Reprogramming of Monocytes by GM-CSF Contributes...

The Journal of Immunology

Reprogramming of Monocytes by GM-CSF Contributes toRegulatory Immune Functions during IntestinalInflammation

Jan Dabritz,*,†,‡,x,1 Toni Weinhage,*,1 Georg Varga,* Timo Wirth,* Karoline Walscheid,*Anne Brockhausen,{,‖ David Schwarzmaier,* Markus Br!uckner,# 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-CSFmediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatorypotential 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 toincrease adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbialstimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell–induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production ofIL-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-neously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limitexcessive inflammation. The Journal of Immunology, 2015, 194: 2424–2438.

O ur concepts of immunology have changed dramaticallyover the past decades. The postulates of primary func-tions assigned to innate or adaptive immunity have been

challenged by the recognition of a complex interplay between thedifferent cellular and humoral factors that all together constituteour immune system. This helped in understanding how we areprotected from infections, but it also enabled discovering keyaspects of autoimmunity and chronic inflammation including reg-ulatory mechanisms that counteract a perpetuated immune acti-vation. Although different functions of adaptive immune cells,including regulatory T cells (Tregs), are already consolidated, ourunderstanding of different functions of innate immune cells hasonly recently been enriched. As an example, phagocytes were

traditionally seen solely as effector cells of innate immunitypromoting host defense and driving chronic inflammation. It isnow accepted that monocytes can differentiate into macrophageswith various activation patterns ranging from classically activatedproinflammatory to anti-inflammatory phenotypes. These cells(often referred to as M1 and M2 macrophages) represent the outermargins of a broad spectrum of numerous activation and differ-entiation patterns of heterogeneous monocyte-derived cells (1–3).As the concepts of immunity evolve, the pathophysiology of

chronic inflammatory diseases is also being revisited. As a strikingexample, our view of Crohn’s disease (CD) is constantly chal-lenged. Traditionally, CD has been associated with a Th1 cytokineprofile. In addition, because CD is a chronic granulomatous dis-

*Department of Pediatric Rheumatology and Immunology, University Children’sHospital M!unster, M!unster 48149, Germany; †Interdisciplinary Center of ClinicalResearch, University Hospital M!unster, M!unster 48149, Germany; ‡GastrointestinalResearch in Inflammation & Pathology, Murdoch Children’s Research Institute,The Royal Children’s Hospital Melbourne, Parkville 3052, Victoria, Australia;xDepartment of Pediatrics, University of Melbourne, Melbourne Medical School,Parkville 3052, Victoria, Australia; {Department of Dermatology, UniversityHospital M!unster, M!unster 48149, Germany; ‖Institute of Immunology, UniversityHospital M!unster, M!unster 48149, Germany; and #Department of Medicine B,University Hospital M!unster, M!unster 48149, Germany1J.D. and T. Weinhage contributed equally and should be considered cofirst authors.

Received for publication June 11, 2014. Accepted for publication January 4, 2015.

This work was supported by the Broad Medical Research Program of the Eli andEdythe Broad Foundation (Grant IBD0201 to D.F., J.D., and J.M.E.), the GermanResearch Foundation (Grant DFG DA1161/4-1 to J.D. and D.F., Grant DFG SU195/3-2 to G.V., Grant DFG SF1009B08 to M.B.), the Innovative Medical ResearchProgram of the University of M!unster (Grants IMF DA120904 and DA3!U21003 toJ.D. and D.F.), the Interdisciplinary Center for Clinical Research of the University ofM!unster (Grant IZKF Eh2/019/11 to J.M.E.), the European Union’s Seventh Frame-work Programme (Grant EC-GA305266 ‘MIAMI’ to D.F.), and a research fellowshipfrom the German Research Foundation (Grant DFG DA1161/5-1 to J.D.).

Portions of this work were presented at the 50th Digestive Disease Week AnnualMeeting, May 30–June 4, 2009, Chicago, IL and the 51st Digestive Disease WeekAnnual Meeting, May 1–5, 2010, New Orleans, LA.

J.D. and D.F. developed the concept, designed the experiments, and supervised theexperiments; J.M.E., G.V., and J.R. gave technical support and conceptual advice;M.R., G.V., and J.D. obtained ethical approval from the competent animal welfareauthorities; J.D., T. Weinhage, T. Wirth, K.W., A.B., and D.S. performed the experi-ments and collected data; M.R., G.V., M.B., D.B., and T. Wirth helped with animalmodels of experimental colitis; J.D., T. Weinhage, G.V., and D.F. analyzed the dataand interpreted results; J.D. wrote the manuscript; and each author has approved thefinal version of the report and takes full responsibility for the manuscript.

The sequences presented in this article have been submitted to the Gene Expres-sion Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession numberGSE63662.

Address correspondence and reprint requests to Dr. Jan Dabritz, Department of PediatricRheumatology and Immunology, University Children’s Hospital M!unster, Rontgen-strasse 21, M!unster 48149, Germany. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: CD, Crohn’s disease; DSS, dextran sulfate sodium;GCsM, glucocorticoid-stimulated monocyte; GMaM, GM-CSF–activated monocyte;LPMC, lamina propria mononuclear cell; LTB4, leukotriene B4; MEICS, murineendoscopic score of colitis severity; MFI, mean fluorescence intensity; MLN, mes-enteric lymph node; qRT-PCR, quantitative real-time RT-PCR; ROS, reactive oxygenspecies; Treg, regulatory T cell.

Copyright! 2015 by TheAmerican Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401482

ease and anti-inflammatory therapies targeting innate immunityhave proved effective, it was a paradigm that overactive phag-ocytes are involved. More recently, however, emerging evidencehas consolidated the view of CD as a form of innate immunode-ficiency (4–6). Central to this hypothesis were the observations ofdiminished neutrophil accumulation in patients with CD withimpaired clearance of bacteria from tissues (7, 8). The underlyingproblem appeared to be a primary immunodeficiency of macro-phages, which secreted insufficient concentrations of proinflammatorycytokines and chemokines upon bacterial challenge (9). The viewof defective macrophage functions in CD is further supported byan inappropriate mucosal healing (10, 11). Resolution of inflam-mation and healing relies on the infiltration of monocytes ascrucial regulators of tissue repair processes (12–14).Given the changing concepts on immunity and inflammation,

changes in therapeutic strategies appear as a logical consequence.As a therapy that could help in overcoming insufficient macrophagefunctions, GM-CSF has been shown to alleviate acute dextran sulfatesodium (DSS)-induced colitis in mice (15, 16). Even more important,it is conceivable that GM-CSF–driven modulation of innate immunecells involved in mucosal repair and/or dampening of inflammatoryreactions might contribute to the benefits of GM-CSF therapy ob-served in some CD patients (17). These findings may link the novelconcepts of monocyte biology with that of CD pathogenesis, becauserecent developments in immunology and genetics suggest thatmonocytes and their derivative cells play an important role in thepathophysiology of CD. It is noteworthy that blood monocytes are theexclusive source of macrophages in inflamed intestinal mucosa (18).Undoubtedly, monocytes carry out specific effector functions

during inflammation (19). Recent studies underpin the dualfunction of monocytes: on one hand, the impaired monocytefunction initiating CD, and on the other hand, the overactivation ofmonocytes and adaptive immunity maintaining the disease (20).Cells of the monocyte/macrophage lineage are characterized byconsiderable diversity and plasticity (21). Furthermore, monocytescan drive modulation of adaptive immunity by regulating T cellresponses (22). GM-CSF functions both as a growth factor formyeloid progenitors and as a cytokine acting directly on maturingcells. Data from animal models indicate an important role in in-flammation and autoimmunity, with varying consequences thatlikely depend on the disease-specific context (23).We thus hypothesized that GM-CSF might activate monocytes

in a way that modulates their function during intestinal inflam-mation. To this end, we chose an unbiased but comprehensiveapproach taking all potential functions of GM-CSF–activatedmonocytes (GMaMs) into account (gene expression, innate im-mune functions, interplay with adaptive immunity, wound heal-ing) rather than focusing on polarizing edges. We show in thisarticle that beneficial effects of GM-CSF in CD could be explainedby a complex reprogramming of altered monocyte/macrophagefunctions. These findings suggest the exploration of stimulating,rather than suppressive, therapies with the potential to more spe-cifically reprogram monocytes to modulate immune functions.

Materials and MethodsHuman monocytes

Blood samples from individual healthy donors were purchased from theDepartment of Transfusion Medicine at the University Hospital M!unster,M!unster, Germany. Peripheral blood monocytes were obtained fromdonors by leukapheresis and isolated to .90% purity as previously de-scribed (24). Monocytes were cultured (1 3 106 cells/ml) in hydrophobicTeflon bags (Heraeus, Hanau, Germany) in McCoy’s 5a medium supple-mented with 5% human AB serum, 2 mM L-glutamine, 200 IU/ml peni-cillin, 100 mg/ml streptomycin, and 13 nonessential amino acids (all fromBiochrom, Berlin, Germany). Monocytes were allowed to rest for 16 h

before stimulation. Monocytes from at least three different individualswere assessed with each experiment.

Patients

Clinical and demographic characteristics of the study subjects and methodshave been reported in detail previously (25). Ethical approval was obtainedfrom the Ethics Committee of the University of M!unster (reference no.2006-267-f-S, obtained by Jan Dabritz), and fully written informed consentwas obtained from all patients or legal guardians.

DNA microarray hybridization

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 ofexperiments to analyze changes in gene expression patterns induced byGM-CSF. Using high-density microarrays with .22,000 oligonucleotidesets, we obtained the expression levels of at least 13,000 independenttranscripts. RNA preparation, sample preparation, and hybridization toAffymetrix (Santa Clara, CA) Human Genome 133 A Gene Chip arrays formicroarray analysis were performed as described previously (26).

Statistical analysis of microarray data

For analysis of data from individual donors, raw data of GM-CSF–treatedsamples were processed by MicroArray Suite Software (Affymetrix) usingdata from corresponding control samples as baseline. Signals were scaledto a target intensity of 500 and log-transformed. Detection and change callsusing perfect match and mismatching probes were assigned using a signedrank test as described previously (26–28). Data were submitted to the GeneExpression Omnibus database under accession number GSE63662 (http://www.ncbi.nlm.nih.gov/geo). We retained only genes that were significantlyregulated in every single experiment (change p , 0.05, fold-change $ 2.0,expression over background). The data of the complete set of experimentswere further studied applying the Expressionist Suite software package(GeneData), which allows identification of genes that are significantly reg-ulated in multiple independent experiments as described previously (26).Being aware of the low significance at low-intensity levels, we filtered forgenes with an expression over background in at least one of the two ex-perimental groups (GMaM versus monocytes). We finally retained onlygenes that were significantly regulated in every single experiment (changep , 0.05, fold-change $ 2.0, expression over background), as well as in thecomplete set of experiments (expression over background, fold-change $2.0, p , 0.05, paired t test). Reproducibility of the results was confirmedusing RT-PCR for selected genes and three new independent experiments.

Quantitative real-time PCR

Expression of selected genes in human and mouse (C57BL/6) monocyteswas analyzed by quantitative real-time RT-PCR (qRT-PCR) as describedpreviously (29). PCRs were performed and measured on a CFX384 Touchreal-time PCR detection system (Bio-Rad, Munich, Germany). The relativeexpression was calculated using ribosomal protein L13a as endogenoushousekeeping control gene. The primers used for PCR analysis are given inSupplemental Table III.

Flow cytometry

FACS measurements were performed using a Cyflow space equipped withFlowMax 2.8 (both Partec, M!unster, Germany), and analysis was per-formed using FlowJo software (TreeStar, Ashland, OR). Ab staining ofcells was routinely done with 1 mg/ml of the according Ab. For detectionof cell-surface molecules, flow cytometry was performed as describedearlier (26). All intracellular stains were performed using the transcriptionfactor staining buffer set (eBioscience, San Diego, CA). mAbs used aregiven in Supplemental Table IV.

Chemokine production of GMaMs

Chemokine concentrations of CCL18 and CCL23 were determined incell culture supernatants of monocytes treated for 24 h with GM-CSF(10 ng/ml) or untreated control cells by an ELISA system according tothe manufacturer’s instructions (CCL18; Sigma-Aldrich, Steinheim,Germany; CCL23; Raybiotech, Norcross, GA).

Monocyte/macrophage polarization

Human monocytes were stimulated for 4 and 16 h with IL-4 (100 mg/ml),IFN-g (100 mg/ml), or left untreated. Alternatively, human monocyteswere polarized with GM-CSF (10 ng/ml) 6 IFN-g (100 mg/ml) or leftuntreated as a negative control. Expression of selected genes was analyzedby qRT-PCR as described earlier.

The Journal of Immunology 2425

In additional experiments, human monocytes were polarized for 24 hwith IFN-g (M1; 50 ng/ml) or IL-4 (M2; 50 ng/ml). After polarization,monocytes were stimulated with GM-CSF (10 ng/ml) or left untreated foran additional 24 h. IL-1b, TNF-a, IL-10, and CD206 expression weremeasured by flow cytometry as described earlier.

Finally, human monocytes were stimulated6 GM-CSF (10 ng/ml) for 0,30, 60, and 120 min, and expression of IFN-a, IFN-b, IFN-g, and IL-4 wasanalyzed by qRT-PCR as described earlier.

Migration, chemotaxis, monocyte trafficking, and adherence

Monocyte assays in Transwell plates (Costar, New York, NY) were per-formed as described previously using MCP-1 (10 ng/ml; Immunotools,Friesoythe, Germany), IL-8 (25 ng/ml; Immunotools), and leukotriene B4

(LTB4; 100 nM; Biozol, Eching, Germany) (30). Cells were allowed tomigrate for 4 h. T cell migration was analyzed using the Cultrex 96-wellcell migration assay according to the manufacturer’s protocol (Trevigen,Gaithersburg, MD). T cells were isolated from fresh PBMCs using anEasySep human T cell enrichment kit according to the manufacturer’sprotocol (STEMCELL Technologies, Vancouver, BC, Canada). T cells (5 3104) were added to top-chamber and cell culture supernatants of untreatedmonocytes or GMaMs as well as CCL18 (1 ng/ml), CCL23 (8 ng/ml; bothPeproTech, Rocky Hill, NJ), and medium (control) were added to the bottomchamber. T cells that had migrated into the lower compartment within 4 hwere measured using an Infinite M200 Pro reader (TECAN, Crailsheim,Germany).

Expression of intestinal-associated homing molecules in human mono-cytes treated 6 GM-CSF (10 ng/ml) for 24 h was analyzed by flow cytom-etry (gated on CD14+ cells) as described earlier.

For determination of cell adhesion, monocytes (23 105) were stimulatedwith GM-CSF (10 ng/ml) for 24 h or left untreated. Monocytes wereseeded in triplicates into 96-well flat-bottom plastic tissue-culture platesand 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%glutaraldehyde (Sigma-Aldrich, Taufkirchen, Germany) for 10 min. Wellswere washed two times with H2O and subsequently stained with 0.5%crystal violet (Merck, Darmstadt, Germany) in 2% EtOH (pH 6.0) for anadditional 15 min at room temperature. Finally, wells were washed threetimes and cells were lysed. Ten percent acetic acid was added, and stainingwas quantified measuring the OD at 560 nm using an Asys Expert 96Microplate ELISA reader (Anthos Mikrosysteme, Krefeld, Germany) (25).

GM-CSF priming

For priming experiments, human monocytes were stimulated for 24 h withGM-CSF (10 ng/ml) or left untreated. After pretreatment, monocytes werestimulated with medium containing LPS (10 ng/ml) or left untreated for anadditional 4 h. TNF-a and IL-1b content were measured in culturesupernatants by ELISA (OptEIA ELISA kits; BD Pharmingen, Heidelberg,Germany). Expression of selected genes was confirmed by qRT-PCR asdescribed earlier.

Phagocytosis and oxidative burst

For detection of phagocytic capacity, cells were incubated with carboxy-fluorescein diacetate (Invitrogen, Karlsruhe, Germany)–labeled Leishmaniamajor parasites (ratio cells: L. major = 1:5) or FITC (MoBiTec, Gottingen,Germany)-labeled latex beads (ratio cells: beads = 1:10) for 4 h (31). Rateof phagocytosis was determined by flow cytometry as described previously(30). Cells were incubated with or without PMA (50 nM; Sigma-Aldrich,Taufkirchen, Germany) in addition to 10 ng/ml GM-CSF to investigate theinduction of oxidative burst. The extracellular chemiluminescence re-sponse was measured in the presence of isoluminol (50 mM; Sigma-Aldrich, Taufkirchen, Germany) as described previously (24).

In vitro scratch closure assay

Cells of the Caco-2 human colon adenocarcinoma cell line (ATCCHTB-37)were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml),streptomycin (100 mg/ml), 15 mM HEPES (pH 7.4), 2 mM L-glutamine, and1% nonessential amino acids at 37˚C and 5% CO2 in a humidified incubator.For the scratch closure assay, cells were grown to confluence in 12-wellplates and serum deprived (0.1% FBS) for 24 h before scratch wounding.Monolayers were scratched using a sterile pipette tip and washed twice.Thereafter the wounded monolayers were cultured in fresh serum-deprivedmedium in the presence or absence of 2.5 3 105 untreated monocytes orGMaMs. The initial wound size was determined by microscopy, and thearea of the scratch was calculated with ImageJ software (Version 1.45s;National Institutes of Health). Additional photographs were taken usinga reference line 24 h after wounding, and the rate of wound closure was

analyzed by measuring the scratch area relative to the initial wound areaafter each time point.

Influence of human GMaMs on T cell fate

Human T cells were purified from donor-specific PBMCs using positiveselection of CD2-expressing T cells by MACS technology according to themanufacturer’s protocol (Miltenyi Biotec, Bergisch-Gladbach, Germany).A total of 1 3 106 T cells were cocultured with 1 3 105 monocytes (Mo)for 7 d (ratio T/Mo = 10:1). Cells were harvested and stained using mAbsraised against CD4, CD25, and Foxp3 (Supplemental Table IV). Subse-quent flow cytometry was performed as described earlier.

Mice

Experiments were performed in accordance with approved protocols ofthe animal welfare committee of the North Rhine-Westphalia State Agencyfor Nature, Environment and Consumer Protection, Recklinghausen,Germany (LANUV NRW Reference No. 87-51.04.2010.A113). C57BL/6and Rag12/2 mice were kept under specific pathogen-free conditions andaccording to federal regulations. Mice were purchased from Harlan (Paris,France) and used for experiments at the age of 10–12 wk.

Murine monocytes

Freshly isolated monocytic bone marrow cells were prepared as describedearlier (32). Cells were cultured for 48 h with 150 U/ml GM-CSF(Immunotools) or left untreated as control in 20% L929 cell supernatant(containing M-CSF) conditioned DMEM supplemented with 2 mM glu-tamine, 0.1 mM nonessential amino acids (all Invitrogen, Karlsruhe,Germany), 100 mg/ml penicillin/streptomycin, and 10% heat-inactivatedFCS (both Biochrom, Berlin, Germany). After culture, cells were washedthree times and subsequently used for analyses and coculture experiments.

T cell transfer colitis

To induce colitis, we adoptively transferred 1 3 106 syngeneic CD4+CD252

T cells i.v. into Rag12/2 mice (on C57BL/6 background). Body weight ofanimals was monitored daily, and around day 40 animals that establishedcolitis by weight loss on consecutive days received GMaMs or untreatedmonocytes (2 3 106 per mouse) i.v. Alternatively, 5 mg GM-CSF (Immu-notools) diluted in PBS or PBS alone was administered i.p. on a daily basis.Body weight of mice was monitored for an additional 12 d. Finally, micewere euthanized by CO2 inhalation, and their colons were prepared, mea-sured, and preserved for histology.

Isolation of murine T cells from spleen

T cells were isolated from spleens as described previously (33). T cells usedfor induction of transfer colitis were further purified for CD4+ and depletedof CD25+ cells by MACS technology according to the manufacturer’sinstructions (Miltenyi Biotech).

Histopathologic analysis

For histopathologic analysis, tissue specimens from the proximal and distalcolon were fixed in 10% buffered formalin phosphate and embedded inparaffin. Sections were cut at 3–5 mm and stained with H&E. Inflammationwas graded from 0 to 4 in a blinded fashion: 0, no signs of inflammation; 1,low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyteinfiltration, moderate fibrosis, high vascular density, thickening of the colon wall,moderate goblet cell loss, and focal loss of crypts; and 4, transmural infiltrations,massive loss of goblet cell, extensive fibrosis, and diffuse loss of crypts.

High-resolution colonoscopy

Mice were anesthetized with isoflurane (100% v/v, 1.5 vol %, 1.5 L/min;Florene; Abbott, Wiesbaden, Germany) and administered an enema (Freka-Clyss; Fresenius Kabi, Sevres, France). High-resolution colonoscopy wasperformed using a veterinary endoscopy workstation (Coloview; KarlStorz, Tuttlingen, Germany) to assess colitis. Under visual control, therigid miniature endoscope (1.9-mm outer diameter) was inserted !4 cmaccording to anatomic conditions. The modified murine endoscopic scoreof colitis severity (MEICS) observes thickening of the colon, changing ofvascular pattern, presence of fibrin, granular mucosa surface, and stoolconsistence (0–3 points each, maximum of 15 points); it was used toevaluate colonic inflammation (34).

In vivo cell tracking

For invivo cell tracking ofGMaMs, cells were stainedwith a commercially avail-able lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine

2426 GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS

iodide (Life Technologies, Darmstadt, Germany) with an emission maximumof 782 nm as described elsewhere (35). In vivo distribution of labeled cellsacross the intestine was studied 24 h after i.v. injection using a planar small-animal FMT system (FMT 2500; VisEnMedical) as described previously (35).

Isolation of lamina propria mononuclear cells and cells frommesenteric lymph nodes

Lamina propria mononuclear cells (LPMCs) were isolated from the colonof colitogenic mice by a standard method (36). In brief, the colon wasremoved, opened longitudinally and cut into 5-mm pieces, and washedwith cold Ca2+/Mg2+-free HBSS. The intestinal tissue specimens weretransferred into HBSS with EDTA to remove intraepithelial lymphocytes.After 30 min of gentle shaking at 37˚C, the samples were vortexed andintraepithelial lymphocyte–containing supernatant was removed. This stepwas repeated twice. LPMC suspensions were prepared from the EDTA-treated de-epithelialized intestinal tissue by further incubation with 100 U/mlcollagenase and 5 U/ml DNase for 30 min at 37˚C. LPMCs were washed,resuspended in 44% Percoll solution (Amersham Pharmacia Biotech, Pis-cataway, NJ), underlaid with 66% Percoll solution, and centrifuged for30 min at 600 3 g. The LPMC fraction was harvested from the interface.

For cell isolation from mesenteric lymph nodes (MLNs), the lymphnodes of treated mice and control mice were carefully isolated, pooled, andpassed through a 40-mm cell strainer, and the resulting single-cell sus-pension was washed once with PBS.

Coculture of naive T cells and restimulation of LPMCs andMLNs

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 werecocultured for 4 d (ratio 10:1) with respective monocytes in triplicatesfor each condition in anti-CD3e and anti-CD28 Abs (5 mg/ml each; Sup-plemental Table IV) coated to 96-well, round-bottom plates in RPMI 1640supplemented with 2 mM glutamine, 0.1 mM nonessential amino acids(all Invitrogen, Karlsruhe, Germany), 100 mg/ml penicillin/streptomycin,and 10% heat-inactivated FCS (both Biochrom, Berlin, Germany) at 37˚Cand 5% CO2. To further test for cytokine production, we stimulated iso-lated LPMCs and MLN cells (23 105 per well) from mice used in transfercolitis experiments with anti-CD3e and anti-CD28 Abs (5 mg/ml each;Supplemental Table IV) coated to 96-well, round-bottom plates for 24(LPMCs) or 96 h (MLNs). A total of 100 ml cell supernatants was storeduntil cytokine analysis was performed using a bead-based multiplex assay(mouse Th1/Th2 10plex FlowCytomix; eBioscience), according to man-ufacturer’s instructions.

Induction of Tregs by murine GMaMs

LPMC and MLN single-cell suspensions from mice used in transfer coli-tis experiments were stained with Abs raised against CD4 and Foxp3(Supplemental Table IV). Cells were measured by flow cytometry as de-scribed earlier.

T cell coculture experiments were performed to evaluate the function ofGMaM to induce Tregs. Therefore, 1 3 105 splenic T cells (describedearlier) were cocultured with 1 3 104 monocytes (GM-CSF–activated oruntreated) in 96-well, round-bottom plates for 7 d in RPMI 1640 supple-mented with 2 mM glutamine, 0.1 mM nonessential amino acids (allInvitrogen, Karlsruhe, Germany), 100 mg/ml penicillin/streptomycin, and10% heat-inactivated FCS (both Biochrom, Berlin, Germany) at 37˚C and5% CO2. Cells were harvested and stained with 1 mg anti-CD4 and anti-Foxp3. Used Ab clones are given in Supplemental Table IV. Flowcytometry was performed as described earlier.

Statistics

Data are expressed as mean 6 SEM unless stated otherwise and wereassessed using the Student t test. The p values,0.05 were considered to bestatistically significant. All calculations were performed using SPSS ver-sion 14 (SPSS, Chicago, IL).

ResultsGene expression and phenotype shift of GMaMs

GM-CSF provokes nonclassical monocyte activation. To analyzemonocyte activation comprehensively and unbiased, we performeda global RNA expression analysis. The microarray data were fil-tered using strict statistical criteria and revealed a significantregulation of genes involved in immune/inflammatory responses

(especially chemokines), cell motility, chemotaxis, regulation ofcell growth, endocytosis, and Ag processing and presentation(Table I). Furthermore, we analyzed statistical overrepresentationof transcription factor binding sites in GM-CSF–regulated genesusing CARRIE software (Supplemental Table I). Overall, wefound that in human monocytes, 190 genes were significantlyupregulated, whereas 212 were downregulated after 16-h stimu-lation with GM-CSF (Supplemental Table II). Raw data have beensubmitted to the Gene Expression Omnibus under accessionnumber GSE63662 (http://www.ncbi.nlm.nih.gov/geo). We con-firmed microarray expression data by qRT-PCR for selected genes(Fig. 1A). Analyses of expression levels at different time points ofGM-CSF activation confirmed that gene regulation in GMaMis most relevant after 16–24 h (Fig. 1B). However, we alsoshow that the expression of most of these genes is alreadysignificantly regulated after 4 h of GM-CSF stimulation. Genesthat were not significantly regulated after 24 h of GM-CSF acti-vation also showed no significant upregulation or downregulation oftheir expression after 4, 16, 48, or 120 h (Fig. 1B). In agreementwith the microarray data, flow cytometry confirmed upregulationof CD80 and downregulation of CD9 upon GM-CSF activation(Fig. 1C, 1D).

GM-CSF drives monocytes toward M2-like phenotype. Interest-ingly, the microarray expression data of GMaM chemokine ligandsand receptors showed a gene expression pattern indicative of a GM-CSF–induced shift toward a M2-like phenotype (Fig. 2A). This wasconfirmed by protein quantification (ELISA) for selected chemo-kines (Fig. 2B) and by gene expression analyses (qRT-PCR) fordifferentially expressed genes that were assigned to M1- and M2-like monocytes based on currently accepted annotations (Table II)(2, 38, 39).

Gene expression in GMaM is similar to IL-4–induced geneexpression profiles. Our gene expression analyses (Fig. 1B) sug-gest that the observed gene expression in GMaM is similar toIL-4–induced gene expression profiles in monocytes. We stimu-lated human monocytes from healthy donors with or without GM-CSF for 0, 30, 60, and 120 min and analyzed gene expression ofIFN-a, IFN-b, IFN-g, and IL-4 by qRT-PCR. The expression ofIFNs and IL-4 in monocytes was not upregulated at any time afterGM-CSF stimulation when compared with untreated monocytes(data not shown). Interestingly, we found that the GM-CSF–in-duced gene expression pattern in human monocytes within thefirst 24 h is similar to the gene expression pattern of IL-4– but notIFN-g–stimulated monocytes (Fig. 2C, 2D). We assume that ourtranscriptomic data of human GMaMs rather reflect a primaryeffect, which, however, has similarities with the expression pat-tern of IL-4–induced M2a macrophages.It has been shown that endogenous type I IFN regulates the basal

gene expression of bone marrow–derived macrophages grown inGM-CSF (40). Fifty GM-CSF–specific type I IFN–dependentregulated genes were identified, of which we found only five tobe significantly regulated in GMaMs (IFIT3, CBX6, ISG20,ABCA9, COLEC12; Supplemental Table II). In human cells, itwas found that the expression of 154 type I IFN–regulated geneswere also different between monocyte-derived macrophagescultured in GM-CSF (GM-MDM) or M-CSF (MDM) (41). Wefound that only 7 of the top 50 type I IFN–dependent genesdifferentially expressed between human GM-MDM and MDMwere significantly regulated in GMaMs (EDNRB, LGMN,P2RY14, CH25H, CDKN1C, GGTLA1, CD69; SupplementalTable II). In addition, we found no significant GM-CSF–de-pendent gene regulation of type I IFNs in GMaMs. Likewise,the gene expression of IFN-g in human monocytes was not


significantly regulated by GM-CSF after 16 h nor after 24 h(Table I, II). In addition, GM-CSF induced neither IRF4 (M2polarization) nor IRF5 (M1 polarization) in human monocyteswhen activated with GM-CSF for 24 h (Table II). However,

activation of NF-kB, which promotes M1 macrophage polari-zation, was significantly downregulated by GM-CSF stimula-tion, whereas the activity of C/EBPb, which is crucial for ex-pression of M2-regulated genes, was significantly upregulated

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

Gene Description (NCBI Gene) n-Fold p

Upregulated by GM-CSF Activation

Inflammatory responseGGTLA1 g-Glutamyltransferase-like activity 1 50.5 ,0.001CFH Complement factor H 44.9 0.013CD80 CD80 molecule 8.4 ,0.001PROCR Protein C receptor, endothelial (EPCR) 7.5 ,0.001CD69 CD69 molecule 6.4 0.002BANK1 B-cell scaffold protein with ankyrin repeats 1 5.2 0.011SLAMF1 Signaling lymphocytic activation molecule 5.0 0.041IL7R IL 7 receptor 4.8 0.001CLEC5A C-type lectin domain family 5, member A 4.5 0.011IL1RAP IL 1 receptor accessory protein 4.4 0.008ALOX5AP Arachidonate 5-lipoxygenase-activating protein 4.1 0.003LTB Lymphotoxin b (TNF superfamily, member 3) 3.6 0.002PTGS1 PG-endoperoxide synthase 1 3.3 0.003

ChemotaxisCCL13 Chemokine (C-C motif) ligand 13 16.7 ,0.001CCL23 Chemokine (C-C motif) ligand 23 8.3 0.002PPBP Proplatelet basic protein (CXCL7) 7.2 0.001CXCL5 Chemokine (C-X-C motif) ligand 5 5.8 0.025CCL17 Chemokine (C-C motif) ligand 17 5.5 0.003IL8RB IL 8 receptor, b 5.1 0.002CCR6 Chemokine (C-C motif) receptor 6 4.3 ,0.001SPN Sialophorin (leukosialin, CD43) 3.8 0.007

Ag processing and presentationCD1C CD1c molecule 18.4 ,0.001CD1B CD1b molecule 15.5 0.008CD1E CD1e molecule 9.8 ,0.001CD1A CD1a molecule 6.4 ,0.001

Regulation of cell growthTGFB2 Transforming growth factor, b2 26.8 0.006FGF13 Fibroblast growth factor 13 19.8 0.018CISH Cytokine inducible SH2-containing protein 5.7 ,0.001

Downregulated by GM-CSF Activation

Immune responseFCGR1B Fc fragment of IgG, high affinity Ib, receptor (CD64) 29.0 0.011AQP9 Aquaporin 9 25.3 0.004IFIT1 IFN-induced protein with tetratricopeptide repeats 1 25.0 0.037GBP5 Guanylate binding protein 5 25.0 0.009MX1 Myxovirus (influenza virus) resistance 1 24.2 0.023CD28 CD28 molecule 24.2 0.008OAS1 2’,59-oligoadenylate synthetase 1, 40/46kDa 24.0 0.049OAS2 2’-59-oligoadenylate synthetase 2, 69/71kDa 23.7 0.043HPSE Heparanase 23.6 0.003GBP2 Guanylate binding protein 2, IFN-inducible 23.5 0.002MGLL Monoglyceride lipase 23.4 ,0.001

ChemokinesCXCL12 Chemokine (C-X-C motif) ligand 12 216.9 0.009CXCL11 Chemokine (C-X-C motif) ligand 11 27.1 0.046CXCL13 Chemokine (C-X-C motif) ligand 13 26.4 0.019CXCR4 Chemokine (C-X-C motif) receptor 4 26.2 0.001CXCL10 Chemokine (C-X-C motif) ligand 10 23.5 0.007

Phagocytosis/EndocytosisFCGR1A Fc fragment of IgG, high affinity Ia, receptor (CD64) 27.7 0.022MSR1 Macrophage scavenger receptor 1 24.7 0.041

OtherCADM1 Cell adhesion molecule 1 28.5 0.001CD9 CD9 molecule 26.4 0.012IGF1 Insulin-like growth factor 1 (somatomedin C) 25.0 ,0.001ITGB8 Integrin, b 8 25.0 0.006NID1 Nidogen 1 24.7 ,0.001KITLG KIT ligand 23.1 0.002LEP Leptin (obesity homolog, mouse) 29.1 ,0.001CDKN1C Cyclin-dependent kinase inhibitor 1C (p57, Kip2) 23.5 0.041STAT1 Signal transducer and activator of transcription 1 23.2 0.034


by GM-CSF stimulation in the first 24 h (Supplemental Table I).In addition, further quantitative PCR analysis suggests thatIFN-g does not contribute to our transcriptomic data provided forGMaMs. The gene expression of chemokines (CXCL10, CXCL11,CCL1, CCL13, CCL23, CXCL5), CD206, CD209, and IL-1b inhuman monocytes was specifically and significantly upregulated ordownregulated by either GM-CSF or IFN-g after 24 h of stimula-tion (data not shown).Finally, we analyzed whether GM-CSF is affecting distinct

monocyte subsets within the overall monocyte population differ-entially. Our results showed a homogenous shift of GM-CSF–induced cell-surface markers within the overall monocyte pop-ulation using FACS (data not shown). Thus, we found no evidencethat GM-CSF is affecting distinct monocyte subpopulations. Nev-ertheless, we have performed additional monocyte/macrophagepolarization experiments. To this end, we stimulated human mono-cytes from healthy donors for 24 h with or without IFN-g (M1macrophage polarization) or with or without IL-4 (M2 macrophagepolarization), and cells were subsequently stimulated with orwithout GM-CSF for further 24 h. Expression of IL-1b, TNF-a,IL-10, and CD206 was analyzed by flow cytometry (Fig. 2E). Thedata suggest that GM-CSF is affecting both M1- and M2-polarizedmonocytes and that GM-CSF is not affecting distinct monocytesubsets within the overall population differentially.

Comprehensive characterization of GMaM innate immunefunctions

GM-CSF promotes adherence and migration of monocytes. Majorfunctions of monocytes include the capacity to adhere and migrate,which is crucial for their recruitment into tissue. Adherence ofGMaMs to plastic surfaces was enhanced after 24 and 48 h comparedwith untreated control cells (Fig. 3A). We tested whether GM-CSFactivation would affect migration and chemotaxis of monocytes ingeneral and also specifically in response to MCP1/CCL2, IL-8/CXCL8, and LTB4. By using a modified Boyden chamber assay,we detected that spontaneous migration of GMaM and migrationtoward MCP1 and LTB4 were significantly enhanced after 4 h(Fig. 3B). The chemotactic effect was specific because migrationdid not occur when MCP1 or LTB4 was added to the upper com-partment of the Boyden chamber (data not shown). In addition,GM-CSF–induced increase in chemotaxis was also specific to thestimulus because we did not find increased migration toward IL-8(Fig. 3B). Integrins and CC chemokine receptors play a potentialrole in monocyte trafficking into the mucosa in the context ofmucosal homeostasis at the intestinal epithelial barrier. Thesemolecules are also known to play a role in the pathogenesis ofhuman inflammatory bowel diseases. Expression of integrins andCC chemokine receptors was analyzed by flow cytometry gated on

FIGURE 1. Confirmation of GM-CSF–regulated gene expression in monocytes (microarray data) by real-time PCR and flow cytometry. (A) Results obtainedfrom microarray analysis of GM-CSF–dependent gene regulation in human monocytes (after 16 h) compared with unstimulated monocytes were confirmed byqRT-PCR. Genes analyzed were: lymphotoxin b (LTB); complement factor H (CFH); g-glutamyltransferase-like activity 1 (GGTLA); the chemokinesCXCL10, CXCL12, CCL23, and CCL13; and the CD1c and CD80 molecule. Shown are the mean relative n-fold regulation (6 SEM) of three independentexperiments. (B) GM-CSF–dependent gene expression analysis by qRT-PCR of selected genes at different time points. Shown is the mean relative n-foldregulation (6 SEM) of three independent experiments. (C and D) Expression of selected cell-surface molecules (C, CD80; D, CD9) found to be differentiallyexpressed 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 shiftsbetween control and GMaMs were in accordance with microarray data. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes.


CD14+ cells, and expression is stated as mean fluorescence intensity(MFI; geo-mean) 6 SEM of three independent experiments.Expression of CCR2 and CCR6 was significantly increased inGM-CSF–stimulated monocytes (CCR2 150.2 6 31.1; CCR624.5 6 4.4) compared with untreated cells (CCR2 88.4 6 22.5,p , 0.05; CCR6 10.0 6 0.4, p , 0.05), whereas expression ofCCR7 was significantly reduced in GM-CSF–treated monocytes(22.2 6 1.6) compared with unstimulated cells (49.2 6 6.9, p ,0.05). Expression of b7, CCR1, CCR4, CCR9, and CX3CR1was similar in GM-CSF–stimulated and untreated cells (data notshown).

Production of reactive oxygen species is increased andphagocytosis is unimpaired in GMaMs. Another important func-tion of monocytes after being recruited, for example, to sites ofinfection or defective barriers, is the phagocytosis and killing ofpathogens (e.g., via production of reactive oxygen species [ROS]).Spontaneous and PMA-induced production of ROS was signifi-cantly enhanced in GMaMs (Fig. 3C). A few molecules involvedin phagocytosis/endocytosis were significantly downregulated byGM-CSF stimulation (Table I). We therefore tested phagocytosisof latex beads and of complement opsonized living L. majorparasites after activation of monocytes with GM-CSF. We detected

no significant difference in phagocytosis of latex beads byGMaMs, and also phagocytosis of L. major promastigotes was notaltered compared with control monocytes (data not shown).

GM-CSF primes the monocyte response to a secondary microbialstimulus. To test the hypothesis that GM-CSF specifically activatesmonocyte functions by augmenting anti-infectious/antimicrobialdefense and bacterial clearance, we analyzed GMaMs for an in-creased response to a secondary microbial stimulus. Therefore, weanalyzed the influence of GM-CSF stimulation (24 h) on cytokineproduction and gene expression in human monocytes after 4 h ofcostimulation with bacterial endotoxin (LPS). Compared with con-trol monocytes, GMaMs exposed to LPS produced significantlymore IL-1b and TNF-a (Fig. 3D). LPS stimulation of GMaMsalso resulted in a much more pronounced expression of inflam-matory genes as revealed by qRT-PCR (Fig. 3E). This confirmsa GM-CSF–induced priming effect on monocytes, leading to anincrease in vitro response to other stimuli (42).

GMaM impact on epithelial healing and on adaptive immunity

GMaMs accelerate epithelial healing. In addition to their innatephagocytic and killing activity in antimicrobial defense, monocytesare also involved in wound repair. Because we observed phenotypic

FIGURE 2. Polarization of GMaMs. (A) The gene expression of chemokine ligands and receptors in GMaMs (16 h) was analyzed by microarray analysis.The status of differentiation and polarization was classified according to characteristics of classically activated M1-like and alternatively activated M2-likemonocyte/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 withuntreated monocytes. (E) Effects of GM-CSF activation on already primed monocytes. Monocytes were primed for 24 h toward M1 or M2 and subsequentlytreated 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 independentexperiments. *p , 0.05, **p , 0.01, ***p , 0.001.


similarities between GMaMs and alternatively activated (M2-like)macrophages, which have been originally described as “wound-healing macrophages,” we analyzed the influence of GMaMs onepithelial healing. In epithelial cell (Caco-2) monolayers, pre-activation of monocytes with GM-CSF significantly acceleratedwound closure compared with unstimulated monocytes (Fig. 3F).

GMaMs attract T cells and induce Tregs. Another importantfunction of monocytes is the cross talk to adaptive immunity.Monocytes and macrophages serve as APCs, but they alsoshape lymphocyte activation by a whole battery of different co-stimulatory molecules and cytokines. As shown in Fig. 2, geneexpression (Fig. 2A) and protein production (Fig. 2B) of chemo-kines CCL18 and CCL23 were strongly increased in GMaMs.Because CCL18 and CCL23 have been shown to attract naive andresting T cells (43, 44), we analyzed the capacity of GMaMs toattract T cells by using a modified Boyden chamber. T cells wereallowed to migrate toward the culture supernatants of GMaMs oruntreated monocytes. We observed a significantly increased T cellmigration toward cell supernatants of GMaMs (Fig. 3G). In ad-dition, we have addressed effects of GM-CSF on Treg differen-tiation. To this end, we stimulated human monocytes with GM-CSF and cocultured monocytes and autologous T cells for 7 d, andanalyzed resulting T cells for Foxp3 expression to evaluate Tregdifferentiation. CD25 and Foxp3 expression in T cells were al-ready increased upon interaction with monocytes that had beenstimulated with GM-CSF for only 24 h. The induction of Foxp3expression in T cells cocultured with GMaMs was further in-creased when monocytes were stimulated for 48 h with GM-CSF(Fig. 3H, 3I).

In vivo effects of GMaMs in experimental T cell–induced colitis

GMaMs alleviate CD4+ T cell–induced colitis. Having confirmeda specific activation pattern of human monocytes in response toGM-CSF, with the augmentation of host immune defense functions,

tissue repair capacities, and a positive effect on T cell recruitment,we sought to address the functionality of these cells in vivo in thecontext of CD. Because systematic analyses in the human systemare not feasible, we went on analyzing the effects of GMaMs inthe murine system. Analysis by qRT-PCR showed that murineGMaMs had a similar gene regulation profile when comparedwith the GM-CSF–dependent gene expression in human monocytes(Fig. 4A). We chose the CD4+ T cell–dependent experimental co-litis model as an acceptable surrogate of human CD. In this model,adoptive transfer of syngeneic CD4+CD252 T cells into Rag12/2

mice (which lack mature T cells) induces severe colitis (45). Theonset of colitis is monitored clinically by weight loss. Untreatedmonocytes (control) or ex vivo GMaMs were injected i.v. aftermice had lost weight on consecutive days (!5–6 wk after elicitingcolitis by injection of CD4+CD252 T cells). Migration of injectedGMaMs to the inflamed gut was confirmed by in vivo imaging. Inagreement with the in vitro data on cell migration, we observed anincreased infiltration of GMaMs into MLNs compared with con-trol monocytes in two independent experiments (Fig. 5). Mice thathad received GMaMs showed no weight loss at all over a period of12 d after monocyte transfer (Fig. 4B). Normally, the inflamedcolon becomes shorter and presents with reduced length, and thusshortening of the colon is a measure of inflammation. Also, in thisstudy, mice that received GMaMs did not show relevant shorten-ing of the colon, whereas all other groups were not protected fromcolitis (Fig. 4B, 4C). Control mice that had received untreatedmonocytes or no monocytes showed progressive weight loss andsigns of intestinal inflammation, and had to be euthanized on day12 (Fig. 4B, 4C). Animals that had received untreated monocyteshad significantly severe histopathologic alterations of the colon,most evident in the distal part (Fig. 4D, 4E). In addition, weperformed high-resolution colonoscopy and graded inflammation(MEICS-Score) (34). The colon of mice receiving no treatmentpresented with a vulnerable and bleeding mucosa, rarefication of

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 0.005Chemokine (C-X-C motif) ligand 11 CXCL11 M1 ! 22.75 0.004Chemokine (C-X-C motif) ligand 13 CXCL13 M1 !! 25.27 0.000IFN, g IFNg M1 " 21.20 0.700TNF a TNFa M1 # 4.50 0.000TNF ligand superfamily TRAIL M1 ! 22.03 0.001NO synthase 2, inducible iNOS M1 " 1.94 0.230B7-1 CD80 M1 # 4.22 0.005IL 1 b IL1b M1 # 2.81 0.000IL 6 IL6 M1 # 2.42 0.005IL 8 IL8 M1 " 1.15 1.000IL 12 IL12 M1 " 1.43 0.700IL 18 IL18 M1 " 1.59 0.100IL 23 IL23 M1 " 21.05 1.000IFN regulatory factor 5 IRF5 M1 " 21.07 1.000PG-endoperoxidase synthase 2 COX2 M1 " 21.03 1.000Colony stimulating factor 2 GM-CSF M1 ! 21.36 0.000Colony stimulating factor 3 G-CSF M1 ! 23.81 0.000Chemokine (C-X-C motif) ligand CXCL5 M2 # 3.87 0.015Chemokine ligand 1 CCL1 M2 ## 6.82 0.000Chemokine ligand 13 CCL13 M2 ### 21.06 0.000Chemokine ligand 23 CCL23 M2 ## 6.87 0.000TGF b TGFb M2 " 1.12 0.694Arginase ARG1 M2 # 3.90 0.005Mannose receptor C type 1 (MRC1) CD206 M2 ## 6.45 0.000CD163 molecule CD163 M2 " 21.90 0.113DC-SIGN CD209 M2 # 2.65 0.009IL 1 receptor, type 2 CD121b M2 # 2.70 0.001IL 10 IL10 M2 " 21.25 0.206IFN regulatory factor 4 IRF4 M2 " 1.01 1.000


vascular pattern, presence of fibrin, and ulcerations. Mice treatedwith GMaMs depicted a transparent colonic mucosa with a regularvascular pattern resembling healthy animals (Fig. 4F). Taken to-gether, treatment of an established CD4+ T cell–induced colitiswith GMaMs alleviates inflammation of the colon, resulting insignificantly improved clinical parameters and histology, sug-gesting that GMaMs potentially exert regulatory effects on T cellsin vivo.

GMaMs regulate T cell responses in vivo and in vitro. After ter-mination of colitis experiments, LPMCs and MLNs were harvested.Migration of injected GMaMs to gut tissue and MLNs was con-firmed by in vivo imaging (Fig. 5). Single-cell suspensions wererestimulated with anti-CD3/anti-CD28 to explore how the capac-ity and strength of T cell cytokine production has changed in vivo

during colitis and treatment with GMaMs. After restimulation for24 h, we tested supernatants for production of IFN-g (Th1 cellresponse) and IL-4, IL-10, and IL-13 (Th2 cell response). Asshown in Fig. 6, GMaM transfer led to significantly reduced IFN-gproduction in T cells from LPMCs (Fig. 6A) and MLNs (Fig. 6B).The Th2 cytokines IL-4, IL-10, and IL-13, however, were slightlyincreased in supernatants from LPMCs of animals treated withGMaMs (Fig. 6A), and IL-4 and IL-13 were also increased insupernatants from MLNs of animals treated with GMaMs (Fig.6B). In summary, treatment of mice suffering from colitis withGMaM results in a shift in cytokine production of T cells in vivo.To confirm these in vivo data, we performed coculture experi-ments with anti-CD3e/anti-CD28–stimulated T cells in vitro.Also, in this study, GMaMs skewed the T cell response and led

FIGURE 3. Functional properties of GMaMs and interaction of human GMaMs with T cells. (A) Human monocytes were activated with GM-CSF or leftuntreated (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 placedinto 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 noattractants. After 4 h cells that had migrated into the lower compartment were counted, and numbers are presented as the percentage of cells, whichmigrated in the absence of any chemotactic stimulus. The p values refer to the migration of untreated (control) cells in the absence of any chemotacticstimuli (w/o). (C) Oxidative burst of GMaMs or untreated monocytes was initiated by the addition of PMA. Isoluminol chemiluminescence was measured inPMA-treated cells and control cells after induction of oxidative burst. (D) Cytokine secretion was measured in supernatants of GMaMs (24 h) after exposureto LPS (4 h) and compared with untreated monocytes exposed to LPS. **p , 0.01, ***p , 0.001 compared with LPS-treated control. (E) The geneexpression 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-2monolayers at 24 and 48 h in the absence of monocytes (control; n = 30), the presence of untreated monocytes (monocytes; n = 45), or the presence ofmonocytes preactivated for 48 h with GM-CSF (GMaM; n = 45). (G) T cell migration was analyzed. Monocytes were activated with GM-CSF or leftuntreated for 24 h in Teflon bags. Fifty thousand T cells were added to top chamber, and cell culture supernatants of untreated monocytes or GMaMs, aswell as CCL18, CCL23, and medium (control), were added to the bottom chamber. T cells that had migrated into the lower compartment within 4 h werecounted. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes. (H and I) Human autologous T cells were cocultured with untreatedmonocytes (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 andanalyzed by flow cytometry. (H) Representative dot plots are shown for 48 h. (I) Cells were gated on CD4+ cells and analyzed for CD25 and Foxp3expression. Data shown are the means (6 SEM) of three independent experiments. (A–I) *p , 0.05, **p , 0.01, ***p , 0.001.


to significant upregulation of Th2 cytokines IL-4, IL-13, andIL-10, whereas the Th1 cytokine IFN-g was downregulated(Fig. 6C). These data demonstrate that GMaM cross talk withT cells results in a phenotype shift that attenuates classical Th1responses, which may contribute to an immunomodulatory effect.We did not observe an increase of Tregs, represented by Foxp3expression in CD4+ T cells, in MLNs or LPMCs of mice withexperimental T cell transfer colitis after the transfer of GMaMs(Fig. 6D, 6E). However, we were able to demonstrate that murineGMaMs induce Tregs in vitro (Fig. 6F).

Peripheral blood monocytes from patients with CD behave likeGMaMs

We next studied phenotypic and functional features of untreatedversus GM-CSF–activated peripheral blood monocytes of 18patients with quiescent CD by analyses of cell adherence, mi-gration, chemotaxis, phagocytosis, oxidative burst, and cytokineexpression and secretion. Collectively, our data suggest that theeffects of GM-CSF activation of peripheral monocytes of patientswith CD (Fig. 7) are similar to the observed effects in GMaMsfrom healthy donors (Figs. 1–3). This includes the GM-CSF–in-duced increase in adherence, migration, chemotaxis, and oxidativeburst, as well as the priming of monocytes to secondary microbialstimuli (Fig. 7A–E). In addition, changes in GM-CSF–dependentmRNA expression of selected key inflammatory cytokines were inagreement with our transcriptomic data obtained from GMaMs ofhealthy individuals (Fig. 7F). Importantly, there was no evidencethat GM-CSF activation had different effects on monocytes whencompared between individual patients.

DiscussionDespite the fact that the concept of CD as a chronic granulomatousTh1-driven disease shifts toward a theory of CD as an immuno-deficiency of macrophages, while we only begin to understand theregulatory or suppressive functions of our immune system, ourgeneral approach to chronic inflammatory diseases including CD isstill mainly based upon the paradigm of immunosuppression as theprimary therapeutic intervention. In line with that, phagocytes areprimarily seen as driving forces of inflammation that need to beinhibited. This traditional view of immune interventions, however, isin sharp contrast with our currently changing view of immunity. It isnow accepted that cells of the monocyte–macrophage lineage arecharacterized by considerable diversity and plasticity that may en-compass, as an example, classical M1-macrophage differentiation(when stimulated by IFN-g) or alternative M2 differentiation (whenstimulated by IL-4/IL-13) as outer margins of a broad phenotypicalplasticity (2). Serving another example, the population resulting fromGM-CSF–stimulated human monocytes has been referred to as M1-like macrophages with a proinflammatory cytokine profile (41, 46).As an attempt to introduce a novel concept based on stimulating

rather than suppressing immunity, GM-CSF has been used both inanimal IBD models and in human patients with CD (47). Intra-peritoneal administration of GM-CSF alleviated acute DSS-induced colitis in mice, resulting in decreased proinflammatorycytokine release, improved clinical and histologic parameters, aswell as more rapid ulcer healing, and facilitated epithelial re-generation (15, 16). Importantly, transfer of splenic GM-CSF–induced CD11b+ myeloid cells into DSS-exposed mice improved

FIGURE 4. Treatment with GMaMs protects from experimental colitis. (A) GM-CSF–dependent gene regulation in murine monocytes derived from bonemarrow of C57BL/6 mice and human peripheral blood monocytes compared with unstimulated monocytes. Shown is the mean relative n-fold regulation (6SEM) of three independent experiments. *p , 0.05 compared with GM-CSF–treated human monocytes. (B) Rag12/2 mice were injected i.v. withCD4+CD252 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.(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 magnification3100. (E) Intestinal inflammation scores ofthe proximal and distal colon of mice with colitis injected with control monocytes or GMaM (0, no inflammation; 1, mild inflammation; 2, moderateinflammation; 3, severe inflammation; 4, extreme inflammation). (F) MEICS score and representative pictures of high-resolution colonoscopy showing thecolon of a mouse receiving no treatment with a vulnerable and bleeding mucosa, rarefication of vascular pattern, fibrin, and ulcerations. Mice treated withGMaMs depicted a transparent mucosa with a regular vascular pattern resembling healthy animals. Graphs in (B)–(E) show mean values (6 SEM) of 10control mice and 14 mice injected with GMaMs from 3 independent experiments. *p , 0.05, **p , 0.01 compared with untreated monocytes; #p , 0.05compared with T cells only.


colitis, and GM-CSF–expanded CD11b+ splenocytes were shownto promote in vitro wound repair (16). Furthermore, it has beenshown that: 1) neutralization of GM-CSF increases intestinalpermeability and bacterial translocation in mice; and 2) increasedlevels of GM-CSF autoantibodies are associated with an increasein bowel permeability, disease relapse, stricturing ileal disease,and surgery in patients with CD (48–50). As a therapy that couldhelp in overcoming insufficient macrophage functions, GM-CSFhad strikingly beneficial effects in subgroups of CD patients (17).Seemingly, these beneficial effects were rather unexpected in lightof the previously reported proinflammatory polarization of mac-rophages upon stimulation with GM-CSF (41, 46).We speculated that GM-CSF exerts its beneficial effects in in-

testinal inflammation in vivo by specific activation of monocytesthat combines innate immune activation, thus facilitating anti-infectious defense, with a simultaneous regulatory function serv-ing to limit adaptive immunity and excessive inflammation. Wethus set off in an unbiased systems biology approach to compre-hensively study the many facets of monocyte activation in vitro,ranging from gene expression to innate immune functions and theinterplay with adaptive immunity. All these aspects have previ-ously been studied on monocytes but separately and independentlyfrom each other (41, 46, 51–54). Our findings suggest that theearly imprinting of monocytes after activation with GM-CSF isof crucial importance, because monocytes play an important roleduring the recruitment phase of the innate immune response andhave the potential to regulate adaptive immune mechanisms. GM-CSF has been shown to have a pleiotropic role in inflammationand autoimmunity (23). Data from other groups suggest that cellculture conditions, concentration, time point, and duration chosenfor GM-CSF stimulation of human monocytes may determinetranscriptional outcomes relating to M1/M2 polarity (53–55). Inthis respect, it has been described that different biologic responses

induced by GM-CSF depend on its concentration (53), and that thetime point chosen for the CSF treatment of human monocytes canmarkedly determine the relative expression of cytokine genes (41).Collectively, it is conceivable that the described population ofGMaMs in this study represents an intermediate cell type, com-bining cell-surface expression characteristics and functional fea-tures of different M2 macrophage subsets including CD206 andCD209 cell-surface expression (M2a), Th2 responses/activation(M2a/b), killing, and type II inflammation (M2a) and immuno-regulation (M2b). In contrast with proinflammatory and antimi-crobial responses of classically activated monocytes, M2-likephenotypes are broadly anti-inflammatory and play importantroles in wound healing (54). GMaMs combine: 1) features ofaugmented host defense functions; 2) the ability to facilitate epi-thelial healing; and 3) the regulatory potential on adaptive im-munity. Specifically, we found a GMaM-dependent acceleratedwound closure in Caco-2 monolayers using an in vitro scratchclosure assay and in addition an upregulation of genes involved incell proliferation (e.g., FGF13, CDKN1C, TGF-b). Furthermore,we found that the expression of a number of genes that are as-sociated with M2 polarization is increased in human monocytesafter activation with GM-CSF. In particular, we found a significantregulation of chemokines and chemokine receptors in humanmonocytes. In this study, we found a GM-CSF–dependent downreg-ulation of the M1 chemokines CXCL9, CXCL10, CXCL11, CXCL13,and CXCL16 in monocytes. At the same time, GM-CSF significantlyinduced expression of chemokines CXCL3, CCL1, CCL13, CCL17,CCL18, CCL23, and CCL24 in monocytes, which are characteristic forM2 macrophages (39, 54, 56). In addition, we showed that a numberof other M2a/b macrophage markers were significantly upregulatedin human monocytes after GM-CSF activation (e.g., CD206,CD209, CD121b, ARG1). As mentioned earlier, we also found anenhanced production of proinflammatory cytokines (IL-1b, TNF-a,

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 twoindependent experiments are shown as percent infiltrated donor CD45.1+ cells. *p , 0.05 compared with untreated monocytes. (C) GMaMs were labeledwith 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, and 23 106 cells were injected according to the standard treatment regimen. Monocyteinfiltration in the intestine was visualized after 24 h by a planar small-animal fluorescence-mediated tomography system, and representative pictures aredepicted.


and IL-6) in GMaMs, a feature also seen in M2b macrophagesupon exposure to immune complexes and LPS (56). In addition tothese strengthened innate immune functions, GMaMs simulta-neously indeed have a regulatory potential on adaptive immunity.Overall, our data indicate that GMaMs represent a distinctive cellpopulation with characteristics of both M1 and M2 cells.GM-CSF also stimulated functions that are typically assigned to

classically activated monocytes. It has previously been publishedthat GM-CSF increases the adherence of purified peripheral bloodmonocytes to plastic surfaces and to monolayers of HUVECs (55),and that GM-CSF can prime monocytes for increased trans-endothelial migration (57). Furthermore, reported results on GM-CSF effects on other functions such as oxidative metabolism,cytotoxicity, phagocytosis, and the in vitro response to otherstimuli are conflicting to some extent (23, 52, 58, 59). We confirmin this study that short-term treatment (24 h) of human monocyteswith GM-CSF promotes: 1) cell adherence and migration, 2)production of ROS, and 3) response to a second microbial stim-ulus (LPS). Thus, GM-CSF enhances selective effector functionsof monocytes, an effect of GM-CSF previously described for tis-sue-derived macrophages (60). In addition, our data indicate thatGM-CSF–activated peripheral blood monocytes from patientswith CD behave the same way as GMaMs (Fig. 7) and that GM-CSF may regulate the homing molecules CCR2 and CCR6, whichare involved in regulating several aspects of mucosal immunity,including the ability to mediate the recruitment of innate immunecells to the sites of epithelial inflammation (61, 62).

Monocytes may also significantly regulate the immune contextby interaction with other cells. In particular, chemokines andchemokine receptors have a key role in intestinal epithelial bar-rier repair and maintenance (63, 64). We found a significant regula-tion of chemokines and chemokine receptors with GM-CSF–depen-dent downregulation of the chemokines CXCL9, CXCL10, andCXCL11 in monocytes. These factors are known to be increasedin IBD and to attract Th1 and NK cells (65). At the same time,GM-CSF significantly induced a short-termed expression of thechemokines CCL13, CCL17, CCL18, CCL23, and CCL24 inmonocytes, which are known to attract naive T cells, Th2 cells,and/or Tregs (43, 44, 63, 65). Because of the observed upregula-tion of costimulatory molecule CD80 and the chemotactic factorsfor naive and quiescent T cells CCL18 and CCL23 (43, 44), wenext analyzed the interaction of GMaMs with T cells. Indeed,migration of naive, autologous T cells toward GMaMs was ac-celerated. Our data suggest that particularly CCL18 and CCL23might be responsible for the increased T cell migration. However,our transcriptomic data suggest that other GM-CSF–inducedchemokines (e.g., CCL13, CCL17, CCL24) might also be re-sponsible for the increased T cell migration because they areknown to attract T cells. Studies to address this question morespecifically are beyond the scope of this study. It has been shownthat treatment of human monocytes with GM-CSF generates asubtype of cells that regulate CD4+ T cell proliferation partiallyvia production of IL-10 (52), and that GM-CSF may sustain Treghomeostasis and enhance their suppressive functions (47), indi-

FIGURE 6. GMaMs alter T cell cytokineresponses in vivo and in vitro. LPMCs and MLNswere isolated from the colon of mice that weretreated as indicated. Expression of cytokines after24-h stimulation with anti-CD3e/anti-CD28 Abs isshown 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 withGMaM, control monocytes, or left alone. T cellswere stimulated with anti-CD3e/anti-CD28 Abs.Ratio of respective monocytes to T cells was 1:10,and cytokines were measured in supernatants. Datashown are the means (6 SEM) of nine independentexperiments. *p , 0.05, **p , 0.01, ***p , 0.001compared with untreated monocytes. Cell pop-ulations from the lamina propria (D) and MLNs (E)were stained for CD4 and Foxp3 expression andanalyzed by flow cytometry. Bars refer to mean 6SEM of three independent experiments. (F) MurineT cells were cocultured with GMaMs or controlmonocytes at a ratio of 10:1 (T cells/monocytes). Cellswere stained for CD4 and Foxp3 expression and an-alyzed by flow cytometry. Bars refer to mean 6 SEMof three independent experiments. **p , 0.01 com-pared with untreated monocytes (control).


cating the regulatory potential of GMaMs toward CD4+ T cellsand Tregs. When we cocultured GMaMs with syngeneic CD4+

T cells, we observed that GMaMs shape T cell response towarda Th2 phenotype and induce Tregs.After having analyzed the programming of monocytes with

GM-CSF in vitro, we next aimed at analyzing the therapeuticeffects of this cell population in a model of CD. We found that Tand B cell–deficient (Rag12/2) mice in which Crohn-like colitis wasinduced with the CD4+CD252 T cell transfer model (66, 67) wereprotected from disease progression when they received GMaMs(but not untreated monocytes). Interestingly, Rag12/2 mice thatdid not receive GMaMs but i.p. GM-CSF injections after thetransfer of T cells were not completely protected from colitis butshowed reduced disease severity. This is in agreement with earlierwork demonstrating positive effects of GM-CSF administration(i.p.) in DSS-induced colitis in BALB/c, and more importantly, inRag12/2 mice (15, 16). We postulate that the alleviating effects ofGM-CSF in experimental colitis are due to direct modulation ofmonocyte/macrophage functions including accelerated epithelialhealing. Our data demonstrate that the protective effect ofmonocytes depends on their GM-CSF prestimulation in a T cell–dependent model of colitis. The therapeutic mechanism of actionof GMaM thus involves the regulation of T cell responses, which

includes a down-toning of classical Th1 responses. In this regard,our results reconfirm that monocytes harbor important functionsregarding polarization and expansion of lymphocytes and mayalso contribute to shaping T cell responses (22). The in vivoeffects of GMaMs showed increased levels of Th2 cytokines inLPMCs (IL-4, IL-10, IL-13) and MLNs (IL-4, IL-13), but thistrend was not statistically significant. However, together with theobserved significantly reduced production of IFN-g in T cellsfrom LMPCs and MLNs, we concluded that treatment of micesuffering from colitis with GMaMs results in a shift toward Th2cytokine production of T cells in vivo. Our in vitro experimentsconfirmed that GMaMs skew the T cell response and lead to sig-nificant upregulation of Th2 cytokines (IL-4, IL-10, IL-13), whereasthe Th1 cytokine IFN-g was significantly downregulated. Thismight be explained by our observation that GMaMs display char-acteristics of both M2-like/IL-4–induced macrophages and M1-like/IFN-g–induced macrophages (as discussed earlier).Interestingly, it has recently been shown that the therapeutic

transfer of glucocorticoid-stimulated monocytes (GCsMs) in theT cell transfer colitis model also resulted in a strongly downreg-ulated release of IFN-g by T cells from LPMCs and MLNs. Theproduction of IL-4 and IL-13 was not influenced in single-cellsuspensions from LPMCs and MLNs after treatment of mice

FIGURE 7. Features of GM-CSF–activated peripheral blood monocytes of patients with quiescent CD (n = 18). (A) Adhesion of untreated (w/o) versusGM-CSF–activated patient monocytes (24 h) to fibronectin-coated plastic surface. Adhering cells were stained with 0.5% crystal violet, and staining wasquantified measuring the OD at 560 nm. (B) Migration and chemotaxis studies of untreated (w/o) versus GMaMs (24 h) using a modified Boyden chamberand LTB4 (100 nM) as an additional chemoattractant. After 4 h, cells that had migrated into the lower compartment were counted, and numbers arepresented as the percentage of untreated cells, which migrated in the absence of any chemotactic stimuli. (C) Phagocytosis of fluorescein-labeled E. coli byuntreated (w/o) versus GMaMs (24 h). E. coli phagocytosis was analyzed by flow cytometry, and phagocytic internalization was confirmed by fluorescencemicroscopy. (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 rhodaminefor the final 15 min. Oxidative burst was analyzed by flow cytometry. (E) Cytokine secretion of untreated (w/o) versus GMaMs (24 h) with and withoutfurther 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.


with injection of GCsMs, which is also in contrast with the in vitrosystem, where cytokine production of IFN-g, IL-4, and IL-13 byT cells was significantly regulated in cocultures with GCsMs (68).The same study also showed that in the T cell transfer colitismodel, CD4+Foxp3+ Tregs accumulate locally in the colon aftertreatment with GCsMs and that repetitive stimulation of naivesplenic T cells with GCsMs induces Tregs in vitro. However,Tregs also did not expand in draining MLNs and LPMCs of ani-mals treated with GCsMs during T cell transfer colitis (68).Collectively, GMaMs induce the differentiation of CD4+Foxp3+

Tregs in vitro, but we found no evidence that the GMaM-mediatedprotection from colitis is facilitated via expansion of mucosalTregs in vivo.Limitations of our study include the lack of in vivo data ex-

ploring specifically GMaMs in human CD, which may becomefeasible in the future. Although we address the tissue context byanalyzing LPMCs and MLN, the further differentiation of ex vivo–activated monocytes after transfer in vivo, especially after infil-trating the inflamed tissue, is certainly a complex issue. The cir-culating monocyte population is not homogenous but consists ofboth inflammatory and regulatory populations that counterbalanceeach other. We speculate that GM-CSF exerts its beneficial ef-fects in intestinal inflammation in vivo by specific activation ofmonocytes that combines innate immune activation, facilitatinganti-infectious defense and a simultaneous regulatory functionserving to limit adaptive immunity and excessive inflammationrather short term. The pleiotropic GM-CSF functions on monocyteactivation and their consequences for innate and adaptive immu-nity range from activation of M2-like monocytes, chemotacticmigration, and antimicrobial response to mucosal healing, but alsoencompasses regulation of adaptive immunity by attraction of, forexample, T cells with the possibility to differentiate Th cells andsubsequently limit inflammation induced by adaptive immunity.However, it remains a question for further studies whether andhow monocytes differentiate long term at sites of inflammationand within damaged tissue. It is also important to investigate theimmunomodulatory properties of monocytes in other animalmodels of experimental colitis, for example, in DSS-induced co-litis. Recent work by Kurmaeva et al. (69) has demonstrated thatimmunosuppressive monocytes (CD11b+Ly6GnegLy6Chigh cells)accumulate in the spleen and inflamed intestine during experi-mental colitis not only in T cell–induced colitis but also in anacute model induced by administration of DSS and a TNFDARE

model of chronic ileitis.In conclusion, while taking the shifting paradigms of CD

pathogenesis and immune regulation into account, our findingssupport the exploration of stimulating rather than suppressivetherapies with the potential to more specifically reprogram mono-cytes toward immunomodulatory functions to alleviate chronicinflammatory bowel disease.

AcknowledgmentsWe thank Melanie Saers, Susanne Schleifenbaum, Andrea Dick, ClaudiaSole, Eva Nattkemper, Andrea Stadtbaumer, and Sonja Dufentester forexcellent technical work. We also thank Dr. Tilmann Spieker (Departmentof Pathology, University Hospital M!unster, Germany, and St. Franziskus-Hospital, Institute of Pathology, M!unster, Germany), who helped withhistopathology. We also thank the American Gastroenterological Associa-tion Research Foundation for the Moti L and Kamla Rustgi Awards, whichsupported the presentation of the research at the 50th and 51st DigestiveDisease Week annual meetings.

DisclosuresThe authors have no financial conflicts of interest.

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68. Varga, G., J. Ehrchen, A. Brockhausen, T. Weinhage, N. Nippe, M. Belz,A. Tsianakas, M. Ross, D. Bettenworth, T. Spieker, et al. 2014. Immune sup-pression via glucocorticoid-stimulated monocytes: a novel mechanism to copewith inflammation. J. Immunol. 193: 1090–1099.

69. Kurmaeva, E., D. Bhattacharya, W. Goodman, S. Omenetti, A. Merendino,S. Berney, T. Pizarro, and D. V. Ostanin. 2014. Immunosuppressive monocytes:possible homeostatic mechanism to restrain chronic intestinal inflammation. J.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


2

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


3

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


4

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


5

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


6

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


7

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


8

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


9

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


10

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


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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


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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


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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


Human CCR4 L291H4

Biolegend, San Diego, CA, USA Human CCR6 G034E3


Human CCR7 G043H7

Biolegend, San Diego, CA, USA Human CCR9 L053E8


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

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