M total count 2000 TSG-6 **** **** WT **** HT KO p=0.13 p=0.29 p=0.34 50 PBS 1 dpi 4 dpi 6 dpi (LPS) PBS 1 dpi 4 dpi 6 dpi (LPS) + + BALF CD4 T cell total count BALF CD8 T cell total count ef **** **** 150 TSG-6 **** **** WT HT 100 KO ** p=0.82 PBS 1 dpi 4 dpi 6 dpi (LPS) PBS 1 dpi 4 dpi 6 dpi (LPS) g BALF albumin BALF RAGE 300 **** **** TSG-6 **** **** WT HT KO 0 0 PBS 1 dpi 4 dpi (LPS) PBS 1 dpi 4 dpi 6 dpi (LPS) ALI scoring 0.6 *** TSG-6 p=0.89 WT KO 0.4 0.2 PBS 4 dpi (LPS) Fig. 6 Effect of TSG-6 deficiency on severity and resolution of LPS induced ALI. a. Daily weight loss over six days following LPS instillation shown as % + + + + change from baseline (mean +/− SD). b-f. Total bronchoalveolar lavage counts of CD45 leukocytes, Ly6G neutrophils, CD11b macrophages, CD4 T-cells, and CD8 T-Cells determined by flow cytometry following PBS or LPS instillation for the indicated time. g-h. Levels of albumin and RAGE in bronchoalveolar lavage were determined by ELISA for mice treated with PBS or LPS. i. Lung injury scores were calculated from hematoxylin and eosin stained lung sections to assess the rate of ALI resolution in TSG-6 KO and WT mice treated with LPS. n =3–7 per group. ANOVA with Tukey’smultiple comparisons. **P < 0.01, ****P < .0001. SD, standard deviation depends on CD44 binding of HA at the cell surface Function of HC-HA formation during ALI [43, 44], we assessed CD44 expression and noted that HC-modified HA is critical for neutrophil sequestration in it was highly expressed and increased during reso- liver sinusoids during sepsis induced by systemic LPS expo- lution reaching peak expression on day 12 in both re- sures [6, 7]. We investigated the role of HC-HA formation cruited and resident macrophages (Fig. 5e). on outcomes of lung injury induced by direct lung Total cells (10 ) Histological score Total cells (10 ) Albumin (µg/mL) /mouse Weight loss (%) /mouse Total cells (10 ) Total cells (10 ) Total cells (10 ) /mouse RAGE (ng/mL) /mouse /mouse Ni et al. Respiratory Research (2018) 19:107 Page 12 of 17 instillation of LPS in TSG-6 sufficient and TSG-6 deficient evolutionarily conserved ability of TSG-6 to form mice. TSG-6-KO mice exhibited similar body weight loss HC-modified HA. In this context, alveolar macrophages and recovery following lung instillation of LPS (Fig. 6a). To may be an important source of secreted TSG-6 in the lung. assess the impact of TSG-6 on lung inflammation, we mea- Concomitant with HC-HA formation, we noted robust HA sured leukocyte abundance in the airway and airspaces fragmentation which was independent of TSG-6. Both using flow cytometry on cells harvested by bronchoalveolar HC-modified HA and airway HA levels subsided with the lavage. (Additional file 7). In response to lung instillation of resolution of lung injury. However, the TSG-6-dependent LPS, TSG-6-KO mice had similar levels of total CD45 leu- formation of HC-HA in this model did not have a major + + kocytes, neutrophils, CD11b macrophages, CD4 T cells, impact on the extent of lung inflammation and injury in and CD8 T cells in the bronchoalveolar lavage as their lit- this model. termate controls at almost all time points studied (Fig. Unlike other glycosaminoglycans (e.g. heparan sulfate, 6b-f). Compared to wild type or heterozygous mice, CD45 dermatan sulfate, chondroitin sulfate, and keratan sulfate), leukocytes were significantly or tended to be higher in HA’s dissacharide backbone exhibits the least diversity, TSG-6-KO mice only at day 4 following LPS. A similar because HA cannot be covalently modified by sulfation, trend was observed for neutrophils. To gauge the role of deacetylation, epimerization, and membrane-bound core endogenous TSG-6 on the severity of lung injury in this proteins. Instead, the evolutionarily conserved TSG-6 me- model, we measured albumin and RAGE in the bronchoal- diated modification of HA’s N-acetylglucosamine with veolar lavage as markers of endothelial and epithelial barrier serum IαI’s HC is the only known HA covalent modifica- integrity, respectively (Fig. 6g-h). Both markers were signifi- tion [5, 45]. Preclinical LPS-induced endotoxic shock cantly elevated at day 1 following LPS injury, followed by models of sepsis showed that HC-modified HA was re- return to baseline, to similar extent and kinetics in quired for neutrophil sequestration in the liver [6, 7]and TSG-6-KO and littermate control mice. To confirm these was overall protective for animal survival [8, 9], indicating findings, lungs at day 4 following LPS were formalin-fixed, that endogenous TSG-6 secretion is important for the paraffin-embedded, hematoxylin and eosin stained, and control of bacteria-induced inflammation and injury. scored for lung injury severity (Fig. 6i, Additional file 8) TSG-6-and IαI-KO mice not only had worse endotoxic using current guidelines . Lung injury scores were not shock outcomes [8, 9], but they also had markedly in- significantly different between TSG-6-KO and -WT mice. creased lung neutrophil infiltration [8, 46], suggesting a To extend our findings to gram negative bacterial in- protective role for TSG-6-mediated formation of HC-HA fection, total neutrophil counts and albumin in broncho- in the lung in controlling local neutrophil recruitment and alveolar lavage were similarly assessed following PA accumulation, and/or a major reduction in the pool of re- (Fig.7a-b). Compared to heterozygous littermates, lavage cruitable neutrophils to other organs due to liver seques- neutrophils and albumin were also significantly or tration and removal of circulating neutrophils . tended to be higher in TSG-6-KO mice. Together with Our data indicate a modest impact of TSG-6 on neutro- the data obtained using LPS, these results suggest a phil levels in the bronchoalveolar lavage following lung in- modest role for HC-HA formation in the onset, severity, stillation of LPS, suggesting that the lung production of or resolution of lung inflammation and injury during re- TSG-6 may not be a major determinant of acute inflam- spiratory infection-induced ALI. matory cell accumulation, at least in ALI induced by lung rather than systemic endotoxin exposure. It is possible Discussion that in this more localized injury model, the levels of sys- This study indicates that lung infections induce rapid temically absorbed LPS were insufficient to cause major covalent modification of HA, which was dependent on the formation of HC-modified HA in the liver with a + b BALF albumin BALF Ly6G total count TSG-6 **** **** **** 20000 **** p=0.049 p=0.086 HT KO 2 dpi PBS 4 dpi 6 dpi (PA) PBS 2 dpi 4 dpi (PA) Fig. 7 Effect of TSG-6 deficiency on severity and resolution of PA induced ALI. a. Total bronchoalveolar lavage counts of Ly6G neutrophils were determined by flow cytometry following PBS or LPS instillation at the indicated times. b. Level of albumin in bronchoalveolar lavage was determined by ELISA for mice treated with PBS or LPS. n =3–4 per group. ANOVA with Tukey’s multiple comparisons. ****P < .0001 Total cells (10 ) /mouse Albumin (µg/mL) Ni et al. Respiratory Research (2018) 19:107 Page 13 of 17 subsequent entrapment of neutrophils. In addition, since determinants of acute lung inflammation and injury follow- the HA expression in liver sinusoidal vasculature is 500- ing LPS instillation. The only significant impact of TSG-6 and 600-fold higher than in the lung vasculature both at deficiency in our study was that of a persistent increase in baseline and during endotoxemia , much higher levels total inflammatory cell counts in bronchoalveolar lavage, of TSG-6 than those locally produced in ALI may be re- with a trend of affecting particularly neutrophils during the quired to significantly modulate inflammation, including resolution of inflammation. The functional significance of neutrophil trafficking across lung tissue barriers. This this effect remains to be determined in chronic or irrevers- notion is further supported by the remarkable ible models of lung injury. Given the lack of differences in anti-inflammatory effects following treatment with ex- acute lung injury indices in TSG-6-KO mice, we did not ex- ogenous recombinant TSG-6 in several conditions, includ- plore the role of TSG-6 on monocyte and macrophage ing models of ALI [8, 47]. These protective effects of function. This area has received recent attention in endo- exogenous TSG-6 have been ascribed to its ability to bind toxic shock models of systemic sepsis, where TSG-6 has to and inhibit neutrophilic chemokines [48–50], and may been implicated in lung macrophage polarization [8, 60], at- also be linked to an effect on bone marrow myeloid pro- tributed to its modulatory effects on HA interactions with genitor cell function  and stromal cell differentiation its receptor CD44 on monocytes [61–63], or to a marked [51–53]. inflammatory milieu in TSG-6-KO mice that could also im- Our report is the first to use knockout mice to investigate pact macrophage functionality and programming . thespecific roleof TSG-6 andHC-modified HA during To our knowledge, this report is the first to characterize bacterial lung infection. We found that there are similar the kinetics of HC-HA formation and HA fragmentation trends toward greater neutrophilic inflammation during and remodeling during LPS and PA-induced reversible TSG-6 deficiency in both LPS and gram negative bacterial lung injury in mice. We noted HC-HA covalent interac- infection models of localized lung injury (Figs. 6a-b and 7). tions followed by rapid clearance of HC-modified HA dur- Since these trends were observed at time points of inflam- ing lung inflammation, suggesting a high HA turnover mation that directly followed (4 dpi, LPS) or coincided with during ALI, which resolves within 4 days after LPS intra- (2 dpi, PA) peak HC-HA levels, our data suggest that tracheal instillation. Ability to form HC-HA paralleled the TSG-6 formation of HC-modified HA has modest effect on increase in lung levels of TSG-6 and the availability of the abundance of inflammatory cells in the lung during serum IαI (the source for HC) in the lung interstitium, as acute infections. Considering the magnitude of the differ- measured by correlations with markers of endothelial per- ences in BAL neutrophils and albumin levels, our results meability (Additional file 9). The fact that the highest suggest that TSG-6 has a mild, protective role during acute HC-HA levels coincide with peak alveolar permeability lung inflammation. Given the well-established role of neu- (BAL albumin and RAGE levels, 1 dpi) supports the hy- trophils in antibacterial defense [54–56], future studies are pothesis that vascular leak of serum-derived HC substrate needed to carefully dissect whether induction of TSG-6 and into tissue is a critical step in the formation of HC-HA in HC-modified HA in the lung has a protective or deleterious lung tissue, as we have described before . Since TSG-6 role in eliminating the gram negative bacterial infection. was not required for the control of lung injury and barrier Additionally, the role of TSG-6 during gram negative bac- function, it is possible HC-HA formation is not required terial sepsis has not been investigated and remains unclear, for control of lung injury, but necessary for other pro- since the published reports on the role of TSG-6 and its co- cesses that were not investigated, such as airway epithelial valent modification of HA during sepsis have been per- cell survival and homeostasis [64, 65]. formed using systemic administration of LPS. Whereas HC-modified HA was not critical for the out- Our investigations expand on previous studies that impli- comes of ALI measured, future investigations will have to es- cated PBMCs as sources of TSG-6 in response to TNFα or tablish the functional role of HA fragmentation and LPS [35, 36], by showing that terminally differentiated remodeling in lung injury and repair. Unlike the accumula- hAM are more versatile producers of TSG-6 compared to tion and persistence of HC-modified HA observed in histo- bronchoepithelial cells or lung fibroblasts. Furthermore, pathological lesions of various chronic lung diseases, our comparative studies using adipose stem/progenitor cell HC-modified HA in our models of ALI did not accumulate suggestedthathAM maybequitepotent secretors of and was accompanied by markedly increased fragmentation TSG-6 during acute inflammation. Locally produced of high molecular weight HA and/or de novo production of TSG-6 may be essential for HC-HA formation in various medium molecular weight HA products. Our study design lung compartments during bacterial or other types of in- could not differentiate between these two processes, nor did flammation associated with high TNFα levels, a cytokine it carefully characterize the production of small molecular implicated in the pathogenesis of a variety of acute and weight HA. The latter, however, were included in the total chronic lung diseases in humans [57–59]. The local levels HA levels we measured in the bronchoalveolar lavage. These of TSG-6 produced in the lungs were not major results suggest a key role of hyaluronidases in clearing HA Ni et al. Respiratory Research (2018) 19:107 Page 14 of 17 to ensure resolution of acute lung inflammation, since hyal- 6 pro-protein (residues 1–47 depicted, hTSG-6 numbering) containing the uronidase deficiency is associated with failure to clear HA signal peptide (highlighted in gray) and start of the HA-binding Link domain (highlighted in green). The serine residue (highlighted in yellow) responsible and development of lung fibrosis . However, the tran- for removing HC from serum IaI and transferring HC onto HA is evolutionar- scription of both HYAL1 and HYAL2, which encode the ily conserved across all vertebrates including fish, reptile, and bird: Homo principal hyaluronidases in human and mice were de- sapiens (human), Mus musculus (mouse), Equus caballus (horse), Bos taurus (cattle), Pelodiscus sinensis (chinese softshell turtle), and Danio rerio creased in ALI lungs. Although the actual hyaluronidase ac- (zebrafish). CLUSTAL multiple sequence alignment by MUSCLE 3.8 (MUltiple tivity may diverge from the abundance of its mRNA , Sequence Comparison by Log-Expectation); “*” (asterisk) indicates fully our data indicate that reactive oxygen species or other hyal- conserved residue. “:” (colon) indicates residues with strongly similar properties (>0.5 in Gonnet PAM 250); “.” (period) indicates residues with uronidases may cause the marked HA fragmentation noted weakly similar properties (≤0.5 in Gonnet PAM 250). (DOCX 95 kb) during ALI. We focused on two proteins that impact HA Additional file 2: Human TSG-6 (hTSG-6) ELISA standard curves. hTSG-6 turnover whose genes are abundantly expressed in the lung: standard curves were obtained using recombinant hTSG-6 (R&D) in the absence TMEM2 and KIAA1199/CEMIP [39–41]. Of these, the ex- and presence of FBS. FBS reduced the magnitude of HRP substrate color change. Of note, TSG-6 readily forms covalent complex with HC (TSG-6-HC intermediate, pression of CEMIP, a HA-binding protein that promotes HA Fig. 2a-b) at a conserved serine residue (Additional file 1) in the presence of degradation via clathrin-mediated endocytosis inthe serum IαI source (e.g. FBS). Formation of the TSG-6-HC intermediate may whole lung paralleled the kinetics of HA fragmentation in sterically hinder the binding of TSG-6 by the capture and detection antibodies of TSG-6 sandwich ELISA. OD, optical density. (DOCX 43 kb) ALI. In turn, in alveolar macrophages, it was the expression Additional file 3: HC-modification of HA after LPS and PA injury. A. HC- of CD44 that increased during the resolution phase of ALI, modified HA following LPS injury at 1 and 4 dpi measured as in Fig. 1a. whichmayindicatearoleintheproper clearanceofHA 6 B. HC-modified HA following PA injury (2*10 CFU) at 2 and 4 dpi fragments. Future studies should investigate the relative con- measured as in Fig. 1b. HT denotes heterozygous TSG-6 control littermate. (DOCX 110 kb) tribution of enzymatic vs. non-enzymatic (i.e. reactive oxygen Additional file 4: TSG-6 expression in resident and recruited mouse species) regulation of HA turnover in ALI. alveolar macrophages. Resident and recruited mouse alveolar macrophages (msAM) were isolated from bronchoalveolar lavage of LPS treated mice (20 μg intratracheal; 0, 3, 6, 9, and 12 dpi). Expression level Conclusions of msTSG-6 was assessed by RNA-seq and shown as transcripts per million Our study indicates that both HC-HA formation and (TPM). Mean (n = 3 independently pooled samples per time point, 4–7 HA degradation are rapidly and transiently induced in mice for each pool) and error bar (SD) plotted. ND, not detected; NA, not applicable; SD, standard deviation. (DOCX 42 kb) models of gram-negative bacterial respiratory infec- Additional file 5: Effect of TSG-6 deficiency on lung HA molecular weight tions that cause resolving ALI. The rapid HA turn- distribution and lavage HA levels following LPS injury. A. HA was extracted over was associated with increased TSG-6 production, from lung tissue (LPS 1 dpi), separated by agarose gel, and visualized as with induction of HA synthases expression, and with described in Fig. 3. HT and KO denote TSG-6 heterozygous and knockout mice. Select-HA consisting of 2500, 1000, 500, and 250 kDa HA was used to increased HA degradation-promoting CEMIP expres- determine the molecular weight. B. HA levels in bronchoalveolar lavage sion. While alveolar macrophages are likely sources of were measured by ELISA. n =5–10 mice per group. (DOCX 87 kb) TSG-6 secretion following lung endotoxin exposure, Additional file 6: Effect of LPS on HA staining. A.Representativeimagesof the endogenous TSG-6-dependent HC-HA formation paraformaldehyde-fixed, frozen lung sections immunostained as described for Fig. 4 shown here in detail. Changes in HA staining can be seen in the peri- had a modest effect on reducing neutrophilic inflam- broncho-vascular interstitium (white arrow). B. Representative sections from matory cell abundance in the bronchoalveolar lavage independent animals (n = 3 mice per group). Br depicts a bronchial airway during the resolving phases of ALI. TSG-6 is dispens- and V pulmonary vessels. Scale bar 50 μm. (DOCX 821 kb) able for the inflammatory response to transient ALI Additional file 7: Schematic of flow strategy applied to lavaged cells from PBS and LPS treated mice. Bronchoalveolar lavage from mice at induced by lung infection, but its major role in form- 4 day post LPS instillation is depicted to illustrate all the cell populations ing HC-modified HA suggests that it may play a role assessed. A. Total leukocytes were identified by excluding debris and + + in non-resolving inflammatory lung conditions associ- doublets and using CD45 staining. T cells were identified by CD3 + + staining and differentiated by CD4 and CD8 staining. Neutrophils were ated with abnormal HA turnover. + − identified by Ly6G CD64 staining. Macrophages were identified by + + + low CD64 F4/80 and classified as recruited (CD11b CD11c ) or resident low + + (CD11b CD11c SiglecF ). B. Counting beads were identified by high Additional files SSC and low FSC and high fluorescence in FITC and PE. (DOCX 154 kb) Additional file 8: Histologic scoring of ALI lungs.A. Formalin-fixed, paraffin- Additional file 1: TSG-6 is conserved for catalyzing HC-modification of embedded mice lungs (4 day post LPS instillation) were stained with HA. A. Modifying HA with heavy chains (HC) of the serum protein inter hematoxylin and eosin and scored at high power fields (400X magnification). alpha inhibitor (IaI), also known as serum-derived HA-associated protein Neutrophils marked with blue arrowhead. B.Representativeimagesoflung (SHAP), is the only covalent modification HA can undergo. B.TNFα- sections from LPS injured TSG-6 KO and WT mice compared to PBS control stimulated gene-6 (TSG-6) is an inflammation-induced secreted protein that taken at lower power fields (100X and 200X). Scale bars: 25 μm (400X), 50 μm exclusively mediates formation of HC-modified HA. Through two transesteri- (200X), and 100 μm (100X). (DOCX 984 kb) fication reactions, TSG-6 transfers HC from IαI onto itself and then onto HA. To form the TSG-6-HC intermediate, HC is covalently linked to a conserved Additional file 9: Correlation between HC-HA and markers of alveolar serine residue adjacent to the TSG-6 Link domain that binds HA and permeability. Correlation plot of HC-HA abundance, measured in arbitrary units (AU) versus BAL fluid albumin (A)orRAGE(B) levels in individual mice facilitates HC transfer [1–5]. The serum protein IaI consists of a chondroitin sulfate that is covalently linked to the light chain bikunin and two heavy exposed to LPS for 1 or 4 days compared to PBS control with simple linear chains (HC1 and HC2) that can be removed by TSG-6. C. Alignment of TSG- regression line and coefficient of determination (R-squared). (DOCX 36 kb) Ni et al. Respiratory Research (2018) 19:107 Page 15 of 17 Abbreviations Authors’ information ALI: Acute lung injury; AM: Alveolar macrophage; ASC: Adipose stromal/ KN is MD/PhD (MSTP) predoctoral student at Indiana University School of progenitor cell; AU: Arbitrary unit; BALF: Bronchoalveolar lavage fluid; Medicine, performing his PhD training at National Jewish Health. BSA: Bovine serum albumin; C: Celsius; cDNA: Complementary DNA; CFU: Colony forming unit; DAPI: 4′,6-diamidino-2-phenylindole; Ethics approval and consent to participate DMEM: Dulbecco’s Modified Eagle Medium; DNAse: Deoxyribonuclease; All collection of human adipose tissue was approved by the Indiana dpi: Day(s) post-instillation; E. coli: Escherichia coli; EBM2: Endothelial growth University School of Medicine Institutional Review Board. basal medium; ECM: Extracellular matrix; EDTA: Ethylenediaminetetraacetic acid; EGM2-MV: Endothelial cell growth medium; ELISA: Enzyme-linked Competing interests immunosorbent assay; FBS: Fetal bovine serum; g: Gram; h: Hour; IP and KLM have patents applications related to therapeutic use of ASC. HA: Hyaluronic acid (hyaluronan); hAM: Human alveolar macrophages; HAS: HA synthase; HC: Heavy chain (IαI); HMW: High molecular weight; Publisher’sNote hPBDM: Human PBMC derived macrophage; HSPC: Hematopoietic stem and Springer Nature remains neutral with regard to jurisdictional claims in progenitor cell; HT: Heterozygous; hTSG-6: Human TSG-6; published maps and institutional affiliations. HYAL: Hyaluronidase; IACUC: Institutional Animal Care and Use Committee; IαI: Inter-alpha-inhibitor; kDa: kiloDalton; KO: Knockout; LB: Lysogeny broth; Author details LPS: Lipopolysaccharide; M: Molar concentration; mAM: mouse alveolar Department of Medicine, National Jewish Health, 1400 Jackson Street, Molly macrophage; MEM: Minimum essential media; mg: milligram; mL: milliliter; Blank Building, J203, Denver, CO 80206, USA. Department of Biochemistry mM: millimolar; mRNA: messenger RNA; MSC: Mesenchymal stem cell; and Molecular Biology, Indiana University School of Medicine, Indianapolis, msCEMIP: Mouse cell migration inducing hyaluronan binding protein; IN, USA. Department of Pediatrics, University of Colorado School of msTSG-6: Mouse TSG-6; NCBI: National Center for Biotechnology Information; Medicine, Aurora, CO, USA. Department of Medicine, University of Colorado ng: nanogram; OCT: optimal cutting temperature; PA: Pseudomonas School of Medicine, Aurora, CO, USA. Department of Pathology, University aeruginosa; PBDM: PBMC derived macrophage; PBMC: Pperipheral blood of Colorado School of Medicine, Aurora, CO, USA. Department of mononuclear cell; PBS: Phosphate buffered saline; PFA: Paraformaldehyde; Biomedical Research, National Jewish Health, Denver, CO, USA. Department pH: Potential of hydrogen; qPCR: Quantitative polymerase chain reaction; of Medicine, University of Florida College of Medicine, Gainesville, FL, USA. qPCR: Quantitative real-time polymerase chain reaction; RAGE: Receptor for National Institute of Environmental Health Services, Durham, NC, USA. advanced glycation end products; RNA: Ribonucleic acid; RNA-Seq: RNA- sequencing; RPMI: Roswell Park Memorial Institute; SD: Standard deviation; Received: 30 November 2017 Accepted: 14 May 2018 SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; siRNA: Small interfering RNA; TMEM2: Transmembrane protein 2; TNFAIP6: Tumor necrosis factor-inducible gene 6 protein; TNFα: Tumor References necrosis factor α; TRU: Turbidity reducing units; TSG-6: TNFα-stimulated 1. Sanggaard KW, Karring H, Valnickova Z, Thogersen IB, Enghild JJ. The TSG-6 gene-6; U: Unified atomic mass unit; WT: Wild type; ΔΔCt: Comparative CT; and I alpha I interaction promotes a transesterification cleaving the protein- μg: Microgram; μL: Microliter; μm: Micrometer glycosaminoglycan-protein (PGP) cross-link. J Biol Chem. 2005;280(12): 11936–42. 2. Sanggaard KW, Sonne-Schmidt CS, Jacobsen C, Thogersen IB, Valnickova Z, Acknowledgements Wisniewski HG, Enghild JJ. Evidence for a two-step mechanism involved in the The authors wish to thank the Cleveland Clinic Program of Excellence in formation of covalent HC x TSG-6 complexes. Biochemistry. 2006;45(24):7661–8. Glycoscience Resource Core (PO1HL107147) for providing useful protocols and 3. Sanggaard KW, Sonne-Schmidt CS, Krogager TP, Kristensen T, Wisniewski on-site training. The authors are grateful to Matthew J. Justice for assistance HG, Thogersen IB, Enghild JJ. TSG-6 transfers proteins between with PA infection experiments and to Sophie Gibbings and Kelly Corell for glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism. J providing alveolar macrophages. The authors wish to thank Alexandra L. Biol Chem. 2008;283(49):33919–26. McCubbrey and Lea Barthel for their assistance with antibody panel design. 4. Day AJ, de la Motte CA. Hyaluronan cross-linking: a protective mechanism We thank Lauryn Bennett for assistance with manuscript editing. in inflammation? Trends Immunol. 2005;26(12):637–43. 5. Sanggaard KW, Hansen L, Scavenius C, Wisniewski HG, Kristensen T, Thogersen IB, Enghild JJ. Evolutionary conservation of heavy chain protein Funding transfer between glycosaminoglycans. Biochim Biophys Acta. 2010;1804(4): This project was supported by 1R01HL105772-01A1 to IP and KLM, 1011–9. 2R01HL109517–06 to WJJ, 2 R01 HL086680–09 to ENG, and partly through the 6. McDonald B, Jenne CN, Zhuo L, Kimata K, Kubes P. Kupffer cells and Division of Intramural Research, NIEHS (ZIAES102605 to SG). KN was supported activation of endothelial TLR4 coordinate neutrophil adhesion within liver by T32HL091816–07 (IUSM Training program in Lung Diseases), sinusoids during endotoxemia. Am J Physiol Gastrointest Liver Physiol. 2013; 5T32GM077229–03 (IUSM MSTP), and 1F30HL136169-01A1 (NRSA to KN). 305(11):G797–806. 7. McDonald B, McAvoy EF, Lam F, Gill V, de la Motte C, Savani RC, Kubes P. Interaction of CD44 and hyaluronan is the dominant mechanism for Availability of data and materials neutrophil sequestration in inflamed liver sinusoids. J Exp Med. 2008;205(4): Data can be requested from KN and IP. 915–27. 8. Mittal M, Tiruppathi C, Nepal S, Zhao YY, Grzych D, Soni D, Prockop DJ, Authors’ contributions Malik AB. TNFalpha-stimulated gene-6 (TSG6) activates macrophage KN designed and carried out the experiments, collected and analyzed the data, phenotype transition to prevent inflammatory lung injury. Proc Natl Acad performed the statistical analysis, and wrote the manuscript. AG performed qPCR, Sci U S A. 2016;113(50):E8151–8. HC-modified HA, ELISA, and immunofluorescence experiments and analyzed data. 9. Wakahara K, Kobayashi H, Yagyu T, Matsuzaki H, Kondo T, Kurita N, Sekino H, VT and ENG carried out HA fragmentation and remodeling experiments and Inagaki K, Suzuki M, Kanayama N, et al. Bikunin suppresses analyzed and scored HA immunostaining. AMM and KAS carried out IF staining, lipopolysaccharide-induced lethality through down-regulation of tumor imaging, and analysis. KK, ELB, MJJ, FG assisted with or performed animal necrosis factor- alpha and interleukin-1 beta in macrophages. J Infect Dis. experiments and data analysis. CYX assisted with histological scoring. DC assisted 2005;191(6):930–8. with collection and processing of flow cytometry samples. KJM and WJJ provided 10. Shi J, Gilbert GE, Kokubo Y, Ohashi T. 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Published: May 31, 2018