sign in sign up
<<
Home , CCL13, CLEC5A, Lymphotoxin

Reprogramming of by GM-CSF Contributes to Regulatory Immune Functions during Intestinal Inflammation

This information is current as Jan Däbritz, Toni Weinhage, Georg Varga, Timo Wirth, of October 2, 2021. Karoline Walscheid, Anne Brockhausen, David Schwarzmaier, Markus Brückner, Matthias Ross, Dominik Bettenworth, Johannes Roth, Jan M. Ehrchen and Dirk Foell J Immunol published online 4 February 2015 http://www.jimmunol.org/content/early/2015/02/04/jimmun ol.1401482 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2015/02/04/jimmunol.140148

Material 2.DCSupplemental http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 4, 2015, doi:10.4049/jimmunol.1401482 The Journal of Immunology

Reprogramming of Monocytes by GM-CSF Contributes to Regulatory Immune Functions during Intestinal Inflammation

Jan Da¨britz,*,†,‡,x,1 Toni Weinhage,*,1 Georg Varga,* Timo Wirth,* Karoline Walscheid,* Anne Brockhausen,{,‖ David Schwarzmaier,* Markus Bruckner,€ # Matthias Ross,# Dominik Bettenworth,# Johannes Roth,†,‖ Jan M. Ehrchen,†,{ and Dirk Foell*,†

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF–activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to charac- terize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to Downloaded from increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental – induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-g in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn’s disease may possibly be mediated through reprogramming of monocytes to simulta- http://www.jimmunol.org/ neously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation. The Journal of Immunology, 2015, 194: 000–000.

ur concepts of immunology have changed dramatically traditionally seen solely as effector cells of innate immunity over the past decades. The postulates of primary func- promoting host defense and driving chronic inflammation. It is O tions assigned to innate or adaptive immunity have been now accepted that monocytes can differentiate into challenged by the recognition of a complex interplay between the with various activation patterns ranging from classically activated different cellular and humoral factors that all together constitute proinflammatory to anti-inflammatory phenotypes. These cells

our immune system. This helped in understanding how we are (often referred to as M1 and M2 macrophages) represent the outer by guest on October 2, 2021 protected from infections, but it also enabled discovering key margins of a broad spectrum of numerous activation and differ- aspects of autoimmunity and chronic inflammation including reg- entiation patterns of heterogeneous monocyte-derived cells (1–3). ulatory mechanisms that counteract a perpetuated immune acti- As the concepts of immunity evolve, the pathophysiology of vation. Although different functions of adaptive immune cells, chronic inflammatory diseases is also being revisited. As a striking including regulatory T cells (Tregs), are already consolidated, our example, our view of Crohn’s disease (CD) is constantly chal- understanding of different functions of innate immune cells has lenged. Traditionally, CD has been associated with a Th1 only recently been enriched. As an example, phagocytes were profile. In addition, because CD is a chronic granulomatous dis-

*Department of Pediatric Rheumatology and Immunology, University Children’s J.D. and D.F. developed the concept, designed the experiments, and supervised the Hospital Munster,€ Munster€ 48149, Germany; †Interdisciplinary Center of Clinical experiments; J.M.E., G.V., and J.R. gave technical support and conceptual advice; Research, University Hospital Munster,€ Munster€ 48149, Germany; ‡Gastrointestinal M.R., G.V., and J.D. obtained ethical approval from the competent animal welfare Research in Inflammation & Pathology, Murdoch Children’s Research Institute, authorities; J.D., T. Weinhage, T. Wirth, K.W., A.B., and D.S. performed the experi- The Royal Children’s Hospital Melbourne, Parkville 3052, Victoria, Australia; ments and collected data; M.R., G.V., M.B., D.B., and T. Wirth helped with animal xDepartment of Pediatrics, University of Melbourne, Melbourne Medical School, models of experimental colitis; J.D., T. Weinhage, G.V., and D.F. analyzed the data Parkville 3052, Victoria, Australia; {Department of Dermatology, University and interpreted results; J.D. wrote the manuscript; and each author has approved the Hospital Munster,€ Munster€ 48149, Germany; ‖Institute of Immunology, University final version of the report and takes full responsibility for the manuscript. Hospital Munster,€ Munster€ 48149, Germany; and #Department of Medicine B, € € The sequences presented in this article have been submitted to the Expres- University Hospital Munster, Munster 48149, Germany sion Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession number 1J.D. and T. Weinhage contributed equally and should be considered cofirst authors. GSE63662. Received for publication June 11, 2014. Accepted for publication January 4, 2015. Address correspondence and reprint requests to Dr. Jan Da¨britz, Department of Pediatric Rheumatology and Immunology, University Children’s Hospital Munster,€ Ro¨ntgen- This work was supported by the Broad Medical Research Program of the Eli and strasse 21, Munster€ 48149, Germany. E-mail address: [email protected] Edythe Broad Foundation (Grant IBD0201 to D.F., J.D., and J.M.E.), the German Research Foundation (Grant DFG DA1161/4-1 to J.D. and D.F., Grant DFG SU195/ The online version of this article contains supplemental material. 3-2 to G.V., Grant DFG SF1009B08 to M.B.), the Innovative Medical Research € ¨ ¨ € Abbreviations used in this article: CD, Crohn’s disease; DSS, dextran sulfate sodium; Program of the University of Munster (Grants IMF DA120904 and DA3U21003 to GCsM, glucocorticoid-stimulated monocyte; GMaM, GM-CSF–activated monocyte; J.D. and D.F.), the Interdisciplinary Center for Clinical Research of the University of € LPMC, lamina propria mononuclear cell; LTB4, leukotriene B4; MEICS, murine Munster (Grant IZKF Eh2/019/11 to J.M.E.), the European Union’s Seventh Frame- endoscopic score of colitis severity; MFI, mean fluorescence intensity; MLN, mes- work Programme (Grant EC-GA305266 ‘MIAMI’ to D.F.), and a research fellowship enteric lymph node; qRT-PCR, quantitative real-time RT-PCR; ROS, reactive oxygen from the German Research Foundation (Grant DFG DA1161/5-1 to J.D.). species; Treg, regulatory T cell. Portions of this work were presented at the 50th Digestive Disease Week Annual Meeting, May 30–June 4, 2009, Chicago, IL and the 51st Digestive Disease Week Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Annual Meeting, May 1–5, 2010, New Orleans, LA.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401482 2 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS ease and anti-inflammatory therapies targeting innate immunity before stimulation. Monocytes from at least three different individuals have proved effective, it was a paradigm that overactive phag- were assessed with each experiment. ocytes are involved. More recently, however, emerging evidence Patients has consolidated the view of CD as a form of innate immunode- ficiency (4–6). Central to this hypothesis were the observations of Clinical and demographic characteristics of the study subjects and methods have been reported in detail previously (25). Ethical approval was obtained diminished accumulation in patients with CD with from the Ethics Committee of the University of Munster€ (reference no. impaired clearance of bacteria from tissues (7, 8). The underlying 2006-267-f-S, obtained by Jan Da¨britz), and fully written informed consent problem appeared to be a primary immunodeficiency of macro- was obtained from all patients or legal guardians. phages, which secreted insufficient concentrations of proinflammatory DNA microarray hybridization and upon bacterial challenge (9). The view of defective functions in CD is further supported by Human monocytes were exposed to GM-CSF (10 ng/ml; MP Biomedicals, Santa Ana, CA) for 16 h or left untreated in three independent sets of an inappropriate mucosal healing (10, 11). Resolution of inflam- experiments to analyze changes in gene expression patterns induced by mation and healing relies on the infiltration of monocytes as GM-CSF. Using high-density microarrays with .22,000 oligonucleotide crucial regulators of tissue repair processes (12–14). sets, we obtained the expression levels of at least 13,000 independent Given the changing concepts on immunity and inflammation, transcripts. RNA preparation, sample preparation, and hybridization to changes in therapeutic strategies appear as a logical consequence. Affymetrix (Santa Clara, CA) 133 A Gene Chip arrays for microarray analysis were performed as described previously (26). As a therapy that could help in overcoming insufficient macrophage functions, GM-CSF has been shown to alleviate acute dextran sulfate Statistical analysis of microarray data sodium (DSS)-induced colitis in mice (15, 16). Even more important, For analysis of data from individual donors, raw data of GM-CSF–treated Downloaded from it is conceivable that GM-CSF–driven modulation of innate immune samples were processed by MicroArray Suite Software (Affymetrix) using cells involved in mucosal repair and/or dampening of inflammatory data from corresponding control samples as baseline. Signals were scaled reactions might contribute to the benefits of GM-CSF therapy ob- to a target intensity of 500 and log-transformed. Detection and change calls using perfect match and mismatching probes were assigned using a signed served in some CD patients (17). These findings may link the novel rank test as described previously (26–28). Data were submitted to the Gene concepts of monocyte biology with that of CD pathogenesis, because Expression Omnibus database under accession number GSE63662 (http:// recent developments in immunology and genetics suggest that www.ncbi.nlm.nih.gov/geo). We retained only that were significantly monocytes and their derivative cells play an important role in the regulated in every single experiment (change p , 0.05, fold-change $ 2.0, http://www.jimmunol.org/ pathophysiology of CD. It is noteworthy that blood monocytes are the expression over background). The data of the complete set of experiments were further studied applying the Expressionist Suite software package exclusive source of macrophages in inflamed intestinal mucosa (18). (GeneData), which allows identification of genes that are significantly reg- Undoubtedly, monocytes carry out specific effector functions ulated in multiple independent experiments as described previously (26). during inflammation (19). Recent studies underpin the dual Being aware of the low significance at low-intensity levels, we filtered for function of monocytes: on one hand, the impaired monocyte genes with an expression over background in at least one of the two ex- perimental groups (GMaM versus monocytes). We finally retained only function initiating CD, and on the other hand, the overactivation of genes that were significantly regulated in every single experiment (change monocytes and adaptive immunity maintaining the disease (20). p , 0.05, fold-change $ 2.0, expression over background), as well as in the Cells of the monocyte/macrophage lineage are characterized by complete set of experiments (expression over background, fold-change $ by guest on October 2, 2021 considerable diversity and plasticity (21). Furthermore, monocytes 2.0, p , 0.05, paired t test). Reproducibility of the results was confirmed can drive modulation of adaptive immunity by regulating T cell using RT-PCR for selected genes and three new independent experiments. responses (22). GM-CSF functions both as a for Quantitative real-time PCR myeloid progenitors and as a cytokine acting directly on maturing Expression of selected genes in human and mouse (C57BL/6) monocytes cells. Data from animal models indicate an important role in in- was analyzed by quantitative real-time RT-PCR (qRT-PCR) as described flammation and autoimmunity, with varying consequences that previously (29). PCRs were performed and measured on a CFX384 Touch likely depend on the disease-specific context (23). real-time PCR detection system (Bio-Rad, Munich, Germany). The relative We thus hypothesized that GM-CSF might activate monocytes expression was calculated using ribosomal protein L13a as endogenous housekeeping control gene. The primers used for PCR analysis are given in in a way that modulates their function during intestinal inflam- Supplemental Table III. mation. To this end, we chose an unbiased but comprehensive approach taking all potential functions of GM-CSF–activated Flow cytometry monocytes (GMaMs) into account (gene expression, innate im- FACS measurements were performed using a Cyflow space equipped with mune functions, interplay with adaptive immunity, wound heal- FlowMax 2.8 (both Partec, Munster,€ Germany), and analysis was per- ing) rather than focusing on polarizing edges. We show in this formed using FlowJo software (TreeStar, Ashland, OR). Ab staining of cells was routinely done with 1 mg/ml of the according Ab. For detection article that beneficial effects of GM-CSF in CD could be explained of cell-surface molecules, flow cytometry was performed as described by a complex reprogramming of altered monocyte/macrophage earlier (26). All intracellular stains were performed using the transcription functions. These findings suggest the exploration of stimulating, factor staining buffer set (eBioscience, San Diego, CA). mAbs used are rather than suppressive, therapies with the potential to more spe- given in Supplemental Table IV. cifically reprogram monocytes to modulate immune functions. production of GMaMs Chemokine concentrations of CCL18 and CCL23 were determined in Materials and Methods cell culture supernatants of monocytes treated for 24 h with GM-CSF Human monocytes (10 ng/ml) or untreated control cells by an ELISA system according to the manufacturer’s instructions (CCL18; Sigma-Aldrich, Steinheim, Blood samples from individual healthy donors were purchased from the Germany; CCL23; Raybiotech, Norcross, GA). Department of Transfusion Medicine at the University Hospital Munster,€ Munster,€ Germany. Peripheral blood monocytes were obtained from Monocyte/macrophage polarization donors by leukapheresis and isolated to .90% purity as previously de- scribed (24). Monocytes were cultured (1 3 106 cells/ml) in hydrophobic Human monocytes were stimulated for 4 and 16 h with IL-4 (100 mg/ml), Teflon bags (Heraeus, Hanau, Germany) in McCoy’s 5a medium supple- IFN-g (100 mg/ml), or left untreated. Alternatively, human monocytes mented with 5% human AB serum, 2 mM L-glutamine, 200 IU/ml peni- were polarized with GM-CSF (10 ng/ml) 6 IFN-g (100 mg/ml) or left cillin, 100 mg/ml streptomycin, and 13 nonessential amino acids (all from untreated as a negative control. Expression of selected genes was analyzed Biochrom, Berlin, Germany). Monocytes were allowed to rest for 16 h by qRT-PCR as described earlier. The Journal of Immunology 3

In additional experiments, human monocytes were polarized for 24 h analyzed by measuring the scratch area relative to the initial wound area with IFN-g (M1; 50 ng/ml) or IL-4 (M2; 50 ng/ml). After polarization, after each time point. monocytes were stimulated with GM-CSF (10 ng/ml) or left untreated for an additional 24 h. IL-1b, TNF-a, IL-10, and CD206 expression were Influence of human GMaMs on T cell fate measured by flow cytometry as described earlier. 6 Human T cells were purified from donor-specific PBMCs using positive Finally, human monocytes were stimulated GM-CSF (10 ng/ml) for 0, selection of CD2-expressing T cells by MACS technology according to the a b g 30, 60, and 120 min, and expression of IFN- , IFN- , IFN- , and IL-4 was manufacturer’s protocol (Miltenyi Biotec, Bergisch-Gladbach, Germany). analyzed by qRT-PCR as described earlier. A total of 1 3 106 T cells were cocultured with 1 3 105 monocytes (Mo) Migration, chemotaxis, monocyte trafficking, and adherence for 7 d (ratio T/Mo = 10:1). Cells were harvested and stained using mAbs raised against CD4, CD25, and Foxp3 (Supplemental Table IV). Subse- Monocyte assays in Transwell plates (Costar, New York, NY) were per- quent flow cytometry was performed as described earlier. formed as described previously using MCP-1 (10 ng/ml; Immunotools, Friesoythe, Germany), IL-8 (25 ng/ml; Immunotools), and leukotriene B4 Mice (LTB4; 100 nM; Biozol, Eching, Germany) (30). Cells were allowed to Experiments were performed in accordance with approved protocols of migrate for 4 h. T cell migration was analyzed using the Cultrex 96-well the animal welfare committee of the North Rhine-Westphalia State Agency cell migration assay according to the manufacturer’s protocol (Trevigen, for Nature, Environment and Consumer Protection, Recklinghausen, Gaithersburg, MD). T cells were isolated from fresh PBMCs using an Germany (LANUV NRW Reference No. 87-51.04.2010.A113). C57BL/6 EasySep human T cell enrichment kit according to the manufacturer’s and Rag12/2 mice were kept under specific pathogen-free conditions and protocol (STEMCELL Technologies, Vancouver, BC, Canada). T cells (5 3 4 according to federal regulations. Mice were purchased from Harlan (Paris, 10 ) were added to top-chamber and cell culture supernatants of untreated France) and used for experiments at the age of 10–12 wk. monocytes or GMaMs as well as CCL18 (1 ng/ml), CCL23 (8 ng/ml; both PeproTech, Rocky Hill, NJ), and medium (control) were added to the bottom Murine monocytes

chamber. T cells that had migrated into the lower compartment within 4 h Downloaded from were measured using an Infinite M200 Pro reader (TECAN, Crailsheim, Freshly isolated monocytic bone marrow cells were prepared as described Germany). earlier (32). Cells were cultured for 48 h with 150 U/ml GM-CSF Expression of intestinal-associated homing molecules in human mono- (Immunotools) or left untreated as control in 20% L929 cell supernatant cytes treated 6 GM-CSF (10 ng/ml) for 24 h was analyzed by flow cytom- (containing M-CSF) conditioned DMEM supplemented with 2 mM glu- etry (gated on CD14+ cells) as described earlier. tamine, 0.1 mM nonessential amino acids (all Invitrogen, Karlsruhe, For determination of cell adhesion, monocytes (2 3 105) were stimulated Germany), 100 mg/ml penicillin/streptomycin, and 10% heat-inactivated with GM-CSF (10 ng/ml) for 24 h or left untreated. Monocytes were FCS (both Biochrom, Berlin, Germany). After culture, cells were washed seeded in triplicates into 96-well flat-bottom plastic tissue-culture plates three times and subsequently used for analyses and coculture experiments. http://www.jimmunol.org/ and incubated at 37˚C and 7% CO2 for 4 h. Nonadhering cells were re- moved by washing twice; remaining adherent cells were fixed with 2% T cell transfer colitis glutaraldehyde (Sigma-Aldrich, Taufkirchen, Germany) for 10 min. Wells 6 + 2 To induce colitis, we adoptively transferred 1 3 10 syngeneic CD4 CD25 were washed two times with H O and subsequently stained with 0.5% 2/2 2 T cells i.v. into Rag1 mice (on C57BL/6 background). Body weight of crystal violet (Merck, Darmstadt, Germany) in 2% EtOH (pH 6.0) for an animals was monitored daily, and around day 40 animals that established additional 15 min at room temperature. Finally, wells were washed three colitis by weight loss on consecutive days received GMaMs or untreated times and cells were lysed. Ten percent acetic acid was added, and staining monocytes (2 3 106 per mouse) i.v. Alternatively, 5 mgGM-CSF(Immu- was quantified measuring the OD at 560 nm using an Asys Expert 96 notools) diluted in PBS or PBS alone was administered i.p. on a daily basis. Microplate ELISA reader (Anthos Mikrosysteme, Krefeld, Germany) (25). Body weight of mice was monitored for an additional 12 d. Finally, mice GM-CSF priming were euthanized by CO2 inhalation, and their colons were prepared, mea- by guest on October 2, 2021 sured, and preserved for histology. For priming experiments, human monocytes were stimulated for 24 h with GM-CSF (10 ng/ml) or left untreated. After pretreatment, monocytes were Isolation of murine T cells from spleen stimulated with medium containing LPS (10 ng/ml) or left untreated for an additional 4 h. TNF-a and IL-1b content were measured in culture T cells were isolated from spleens as described previously (33). T cells used for induction of transfer colitis were further purified for CD4+ and depleted supernatants by ELISA (OptEIA ELISA kits; BD Pharmingen, Heidelberg, of CD25+ cells by MACS technology according to the manufacturer’s Germany). Expression of selected genes was confirmed by qRT-PCR as described earlier. instructions (Miltenyi Biotech). Phagocytosis and oxidative burst Histopathologic analysis For detection of phagocytic capacity, cells were incubated with carboxy- For histopathologic analysis, tissue specimens from the proximal and distal fluorescein diacetate (Invitrogen, Karlsruhe, Germany)–labeled Leishmania colon were fixed in 10% buffered formalin phosphate and embedded in paraffin. Sections were cut at 3–5 mm and stained with H&E. Inflammation major parasites (ratio cells: L. major = 1:5) or FITC (MoBiTec, Go¨ttingen, was graded from 0 to 4 in a blinded fashion: 0, no signs of inflammation; 1, Germany)-labeled latex beads (ratio cells: beads = 1:10) for 4 h (31). Rate of phagocytosis was determined by flow cytometry as described previously low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte (30). Cells were incubated with or without PMA (50 nM; Sigma-Aldrich, infiltration, moderate fibrosis, high vascular density, thickening of the colon wall, Taufkirchen, Germany) in addition to 10 ng/ml GM-CSF to investigate the moderate goblet cell loss, and focal loss of crypts; and 4, transmural infiltrations, induction of oxidative burst. The extracellular chemiluminescence re- massive loss of goblet cell, extensive fibrosis, and diffuse loss of crypts. sponse was measured in the presence of isoluminol (50 mM; Sigma- High-resolution colonoscopy Aldrich, Taufkirchen, Germany) as described previously (24). Mice were anesthetized with isoflurane (100% v/v, 1.5 vol %, 1.5 L/min; In vitro scratch closure assay Florene; Abbott, Wiesbaden, Germany) and administered an enema (Freka- Clyss; Fresenius Kabi, Se`vres, France). High-resolution colonoscopy was Cells of the Caco-2 human colon adenocarcinoma cell line (ATCC HTB-37) performed using a veterinary endoscopy workstation (Coloview; Karl were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), Storz, Tuttlingen, Germany) to assess colitis. Under visual control, the streptomycin (100 mg/ml), 15 mM HEPES (pH 7.4), 2 mM L-glutamine, and rigid miniature endoscope (1.9-mm outer diameter) was inserted ∼4cm 1% nonessential amino acids at 37˚C and 5% CO in a humidified incubator. 2 according to anatomic conditions. The modified murine endoscopic score For the scratch closure assay, cells were grown to confluence in 12-well of colitis severity (MEICS) observes thickening of the colon, changing of plates and serum deprived (0.1% FBS) for 24 h before scratch wounding. vascular pattern, presence of fibrin, granular mucosa surface, and stool Monolayers were scratched using a sterile pipette tip and washed twice. consistence (0–3 points each, maximum of 15 points); it was used to Thereafter the wounded monolayers were cultured in fresh serum-deprived evaluate colonic inflammation (34). medium in the presence or absence of 2.5 3 105 untreated monocytes or GMaMs. The initial wound size was determined by microscopy, and the In vivo cell tracking area of the scratch was calculated with ImageJ software (Version 1.45s; National Institutes of Health). Additional photographs were taken using For in vivo cell tracking of GMaMs, cells were stained with a commercially avail- a reference line 24 h after wounding, and the rate of wound closure was able lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine 4 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS iodide (Life Technologies, Darmstadt, Germany) with an emission maximum (especially chemokines), cell motility, chemotaxis, regulation of of 782 nm as described elsewhere (35). In vivo distribution of labeled cells cell growth, endocytosis, and Ag processing and presentation across the intestine was studied 24 h after i.v. injection using a planar small- animal FMT system (FMT 2500; VisEn Medical) as described previously (35). (Table I). Furthermore, we analyzed statistical overrepresentation of transcription factor binding sites in GM-CSF–regulated genes Isolation of lamina propria mononuclear cells and cells from using CARRIE software (Supplemental Table I). Overall, we mesenteric lymph nodes found that in human monocytes, 190 genes were significantly Lamina propria mononuclear cells (LPMCs) were isolated from the colon upregulated, whereas 212 were downregulated after 16-h stimu- of colitogenic mice by a standard method (36). In brief, the colon was lation with GM-CSF (Supplemental Table II). Raw data have been removed, opened longitudinally and cut into 5-mm pieces, and washed submitted to the Gene Expression Omnibus under accession with cold Ca2+/Mg2+-free HBSS. The intestinal tissue specimens were transferred into HBSS with EDTA to remove intraepithelial . number GSE63662 (http://www.ncbi.nlm.nih.gov/geo). We con- After 30 min of gentle shaking at 37˚C, the samples were vortexed and firmed microarray expression data by qRT-PCR for selected genes intraepithelial –containing supernatant was removed. This step (Fig. 1A). Analyses of expression levels at different time points of was repeated twice. LPMC suspensions were prepared from the EDTA- GM-CSFactivationconfirmedthatgeneregulationinGMaM treated de-epithelialized intestinal tissue by further incubation with 100 U/ml collagenase and 5 U/ml DNase for 30 min at 37˚C. LPMCs were washed, is most relevant after 16–24 h (Fig. 1B). However, we also resuspended in 44% Percoll solution (Amersham Pharmacia Biotech, Pis- show that the expression of most of these genes is already cataway, NJ), underlaid with 66% Percoll solution, and centrifuged for significantly regulated after 4 h of GM-CSF stimulation. Genes 3 g 30 min at 600 . The LPMC fraction was harvested from the interface. that were not significantly regulated after 24 h of GM-CSF acti- For cell isolation from mesenteric lymph nodes (MLNs), the lymph nodes of treated mice and control mice were carefully isolated, pooled, and vation also showed no significant upregulation or downregulation of passed through a 40-mm cell strainer, and the resulting single-cell sus- their expression after 4, 16, 48, or 120 h (Fig. 1B). In agreement Downloaded from pension was washed once with PBS. with the microarray data, flow cytometry confirmed upregulation Coculture of naive T cells and restimulation of LPMCs and of CD80 and downregulation of CD9 upon GM-CSF activation MLNs (Fig. 1C, 1D). GM-CSF drives monocytes toward M2-like phenotype. Interest- Naive T cells were isolated from splenocytes using a pan T cell kit II (Miltenyi Biotec) as described previously (37), and 1 3 105 T cells were ingly, the microarray expression data of GMaM chemokine ligands cocultured for 4 d (ratio 10:1) with respective monocytes in triplicates and receptors showed a gene expression pattern indicative of a GM- http://www.jimmunol.org/ for each condition in anti-CD3e and anti-CD28 Abs (5 mg/ml each; Sup- CSF–induced shift toward a M2-like phenotype (Fig. 2A). This was plemental Table IV) coated to 96-well, round-bottom plates in RPMI 1640 confirmed by protein quantification (ELISA) for selected chemo- supplemented with 2 mM glutamine, 0.1 mM nonessential amino acids (all Invitrogen, Karlsruhe, Germany), 100 mg/ml penicillin/streptomycin, kines (Fig. 2B) and by gene expression analyses (qRT-PCR) for and 10% heat-inactivated FCS (both Biochrom, Berlin, Germany) at 37˚C differentially expressed genes that were assigned to M1- and M2- and 5% CO2. To further test for cytokine production, we stimulated iso- like monocytes based on currently accepted annotations (Table II) 5 lated LPMCs and MLN cells (2 3 10 per well) from mice used in transfer (2, 38, 39). colitis experiments with anti-CD3e and anti-CD28 Abs (5 mg/ml each; Supplemental Table IV) coated to 96-well, round-bottom plates for 24 Gene expression in GMaM is similar to IL-4–induced gene (LPMCs) or 96 h (MLNs). A total of 100 ml cell supernatants was stored expression profiles. Our gene expression analyses (Fig. 1B) sug- by guest on October 2, 2021 until cytokine analysis was performed using a bead-based multiplex assay gest that the observed gene expression in GMaM is similar to (mouse Th1/Th2 10plex FlowCytomix; eBioscience), according to man- IL-4–induced gene expression profiles in monocytes. We stimu- ufacturer’s instructions. lated human monocytes from healthy donors with or without GM- Induction of Tregs by murine GMaMs CSF for 0, 30, 60, and 120 min and analyzed gene expression of IFN-a, IFN-b, IFN-g, and IL-4 by qRT-PCR. The expression of LPMC and MLN single-cell suspensions from mice used in transfer coli- tis experiments were stained with Abs raised against CD4 and Foxp3 IFNs and IL-4 in monocytes was not upregulated at any time after (Supplemental Table IV). Cells were measured by flow cytometry as de- GM-CSF stimulation when compared with untreated monocytes scribed earlier. (data not shown). Interestingly, we found that the GM-CSF–in- T cell coculture experiments were performed to evaluate the function of duced gene expression pattern in human monocytes within the GMaM to induce Tregs. Therefore, 1 3 105 splenic T cells (described earlier) were cocultured with 1 3 104 monocytes (GM-CSF–activated or first 24 h is similar to the gene expression pattern of IL-4– but not untreated) in 96-well, round-bottom plates for 7 d in RPMI 1640 supple- IFN-g–stimulated monocytes (Fig. 2C, 2D). We assume that our mented with 2 mM glutamine, 0.1 mM nonessential amino acids (all transcriptomic data of human GMaMs rather reflect a primary Invitrogen, Karlsruhe, Germany), 100 mg/ml penicillin/streptomycin, and effect, which, however, has similarities with the expression pat- 10% heat-inactivated FCS (both Biochrom, Berlin, Germany) at 37˚C and tern of IL-4–induced M2a macrophages. 5% CO2. Cells were harvested and stained with 1 mg anti-CD4 and anti- Foxp3. Used Ab clones are given in Supplemental Table IV. Flow It has been shown that endogenous type I IFN regulates the basal cytometry was performed as described earlier. gene expression of bone marrow–derived macrophages grown in GM-CSF (40). Fifty GM-CSF–specific type I IFN–dependent Statistics regulated genes were identified, of which we found only five to Data are expressed as mean 6 SEM unless stated otherwise and were be significantly regulated in GMaMs (IFIT3, CBX6, ISG20, assessed using the Student t test. The p values ,0.05 were considered to be ABCA9, COLEC12; Supplemental Table II). In human cells, it statistically significant. All calculations were performed using SPSS ver- was found that the expression of 154 type I IFN–regulated genes sion 14 (SPSS, Chicago, IL). were also different between monocyte-derived macrophages cultured in GM-CSF (GM-MDM) or M-CSF (MDM) (41). We Results found that only 7 of the top 50 type I IFN–dependent genes Gene expression and phenotype shift of GMaMs differentially expressed between human GM-MDM and MDM GM-CSF provokes nonclassical monocyte activation. To analyze were significantly regulated in GMaMs (EDNRB, LGMN, monocyte activation comprehensively and unbiased, we performed P2RY14, CH25H, CDKN1C, GGTLA1, CD69; Supplemental a global RNA expression analysis. The microarray data were fil- Table II). In addition, we found no significant GM-CSF–de- tered using strict statistical criteria and revealed a significant pendent gene regulation of type I IFNs in GMaMs. Likewise, regulation of genes involved in immune/inflammatory responses the gene expression of IFN-g in human monocytes was not The Journal of Immunology 5

Table I. Selected genes upregulated and downregulated by GM-CSF activation

Gene Description (NCBI Gene) n-Fold p

Upregulated by GM-CSF Activation Inflammatory response GGTLA1 g-Glutamyltransferase-like activity 1 50.5 ,0.001 CFH Complement factor H 44.9 0.013 CD80 CD80 molecule 8.4 ,0.001 PROCR Protein C receptor, endothelial (EPCR) 7.5 ,0.001 CD69 CD69 molecule 6.4 0.002 BANK1 B-cell scaffold protein with ankyrin repeats 1 5.2 0.011 SLAMF1 Signaling lymphocytic activation molecule 5.0 0.041 IL7R IL 7 receptor 4.8 0.001 CLEC5A C-type lectin domain family 5, member A 4.5 0.011 IL1RAP IL 1 receptor accessory protein 4.4 0.008 ALOX5AP Arachidonate 5-lipoxygenase-activating protein 4.1 0.003 LTB b (TNF superfamily, member 3) 3.6 0.002 PTGS1 PG-endoperoxide synthase 1 3.3 0.003 Chemotaxis CCL13 Chemokine (C-C motif) ligand 13 16.7 ,0.001 CCL23 Chemokine (C-C motif) ligand 23 8.3 0.002 PPBP Proplatelet basic protein (CXCL7) 7.2 0.001 Downloaded from CXCL5 Chemokine (C-X-C motif) ligand 5 5.8 0.025 CCL17 Chemokine (C-C motif) ligand 17 5.5 0.003 IL8RB IL 8 receptor, b 5.1 0.002 CCR6 Chemokine (C-C motif) receptor 6 4.3 ,0.001 SPN Sialophorin (leukosialin, CD43) 3.8 0.007 Ag processing and presentation CD1C CD1c molecule 18.4 ,0.001 http://www.jimmunol.org/ CD1B CD1b molecule 15.5 0.008 CD1E CD1e molecule 9.8 ,0.001 CD1A CD1a molecule 6.4 ,0.001 Regulation of cell growth TGFB2 Transforming growth factor, b2 26.8 0.006 FGF13 Fibroblast growth factor 13 19.8 0.018 CISH Cytokine inducible SH2-containing protein 5.7 ,0.001

Downregulated by GM-CSF Activation Immune response FCGR1B Fc fragment of IgG, high affinity Ib, receptor (CD64) 29.0 0.011 by guest on October 2, 2021 AQP9 Aquaporin 9 25.3 0.004 IFIT1 IFN-induced protein with tetratricopeptide repeats 1 25.0 0.037 GBP5 Guanylate binding protein 5 25.0 0.009 MX1 Myxovirus (influenza virus) resistance 1 24.2 0.023 CD28 CD28 molecule 24.2 0.008 OAS1 2’,59-oligoadenylate synthetase 1, 40/46kDa 24.0 0.049 OAS2 2’-59-oligoadenylate synthetase 2, 69/71kDa 23.7 0.043 HPSE Heparanase 23.6 0.003 GBP2 Guanylate binding protein 2, IFN-inducible 23.5 0.002 MGLL Monoglyceride lipase 23.4 ,0.001 Chemokines CXCL12 Chemokine (C-X-C motif) ligand 12 216.9 0.009 CXCL11 Chemokine (C-X-C motif) ligand 11 27.1 0.046 CXCL13 Chemokine (C-X-C motif) ligand 13 26.4 0.019 CXCR4 Chemokine (C-X-C motif) receptor 4 26.2 0.001 CXCL10 Chemokine (C-X-C motif) ligand 10 23.5 0.007 Phagocytosis/Endocytosis FCGR1A Fc fragment of IgG, high affinity Ia, receptor (CD64) 27.7 0.022 MSR1 Macrophage scavenger receptor 1 24.7 0.041 Other CADM1 Cell adhesion molecule 1 28.5 0.001 CD9 CD9 molecule 26.4 0.012 IGF1 Insulin-like growth factor 1 (somatomedin C) 25.0 ,0.001 ITGB8 Integrin, b 8 25.0 0.006 NID1 Nidogen 1 24.7 ,0.001 KITLG KIT ligand 23.1 0.002 LEP Leptin (obesity homolog, mouse) 29.1 ,0.001 CDKN1C Cyclin-dependent kinase inhibitor 1C (p57, Kip2) 23.5 0.041 STAT1 Signal transducer and activator of transcription 1 23.2 0.034 significantlyregulatedbyGM-CSFafter16hnorafter24h activation of NF-kB, which promotes M1 macrophage polari- (Table I, II). In addition, GM-CSF induced neither IRF4 (M2 zation, was significantly downregulated by GM-CSF stimula- polarization) nor IRF5 (M1 polarization) in human monocytes tion, whereas the activity of C/EBPb, which is crucial for ex- when activated with GM-CSF for 24 h (Table II). However, pression of M2-regulated genes, was significantly upregulated 6 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS Downloaded from http://www.jimmunol.org/

FIGURE 1. Confirmation of GM-CSF–regulated gene expression in monocytes (microarray data) by real-time PCR and flow cytometry. (A) Results obtained from microarray analysis of GM-CSF–dependent gene regulation in human monocytes (after 16 h) compared with unstimulated monocytes were confirmed by qRT-PCR. Genes analyzed were: lymphotoxin b (LTB); complement factor H (CFH); g-glutamyltransferase-like activity 1 (GGTLA); the chemokines CXCL10, CXCL12, CCL23, and CCL13; and the CD1c and CD80 molecule. Shown are the mean relative n-fold regulation (6 SEM) of three independent by guest on October 2, 2021 experiments. (B) GM-CSF–dependent gene expression analysis by qRT-PCR of selected genes at different time points. Shown is the mean relative n-fold regulation (6 SEM) of three independent experiments. (C and D) Expression of selected cell-surface molecules (C, CD80; D, CD9) found to be differentially expressed in GMaMs by microarray analysis, confirmed by flow cytometry. Specific profiles are shown by thick lines; isotype controls appear as thin lines. Numbers show the quotient of specific/isotype control MFI. The experiment was repeated three times with similar results, and the differences in MFI shifts between control and GMaMs were in accordance with microarray data. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes. by GM-CSF stimulation in the first 24 h (Supplemental Table I). Comprehensive characterization of GMaM innate immune In addition, further quantitative PCR analysis suggests that functions IFN-g does not contribute to our transcriptomic data provided for GM-CSF promotes adherence and migration of monocytes. Major GMaMs. The gene expression of chemokines (CXCL10, CXCL11, functions of monocytes include the capacity to adhere and migrate, CCL1, CCL13, CCL23, CXCL5), CD206, CD209, and IL-1b in which is crucial for their recruitment into tissue. Adherence of human monocytes was specifically and significantly upregulated or GMaMs to plastic surfaces was enhanced after 24 and 48 h compared downregulated by either GM-CSF or IFN-g after 24 h of stimula- with untreated control cells (Fig. 3A). We tested whether GM-CSF tion (data not shown). activation would affect migration and chemotaxis of monocytes in Finally, we analyzed whether GM-CSF is affecting distinct general and also specifically in response to MCP1/CCL2, IL-8/ monocyte subsets within the overall monocyte population differ- CXCL8, and LTB4. By using a modified Boyden chamber assay, entially. Our results showed a homogenous shift of GM-CSF– induced cell-surface markers within the overall monocyte pop- we detected that spontaneous migration of GMaM and migration ulation using FACS (data not shown). Thus, we found no evidence toward MCP1 and LTB4 were significantly enhanced after 4 h that GM-CSF is affecting distinct monocyte subpopulations. Nev- (Fig. 3B). The chemotactic effect was specific because migration ertheless, we have performed additional monocyte/macrophage did not occur when MCP1 or LTB4 was added to the upper com- polarization experiments. To this end, we stimulated human mono- partment of the Boyden chamber (data not shown). In addition, cytes from healthy donors for 24 h with or without IFN-g (M1 GM-CSF–induced increase in chemotaxis was also specific to the macrophage polarization) or with or without IL-4 (M2 macrophage stimulus because we did not find increased migration toward IL-8 polarization), and cells were subsequently stimulated with or (Fig. 3B). Integrins and CC chemokine receptors play a potential without GM-CSF for further 24 h. Expression of IL-1b,TNF-a, role in monocyte trafficking into the mucosa in the context of IL-10, and CD206 was analyzed by flow cytometry (Fig. 2E). The mucosal homeostasis at the intestinal epithelial barrier. These data suggest that GM-CSF is affecting both M1- and M2-polarized molecules are also known to play a role in the pathogenesis of monocytes and that GM-CSF is not affecting distinct monocyte human inflammatory bowel diseases. Expression of integrins and subsets within the overall population differentially. CC chemokine receptors was analyzed by flow cytometry gated on The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 2. Polarization of GMaMs. (A) The gene expression of chemokine ligands and receptors in GMaMs (16 h) was analyzed by microarray analysis. by guest on October 2, 2021 The status of differentiation and polarization was classified according to characteristics of classically activated M1-like and alternatively activated M2-like monocyte/macrophage subsets in humans. (B) Microarray analysis data were confirmed by protein quantification (ELISA) for two selected chemokines (CCL 18 and CCL 23). (C and D) IL-4– (C) and IFN-g–dependent (D) gene expression analysis (RT-PCR) in human monocytes at different time points. Bars represent the relative n-fold regulation (mean 6 SEM) of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes. (E) Effects of GM-CSF activation on already primed monocytes. Monocytes were primed for 24 h toward M1 or M2 and subsequently treated for 24 h 6 GM-CSF. Expression (mean 6 SEM) of IL-1b, TNF-a, IL-10, and CD206 is shown gated on CD14+ cells of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

CD14+ cells, and expression is stated as mean fluorescence intensity no significant difference in phagocytosis of latex beads by (MFI; geo-mean) 6 SEM of three independent experiments. GMaMs, and also phagocytosis of L. major promastigotes was not Expression of CCR2 and CCR6 was significantly increased in altered compared with control monocytes (data not shown). 6 GM-CSF–stimulated monocytes (CCR2 150.2 31.1; CCR6 GM-CSF primes the monocyte response to a secondary microbial 6 6 24.5 4.4) compared with untreated cells (CCR2 88.4 22.5, stimulus. To test the hypothesis that GM-CSF specifically activates , 6 , p 0.05; CCR6 10.0 0.4, p 0.05), whereas expression of monocyte functions by augmenting anti-infectious/antimicrobial CCR7 was significantly reduced in GM-CSF–treated monocytes defense and bacterial clearance, we analyzed GMaMs for an in- 6 6 , (22.2 1.6) compared with unstimulated cells (49.2 6.9, p creased response to a secondary microbial stimulus. Therefore, we 0.05). Expression of b7, CCR1, CCR4, CCR9, and CX3CR1 analyzed the influence of GM-CSF stimulation (24 h) on cytokine was similar in GM-CSF–stimulated and untreated cells (data not production and gene expression in human monocytes after 4 h of shown). costimulation with bacterial endotoxin (LPS). Compared with con- Production of reactive oxygen species is increased and trol monocytes, GMaMs exposed to LPS produced significantly phagocytosis is unimpaired in GMaMs. Another important func- more IL-1b and TNF-a (Fig. 3D). LPS stimulation of GMaMs tion of monocytes after being recruited, for example, to sites of also resulted in a much more pronounced expression of inflam- infection or defective barriers, is the phagocytosis and killing of matory genes as revealed by qRT-PCR (Fig. 3E). This confirms pathogens (e.g., via production of reactive oxygen species [ROS]). a GM-CSF–induced priming effect on monocytes, leading to an Spontaneous and PMA-induced production of ROS was signifi- increase in vitro response to other stimuli (42). cantly enhanced in GMaMs (Fig. 3C). A few molecules involved in phagocytosis/endocytosis were significantly downregulated by GMaM impact on epithelial healing and on adaptive immunity GM-CSF stimulation (Table I). We therefore tested phagocytosis GMaMs accelerate epithelial healing. In addition to their innate of latex beads and of complement opsonized living L. major phagocytic and killing activity in antimicrobial defense, monocytes parasites after activation of monocytes with GM-CSF. We detected are also involved in wound repair. Because we observed phenotypic 8 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS

Table II. M1-/M2-like differentiation and polarization of GMaMs

Gene Name Gene Symbol M1/M2 Regulation n-Fold p Chemokine (C-X-C motif) ligand 10 CXCL10 M1 ↓ 22.36 0.005 Chemokine (C-X-C motif) ligand 11 CXCL11 M1 ↓ 22.75 0.004 Chemokine (C-X-C motif) ligand 13 CXCL13 M1 ↓↓ 25.27 0.000 IFN, g IFNg M1 ↔ 21.20 0.700 TNF a TNFa M1 ↑ 4.50 0.000 TNF ligand superfamily TRAIL M1 ↓ 22.03 0.001 NO synthase 2, inducible iNOS M1 ↔ 1.94 0.230 B7-1 CD80 M1 ↑ 4.22 0.005 IL 1 b IL1b M1 ↑ 2.81 0.000 IL 6 IL6 M1 ↑ 2.42 0.005 IL 8 IL8 M1 ↔ 1.15 1.000 IL 12 IL12 M1 ↔ 1.43 0.700 IL 18 IL18 M1 ↔ 1.59 0.100 IL 23 IL23 M1 ↔ 21.05 1.000 IFN regulatory factor 5 IRF5 M1 ↔ 21.07 1.000 PG-endoperoxidase synthase 2 COX2 M1 ↔ 21.03 1.000 Colony stimulating factor 2 GM-CSF M1 ↓ 21.36 0.000 Colony stimulating factor 3 G-CSF M1 ↓ 23.81 0.000 Chemokine (C-X-C motif) ligand CXCL5 M2 ↑ 3.87 0.015 Chemokine ligand 1 CCL1 M2 ↑↑ 6.82 0.000 Downloaded from Chemokine ligand 13 CCL13 M2 ↑↑↑ 21.06 0.000 Chemokine ligand 23 CCL23 M2 ↑↑ 6.87 0.000 TGF b TGFb M2 ↔ 1.12 0.694 Arginase ARG1 M2 ↑ 3.90 0.005 Mannose receptor C type 1 (MRC1) CD206 M2 ↑↑ 6.45 0.000 CD163 molecule CD163 M2 ↔ 21.90 0.113 ↑ DC-SIGN CD209 M2 2.65 0.009 http://www.jimmunol.org/ IL 1 receptor, type 2 CD121b M2 ↑ 2.70 0.001 IL 10 IL10 M2 ↔ 21.25 0.206 IFN regulatory factor 4 IRF4 M2 ↔ 1.01 1.000 similarities between GMaMs and alternatively activated (M2-like) tissue repair capacities, and a positive effect on T cell recruitment, macrophages, which have been originally described as “wound- we sought to address the functionality of these cells in vivo in the healing macrophages,” we analyzed the influence of GMaMs on context of CD. Because systematic analyses in the human system epithelial healing. In epithelial cell (Caco-2) monolayers, pre- are not feasible, we went on analyzing the effects of GMaMs in by guest on October 2, 2021 activation of monocytes with GM-CSF significantly accelerated the murine system. Analysis by qRT-PCR showed that murine wound closure compared with unstimulated monocytes (Fig. 3F). GMaMs had a similar gene regulation profile when compared GMaMs attract T cells and induce Tregs. Another important with the GM-CSF–dependent gene expression in human monocytes + function of monocytes is the cross talk to adaptive immunity. (Fig. 4A). We chose the CD4 T cell–dependent experimental co- Monocytes and macrophages serve as APCs, but they also litis model as an acceptable surrogate of human CD. In this model, + 2 2/2 shape lymphocyte activation by a whole battery of different co- adoptive transfer of syngeneic CD4 CD25 T cells into Rag1 stimulatory molecules and cytokines. As shown in Fig. 2, gene mice (which lack mature T cells) induces severe colitis (45). The expression (Fig. 2A) and protein production (Fig. 2B) of chemo- onset of colitis is monitored clinically by weight loss. Untreated kines CCL18 and CCL23 were strongly increased in GMaMs. monocytes (control) or ex vivo GMaMs were injected i.v. after Because CCL18 and CCL23 have been shown to attract naive and mice had lost weight on consecutive days (∼5–6 wk after eliciting + 2 resting T cells (43, 44), we analyzed the capacity of GMaMs to colitis by injection of CD4 CD25 T cells). Migration of injected attract T cells by using a modified Boyden chamber. T cells were GMaMs to the inflamed gut was confirmed by in vivo imaging. In allowed to migrate toward the culture supernatants of GMaMs or agreement with the in vitro data on cell migration, we observed an untreated monocytes. We observed a significantly increased T cell increased infiltration of GMaMs into MLNs compared with con- migration toward cell supernatants of GMaMs (Fig. 3G). In ad- trol monocytes in two independent experiments (Fig. 5). Mice that dition, we have addressed effects of GM-CSF on Treg differen- had received GMaMs showed no weight loss at all over a period of tiation. To this end, we stimulated human monocytes with GM- 12 d after monocyte transfer (Fig. 4B). Normally, the inflamed CSF and cocultured monocytes and autologous T cells for 7 d, and colon becomes shorter and presents with reduced length, and thus analyzed resulting T cells for Foxp3 expression to evaluate Treg shortening of the colon is a measure of inflammation. Also, in this differentiation. CD25 and Foxp3 expression in T cells were al- study, mice that received GMaMs did not show relevant shorten- ready increased upon interaction with monocytes that had been ing of the colon, whereas all other groups were not protected from stimulated with GM-CSF for only 24 h. The induction of Foxp3 colitis (Fig. 4B, 4C). Control mice that had received untreated expression in T cells cocultured with GMaMs was further in- monocytes or no monocytes showed progressive weight loss and creased when monocytes were stimulated for 48 h with GM-CSF signs of intestinal inflammation, and had to be euthanized on day (Fig. 3H, 3I). 12 (Fig. 4B, 4C). Animals that had received untreated monocytes had significantly severe histopathologic alterations of the colon, In vivo effects of GMaMs in experimental T cell–induced colitis most evident in the distal part (Fig. 4D, 4E). In addition, we GMaMs alleviate CD4+ T cell–induced colitis. Having confirmed performed high-resolution colonoscopy and graded inflammation a specific activation pattern of human monocytes in response to (MEICS-Score) (34). The colon of mice receiving no treatment GM-CSF, with the augmentation of host immune defense functions, presented with a vulnerable and bleeding mucosa, rarefication of The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 3. Functional properties of GMaMs and interaction of human GMaMs with T cells. (A) Human monocytes were activated with GM-CSF or left untreated (control) for 24/48 h in Teflon bags and subsequently allowed to adhere to multiwell plates for 4 h. Adherent cells were stained with crystal violet, and staining was quantified measuring the OD at 560 nm. (B) Monocytes were activated with GM-CSF or left untreated for 24 h in Teflon bags and placed by guest on October 2, 2021 into the upper chamber of a Transwell filter. The lower chamber contained monocyte medium with the addition of LTB4, MCP-1/CCL2, IL-8/CXCL8, or no attractants. After 4 h cells that had migrated into the lower compartment were counted, and numbers are presented as the percentage of cells, which migrated in the absence of any chemotactic stimulus. The p values refer to the migration of untreated (control) cells in the absence of any chemotactic stimuli (w/o). (C) Oxidative burst of GMaMs or untreated monocytes was initiated by the addition of PMA. Isoluminol chemiluminescence was measured in PMA-treated cells and control cells after induction of oxidative burst. (D) Cytokine secretion was measured in supernatants of GMaMs (24 h) after exposure to LPS (4 h) and compared with untreated monocytes exposed to LPS. **p , 0.01, ***p , 0.001 compared with LPS-treated control. (E) The gene expression of GMaMs (24 h) after exposure to LPS (4 h) was assessed by qRT-PCR. (F) Shown is the extent of wound closure in scratch assays of Caco-2 monolayers at 24 and 48 h in the absence of monocytes (control; n = 30), the presence of untreated monocytes (monocytes; n = 45), or the presence of monocytes preactivated for 48 h with GM-CSF (GMaM; n = 45). (G) T cell migration was analyzed. Monocytes were activated with GM-CSF or left untreated for 24 h in Teflon bags. Fifty thousand T cells were added to top chamber, and cell culture supernatants of untreated monocytes or GMaMs, as well as CCL18, CCL23, and medium (control), were added to the bottom chamber. T cells that had migrated into the lower compartment within 4 h were counted. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes. (H and I) Human autologous T cells were cocultured with untreated monocytes (control) or GMaMs (24 and 48 h) at a ratio of 10:1 for 7 d. Cells were stained for CD4, CD25, and intracellular for Foxp3 expression and analyzed by flow cytometry. (H) Representative dot plots are shown for 48 h. (I) Cells were gated on CD4+ cells and analyzed for CD25 and Foxp3 expression. Data shown are the means (6 SEM) of three independent experiments. (A–I)*p , 0.05, **p , 0.01, ***p , 0.001. vascular pattern, presence of fibrin, and ulcerations. Mice treated during colitis and treatment with GMaMs. After restimulation for with GMaMs depicted a transparent colonic mucosa with a regular 24 h, we tested supernatants for production of IFN-g (Th1 cell vascular pattern resembling healthy animals (Fig. 4F). Taken to- response) and IL-4, IL-10, and IL-13 (Th2 cell response). As gether, treatment of an established CD4+ T cell–induced colitis shown in Fig. 6, GMaM transfer led to significantly reduced IFN-g with GMaMs alleviates inflammation of the colon, resulting in production in T cells from LPMCs (Fig. 6A) and MLNs (Fig. 6B). significantly improved clinical parameters and histology, sug- The Th2 cytokines IL-4, IL-10, and IL-13, however, were slightly gesting that GMaMs potentially exert regulatory effects on T cells increased in supernatants from LPMCs of animals treated with in vivo. GMaMs (Fig. 6A), and IL-4 and IL-13 were also increased in GMaMs regulate T cell responses in vivo and in vitro. After ter- supernatants from MLNs of animals treated with GMaMs (Fig. mination of colitis experiments, LPMCs and MLNs were harvested. 6B). In summary, treatment of mice suffering from colitis with Migration of injected GMaMs to gut tissue and MLNs was con- GMaM results in a shift in cytokine production of T cells in vivo. firmed by in vivo imaging (Fig. 5). Single-cell suspensions were To confirm these in vivo data, we performed coculture experi- restimulated with anti-CD3/anti-CD28 to explore how the capac- ments with anti-CD3e/anti-CD28–stimulated T cells in vitro. ity and strength of T cell cytokine production has changed in vivo Also, in this study, GMaMs skewed the T cell response and led 10 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS Downloaded from

FIGURE 4. Treatment with GMaMs protects from experimental colitis. (A) GM-CSF–dependent gene regulation in murine monocytes derived from bone marrow of C57BL/6 mice and human peripheral blood monocytes compared with unstimulated monocytes. Shown is the mean relative n-fold regulation (6 SEM) of three independent experiments. *p , 0.05 compared with GM-CSF–treated human monocytes. (B) Rag12/2 mice were injected i.v. with + 2 CD4 CD25 T cells. After 40 d, when weight loss of the animals was severe, we injected: 1) GMaM i.v., or 2) untreated monocytes i.v., or 3) GM-CSF i.p. http://www.jimmunol.org/ (daily for 7 consecutive days), or, as a control, 4) PBS i.v. or 5) PBS i.p. Body weight of mice was subsequently monitored daily for 12 d. (C) On day 12, colons were removed for histology. The graph shows the mean colon lengths of each experimental group. (D) Representative macroscopic and microscopic (H&E staining) images of mice with colitis injected with control monocytes or GMaMs. Original magnification 3100. (E) Intestinal inflammation scores of the proximal and distal colon of mice with colitis injected with control monocytes or GMaM (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3, severe inflammation; 4, extreme inflammation). (F) MEICS score and representative pictures of high-resolution colonoscopy showing the colon of a mouse receiving no treatment with a vulnerable and bleeding mucosa, rarefication of vascular pattern, fibrin, and ulcerations. Mice treated with GMaMs depicted a transparent mucosa with a regular vascular pattern resembling healthy animals. Graphs in (B)–(E) show mean values (6 SEM) of 10 control mice and 14 mice injected with GMaMs from 3 independent experiments. *p , 0.05, **p , 0.01 compared with untreated monocytes; #p , 0.05 compared with T cells only. by guest on October 2, 2021 to significant upregulation of Th2 cytokines IL-4, IL-13, and Discussion IL-10, whereas the Th1 cytokine IFN-g was downregulated Despite the fact that the concept of CD as a chronic granulomatous (Fig. 6C). These data demonstrate that GMaM cross talk with Th1-driven disease shifts toward a theory of CD as an immuno- T cells results in a phenotype shift that attenuates classical Th1 deficiency of macrophages, while we only begin to understand the responses, which may contribute to an immunomodulatory effect. regulatory or suppressive functions of our immune system, our We did not observe an increase of Tregs, represented by Foxp3 general approach to chronic inflammatory diseases including CD is + expression in CD4 T cells, in MLNs or LPMCs of mice with still mainly based upon the paradigm of immunosuppression as the experimental T cell transfer colitis after the transfer of GMaMs primary therapeutic intervention. In line with that, phagocytes are (Fig. 6D, 6E). However, we were able to demonstrate that murine primarily seen as driving forces of inflammation that need to be GMaMs induce Tregs in vitro (Fig. 6F). inhibited. This traditional view of immune interventions, however, is Peripheral blood monocytes from patients with CD behave like in sharp contrast with our currently changing view of immunity. It is GMaMs now accepted that cells of the monocyte–macrophage lineage are We next studied phenotypic and functional features of untreated characterized by considerable diversity and plasticity that may en- versus GM-CSF–activated peripheral blood monocytes of 18 compass, as an example, classical M1-macrophage differentiation patients with quiescent CD by analyses of cell adherence, mi- (when stimulated by IFN-g) or alternative M2 differentiation (when gration, chemotaxis, phagocytosis, oxidative burst, and cytokine stimulated by IL-4/IL-13) as outer margins of a broad phenotypical expression and secretion. Collectively, our data suggest that the plasticity (2). Serving another example, the population resulting from effects of GM-CSF activation of peripheral monocytes of patients GM-CSF–stimulated human monocytes has been referred to as M1- with CD (Fig. 7) are similar to the observed effects in GMaMs like macrophages with a proinflammatory cytokine profile (41, 46). from healthy donors (Figs. 1–3). This includes the GM-CSF–in- As an attempt to introduce a novel concept based on stimulating duced increase in adherence, migration, chemotaxis, and oxidative rather than suppressing immunity, GM-CSF has been used both in burst, as well as the priming of monocytes to secondary microbial animal IBD models and in human patients with CD (47). Intra- stimuli (Fig. 7A–E). In addition, changes in GM-CSF–dependent peritoneal administration of GM-CSF alleviated acute DSS- mRNA expression of selected key inflammatory cytokines were in induced colitis in mice, resulting in decreased proinflammatory agreement with our transcriptomic data obtained from GMaMs of cytokine release, improved clinical and histologic parameters, as healthy individuals (Fig. 7F). Importantly, there was no evidence well as more rapid ulcer healing, and facilitated epithelial re- that GM-CSF activation had different effects on monocytes when generation (15, 16). Importantly, transfer of splenic GM-CSF– compared between individual patients. induced CD11b+ myeloid cells into DSS-exposed mice improved The Journal of Immunology 11 Downloaded from

FIGURE 5. GMaMs rapidly infiltrate the intestine. (A) Mesenteric lymph node single-cell suspensions of congenic CD45.2 mice, suffering from colitis (and treated as indicated), were analyzed for infiltration of injected donor monocytes (CD45.1+) using CD45.1 Ab by flow cytometry. (B) Results of two http://www.jimmunol.org/ independent experiments are shown as percent infiltrated donor CD45.1+ cells. *p , 0.05 compared with untreated monocytes. (C) GMaMs were labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, and 2 3 106 cells were injected according to the standard treatment regimen. Monocyte infiltration in the intestine was visualized after 24 h by a planar small-animal fluorescence-mediated tomography system, and representative pictures are depicted. colitis, and GM-CSF–expanded CD11b+ splenocytes were shown induced by GM-CSF depend on its concentration (53), and that the to promote in vitro wound repair (16). Furthermore, it has been time point chosen for the CSF treatment of human monocytes can shown that: 1) neutralization of GM-CSF increases intestinal markedly determine the relative expression of cytokine genes (41). by guest on October 2, 2021 permeability and bacterial translocation in mice; and 2) increased Collectively, it is conceivable that the described population of levels of GM-CSF autoantibodies are associated with an increase GMaMs in this study represents an intermediate cell type, com- in bowel permeability, disease relapse, stricturing ileal disease, bining cell-surface expression characteristics and functional fea- and surgery in patients with CD (48–50). As a therapy that could tures of different M2 macrophage subsets including CD206 and help in overcoming insufficient macrophage functions, GM-CSF CD209 cell-surface expression (M2a), Th2 responses/activation had strikingly beneficial effects in subgroups of CD patients (17). (M2a/b), killing, and type II inflammation (M2a) and immuno- Seemingly, these beneficial effects were rather unexpected in regulation (M2b). In contrast with proinflammatory and antimi- of the previously reported proinflammatory polarization of mac- crobial responses of classically activated monocytes, M2-like rophages upon stimulation with GM-CSF (41, 46). phenotypes are broadly anti-inflammatory and play important We speculated that GM-CSF exerts its beneficial effects in in- roles in wound healing (54). GMaMs combine: 1) features of testinal inflammation in vivo by specific activation of monocytes augmented host defense functions; 2) the ability to facilitate epi- that combines innate immune activation, thus facilitating anti- thelial healing; and 3) the regulatory potential on adaptive im- infectious defense, with a simultaneous regulatory function serv- munity. Specifically, we found a GMaM-dependent accelerated ing to limit adaptive immunity and excessive inflammation. We wound closure in Caco-2 monolayers using an in vitro scratch thus set off in an unbiased systems biology approach to compre- closure assay and in addition an upregulation of genes involved in hensively study the many facets of monocyte activation in vitro, cell proliferation (e.g., FGF13, CDKN1C, TGF-b). Furthermore, ranging from gene expression to innate immune functions and the we found that the expression of a number of genes that are as- interplay with adaptive immunity. All these aspects have previ- sociated with M2 polarization is increased in human monocytes ously been studied on monocytes but separately and independently after activation with GM-CSF. In particular, we found a significant from each other (41, 46, 51–54). Our findings suggest that the regulation of chemokines and chemokine receptors in human early imprinting of monocytes after activation with GM-CSF is monocytes. In this study, we found a GM-CSF–dependent downreg- of crucial importance, because monocytes play an important role ulation of the M1 chemokines CXCL9, CXCL10, CXCL11, CXCL13, during the recruitment phase of the innate immune response and and CXCL16 in monocytes. At the same time, GM-CSF significantly have the potential to regulate adaptive immune mechanisms. GM- induced expression of chemokines CXCL3, CCL1, CCL13, CCL17, CSF has been shown to have a pleiotropic role in inflammation CCL18, CCL23, and CCL24 in monocytes, which are characteristic for and autoimmunity (23). Data from other groups suggest that cell M2 macrophages (39, 54, 56). In addition, we showed that a number culture conditions, concentration, time point, and duration chosen of other M2a/b macrophage markers were significantly upregulated for GM-CSF stimulation of human monocytes may determine in human monocytes after GM-CSF activation (e.g., CD206, transcriptional outcomes relating to M1/M2 polarity (53–55). In CD209, CD121b, ARG1). As mentioned earlier, we also found an this respect, it has been described that different biologic responses enhanced production of proinflammatory cytokines (IL-1b,TNF-a, 12 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS

FIGURE 6. GMaMs alter T cell cytokine responses in vivo and in vitro. LPMCs and MLNs were isolated from the colon of mice that were treated as indicated. Expression of cytokines after 24-h stimulation with anti-CD3e/anti-CD28 Abs is shown for LPMCs (A) and after 96 h for MLNs (B). Graphs show mean values (6 SEM) from 3 inde- pendent experiments and n = 6–8 per group. (C) Naive pan T cells were cocultured for 4 d with GMaM, control monocytes, or left alone. T cells were stimulated with anti-CD3e/anti-CD28 Abs. Ratio of respective monocytes to T cells was 1:10, and cytokines were measured in supernatants. Data shown are the means (6 SEM) of nine independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 Downloaded from compared with untreated monocytes. Cell pop- ulations from the lamina propria (D) and MLNs (E) were stained for CD4 and Foxp3 expression and analyzed by flow cytometry. Bars refer to mean 6 SEM of three independent experiments. (F) Murine T cells were cocultured with GMaMs or control monocytes at a ratio of 10:1 (T cells/monocytes). Cells http://www.jimmunol.org/ were stained for CD4 and Foxp3 expression and an- alyzed by flow cytometry. Bars refer to mean 6 SEM of three independent experiments. **p , 0.01 com- pared with untreated monocytes (control). by guest on October 2, 2021

and IL-6) in GMaMs, a feature also seen in M2b macrophages Monocytes may also significantly regulate the immune context upon exposure to immune complexes and LPS (56). In addition to by interaction with other cells. In particular, chemokines and these strengthened innate immune functions, GMaMs simulta- chemokine receptors have a key role in intestinal epithelial bar- neously indeed have a regulatory potential on adaptive immunity. rier repair and maintenance (63, 64). We found a significant regula- Overall, our data indicate that GMaMs represent a distinctive cell tion of chemokines and chemokine receptors with GM-CSF–depen- population with characteristics of both M1 and M2 cells. dent downregulation of the chemokines CXCL9, CXCL10, and GM-CSF also stimulated functions that are typically assigned to CXCL11 in monocytes. These factors are known to be increased classically activated monocytes. It has previously been published in IBD and to attract Th1 and NK cells (65). At the same time, that GM-CSF increases the adherence of purified peripheral blood GM-CSF significantly induced a short-termed expression of the monocytes to plastic surfaces and to monolayers of HUVECs (55), chemokines CCL13, CCL17, CCL18, CCL23, and CCL24 in and that GM-CSF can prime monocytes for increased trans- monocytes, which are known to attract naive T cells, Th2 cells, endothelial migration (57). Furthermore, reported results on GM- and/or Tregs (43, 44, 63, 65). Because of the observed upregula- CSF effects on other functions such as oxidative metabolism, tion of costimulatory molecule CD80 and the chemotactic factors , phagocytosis, and the in vitro response to other for naive and quiescent T cells CCL18 and CCL23 (43, 44), we stimuli are conflicting to some extent (23, 52, 58, 59). We confirm next analyzed the interaction of GMaMs with T cells. Indeed, in this study that short-term treatment (24 h) of human monocytes migration of naive, autologous T cells toward GMaMs was ac- with GM-CSF promotes: 1) cell adherence and migration, 2) celerated. Our data suggest that particularly CCL18 and CCL23 production of ROS, and 3) response to a second microbial stim- might be responsible for the increased T cell migration. However, ulus (LPS). Thus, GM-CSF enhances selective effector functions our transcriptomic data suggest that other GM-CSF–induced of monocytes, an effect of GM-CSF previously described for tis- chemokines (e.g., CCL13, CCL17, CCL24) might also be re- sue-derived macrophages (60). In addition, our data indicate that sponsible for the increased T cell migration because they are GM-CSF–activated peripheral blood monocytes from patients known to attract T cells. Studies to address this question more with CD behave the same way as GMaMs (Fig. 7) and that GM- specifically are beyond the scope of this study. It has been shown CSF may regulate the homing molecules CCR2 and CCR6, which that treatment of human monocytes with GM-CSF generates a are involved in regulating several aspects of mucosal immunity, subtype of cells that regulate CD4+ T cell proliferation partially including the ability to mediate the recruitment of innate immune via production of IL-10 (52), and that GM-CSF may sustain Treg cells to the sites of epithelial inflammation (61, 62). homeostasis and enhance their suppressive functions (47), indi- The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/

FIGURE 7. Features of GM-CSF–activated peripheral blood monocytes of patients with quiescent CD (n = 18). (A) Adhesion of untreated (w/o) versus GM-CSF–activated patient monocytes (24 h) to fibronectin-coated plastic surface. Adhering cells were stained with 0.5% crystal violet, and staining was quantified measuring the OD at 560 nm. (B) Migration and chemotaxis studies of untreated (w/o) versus GMaMs (24 h) using a modified Boyden chamber and LTB4 (100 nM) as an additional chemoattractant. After 4 h, cells that had migrated into the lower compartment were counted, and numbers are presented as the percentage of untreated cells, which migrated in the absence of any chemotactic stimuli. (C) Phagocytosis of fluorescein-labeled E. coli by by guest on October 2, 2021 untreated (w/o) versus GMaMs (24 h). E. coli phagocytosis was analyzed by flow cytometry, and phagocytic internalization was confirmed by fluorescence microscopy. (D) Production of ROS by untreated (w/o) versus GMaMs (24 h) with and without further LPS stimulation for 2 h in the presence of rhodamine for the final 15 min. Oxidative burst was analyzed by flow cytometry. (E) Cytokine secretion of untreated (w/o) versus GMaMs (24 h) with and without further LPS stimulation for 2 h. (F) Gene expression (relative n-fold regulation) in untreated (w/o) versus GMaMs (24 h). Bars refer to mean 6 SEM. *p , ### 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes; p , 0.001 compared with controls without LTB4 or LPS. cating the regulatory potential of GMaMs toward CD4+ T cells includes a down-toning of classical Th1 responses. In this regard, and Tregs. When we cocultured GMaMs with syngeneic CD4+ our results reconfirm that monocytes harbor important functions T cells, we observed that GMaMs shape T cell response toward regarding polarization and expansion of lymphocytes and may a Th2 phenotype and induce Tregs. also contribute to shaping T cell responses (22). The in vivo After having analyzed the programming of monocytes with effects of GMaMs showed increased levels of Th2 cytokines in GM-CSF in vitro, we next aimed at analyzing the therapeutic LPMCs (IL-4, IL-10, IL-13) and MLNs (IL-4, IL-13), but this effects of this cell population in a model of CD. We found that T trend was not statistically significant. However, together with the and –deficient (Rag12/2) mice in which Crohn-like colitis was observed significantly reduced production of IFN-g in T cells induced with the CD4+CD252 T cell transfer model (66, 67) were from LMPCs and MLNs, we concluded that treatment of mice protected from disease progression when they received GMaMs suffering from colitis with GMaMs results in a shift toward Th2 (but not untreated monocytes). Interestingly, Rag12/2 mice that cytokine production of T cells in vivo. Our in vitro experiments did not receive GMaMs but i.p. GM-CSF injections after the confirmed that GMaMs skew the T cell response and lead to sig- transfer of T cells were not completely protected from colitis but nificant upregulation of Th2 cytokines (IL-4, IL-10, IL-13), whereas showed reduced disease severity. This is in agreement with earlier the Th1 cytokine IFN-g was significantly downregulated. This work demonstrating positive effects of GM-CSF administration might be explained by our observation that GMaMs display char- (i.p.) in DSS-induced colitis in BALB/c, and more importantly, in acteristics of both M2-like/IL-4–induced macrophages and M1-like/ Rag12/2 mice (15, 16). We postulate that the alleviating effects of IFN-g–induced macrophages (as discussed earlier). GM-CSF in experimental colitis are due to direct modulation of Interestingly, it has recently been shown that the therapeutic monocyte/macrophage functions including accelerated epithelial transfer of glucocorticoid-stimulated monocytes (GCsMs) in the healing. Our data demonstrate that the protective effect of T cell transfer colitis model also resulted in a strongly downreg- monocytes depends on their GM-CSF prestimulation in a T cell– ulated release of IFN-g by T cells from LPMCs and MLNs. The dependent model of colitis. The therapeutic mechanism of action production of IL-4 and IL-13 was not influenced in single-cell of GMaM thus involves the regulation of T cell responses, which suspensions from LPMCs and MLNs after treatment of mice 14 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS with injection of GCsMs, which is also in contrast with the in vitro References system, where cytokine production of IFN-g, IL-4, and IL-13 by 1. Shi, C., and E. G. Pamer. 2011. Monocyte recruitment during infection and T cells was significantly regulated in cocultures with GCsMs (68). inflammation. Nat. Rev. Immunol. 11: 762–774. 2. Sica, A., and A. Mantovani. 2012. Macrophage plasticity and polarization: The same study also showed that in the T cell transfer colitis in vivo veritas. J. Clin. Invest. 122: 787–795. model, CD4+Foxp3+ Tregs accumulate locally in the colon after 3. Lawrence, T., and G. Natoli. 2011. Transcriptional regulation of macrophage treatment with GCsMs and that repetitive stimulation of naive polarization: enabling diversity with identity. Nat. Rev. Immunol. 11: 750–761. 4. Marks, D. J. 2011. Defective innate immunity in inflammatory bowel disease: splenic T cells with GCsMs induces Tregs in vitro. However, a Crohn’s disease exclusivity? Curr. Opin. Gastroenterol. 27: 328–334. Tregs also did not expand in draining MLNs and LPMCs of ani- 5. Casanova, J. L., and L. Abel. 2009. Revisiting Crohn’s disease as a primary immunodeficiency of macrophages. J. Exp. Med. 206: 1839–1843. mals treated with GCsMs during T cell transfer colitis (68). 6. Rahman, F. Z., D. J. Marks, B. H. Hayee, A. M. Smith, S. L. Bloom, and + + Collectively, GMaMs induce the differentiation of CD4 Foxp3 A. W. Segal. 2008. Phagocyte dysfunction and inflammatory bowel disease. Tregs in vitro, but we found no evidence that the GMaM-mediated Inflamm. Bowel Dis. 14: 1443–1452. 7. Segal, A. W., and G. Loewi. 1976. Neutrophil dysfunction in Crohn’s disease. protection from colitis is facilitated via expansion of mucosal Lancet 2: 219–221. Tregs in vivo. 8. Marks, D. J., M. W. Harbord, R. MacAllister, F. Z. Rahman, J. Young, B. Al- Limitations of our study include the lack of in vivo data ex- Lazikani, W. Lees, M. Novelli, S. Bloom, and A. W. Segal. 2006. Defective acute inflammation in Crohn’s disease: a clinical investigation. Lancet 367: 668– ploring specifically GMaMs in human CD, which may become 678. feasible in the future. Although we address the tissue context by 9. Smith, A. M., F. Z. Rahman, B. Hayee, S. J. Graham, D. J. Marks, G. W. Sewell, C. D. Palmer, J. Wilde, B. M. Foxwell, I. S. Gloger, et al. 2009. Disordered analyzing LPMCs and MLN, the further differentiation of ex vivo– macrophage cytokine secretion underlies impaired acute inflammation and activated monocytes after transfer in vivo, especially after infil- bacterial clearance in Crohn’s disease. J. Exp. Med. 206: 1883–1897. trating the inflamed tissue, is certainly a complex issue. The cir- 10. Maloy, K. J., and F. Powrie. 2011. Intestinal homeostasis and its breakdown in inflammatory bowel disease. Nature 474: 298–306. Downloaded from culating monocyte population is not homogenous but consists of 11. Ja¨ger, S., E. F. Stange, and J. Wehkamp. 2013. Inflammatory bowel disease: an both inflammatory and regulatory populations that counterbalance impaired barrier disease. Langenbecks Arch. Surg. 398: 1–12. each other. We speculate that GM-CSF exerts its beneficial ef- 12. Norling, L. V., and M. Perretti. 2013. Control of myeloid cell trafficking in resolution. J. Innate Immun. 5: 367–376. fects in intestinal inflammation in vivo by specific activation of 13. Ortega-Go´mez, A., M. Perretti, and O. Soehnlein. 2013. Resolution of inflam- monocytes that combines innate immune activation, facilitating mation: an integrated view. EMBO Mol. Med. 5: 661–674. 14. Mantovani, A., S. K. Biswas, M. R. Galdiero, A. Sica, and M. Locati. 2013. anti-infectious defense and a simultaneous regulatory function Macrophage plasticity and polarization in tissue repair and remodelling. J. http://www.jimmunol.org/ serving to limit adaptive immunity and excessive inflammation Pathol. 229: 176–185. rather short term. The pleiotropic GM-CSF functions on monocyte 15. Sainathan, S. K., E. M. Hanna, Q. Gong, K. S. Bishnupuri, Q. Luo, M. Colonna, F. V. White, E. Croze, C. Houchen, S. Anant, and B. K. Dieckgraefe. 2008. activation and their consequences for innate and adaptive immu- Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced nity range from activation of M2-like monocytes, chemotactic experimental colitis. Inflamm. Bowel Dis. 14: 88–99. migration, and antimicrobial response to mucosal healing, but also 16. Bernasconi, E., L. Favre, M. H. Maillard, D. Bachmann, C. Pythoud, H. Bouzourene, E. Croze, S. Velichko, J. Parkinson, P. Michetti, and D. Velin. encompasses regulation of adaptive immunity by attraction of, for 2010. Granulocyte-macrophage colony-stimulating factor elicits bone marrow- example, T cells with the possibility to differentiate Th cells and derived cells that promote efficient colonic mucosal healing. Inflamm. Bowel Dis. subsequently limit inflammation induced by adaptive immunity. 16: 428–441. 17. Roth, L., J. K. MacDonald, J. W. McDonald, and N. Chande. 2012. Sar- However, it remains a question for further studies whether and gramostim (GM-CSF) for induction of remission in Crohn’s disease: a cochrane by guest on October 2, 2021 how monocytes differentiate long term at sites of inflammation inflammatory bowel disease and functional bowel disorders systematic review of randomized trials. Inflamm. Bowel Dis. 18: 1333–1339. and within damaged tissue. It is also important to investigate the 18. Smythies, L. E., A. Maheshwari, R. Clements, D. Eckhoff, L. Novak, H. L. Vu, immunomodulatory properties of monocytes in other animal L. M. Mosteller-Barnum, M. Sellers, and P. D. Smith. 2006. Mucosal IL-8 and models of experimental colitis, for example, in DSS-induced co- TGF-beta recruit blood monocytes: evidence for cross-talk between the lamina propria stroma and myeloid cells. J. Leukoc. Biol. 80: 492–499. litis. Recent work by Kurmaeva et al. (69) has demonstrated that 19. Geissmann, F., M. G. Manz, S. Jung, M. H. Sieweke, M. Merad, and K. Ley. immunosuppressive monocytes (CD11b+Ly6GnegLy6Chigh cells) 2010. Development of monocytes, macrophages, and dendritic cells. Science accumulate in the spleen and inflamed intestine during experi- 327: 656–661. 20. Zhou, L., H. Braat, K. N. Faber, G. Dijkstra, and M. P. Peppelenbosch. 2009. mental colitis not only in T cell–induced colitis but also in an Monocytes and their pathophysiological role in Crohn’s disease. Cell. Mol. Life acute model induced by administration of DSS and a TNFDARE Sci. 66: 192–202. 21. Auffray, C., M. H. Sieweke, and F. Geissmann. 2009. Blood monocytes: de- model of chronic ileitis. velopment, heterogeneity, and relationship with dendritic cells. Annu. Rev. In conclusion, while taking the shifting paradigms of CD Immunol. 27: 669–692. pathogenesis and immune regulation into account, our findings 22. Geissmann, F., C. Auffray, R. Palframan, C. Wirrig, A. Ciocca, L. Campisi, E. Narni-Mancinelli, and G. Lauvau. 2008. Blood monocytes: distinct subsets, support the exploration of stimulating rather than suppressive how they relate to dendritic cells, and their possible roles in the regulation of therapies with the potential to more specifically reprogram mono- T-cell responses. Immunol. Cell Biol. 86: 398–408. cytes toward immunomodulatory functions to alleviate chronic 23. Hamilton, J. A. 2008. Colony-stimulating factors in inflammation and autoim- munity. Nat. Rev. Immunol. 8: 533–544. inflammatory bowel disease. 24. Ehrchen, J., L. Steinmuller,€ K. Barczyk, K. Tenbrock, W. Nacken, M. Eisenacher, U. Nordhues, C. Sorg, C. Sunderko¨tter, and J. Roth. 2007. Glucocorticoids induce differentiation of a specifically activated, anti- Acknowledgments inflammatory subtype of human monocytes. Blood 109: 1265–1274. We thank Melanie Saers, Susanne Schleifenbaum, Andrea Dick, Claudia 25. Schwarzmaier, D., D. Foell, T. Weinhage, G. Varga, and J. Da¨britz. 2013. Pe- ripheral monocyte functions and activation in patients with quiescent Crohn’s Sole´, Eva Nattkemper, Andrea Stadtba¨umer, and Sonja Dufentester for disease. PLoS ONE 8: e62761. excellent technical work. We also thank Dr. Tilmann Spieker (Department 26. Viemann, D., M. Goebeler, S. Schmid, K. Klimmek, C. Sorg, S. Ludwig, and of Pathology, University Hospital Munster,€ Germany, and St. Franziskus- J. Roth. 2004. Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP Hospital, Institute of Pathology, Munster,€ Germany), who helped with kinase-dependent gene expression in TNF-alpha-stimulated primary human en- dothelial cells. Blood 103: 3365–3373. histopathology. We also thank the American Gastroenterological Associa- 27. Viemann, D., A. Strey, A. Janning, K. Jurk, K. Klimmek, T. Vogl, K. Hirono, tion Research Foundation for the Moti L and Kamla Rustgi Awards, which F. Ichida, D. Foell, B. Kehrel, et al. 2005. Myeloid-related proteins 8 and 14 supported the presentation of the research at the 50th and 51st Digestive induce a specific inflammatory response in human microvascular endothelial Disease Week annual meetings. cells. Blood 105: 2955–2962. 28. Quackenbush, J. 2002. Microarray data normalization and transformation. Nat. Genet. 32(Suppl.): 496–501. 29. Da¨britz, J., F. Friedrichs, T. Weinhage, J. Hampe, T. Kucharzik, A. Lugering,€ Disclosures U. Broeckel, S. Schreiber, T. Spieker, M. Stoll, and D. Foell. 2011. The func- The authors have no financial conflicts of interest. tional -374T/A polymorphism of the receptor for advanced glycation end The Journal of Immunology 15

products may modulate Crohn’s disease. Am. J. Physiol. Gastrointest. Liver colony-stimulating factor autoantibodies and increased intestinal permeability in Physiol. 300: G823–G832. Crohn disease. J. Pediatr. Gastroenterol. Nutr. 52: 542–548. 30. Vogl, T., S. Ludwig, M. Goebeler, A. Strey, I. S. Thorey, R. Reichelt, D. Foell, 50. Da¨britz, J., E. Bonkowski, C. Chalk, B. C. Trapnell, J. Langhorst, L. A. Denson, V. Gerke, M. P. Manitz, W. Nacken, et al. 2004. MRP8 and MRP14 control and D. Foell. 2013. Granulocyte macrophage colony-stimulating factor auto- microtubule reorganization during transendothelial migration of phagocytes. antibodies and disease relapse in inflammatory bowel disease. Am. J. Gastro- Blood 104: 4260–4268. enterol. 108: 1901–1910. 31. Ehrchen, J., L. Helming, G. Varga, B. Pasche, K. Loser, M. Gunzer, 51. Sierra-Filardi, E., A. Puig-Kro¨ger, F. J. Blanco, C. Nieto, R. Bragado, C. Sunderko¨tter, C. Sorg, J. Roth, and A. Lengeling. 2007. Vitamin D receptor M. I. Palomero, C. Bernabe´u, M. A. Vega, and A. L. Corbı´. 2011. Activin A signaling contributes to susceptibility to infection with Leishmania major. skews macrophage polarization by promoting a proinflammatory phenotype and FASEB J. 21: 3208–3218. inhibiting the acquisition of anti-inflammatory macrophage markers. Blood 117: 32. Varga, G., J. Ehrchen, A. Tsianakas, K. Tenbrock, A. Rattenholl, S. Seeliger, 5092–5101. M. Mack, J. Roth, and C. Sunderkoetter. 2008. Glucocorticoids induce an acti- 52. Ganguly, D., K. Paul, J. Bagchi, S. Rakshit, L. Mandal, G. Bandyopadhyay, and vated, anti-inflammatory monocyte subset in mice that resembles myeloid- S. Bandyopadhyay. 2007. Granulocyte-macrophage colony-stimulating factor derived suppressor cells. J. Leukoc. Biol. 84: 644–650. drives monocytes to CD14low CD83+ DCSIGN- -10-producing my- 33. Varga, G., S. Balkow, M. K. Wild, A. Stadtbaeumer, M. Krummen, T. Rothoeft, eloid cells with differential effects on T-cell subsets. Immunology 121: 499–507. T. Higuchi, S. Beissert, K. Wethmar, K. Scharffetter-Kochanek, et al. 2007. Active 53. Guthridge, M. A., J. A. Powell, E. F. Barry, F. C. Stomski, B. J. McClure, MAC-1 (CD11b/CD18) on DCs inhibits full T-cell activation. Blood 109: 661–669. H. Ramshaw, F. A. Felquer, M. Dottore, D. T. Thomas, B. To, et al. 2006. Growth 34. Becker, C., M. C. Fantini, S. Wirtz, A. Nikolaev, R. Kiesslich, H. A. Lehr, factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary P. R. Galle, and M. F. Neurath. 2005. In vivo imaging of colitis and colon cancer switch. EMBO J. 25: 479–489. development in mice using high resolution chromoendoscopy. Gut 54: 950–954. 54. Murray, P. J., and T. A. Wynn. 2011. Protective and pathogenic functions of 35. Eisenbla¨tter, M., J. Ehrchen, G. Varga, C. Sunderko¨tter, W. Heindel, J. Roth, macrophage subsets. Nat. Rev. Immunol. 11: 723–737. C. Bremer, and A. Wall. 2009. In vivo optical imaging of cellular inflammatory 55. Gamble, J. R., M. J. Elliott, E. Jaipargas, A. F. Lopez, and M. A. Vadas. 1989. response in granuloma formation using fluorescence-labeled macrophages. J. Regulation of human monocyte adherence by granulocyte-macrophage colony- Nucl. Med. 50: 1676–1682. stimulating factor. Proc. Natl. Acad. Sci. USA 86: 7169–7173. 36. Lugering,€ A., T. Kucharzik, D. Soler, D. Picarella, J. T. Hudson, III, and 56. Mantovani, A., A. Sica, S. Sozzani, P. Allavena, A. Vecchi, and M. Locati. 2004. I. R. Williams. 2003. Lymphoid precursors in intestinal cryptopatches express The chemokine system in diverse forms of macrophage activation and polari- CCR6 and undergo dysregulated development in the absence of CCR6. J. zation. Trends Immunol. 25: 677–686. Downloaded from Immunol. 171: 2208–2215. 57. Shang, X. Z., and A. C. Issekutz. 1999. Enhancement of monocyte trans- 37. Ahlmann, M., G. Varga, K. Sturm, R. Lippe, K. Benedyk, D. Viemann, endothelial migration by granulocyte-macrophage colony-stimulating factor: T. Scholzen, J. Ehrchen, F. U. Muller,€ M. Seidl, et al. 2009. The cyclic AMP requirement for chemoattractant and CD11a/CD18 mechanisms. Eur. J. Immu- response element modulator alpha suppresses CD86 expression and APC func- nol. 29: 3571–3582. tion. J. Immunol. 182: 4167–4174. 58. Armitage, J. O. 1998. Emerging applications of recombinant human granulocyte- 38. Martinez, F. O., S. Gordon, M. Locati, and A. Mantovani. 2006. Transcriptional macrophage colony-stimulating factor. Blood 92: 4491–4508. profiling of the human monocyte-to-macrophage differentiation and polarization: 59. Robin, G., S. Markovich, A. Athamna, and Y. Keisari. 1991. Human recombinant

new molecules and patterns of gene expression. J. Immunol. 177: 7303–7311. granulocyte-macrophage colony-stimulating factor augments viability and cy- http://www.jimmunol.org/ 39. Mellins, E. D., C. Macaubas, and A. A. Grom. 2011. Pathogenesis of systemic totoxic activities of human monocyte-derived macrophages in long-term cul- juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol tures. Cytokine Res. 10: 257–263. 7: 416–426. 60. Coleman, D. L., J. A. Chodakewitz, A. H. Bartiss, and J. W. Mellors. 1988. 40. Fleetwood, A. J., H. Dinh, A. D. Cook, P. J. Hertzog, and J. A. Hamilton. 2009. Granulocyte-macrophage colony-stimulating factor enhances selective effector GM-CSF- and M-CSF-dependent macrophage phenotypes display differential functions of tissue-derived macrophages. Blood 72: 573–578. dependence on type I signaling. J. Leukoc. Biol. 86: 411–421. 61. Ito, T., W. F. Carson, IV, K. A. Cavassani, J. M. Connett, and S. L. Kunkel. 2011. 41. Lacey, D. C., A. Achuthan, A. J. Fleetwood, H. Dinh, J. Roiniotis, G. M. Scholz, CCR6 as a mediator of immunity in the lung and gut. Exp. Cell Res. 317: 613–619. M. W. Chang, S. K. Beckman, A. D. Cook, and J. A. Hamilton. 2012. Defining 62. Thomas, S., and D. C. Baumgart. 2012. Targeting leukocyte migration and adhe- GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro sion in Crohn’s disease and ulcerative colitis. Inflammopharmacology 20: 1–18. models. J. Immunol. 188: 5752–5765. 63. Zimmerman, N. P., R. A. Vongsa, M. K. Wendt, and M. B. Dwinell. 2008. 42. Adib-Conquy, M., and J. M. Cavaillon. 2002. Gamma interferon and Chemokines and chemokine receptors in mucosal homeostasis at the intestinal

granulocyte/monocyte colony-stimulating factor prevent endotoxin tolerance in epithelial barrier in inflammatory bowel disease. Inflamm. Bowel Dis. 14: 1000– by guest on October 2, 2021 human monocytes by promoting interleukin-1 receptor-associated kinase ex- 1011. pression and its association to MyD88 and not by modulating TLR4 expression. 64. Tokuyama, H., S. Ueha, M. Kurachi, K. Matsushima, F. Moriyasu, J. Biol. Chem. 277: 27927–27934. R. S. Blumberg, and K. Kakimi. 2005. The simultaneous blockade of chemokine 43. Adema, G. J., F. Hartgers, R. Verstraten, E. de Vries, G. Marland, S. Menon, receptors CCR2, CCR5 and CXCR3 by a non-peptide an- J. Foster, Y. Xu, P. Nooyen, T. McClanahan, et al. 1997. A dendritic-cell-derived tagonist protects mice from dextran sodium sulfate-mediated colitis. Int. C-C chemokine that preferentially attracts naive T cells. Nature 387: 713–717. Immunol. 17: 1023–1034. 44. Forssmann, U., M. B. Delgado, M. Uguccioni, P. Loetscher, G. Garotta, and 65. Koelink, P. J., S. A. Overbeek, S. Braber, P. de Kruijf, G. Folkerts, M. J. Smit, M. Baggiolini. 1997. CKbeta8, a novel CC chemokine that predominantly acts and A. D. Kraneveld. 2012. Targeting chemokine receptors in chronic inflam- on monocytes. FEBS Lett. 408: 211–216. matory diseases: an extensive review. Pharmacol. Ther. 133: 1–18. 45. Ostanin, D. V., J. Bao, I. Koboziev, L. Gray, S. A. Robinson-Jackson, 66. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, and R. L. Coffman. 1996. A M. Kosloski-Davidson, V. H. Price, and M. B. Grisham. 2009. T cell transfer critical role for transforming growth factor-beta but not in the model of chronic colitis: concepts, considerations, and tricks of the trade. Am. J. suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. Physiol. Gastrointest. Liver Physiol. 296: G135–G146. J. Exp. Med. 183: 2669–2674. 46. Krausgruber, T., K. Blazek, T. Smallie, S. Alzabin, H. Lockstone, N. Sahgal, 67. Mudter, J., S. Wirtz, P. R. Galle, and M. F. Neurath. 2002-2003. A new model of T. Hussell, M. Feldmann, and I. A. Udalova. 2011. IRF5 promotes inflammatory chronic colitis in SCID mice induced by adoptive transfer of CD62L+ CD4+ macrophage polarization and TH1-TH17 responses. Nat. Immunol. 12: 231–238. T cells: insights into the regulatory role of interleukin-6 on apoptosis. Patho- 47. Da¨britz, J. 2014. Granulocyte macrophage colony-stimulating factor and the biology 70: 170–176. intestinal innate immune cell homeostasis in Crohn’s disease. Am. J. Physiol. 68. Varga, G., J. Ehrchen, A. Brockhausen, T. Weinhage, N. Nippe, M. Belz, Gastrointest. Liver Physiol. 306: G455–G465. A. Tsianakas, M. Ross, D. Bettenworth, T. Spieker, et al. 2014. Immune sup- 48. Han, X., K. Uchida, I. Jurickova, D. Koch, T. Willson, C. Samson, pression via glucocorticoid-stimulated monocytes: a novel mechanism to cope E. Bonkowski, A. Trauernicht, M. O. Kim, G. Tomer, et al. 2009. Granulocyte- with inflammation. J. Immunol. 193: 1090–1099. macrophage colony-stimulating factor autoantibodies in murine ileitis and pro- 69. Kurmaeva, E., D. Bhattacharya, W. Goodman, S. Omenetti, A. Merendino, gressive ileal Crohn’s disease. Gastroenterology 136: 1261–1271, e1–3. S. Berney, T. Pizarro, and D. V. Ostanin. 2014. Immunosuppressive monocytes: 49. Nylund, C. M., S. D’Mello, M. O. Kim, E. Bonkowski, J. Da¨britz, D. Foell, possible homeostatic mechanism to restrain chronic intestinal inflammation. J. J. Meddings, B. C. Trapnell, and L. A. Denson. 2011. Granulocyte macrophage- Leukoc. Biol. 96: 377–389.

SUPPLEMENTARY MATERIAL

Reprogramming of monocytes by GM-CSF contributes to regulatory immune functions during intestinal inflammation

Jan Däbritz, Toni Weinhage, Georg Varga, Timo Wirth, Karoline Walscheid, Anne Brockhausen, David Schwarzmaier, Markus Brückner, Matthias Ross, Dominik Bettenworth, Johannes Roth, Jan M. Ehrchen, Dirk Foell

University Hospital Münster Münster, Germany

SUPPLEMENTARY MATERIAL

SUPPLEMENTARY TABLES

Table S1. Computational Ascertainment of Regulatory Relationships (Inferred from Expression)

Pattern name P # Pattern

Up-regulated by GM-CSF STAT 5b 9.63 x 10-11 M00459 STAT 5a 3.44 x 10-8 M00457 c-Ets-2 binding sites 3.38 x 10-7 M00340 cut-like homeodomain protein 3.46 x 10-3 M00104 CCAAT/enhancer binding protein beta 4.84 x 10-2 M00109 androgen receptor 5.86 x 10-4 M00447 PEA3 2.29 x 10-3 M00655 C/EBPbeta 4.89 x 10-2 M00621

Down-regulated by GM-CSF LBP-1 1.54 x 10-2 M00644 NF-kappaB (p65) 7.19 x 10-6 M00052 c-Rel 1.94 x 10-5 M00053 NF-kappaB 5.41 x 10-5 M00774 activator protein 4 3.93 x 10-5 M00175 E2F 1.12 x 10-4 M00803 NF-kappaB (p50) 4.20 x 10-3 M00051 serum response factor 2.76 x 10-2 M00186 LEF1 6.32 x 10-5 M00805

2 SUPPLEMENTARY MATERIAL

Table S2. Complete list of up- and down-regulated genes.

Affymetrix identifier Gene symbol p-value N-fold 205582_s_at GGTLA1 0.0001693 50.50649347 213800_at CFH 0.01343855 44.90593032 218717_s_at LEPREL1 0.00119232 31.26424011 209909_s_at TGFB2 0.00633007 26.84033465 206932_at CH25H 0.00570894 25.42228437 1553311_at C20orf197 0.04907555 24.09862336 201904_s_at CTDSPL 0.00068324 20.2349671 205110_s_at FGF13 0.01835098 19.81260534 205743_at STAC 4.2567E-05 18.5919308 205987_at CD1C 8.1715E-05 18.39333098 206407_s_at CCL13 0.00013093 16.68245451 225987_at STEAP4 0.00599794 15.99445019 220407_s_at TGFB2 0.00332232 15.54808562 206749_at CD1B 0.00825223 15.48920616 235171_at --- 1.0164E-05 14.64152207 223374_s_at B3GALNT1 0.00616719 14.21784293 204529_s_at TOX 0.00012043 14.18535443 221019_s_at COLEC12 0.00037242 14.12670214 1555600_s_at APOL4 0.00946476 14.04685127 217504_at ABCA6 0.00196413 13.78119273 213906_at MYBL1 3.3347E-05 13.60347906 207819_s_at ABCB4 0.0149746 12.31540717 201906_s_at CTDSPL 0.02083693 12.01831432 220187_at STEAP4 0.00566497 11.88489592 230323_s_at TMEM45B 0.00368398 11.74804279 206637_at P2RY14 0.00047201 11.24085437 215784_at CD1E 0.00036557 9.842376733 216375_s_at ETV5 0.0173255 9.690221163 213265_at PGA3 /// PGA4 /// PGA5 0.00541377 9.501098446 202565_s_at SVIL 3.1281E-05 8.967745921 223939_at SUCNR1 0.00069322 8.966488497 240232_at --- 0.00422387 8.593775661 205404_at HSD11B1 0.00033605 8.415616905 1554519_at CD80 0.00016535 8.351698565 210549_s_at CCL23 0.00221019 8.328243908 238439_at ANKRD22 0.00058313 7.987773267 210548_at CCL23 0.02167974 7.849443119 229568_at MOBKL2B 8.2151E-07 7.821421311 204044_at QPRT 0.00550087 7.615064549 211379_x_at B3GALNT1 0.00157536 7.570312236 229566_at LOC645638 0.00379492 7.570145619 225646_at CTSC 1.6407E-05 7.545684655 203650_at PROCR 3.0279E-05 7.481681815 214146_s_at PPBP 0.00149519 7.231768627 202566_s_at SVIL 0.00073725 6.975562537 226844_at MOBKL2B 8.6574E-05 6.820100758 231234_at CTSC 0.00062037 6.764636072

3 SUPPLEMENTARY MATERIAL

228121_at TGFB2 0.00162072 6.508429981 226226_at TMEM45B 0.01204346 6.45310024 210325_at CD1A 0.00073108 6.410251988 209795_at CD69 0.00247926 6.394661483 220144_s_at ANKRD5 0.00079353 6.077013983 242541_at ABCA9 0.00175565 5.840155931 215101_s_at CXCL5 0.02476789 5.752733193 219265_at MOBKL2B 3.0354E-05 5.718559619 223961_s_at CISH 0.00060494 5.684694152 1555689_at CD80 0.00077794 5.653435999 231698_at FLJ36848 0.0031729 5.570477754 207900_at CCL17 0.00336947 5.533766308 223377_x_at CISH 0.00026023 5.229202004 219667_s_at BANK1 0.01092215 5.212358558 225647_s_at CTSC 0.00016548 5.176559771 210375_at PTGER3 0.03294313 5.140865608 207008_at IL8RB 0.00226392 5.083832786 203680_at PRKAR2B 0.00150321 5.058741774 242714_at --- 0.00218649 5.03093753 206181_at SLAMF1 0.04087152 4.964063377 222915_s_at BANK1 0.02367735 4.859928302 205798_at IL7R 0.00144021 4.839081662 205220_at GPR109B 0.00788387 4.672372409 215388_s_at CFH /// CFHR1 0.00996691 4.654094368 213189_at MINA 0.00032132 4.537621679 227856_at C4orf32 2.1238E-05 4.534985676 219890_at CLEC5A 0.01078711 4.518367139 221223_x_at CISH 0.00054497 4.506457024 239196_at ANKRD22 0.00348723 4.492226897 210233_at IL1RAP 0.00674947 4.373316171 228450_at PLEKHA7 0.0008014 4.333857776 206983_at CCR6 0.00064013 4.329658646 201577_at NME1 0.00034558 4.293962423 209994_s_at ABCB1 /// ABCB4 0.00030352 4.27137836 207826_s_at ID3 4.0282E-05 4.1913959 39248_at AQP3 0.00402339 4.17568041 204174_at ALOX5AP 0.00329028 4.131184206 229733_s_at --- 0.01119593 4.119293143 224480_s_at MAG1 0.00061558 4.073673324 227620_at SLC44A1 0.00065128 4.019685784 224596_at SLC44A1 0.0004088 3.845367812 1568964_x_at SPN 0.00713234 3.838904836 226218_at IL7R 0.00313406 3.718450837 228485_s_at SLC44A1 0.00126762 3.718450837 242417_at LOC283278 0.0022643 3.702809602 222364_at SLC44A1 0.01630046 3.683552983 1558662_s_at BANK1 0.00768653 3.677144378 216913_s_at RRP12 0.02004205 3.677144378 207339_s_at LTB 0.00193252 3.568058872 1559065_a_at CLEC4G 0.02320381 3.552553229

4 SUPPLEMENTARY MATERIAL

203349_s_at ETV5 0.00106768 3.552553229 213188_s_at MINA 0.00047061 3.546338607 220581_at C6orf97 0.0007264 3.520186364 224595_at SLC44A1 0.00037046 3.504545129 222457_s_at LIMA1 0.01282761 3.499339724 207113_s_at TNF 0.0029579 3.449986705 206682_at CLEC10A 0.00289933 3.436876459 228486_at SLC44A1 0.00040979 3.430897019 222500_at PPIL1 1.521E-05 3.410050379 205153_s_at CD40 0.02377785 3.405097868 203348_s_at ETV5 0.00028432 3.35914557 211668_s_at PLAU 0.00074962 3.347587115 209567_at RRS1 0.02196573 3.298850568 205786_s_at ITGAM 0.0002406 3.260271937 205128_x_at PTGS1 0.00328404 3.254214991 205479_s_at PLAU 0.00015452 3.186546321 215813_s_at PTGS1 0.00802737 3.186546321 244439_at SPRED1 0.00542101 3.170576976 215346_at CD40 0.01173246 3.155814315 238846_at TNFRSF11A 0.00061909 3.155814315 205659_at HDAC9 0.0058376 3.119293715 203373_at SOCS2 0.00485244 3.114020837 209679_s_at LOC57228 0.01483812 3.096905317 203372_s_at SOCS2 0.03734608 3.092846087 208029_s_at LAPTM4B 0.01760473 3.074502127 221830_at RAP2A 0.00202405 3.064500222 217892_s_at LIMA1 0.02583751 3.064500222 220865_s_at PDSS1 0.0217345 3.046352168 230102_at ETV5 0.00299339 3.036289948 205282_at LRP8 0.00157745 3.020320603 217097_s_at PHTF2 0.01550453 2.978804177 210839_s_at ENPP2 0.03517238 2.937010699 202613_at CTPS 0.00083074 2.931805294 226837_at SPRED1 0.00054464 2.931805294 213268_at CAMTA1 0.00738793 2.919895179 227607_at STAMBPL1 0.00259953 2.914243848 209803_s_at PHLDA2 0.01473078 2.901368672 221724_s_at CLEC4A 0.00339806 2.900627363 221053_s_at TDRKH 0.02137839 2.885727437 204829_s_at FOLR2 0.01145529 2.868611917 211864_s_at FER1L3 0.01082703 2.846575179 229132_at MINA 0.00479289 2.841718364 202314_at CYP51A1 0.03331124 2.838231571 214039_s_at LAPTM4B 0.0075295 2.838231571 205831_at CD2 0.00342397 2.802259969 218984_at PUS7 0.00058368 2.786754325 204601_at N4BP1 0.00060159 2.783438078 208433_s_at LRP8 0.00166523 2.783438078 201798_s_at FER1L3 0.00681365 2.740327878 212110_at SLC39A14 0.00410054 2.735471064

5 SUPPLEMENTARY MATERIAL

209499_x_at TNFSF12-TNFSF13 /// TNFSF13 0.00352244 2.706381025 239648_at DCUN1D3 0.00083273 2.706381025 207419_s_at RAC2 0.01000203 2.673075272 205227_at IL1RAP 0.00047687 2.669565041 211495_x_at TNFSF12-TNFSF13 /// TNFSF13 0.00435195 2.655948555 202047_s_at CBX6 0.00210421 2.643243924 218195_at C6orf211 0.00078258 2.614601002 206148_at IL3RA 0.03245599 2.604665293 205016_at TGFA 0.0282456 2.600133725 203234_at UPP1 0.00170427 2.588279885 227333_at --- 0.00055681 2.580106823 241937_s_at WDR4 0.00043505 2.542075518 220578_at ADAMTSL4 0.03093454 2.542075518 201487_at CTSC 0.00107415 2.536996624 205505_at GCNT1 0.01054675 2.536996624 225438_at NUDCD1 0.00112599 2.536996624 224634_at GPATCH4 0.0064642 2.529370887 209392_at ENPP2 0.00658615 2.474406848 239761_at GCNT1 0.00758671 2.474406848 228955_at --- 0.00395472 2.474406848 202551_s_at CRIM1 0.01264565 2.473859523 204393_s_at ACPP 0.04665685 2.473859523 214487_s_at RAP2A /// RAP2B 0.00202447 2.473859523 201700_at CCND3 0.00077809 2.414950524 211974_x_at RBPJ 6.6175E-05 2.414950524 32069_at N4BP1 0.00226608 2.411269747 201797_s_at VARS 0.00074228 2.407324788 203119_at CCDC86 0.0022327 2.407324788 228372_at C10orf128 0.0079918 2.371840325 218681_s_at SDF2L1 0.0001793 2.359986486 209500_x_at TNFSF12-TNFSF13 /// TNFSF13 0.00045191 2.308703224 210314_x_at TNFSF12-TNFSF13 /// TNFSF13 0.00106288 2.308703224 228231_at --- 0.00190143 2.308703224 1558517_s_at LRRC8C 0.00477875 2.301077487 212295_s_at SLC7A1 3.6157E-05 2.249794226 217809_at BZW2 0.00038262 2.249794226 223533_at LRRC8C 0.00786109 2.249794226 226923_at SCFD2 0.00024058 2.246113448 204744_s_at IARS 0.00094695 2.194830187 219551_at EAF2 0.0061163 2.194830187 225173_at ARHGAP18 0.00555202 2.194830187 235085_at DKFZp761P0423 0.00417984 2.194830187 224516_s_at CXXC5 0.0237143 -2.143546925 207275_s_at ACSL1 0.02708995 -2.194830187 208918_s_at NADK 0.00039223 -2.194830187 221731_x_at VCAN 0.03341251 -2.194830187 225598_at SLC45A4 0.00050444 -2.194830187 204043_at TCN2 0.02674388 -2.249794226 212658_at LHFPL2 0.00319037 -2.249794226 219367_s_at --- 0.00319825 -2.249794226

6 SUPPLEMENTARY MATERIAL

204647_at HOMER3 0.00536832 -2.301077487 208981_at PECAM1 2.2395E-05 -2.301077487 235457_at MAML2 0.00120286 -2.301077487 232231_at RUNX2 0.00344245 -2.308703224 204620_s_at VCAN 0.03173514 -2.352360749 206472_s_at TLE3 0.01436113 -2.352360749 208161_s_at ABCC3 0.01987907 -2.356041526 207574_s_at GADD45B 0.00088207 -2.359986486 208982_at PECAM1 0.00578303 -2.359986486 225956_at LOC153222 0.00166546 -2.359986486 215211_at LOC730092 0.00928518 -2.371840325 233955_x_at CXXC5 0.01714169 -2.407324788 225755_at KLHDC8B 0.00224667 -2.411269747 207167_at IGSF2 0.00028724 -2.414950524 222996_s_at CXXC5 0.00922852 -2.423123587 200897_s_at PALLD 0.00126053 -2.439508994 208983_s_at PECAM1 0.0004589 -2.462288827 209298_s_at ITSN1 0.0315577 -2.466233786 202391_at BASP1 0.00307102 -2.474406848 235556_at --- 0.00478104 -2.478087626 213397_x_at RNASE4 0.03852373 -2.525142784 240137_at --- 0.00445196 -2.529370887 200907_s_at PALLD 0.00047184 -2.536996624 208919_s_at NADK 0.00671747 -2.536996624 224817_at SH3PXD2A 0.00113436 -2.588279885 214581_x_at TNFRSF21 0.04706226 -2.589765368 204526_s_at TBC1D8 0.00046135 -2.639015822 212390_at LOC727893 /// PDE4DIP 0.00189182 -2.643243924 219622_at RAB20 0.00129368 -2.643243924 213241_at PLXNC1 0.00077876 -2.651416986 208626_s_at VAT1 0.00087169 -2.667802394 215646_s_at VCAN 0.04737497 -2.667802394 205382_s_at CFD 0.00781013 -2.669565041 213109_at TNIK 0.01434917 -2.669565041 214129_at LOC727942 0.01629392 -2.669565041 216705_s_at ADA 0.00096118 -2.702152923 225631_at KIAA1706 4.9247E-05 -2.702152923 221563_at DUSP10 0.00354435 -2.706381025 228340_at TLE3 0.00588266 -2.706381025 33304_at ISG20 0.00106415 -2.706381025 204639_at ADA 0.00111826 -2.710912594 221081_s_at DENND2D 0.00595754 -2.710912594 235146_at --- 0.00477377 -2.710912594 200766_at CTSD 0.00318172 -2.719085656 203505_at ABCA1 0.00193347 -2.719085656 217889_s_at CYBRD1 0.02209698 -2.719085656 225762_x_at LOC284801 0.00161705 -2.728474039 241742_at PRAM1 0.00486406 -2.74729593 209122_at ADFP 5.9025E-05 -2.765290024 209831_x_at DNASE2 0.00033775 -2.765290024

7 SUPPLEMENTARY MATERIAL

218729_at LXN 0.03165556 -2.769821592 213388_at LOC727942 0.0036434 -2.774049695 227969_at LOC400960 0.00964696 -2.779322573 41660_at CELSR1 0.00257308 -2.786754325 228933_at NHS 0.00893871 -2.79161114 218856_at TNFRSF21 0.034557 -2.79161114 215111_s_at TSC22D1 0.00113387 -2.832958693 239287_at --- 0.01385525 -2.838231571 213006_at CEBPD 0.00376504 -2.842347076 221246_x_at TNS1 0.00562485 -2.842347076 213061_s_at NTAN1 0.00041726 -2.846575179 1552540_s_at IQCD 0.0172296 -2.868611917 207704_s_at GAS7 0.01181464 -2.900627363 209263_x_at TSPAN4 0.00354284 -2.900627363 213222_at PLCB1 4.0846E-05 -2.900627363 219371_s_at KLF2 0.01766364 -2.901368672 228325_at KIAA0146 0.00328205 -2.927520916 232530_at PLD1 0.00626143 -2.937010699 213902_at ASAH1 0.000186 -2.968621278 205801_s_at RASGRP3 0.00778405 -2.983215066 221042_s_at CLMN 0.00078339 -3.009536183 222857_s_at KCNMB4 0.0339815 -3.033768216 215223_s_at SOD2 0.02198291 -3.03598381 209264_s_at TSPAN4 0.01110871 -3.036289948 216894_x_at CDKN1C 0.02188963 -3.041941278 214099_s_at LOC727927 /// PDE4DIP 0.00261202 -3.046352168 230233_at --- 0.00923389 -3.050883736 211571_s_at VCAN 0.02136148 -3.064500222 1552701_a_at COP1 0.00277034 -3.075284642 222838_at SLAMF7 0.00908658 -3.086355967 213062_at NTAN1 0.00216046 -3.092677214 1553787_at C11orf45 0.00304653 -3.103958617 1555419_a_at ASAH1 0.00243214 -3.103958617 213258_at TFPI 0.00197907 -3.103958617 211067_s_at GAS7 0.00573692 -3.108815432 201212_at LGMN 4.5658E-05 -3.114020837 218999_at TMEM140 0.00051421 -3.12340922 228570_at BTBD11 0.03948206 -3.12340922 227220_at NFXL1 0.00212586 -3.129466166 205466_s_at HS3ST1 0.00591447 -3.13085217 221022_s_at PMFBP1 0.00053595 -3.13419364 226534_at KITLG 0.00180129 -3.137639228 203504_s_at ABCA1 0.00505662 -3.186546321 225757_s_at CLMN 0.01857931 -3.18892249 213839_at KIAA0500 0.00197276 -3.201140109 209969_s_at STAT1 0.03399577 -3.239658426 230139_at --- 0.01517874 -3.251512265 226869_at MEGF6 0.04222651 -3.256591159 1552846_s_at RAB42 0.00010919 -3.26427721 229296_at --- 0.0013362 -3.284450014

8 SUPPLEMENTARY MATERIAL

228274_at SDSL 0.00098447 -3.286620086 203510_at MET 0.02533322 -3.337524895 202191_s_at GAS7 0.00582831 -3.337524895 225102_at MGLL 0.00323484 -3.337524895 210664_s_at TFPI 0.00529179 -3.354078789 221747_at TNS1 0.0014137 -3.35914557 205888_s_at JAKMIP2 0.00030406 -3.404471364 211026_s_at MGLL 0.0004981 -3.404471364 222877_at --- 0.00032681 -3.449986705 229686_at P2RY8 9.4761E-05 -3.482202253 204533_at CXCL10 0.00675986 -3.493838214 219534_x_at CDKN1C 0.04093356 -3.513094833 202748_at GBP2 0.00222671 -3.531675536 219403_s_at HPSE 0.00289568 -3.552553229 201911_s_at FARP1 0.01115936 -3.552747213 200665_s_at SPARC 0.00275603 -3.557410043 202192_s_at GAS7 0.00096308 -3.595037674 203979_at CYP27A1 0.01277469 -3.654801502 204972_at OAS2 0.0431925 -3.678280105 213624_at SMPDL3A 0.00021902 -3.683552983 213348_at CDKN1C 0.00351901 -3.702809602 213182_x_at CDKN1C 0.00418554 -3.775335086 204961_s_at NCF1 /// NCF1B /// NCF1C 0.00286032 -3.79562216 202074_s_at OPTN 0.00442087 -3.799749906 1558397_at --- 0.00210786 -3.833809356 214992_s_at DNASE2 0.00229182 -3.833809356 222881_at HPSE 0.00118532 -3.858644991 204146_at RAD51AP1 0.00658881 -3.923098701 205552_s_at OAS1 0.04871288 -3.978792852 206701_x_at EDNRB 0.00522793 -4.019685784 227618_at --- 0.00110585 -4.081034879 214084_x_at NCF1C 0.00046392 -4.121363174 229620_at SEPP1 0.02556105 -4.124566021 203920_at NR1H3 0.00048322 -4.129613862 209540_at IGF1 0.0117016 -4.135412309 202086_at MX1 0.02266035 -4.175051698 219895_at FAM70A 0.00463366 -4.183601402 209960_at HGF 0.00197154 -4.192167217 228170_at OLIG1 0.00325159 -4.204673079 212912_at RPS6KA2 0.00092461 -4.218903207 206545_at CD28 0.00808069 -4.225234306 221748_s_at TNS1 0.00091278 -4.256126886 208268_at ADAM28 0.00282439 -4.28972232 206756_at CHST7 0.01187277 -4.361874194 210997_at HGF 0.01408447 -4.42052809 201185_at HTRA1 0.01263869 -4.426010548 222173_s_at TBC1D2 4.8979E-05 -4.432419153 201141_at GPNMB 0.00048491 -4.462995405 202464_s_at PFKFB3 0.00060661 -4.500219085 212226_s_at PPAP2B 0.00374695 -4.577949974

9 SUPPLEMENTARY MATERIAL

217028_at CXCR4 1.8921E-06 -4.602154975 209030_s_at CADM1 0.00178361 -4.610147163 202007_at NID1 0.00027978 -4.687878052 214770_at MSR1 0.0407896 -4.693308494 238780_s_at --- 0.00350254 -4.734201426 219452_at DPEP2 0.00030538 -4.771187956 1569403_at --- 0.00119758 -4.815535587 219863_at HERC5 0.01161955 -4.818009986 204271_s_at EDNRB 0.00333527 -4.860477301 226301_at C6orf192 0.00014043 -4.956175251 226702_at LOC129607 0.04907369 -4.957194803 204273_at EDNRB 0.00179274 -4.978295298 226189_at ITGB8 0.00641868 -4.994433515 209541_at IGF1 0.0008278 -5.000122456 204747_at IFIT3 0.03743987 -5.012158842 238581_at GBP5 0.00937433 -5.038506992 224694_at ANTXR1 0.00228002 -5.124914039 212230_at PPAP2B 0.00037949 -5.168669852 208450_at LGALS2 0.031045 -5.244043989 205568_at AQP9 0.00420534 -5.318670262 206134_at ADAMDEC1 0.00524947 -5.324856039 205997_at ADAM28 0.00417381 -5.347695897 225207_at PDK4 0.00456378 -5.412762051 211919_s_at CXCR4 0.00122355 -5.539643184 209355_s_at PPAP2B 0.01837133 -5.652960981 205844_at VNN1 0.00233577 -5.665917386 219697_at HS3ST2 0.00275271 -5.707039394 217897_at FXYD6 0.00116334 -5.713018834 209160_at AKR1C3 0.02049248 -5.848356173 1554018_at GPNMB 0.00010869 -5.927528927 221061_at PKD2L1 0.00666769 -6.007580849 226560_at --- 0.00426252 -6.058693564 239675_at LOC283143 0.00164347 -6.207917234 205695_at SDS 0.00051795 -6.219219895 209201_x_at CXCR4 0.00112863 -6.24681844 221584_s_at KCNMA1 0.02349066 -6.251570778 205242_at CXCL13 0.01926834 -6.37784498 201005_at CD9 0.01166181 -6.389936088 238727_at LOC440934 0.00218154 -6.394661483 203066_at GALNAC4S-6ST 0.00026469 -6.853628479 210163_at CXCL11 0.04645725 -7.105225164 219525_at SLC47A1 0.0059081 -7.435807023 216950_s_at FCGR1A 0.02233428 -7.679580716 206392_s_at RARRES1 0.01824988 -8.105591263 209031_at CADM1 0.00102294 -8.483319219 221872_at RARRES1 0.01653019 -8.779320747 214511_x_at FCGR1B 0.01078419 -8.990724541 207092_at LEP 0.0002238 -9.069971353 210998_s_at HGF 0.03097396 -11.43966736 204698_at ISG20 0.00039274 -12.26609988

10 SUPPLEMENTARY MATERIAL

205922_at VNN2 0.00516081 -14.62125494 209687_at CXCL12 0.00874068 -16.90131603 205960_at PDK4 0.04772079 -19.67973809 224215_s_at DLL1 0.00908821 -25.91147956

11 SUPPLEMENTARY MATERIAL

Table S3. Primer sequences for RT-PCR

Species Gene Forward Primer Sequence (5’-3’) Reverse Primer Sequence (5’-3’)

Human ARG1 GTG GAA ACT TGC ATG GAC AAC CCT GGC ACA TCG GGA ATC TTT Human CCL1 CTC ATT TGC GGA GCA AGA GAT GGA GCT GGT ATT TCT GTA ACA CA Human CD163 TCA GTG CAG AAA TGG CCA ACA GA CGA CGA AAA TGG CCA ACA GA Human CD1a TCA TCT TGG CGG TGA TAG TG GAG GAG GCT CAT GGT GTG TC Human CD1c TGA ATT GGA TTG CCT TGG TAG AGG GGG AAG AGT CTC ACA GG Human CD1e AGT TAC CCT GGT CAT ATT GGT TG GGC TCC CAT GAG AAA GAC AG Human CD206 AGG GGG AAG AGT CTC ACA GG AAA GTC CAA TTC CTC GAT GGT G Human CD209 CAC CTG GAT GGG ACT TTC AG TGT TGG GCT CTC CTC TGT TC Human COX2 GGG TTG CTG GTG GTA GGA ATG AGC ATA AAG CGT TTG CGG TAC TC Human CXCL11 CAG AAT TCC ACT GCC CAA AGG GTA AAC TCC GAT GGT AAC CAG CC Human CXCL13 CTT CCC TTA TCC CTG CTC TGG A CCA TCA GCT CCT GCA AGG TTA TT Human CXCL5 GAT CCA GAA GCC CCT TTT CTA AAG AGA GAC CTC CAG AAA ACT TCT CTG C Human G-CSF ACA AGC AGA GGT GGC CAG AG CAA ACC ATG TCC CAA AAG TCT TAA G Human GMCSF GTC ATC TCA GAA ATG TTT GAC CTC C GTG CTG TTT GTA GTG GCT GGC Human IFNα GCC TCG CCC TTT GCT TTA CT CTG TGG GTC TCA GGG AGA TCA Human IFNβ ATG ACC AAC AAG TGT CTC CTC C GGA ATC CAA GCA AGT TGT AGC TC Human IFNγ TCG GTA ACT GAC TTG AAT GTC CA TCG CTT CCC TGT TTT AGC TGC Human IL12 ATG GCC CTG TGC CTT AGT AGT AGC TTT GCA TTC ATG GTC TTG A Human IL18 TTC AAC TCT CTC CTG TGA GAA CA ATG TCC TGG GAC ACT TCT CTG Human IL1R2 TCC TGA CAT TTG CCC ATG AAG GGA AAT GAT CAC AGG AAT GGT CTC Human IL23 GGA CAA CAG TCA GTT CTG CTT CAC AGG GCT ATC AGG GAG C Human iNOS CCT ACC AAC TGA CGG GAG ATG ATG GCC GAC CTG ATG TTG C Human IRF4 GCT GAT CGA CCA GAT CGA CAG CGG TTG TAG TCC TGC TTG C Human IRF5 TTC TCT CCT GGG CTG TCT CTG CTA TAC AGC TAG GCC CCA GGG Human MRC1 AAG GCG GTG ACC TCA CAA G AAA GTC CAA TTC CTC GAT GGT G Human TRAIL CCG TCA GCT CGT TAG AAA GAC TCC A GCC CAC TCC TTG ATG ATT CCC AGG Human CCL13 ATC TCC TTG CAG AGG CTG AA CTT CTC CTT TGG GTC AGC AC Human CCL23 TTT GAA ACG AAC AGC GAG TG CAG CAT TCT CAC GCA AAC C Human CD1c TGA ATT GGA TTG CCT TGG TAG AGG GGG AAG AGT CTC ACA GG Human CD80 CTG CTT TGC CCC AAG ATG C CAG ATC TTT TCA GCC CCT TGC Human CFH AAG CGC AGA CCA CAG TTA CA TCA AGC TGG AGA GGG ATG AC Human CXCL10 GCA AGC CAA TTT TGT CCA CG ACA TTT CCT TGC TAA CTG CTT TCA G Human CXCL12 CCA ACG TCA AGC ATC TCA AA TAG CTT CGG GTC AAT GCA C Human GGTLA CAG GGG TCG AAG CTA GTG AT CTC TCA GGT CAA AGC CAA GC Human IL10 GCT GAG AAC CAA GAC CCA GAC A CGG CCT TGC TCT TGT TTT CA Human IL1b GCG GCC AGG ATA TAA CTG ACT TC TCC ACA TTC AGC ACA GGA CTC TC Human IL4 CCA ACT GCT TCC CCC TCT G TCT GTT ACG GTC AAC TCG GTG Human IL6 CAA GAA GGG TTT TTG TGA CTG AAT C TCC TTG TTT TGC TCC AAC ACT AAT C Human IL8 CTT GTT CCA CTG TGC CTT GGT T GCT TCC ACA TGT CCT CAC AAC AT Human LTB CCA GAA ACA GAT CTC AGC CCC AAC GCC TGT TCC TTC GTC G Human TGFb ATG GTG TGT GAG ACG TTG ACT GA CGA GAG CCT GTC CAG ATG CT Human TNFa CTT CTC GAA CCC CGA GTG AC TGA GGT ACA GGC CCT CTG ATG Human RPL AGG TAT GCT GCC CCA CAA AAC TGT AGG CTT CAG ACG CAC GAC Mouse CD80 AAA TAT GGA GAT GCT CAC GTG TCA G CTG TTA TTA CTG CGC CGA ATC C Mouse CXCL10 TTC ACC ATG TGC CAT GCC GAA CTG ACG AGC CTG AGC TAG G Mouse CXCL11 AAA ATG GCA GAG ATC GAG AAA GC CAG GCA CCT TTG TCG TTT ATG AG Mouse CXCL12 ATC CTC AAC ACT CCA AAC TGT G TTT CTC CAG GTA CTC TTG GAT C Mouse CXCL13 CAT AGA TCG GAT TCA AGT TAC GCC TCT TGG TCC AGA TCA CAA CTT CA Mouse GMCSF TGC TTT TGT GCC TGC GTA ATG TCC AAG CTG AGT CAG CGT TTT C Mouse LTB AAC ACT TCC CCT CGA GC ATG GCC AGC AGT AGC ATT GC Mouse TGFb GGA CCC TGC CCC TAT ATT TGG TGT TGC AGG TCA TTT AAC CAA GTG Mouse IL1β TGT CTT GGC CGA GGA CTA AGG TGG GCT GGA CTG TTT CTA ATG C Mouse ARG1 CTC CAA GCC AAA GTC CTT AGA G GGA GCT GTC ATT AGG GAC ATC A Mouse MRC1 AGA CGA AAT CCC TGC TAC TGA A TAG AAA GGA ATC CAC GCA GTC T Mouse TNFα AGA AAC ACA AGA TGC TGG GAC AGT CCT TTGCAG AAC TCA GGAATG G Mouse RPL TGG TCC CTG CTG CTC TCA AG GGC CTT TTC CTT CCG TTT CTC

12 SUPPLEMENTARY MATERIAL

Table S4. Antibodies for flow cytometry

Species Molecule Clone Manufacturer

Human CD9 MEM-61 Immunotools, Hamburg, Germany Human CD80 2D10 BD Pharmingen, Heidelberg, Germany Human CD4 RPA-T4 Biolegend, San Diego, CA, USA Human CD25 BC96 Biolegend, San Diego, CA, USA Human Il1β CRM56 eBioscience, San Diego, CA, USA Human TNFα MAb11 eBioscience, San Diego, CA, USA Human IL10 JES3-9D7 Biolegend, San Diego, CA, USA Human CD206 19.2 eBioscience, San Diego, CA, USA Human Beta7 FIB504 Biolegend, San Diego, CA, USA

Human CCR1 5F10B29 Biolegend, San Diego, CA, USA

Human CCR2 K036C2 Biolegend, San Diego, CA, USA

Human CCR4 L291H4 Biolegend, San Diego, CA, USA

Human CCR6 G034E3 Biolegend, San Diego, CA, USA

Human CCR7 G043H7 Biolegend, San Diego, CA, USA

Human CCR9 L053E8 Biolegend, San Diego, CA, USA

Human CX3CR1 528728 R&D Systems, Minneapolis, MN, USA

Human Foxp3 259D Biolegend, San Diego, CA, USA Mouse CD3e 145-2C11 BD Pharmingen, Heidelberg, Germany Mouse CD28 37.51 BD Pharmingen, Heidelberg, Germany Mouse CD4 RM4-5 Biolegend, San Diego, CA, USA Mouse CD11b M1/70 Biolegend, San Diego, CA, USA Mouse CD45.1 A20 Biolegend, San Diego, CA, USA Mouse Foxp3 FJK-16s eBioscience, San Diego, CA, USA

13