The Journal of Immunology
Reprogramming of Monocytes by GM-CSF Contributes to
Regulatory Immune Functions during Intestinal
Inflammation
Jan Däbritz,*,†,‡,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-CSF
mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory
potential of GM-CSF–activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to
increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial
stimuli. 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 of
IL-4, IL-10, and IL-13, and decreased production of IFN-g in lamina propria mononuclear cells in vivo. Confirming this finding,
GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro.
Beneficial effects of GM-CSF in Crohn’s disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit
excessive inflammation. The Journal of Immunology, 2015, 194: 2424–2438.
O
ur concepts of immunology have changed dramatically
over the past decades. The postulates of primary functions assigned to innate or adaptive immunity have been
challenged by the recognition of a complex interplay between the
different cellular and humoral factors that all together constitute
our immune system. This helped in understanding how we are
protected from infections, but it also enabled discovering key
aspects of autoimmunity and chronic inflammation including regulatory mechanisms that counteract a perpetuated immune activation. Although different functions of adaptive immune cells,
including regulatory T cells (Tregs), are already consolidated, our
understanding of different functions of innate immune cells has
only recently been enriched. As an example, phagocytes were
traditionally seen solely as effector cells of innate immunity
promoting host defense and driving chronic inflammation. It is
now accepted that monocytes can differentiate into macrophages
with various activation patterns ranging from classically activated
proinflammatory to anti-inflammatory phenotypes. These cells
(often referred to as M1 and M2 macrophages) represent the outer
margins of a broad spectrum of numerous activation and differentiation 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 striking
example, our view of Crohn’s disease (CD) is constantly challenged. Traditionally, CD has been associated with a Th1 cytokine
profile. In addition, because CD is a chronic granulomatous dis-
*Department of Pediatric Rheumatology and Immunology, University Children’s
Hospital M€
unster, M€
unster 48149, Germany; †Interdisciplinary Center of Clinical
Research, University Hospital M€
unster, M€
unster 48149, Germany; ‡Gastrointestinal
Research in Inflammation & Pathology, Murdoch Children’s Research Institute,
The Royal Children’s Hospital Melbourne, Parkville 3052, Victoria, Australia;
x
Department of Pediatrics, University of Melbourne, Melbourne Medical School,
Parkville 3052, Victoria, Australia; { Department of Dermatology, University
Hospital M€
unster, M€
unster 48149, Germany; ‖Institute of Immunology, University
Hospital M€
unster, M€
unster 48149, Germany; and #Department of Medicine B,
University Hospital M€
unster, M€
unster 48149, Germany
J.D. and D.F. developed the concept, designed the experiments, and supervised the
experiments; 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 welfare
authorities; J.D., T. Weinhage, T. Wirth, K.W., A.B., and D.S. performed the experiments and collected data; M.R., G.V., M.B., D.B., and T. Wirth helped with animal
models of experimental colitis; J.D., T. Weinhage, G.V., and D.F. analyzed the data
and interpreted results; J.D. wrote the manuscript; and each author has approved the
final version of the report and takes full responsibility for the manuscript.
1
J.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 and
Edythe Broad Foundation (Grant IBD0201 to D.F., J.D., and J.M.E.), the German
Research Foundation (Grant DFG DA1161/4-1 to J.D. and D.F., Grant DFG SU195/
3-2 to G.V., Grant DFG SF1009B08 to M.B.), the Innovative Medical Research
€
Program of the University of M€
unster (Grants IMF DÄ120904 and DÄ3U21003
to
J.D. and D.F.), the Interdisciplinary Center for Clinical Research of the University of
M€
unster (Grant IZKF Eh2/019/11 to J.M.E.), the European Union’s Seventh Framework Programme (Grant EC-GA305266 ‘MIAMI’ to D.F.), and a research fellowship
from the German Research Foundation (Grant DFG DA1161/5-1 to J.D.).
Portions of this work were presented at the 50th Digestive Disease Week Annual
Meeting, May 30–June 4, 2009, Chicago, IL and the 51st Digestive Disease Week
Annual Meeting, May 1–5, 2010, New Orleans, LA.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401482
The sequences presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession number
GSE63662.
Address correspondence and reprint requests to Dr. Jan Däbritz, Department of Pediatric
Rheumatology and Immunology, University Children’s Hospital M€
unster, Röntgenstrasse 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, murine
endoscopic score of colitis severity; MFI, mean fluorescence intensity; MLN, mesenteric lymph node; qRT-PCR, quantitative real-time RT-PCR; ROS, reactive oxygen
species; Treg, regulatory T cell.
Copyright ! 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
The Journal of Immunology
ease and anti-inflammatory therapies targeting innate immunity
have proved effective, it was a paradigm that overactive phagocytes are involved. More recently, however, emerging evidence
has consolidated the view of CD as a form of innate immunodeficiency (4–6). Central to this hypothesis were the observations of
diminished neutrophil accumulation in patients with CD with
impaired clearance of bacteria from tissues (7, 8). The underlying
problem appeared to be a primary immunodeficiency of macrophages, which secreted insufficient concentrations of proinflammatory
cytokines and chemokines upon bacterial challenge (9). The view
of defective macrophage functions in CD is further supported by
an inappropriate mucosal healing (10, 11). Resolution of inflammation and healing relies on the infiltration of monocytes as
crucial 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 macrophage
functions, GM-CSF has been shown to alleviate acute dextran sulfate
sodium (DSS)-induced colitis in mice (15, 16). Even more important,
it is conceivable that GM-CSF–driven modulation of innate immune
cells involved in mucosal repair and/or dampening of inflammatory
reactions might contribute to the benefits of GM-CSF therapy observed in some CD patients (17). These findings may link the novel
concepts of monocyte biology with that of CD pathogenesis, because
recent developments in immunology and genetics suggest that
monocytes and their derivative cells play an important role in the
pathophysiology of CD. It is noteworthy that blood monocytes are the
exclusive source of macrophages in inflamed intestinal mucosa (18).
Undoubtedly, monocytes carry out specific effector functions
during inflammation (19). Recent studies underpin the dual
function of monocytes: on one hand, the impaired monocyte
function initiating CD, and on the other hand, the overactivation of
monocytes and adaptive immunity maintaining the disease (20).
Cells of the monocyte/macrophage lineage are characterized by
considerable diversity and plasticity (21). Furthermore, monocytes
can drive modulation of adaptive immunity by regulating T cell
responses (22). GM-CSF functions both as a growth factor for
myeloid progenitors and as a cytokine acting directly on maturing
cells. Data from animal models indicate an important role in inflammation and autoimmunity, with varying consequences that
likely 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 inflammation. To this end, we chose an unbiased but comprehensive
approach taking all potential functions of GM-CSF–activated
monocytes (GMaMs) into account (gene expression, innate immune functions, interplay with adaptive immunity, wound healing) rather than focusing on polarizing edges. We show in this
article that beneficial effects of GM-CSF in CD could be explained
by a complex reprogramming of altered monocyte/macrophage
functions. These findings suggest the exploration of stimulating,
rather than suppressive, therapies with the potential to more specifically reprogram monocytes to modulate immune functions.
Materials and Methods
Human monocytes
Blood samples from individual healthy donors were purchased from the
Department of Transfusion Medicine at the University Hospital M€unster,
M€unster, Germany. Peripheral blood monocytes were obtained from
donors by leukapheresis and isolated to .90% purity as previously described (24). Monocytes were cultured (1 3 106 cells/ml) in hydrophobic
Teflon bags (Heraeus, Hanau, Germany) in McCoy’s 5a medium supplemented with 5% human AB serum, 2 mM L-glutamine, 200 IU/ml penicillin, 100 mg/ml streptomycin, and 13 nonessential amino acids (all from
Biochrom, Berlin, Germany). Monocytes were allowed to rest for 16 h
2425
before stimulation. Monocytes from at least three different individuals
were assessed with each experiment.
Patients
Clinical and demographic characteristics of the study subjects and methods
have been reported in detail previously (25). Ethical approval was obtained
from the Ethics Committee of the University of M€unster (reference no.
2006-267-f-S, obtained by Jan Däbritz), and fully written informed consent
was 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 of
experiments to analyze changes in gene expression patterns induced by
GM-CSF. Using high-density microarrays with .22,000 oligonucleotide
sets, we obtained the expression levels of at least 13,000 independent
transcripts. RNA preparation, sample preparation, and hybridization to
Affymetrix (Santa Clara, CA) Human Genome 133 A Gene Chip arrays for
microarray analysis were performed as described previously (26).
Statistical analysis of microarray data
For analysis of data from individual donors, raw data of GM-CSF–treated
samples were processed by MicroArray Suite Software (Affymetrix) using
data from corresponding control samples as baseline. Signals were scaled
to a target intensity of 500 and log-transformed. Detection and change calls
using perfect match and mismatching probes were assigned using a signed
rank test as described previously (26–28). Data were submitted to the Gene
Expression Omnibus database under accession number GSE63662 (http://
www.ncbi.nlm.nih.gov/geo). We retained only genes that were significantly
regulated in every single experiment (change p , 0.05, fold-change $ 2.0,
expression over background). The data of the complete set of experiments
were further studied applying the Expressionist Suite software package
(GeneData), which allows identification of genes that are significantly regulated in multiple independent experiments as described previously (26).
Being aware of the low significance at low-intensity levels, we filtered for
genes with an expression over background in at least one of the two experimental groups (GMaM versus monocytes). We finally retained only
genes that were significantly regulated in every single experiment (change
p , 0.05, fold-change $ 2.0, expression over background), as well as in the
complete set of experiments (expression over background, fold-change $
2.0, p , 0.05, paired t test). Reproducibility of the results was confirmed
using RT-PCR for selected genes and three new independent experiments.
Quantitative real-time PCR
Expression of selected genes in human and mouse (C57BL/6) monocytes
was analyzed by quantitative real-time RT-PCR (qRT-PCR) as described
previously (29). PCRs were performed and measured on a CFX384 Touch
real-time PCR detection system (Bio-Rad, Munich, Germany). The relative
expression was calculated using ribosomal protein L13a as endogenous
housekeeping control gene. The primers used for PCR analysis are given in
Supplemental Table III.
Flow cytometry
FACS measurements were performed using a Cyflow space equipped with
FlowMax 2.8 (both Partec, M€unster, Germany), and analysis was performed using FlowJo software (TreeStar, Ashland, OR). Ab staining of
cells was routinely done with 1 mg/ml of the according Ab. For detection
of cell-surface molecules, flow cytometry was performed as described
earlier (26). All intracellular stains were performed using the transcription
factor staining buffer set (eBioscience, San Diego, CA). mAbs used are
given in Supplemental Table IV.
Chemokine production of GMaMs
Chemokine concentrations of CCL18 and CCL23 were determined in
cell culture supernatants of monocytes treated for 24 h with GM-CSF
(10 ng/ml) or untreated control cells by an ELISA system according to
the 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 monocytes
were polarized with GM-CSF (10 ng/ml) 6 IFN-g (100 mg/ml) or left
untreated as a negative control. Expression of selected genes was analyzed
by qRT-PCR as described earlier.
2426
In additional experiments, human monocytes were polarized for 24 h
with 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 for
an additional 24 h. IL-1b, TNF-a, IL-10, and CD206 expression were
measured by flow cytometry as described earlier.
Finally, human monocytes were stimulated 6 GM-CSF (10 ng/ml) for 0,
30, 60, and 120 min, and expression of IFN-a, IFN-b, IFN-g, and IL-4 was
analyzed by qRT-PCR as described earlier.
Migration, chemotaxis, monocyte trafficking, and adherence
Monocyte assays in Transwell plates (Costar, New York, NY) were performed 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 to
migrate for 4 h. T cell migration was analyzed using the Cultrex 96-well
cell migration assay according to the manufacturer’s protocol (Trevigen,
Gaithersburg, MD). T cells were isolated from fresh PBMCs using an
EasySep human T cell enrichment kit according to the manufacturer’s
protocol (STEMCELL Technologies, Vancouver, BC, Canada). T cells (5 3
104) were added to top-chamber and cell culture supernatants of untreated
monocytes or GMaMs as well as CCL18 (1 ng/ml), CCL23 (8 ng/ml; both
PeproTech, Rocky Hill, NJ), and medium (control) were added to the bottom
chamber. T cells that had migrated into the lower compartment within 4 h
were measured using an Infinite M200 Pro reader (TECAN, Crailsheim,
Germany).
Expression of intestinal-associated homing molecules in human monocytes treated 6 GM-CSF (10 ng/ml) for 24 h was analyzed by flow cytometry (gated on CD14+ cells) as described earlier.
For determination of cell adhesion, monocytes (2 3 105) were stimulated
with GM-CSF (10 ng/ml) for 24 h or left untreated. Monocytes were
seeded in triplicates into 96-well flat-bottom plastic tissue-culture plates
and incubated at 37˚C and 7% CO2 for 4 h. Nonadhering cells were removed by washing twice; remaining adherent cells were fixed with 2%
glutaraldehyde (Sigma-Aldrich, Taufkirchen, Germany) for 10 min. Wells
were washed two times with H2O and subsequently stained with 0.5%
crystal violet (Merck, Darmstadt, Germany) in 2% EtOH (pH 6.0) for an
additional 15 min at room temperature. Finally, wells were washed three
times and cells were lysed. Ten percent acetic acid was added, and staining
was quantified measuring the OD at 560 nm using an Asys Expert 96
Microplate ELISA reader (Anthos Mikrosysteme, Krefeld, Germany) (25).
GM-CSF priming
For priming experiments, human monocytes were stimulated for 24 h with
GM-CSF (10 ng/ml) or left untreated. After pretreatment, monocytes were
stimulated with medium containing LPS (10 ng/ml) or left untreated for an
additional 4 h. TNF-a and IL-1b content were measured in culture
supernatants by ELISA (OptEIA ELISA kits; BD Pharmingen, Heidelberg,
Germany). Expression of selected genes was confirmed by qRT-PCR as
described earlier.
Phagocytosis and oxidative burst
For detection of phagocytic capacity, cells were incubated with carboxyfluorescein diacetate (Invitrogen, Karlsruhe, Germany)–labeled Leishmania
major parasites (ratio cells: L. major = 1:5) or FITC (MoBiTec, Göttingen,
Germany)-labeled latex beads (ratio cells: beads = 1:10) for 4 h (31). Rate
of 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 the
induction of oxidative burst. The extracellular chemiluminescence response was measured in the presence of isoluminol (50 mM; SigmaAldrich, Taufkirchen, Germany) as described previously (24).
In vitro scratch closure assay
Cells of the Caco-2 human colon adenocarcinoma cell line (ATCC HTB-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, and
1% 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-well
plates 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-deprived
medium in the presence or absence of 2.5 3 105 untreated monocytes or
GMaMs. The initial wound size was determined by microscopy, and the
area of the scratch was calculated with ImageJ software (Version 1.45s;
National Institutes of Health). Additional photographs were taken using
a reference line 24 h after wounding, and the rate of wound closure was
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
analyzed by measuring the scratch area relative to the initial wound area
after each time point.
Influence of human GMaMs on T cell fate
Human T cells were purified from donor-specific PBMCs using positive
selection of CD2-expressing T cells by MACS technology according to the
manufacturer’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 mAbs
raised against CD4, CD25, and Foxp3 (Supplemental Table IV). Subsequent flow cytometry was performed as described earlier.
Mice
Experiments were performed in accordance with approved protocols of
the animal welfare committee of the North Rhine-Westphalia State Agency
for Nature, Environment and Consumer Protection, Recklinghausen,
Germany (LANUV NRW Reference No. 87-51.04.2010.A113). C57BL/6
and Rag12/2 mice were kept under specific pathogen-free conditions and
according 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 described
earlier (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 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). After culture, cells were washed
three 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 of
animals was monitored daily, and around day 40 animals that established
colitis by weight loss on consecutive days received GMaMs or untreated
monocytes (2 3 106 per mouse) i.v. Alternatively, 5 mg GM-CSF (Immunotools) 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, mice
were euthanized by CO2 inhalation, and their colons were prepared, measured, and preserved for histology.
Isolation of murine T cells from spleen
T cells were isolated from spleens as described previously (33). T cells used
for induction of transfer colitis were further purified for CD4+ and depleted
of CD25+ cells by MACS technology according to the manufacturer’s
instructions (Miltenyi Biotech).
Histopathologic analysis
For histopathologic analysis, tissue specimens from the proximal and distal
colon were fixed in 10% buffered formalin phosphate and embedded in
paraffin. Sections were cut at 3–5 mm and stained with H&E. Inflammation
was graded from 0 to 4 in a blinded fashion: 0, no signs of inflammation; 1,
low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte
infiltration, 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 (FrekaClyss; Fresenius Kabi, Sèvres, France). High-resolution colonoscopy was
performed using a veterinary endoscopy workstation (Coloview; Karl
Storz, Tuttlingen, Germany) to assess colitis. Under visual control, the
rigid miniature endoscope (1.9-mm outer diameter) was inserted ∼4 cm
according to anatomic conditions. The modified murine endoscopic score
of colitis severity (MEICS) observes thickening of the colon, changing of
vascular pattern, presence of fibrin, granular mucosa surface, and stool
consistence (0–3 points each, maximum of 15 points); it was used to
evaluate colonic inflammation (34).
In vivo cell tracking
For in vivo cell tracking of GMaMs, cells were stained with a commercially available lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine
The Journal of Immunology
iodide (Life Technologies, Darmstadt, Germany) with an emission maximum
of 782 nm as described elsewhere (35). In vivo distribution of labeled cells
across the intestine was studied 24 h after i.v. injection using a planar smallanimal FMT system (FMT 2500; VisEn Medical) as described previously (35).
Isolation of lamina propria mononuclear cells and cells from
mesenteric lymph nodes
Lamina propria mononuclear cells (LPMCs) were isolated from the colon
of colitogenic mice by a standard method (36). In brief, the colon was
removed, opened longitudinally and cut into 5-mm pieces, and washed
with cold Ca2+/Mg2+-free HBSS. The intestinal tissue specimens were
transferred into HBSS with EDTA to remove intraepithelial lymphocytes.
After 30 min of gentle shaking at 37˚C, the samples were vortexed and
intraepithelial lymphocyte–containing supernatant was removed. This step
was repeated twice. LPMC suspensions were prepared from the EDTAtreated de-epithelialized intestinal tissue by further incubation with 100 U/ml
collagenase and 5 U/ml DNase for 30 min at 37˚C. LPMCs were washed,
resuspended in 44% Percoll solution (Amersham Pharmacia Biotech, Piscataway, NJ), underlaid with 66% Percoll solution, and centrifuged for
30 min at 600 3 g. The LPMC fraction was harvested from the interface.
For cell isolation from mesenteric lymph nodes (MLNs), the lymph
nodes of treated mice and control mice were carefully isolated, pooled, and
passed through a 40-mm cell strainer, and the resulting single-cell suspension was washed once with PBS.
Coculture of naive T cells and restimulation of LPMCs and
MLNs
Naive T cells were isolated from splenocytes using a pan T cell kit II
(Miltenyi Biotec) as described previously (37), and 1 3 105 T cells were
cocultured for 4 d (ratio 10:1) with respective monocytes in triplicates
for each condition in anti-CD3e and anti-CD28 Abs (5 mg/ml each; Supplemental Table IV) coated to 96-well, round-bottom plates in RPMI 1640
supplemented 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˚C
and 5% CO2. To further test for cytokine production, we stimulated isolated LPMCs and MLN cells (2 3 105 per well) from mice used in transfer
colitis experiments with anti-CD3e and anti-CD28 Abs (5 mg/ml each;
Supplemental Table IV) coated to 96-well, round-bottom plates for 24
(LPMCs) or 96 h (MLNs). A total of 100 ml cell supernatants was stored
until cytokine analysis was performed using a bead-based multiplex assay
(mouse Th1/Th2 10plex FlowCytomix; eBioscience), according to manufacturer’s instructions.
Induction of Tregs by murine GMaMs
LPMC and MLN single-cell suspensions from mice used in transfer colitis experiments were stained with Abs raised against CD4 and Foxp3
(Supplemental Table IV). Cells were measured by flow cytometry as described earlier.
T cell coculture experiments were performed to evaluate the function of
GMaM to induce Tregs. Therefore, 1 3 105 splenic T cells (described
earlier) were cocultured with 1 3 104 monocytes (GM-CSF–activated or
untreated) in 96-well, round-bottom plates for 7 d in RPMI 1640 supplemented 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˚C and
5% CO2. Cells were harvested and stained with 1 mg anti-CD4 and antiFoxp3. Used Ab clones are given in Supplemental Table IV. Flow
cytometry was performed as described earlier.
Statistics
Data are expressed as mean 6 SEM unless stated otherwise and were
assessed using the Student t test. The p values ,0.05 were considered to be
statistically significant. All calculations were performed using SPSS version 14 (SPSS, Chicago, IL).
Results
Gene expression and phenotype shift of GMaMs
GM-CSF provokes nonclassical monocyte activation. To analyze
monocyte activation comprehensively and unbiased, we performed
a global RNA expression analysis. The microarray data were filtered using strict statistical criteria and revealed a significant
regulation of genes involved in immune/inflammatory responses
2427
(especially chemokines), cell motility, chemotaxis, regulation of
cell growth, endocytosis, and Ag processing and presentation
(Table I). Furthermore, we analyzed statistical overrepresentation
of transcription factor binding sites in GM-CSF–regulated genes
using CARRIE software (Supplemental Table I). Overall, we
found that in human monocytes, 190 genes were significantly
upregulated, whereas 212 were downregulated after 16-h stimulation with GM-CSF (Supplemental Table II). Raw data have been
submitted to the Gene Expression Omnibus under accession
number GSE63662 (http://www.ncbi.nlm.nih.gov/geo). We confirmed microarray expression data by qRT-PCR for selected genes
(Fig. 1A). Analyses of expression levels at different time points of
GM-CSF activation confirmed that gene regulation in GMaM
is most relevant after 16–24 h (Fig. 1B). However, we also
show that the expression of most of these genes is already
significantly regulated after 4 h of GM-CSF stimulation. Genes
that were not significantly regulated after 24 h of GM-CSF activation also showed no significant upregulation or downregulation of
their expression after 4, 16, 48, or 120 h (Fig. 1B). In agreement
with the microarray data, flow cytometry confirmed upregulation
of CD80 and downregulation of CD9 upon GM-CSF activation
(Fig. 1C, 1D).
GM-CSF drives monocytes toward M2-like phenotype. Interestingly, the microarray expression data of GMaM chemokine ligands
and receptors showed a gene expression pattern indicative of a GMCSF–induced shift toward a M2-like phenotype (Fig. 2A). This was
confirmed by protein quantification (ELISA) for selected chemokines (Fig. 2B) and by gene expression analyses (qRT-PCR) for
differentially expressed genes that were assigned to M1- and M2like monocytes based on currently accepted annotations (Table II)
(2, 38, 39).
Gene expression in GMaM is similar to IL-4–induced gene
expression profiles. Our gene expression analyses (Fig. 1B) suggest that the observed gene expression in GMaM is similar to
IL-4–induced gene expression profiles in monocytes. We stimulated human monocytes from healthy donors with or without GMCSF for 0, 30, 60, and 120 min and analyzed gene expression of
IFN-a, IFN-b, IFN-g, and IL-4 by qRT-PCR. The expression of
IFNs and IL-4 in monocytes was not upregulated at any time after
GM-CSF stimulation when compared with untreated monocytes
(data not shown). Interestingly, we found that the GM-CSF–induced gene expression pattern in human monocytes within the
first 24 h is similar to the gene expression pattern of IL-4– but not
IFN-g–stimulated monocytes (Fig. 2C, 2D). We assume that our
transcriptomic data of human GMaMs rather reflect a primary
effect, which, however, has similarities with the expression pattern 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 in
GM-CSF (40). Fifty GM-CSF–specific type I IFN–dependent
regulated genes were identified, of which we found only five to
be significantly regulated in GMaMs (IFIT3, CBX6, ISG20,
ABCA9, COLEC12; Supplemental Table II). In human cells, it
was found that the expression of 154 type I IFN–regulated genes
were also different between monocyte-derived macrophages
cultured in GM-CSF (GM-MDM) or M-CSF (MDM) (41). We
found that only 7 of the top 50 type I IFN–dependent genes
differentially expressed between human GM-MDM and MDM
were significantly regulated in GMaMs (EDNRB, LGMN,
P2RY14, CH25H, CDKN1C, GGTLA1, CD69; Supplemental
Table II). In addition, we found no significant GM-CSF–dependent gene regulation of type I IFNs in GMaMs. Likewise,
the gene expression of IFN-g in human monocytes was not
2428
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
Table I. Selected genes upregulated and downregulated by GM-CSF activation
Gene
Description (NCBI Gene)
n-Fold
p
Upregulated by GM-CSF Activation
Inflammatory response
GGTLA1
CFH
CD80
PROCR
CD69
BANK1
SLAMF1
IL7R
CLEC5A
IL1RAP
ALOX5AP
LTB
PTGS1
Chemotaxis
CCL13
CCL23
PPBP
CXCL5
CCL17
IL8RB
CCR6
SPN
Ag processing and presentation
CD1C
CD1B
CD1E
CD1A
Regulation of cell growth
TGFB2
FGF13
CISH
g-Glutamyltransferase-like activity 1
Complement factor H
CD80 molecule
Protein C receptor, endothelial (EPCR)
CD69 molecule
B-cell scaffold protein with ankyrin repeats 1
Signaling lymphocytic activation molecule
IL 7 receptor
C-type lectin domain family 5, member A
IL 1 receptor accessory protein
Arachidonate 5-lipoxygenase-activating protein
Lymphotoxin b (TNF superfamily, member 3)
PG-endoperoxide synthase 1
50.5
44.9
8.4
7.5
6.4
5.2
5.0
4.8
4.5
4.4
4.1
3.6
3.3
,0.001
0.013
,0.001
,0.001
0.002
0.011
0.041
0.001
0.011
0.008
0.003
0.002
0.003
Chemokine (C-C motif) ligand 13
Chemokine (C-C motif) ligand 23
Proplatelet basic protein (CXCL7)
Chemokine (C-X-C motif) ligand 5
Chemokine (C-C motif) ligand 17
IL 8 receptor, b
Chemokine (C-C motif) receptor 6
Sialophorin (leukosialin, CD43)
16.7
8.3
7.2
5.8
5.5
5.1
4.3
3.8
,0.001
0.002
0.001
0.025
0.003
0.002
,0.001
0.007
18.4
15.5
9.8
6.4
,0.001
0.008
,0.001
,0.001
Transforming growth factor, b2
Fibroblast growth factor 13
Cytokine inducible SH2-containing protein
26.8
19.8
5.7
0.006
0.018
,0.001
Fc fragment of IgG, high affinity Ib, receptor (CD64)
Aquaporin 9
IFN-induced protein with tetratricopeptide repeats 1
Guanylate binding protein 5
Myxovirus (influenza virus) resistance 1
CD28 molecule
2’,59-oligoadenylate synthetase 1, 40/46kDa
2’-59-oligoadenylate synthetase 2, 69/71kDa
Heparanase
Guanylate binding protein 2, IFN-inducible
Monoglyceride lipase
29.0
25.3
25.0
25.0
24.2
24.2
24.0
23.7
23.6
23.5
23.4
0.011
0.004
0.037
0.009
0.023
0.008
0.049
0.043
0.003
0.002
,0.001
Chemokine (C-X-C motif) ligand 12
Chemokine (C-X-C motif) ligand 11
Chemokine (C-X-C motif) ligand 13
Chemokine (C-X-C motif) receptor 4
Chemokine (C-X-C motif) ligand 10
216.9
27.1
26.4
26.2
23.5
0.009
0.046
0.019
0.001
0.007
Fc fragment of IgG, high affinity Ia, receptor (CD64)
Macrophage scavenger receptor 1
27.7
24.7
0.022
0.041
Cell adhesion molecule 1
CD9 molecule
Insulin-like growth factor 1 (somatomedin C)
Integrin, b 8
Nidogen 1
KIT ligand
Leptin (obesity homolog, mouse)
Cyclin-dependent kinase inhibitor 1C (p57, Kip2)
Signal transducer and activator of transcription 1
28.5
26.4
25.0
25.0
24.7
23.1
29.1
23.5
23.2
0.001
0.012
,0.001
0.006
,0.001
0.002
,0.001
0.041
0.034
CD1c
CD1b
CD1e
CD1a
molecule
molecule
molecule
molecule
Downregulated by GM-CSF Activation
Immune response
FCGR1B
AQP9
IFIT1
GBP5
MX1
CD28
OAS1
OAS2
HPSE
GBP2
MGLL
Chemokines
CXCL12
CXCL11
CXCL13
CXCR4
CXCL10
Phagocytosis/Endocytosis
FCGR1A
MSR1
Other
CADM1
CD9
IGF1
ITGB8
NID1
KITLG
LEP
CDKN1C
STAT1
significantly regulated by GM-CSF after 16 h nor after 24 h
(Table I, II). In addition, GM-CSF induced neither IRF4 (M2
polarization) nor IRF5 (M1 polarization) in human monocytes
when activated with GM-CSF for 24 h (Table II). However,
activation of NF-kB, which promotes M1 macrophage polarization, was significantly downregulated by GM-CSF stimulation, whereas the activity of C/EBPb, which is crucial for expression of M2-regulated genes, was significantly upregulated
The Journal of Immunology
2429
FIGURE 1. Confirmation of GM-CSF–regulated gene expression in monocytes (microarray data) by real-time PCR and flow cytometry. (A) Results obtained
from microarray analysis of GM-CSF–dependent gene regulation in human monocytes (after 16 h) compared with unstimulated monocytes were confirmed by
qRT-PCR. Genes analyzed were: lymphotoxin b (LTB); complement factor H (CFH); g-glutamyltransferase-like activity 1 (GGTLA); the chemokines
CXCL10, CXCL12, CCL23, and CCL13; and the CD1c and CD80 molecule. Shown are the mean relative n-fold regulation (6 SEM) of three independent
experiments. (B) GM-CSF–dependent gene expression analysis by qRT-PCR of selected genes at different time points. Shown is the mean relative n-fold
regulation (6 SEM) of three independent experiments. (C and D) Expression of selected cell-surface molecules (C, CD80; D, CD9) found to be differentially
expressed in GMaMs by microarray analysis, confirmed by flow cytometry. Specific profiles are shown by thick lines; isotype controls appear as thin lines.
Numbers show the quotient of specific/isotype control MFI. The experiment was repeated three times with similar results, and the differences in MFI shifts
between control and GMaMs were in accordance with microarray data. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes.
by GM-CSF stimulation in the first 24 h (Supplemental Table I).
In addition, further quantitative PCR analysis suggests that
IFN-g does not contribute to our transcriptomic data provided for
GMaMs. The gene expression of chemokines (CXCL10, CXCL11,
CCL1, CCL13, CCL23, CXCL5), CD206, CD209, and IL-1b in
human monocytes was specifically and significantly upregulated or
downregulated by either GM-CSF or IFN-g after 24 h of stimulation (data not shown).
Finally, we analyzed whether GM-CSF is affecting distinct
monocyte subsets within the overall monocyte population differentially. Our results showed a homogenous shift of GM-CSF–
induced cell-surface markers within the overall monocyte population using FACS (data not shown). Thus, we found no evidence
that GM-CSF is affecting distinct monocyte subpopulations. Nevertheless, we have performed additional monocyte/macrophage
polarization experiments. To this end, we stimulated human monocytes from healthy donors for 24 h with or without IFN-g (M1
macrophage polarization) or with or without IL-4 (M2 macrophage
polarization), and cells were subsequently stimulated with or
without GM-CSF for further 24 h. Expression of IL-1b, TNF-a,
IL-10, and CD206 was analyzed by flow cytometry (Fig. 2E). The
data suggest that GM-CSF is affecting both M1- and M2-polarized
monocytes and that GM-CSF is not affecting distinct monocyte
subsets within the overall population differentially.
Comprehensive characterization of GMaM innate immune
functions
GM-CSF promotes adherence and migration of monocytes. Major
functions of monocytes include the capacity to adhere and migrate,
which is crucial for their recruitment into tissue. Adherence of
GMaMs to plastic surfaces was enhanced after 24 and 48 h compared
with untreated control cells (Fig. 3A). We tested whether GM-CSF
activation would affect migration and chemotaxis of monocytes in
general 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 migration
toward MCP1 and LTB4 were significantly enhanced after 4 h
(Fig. 3B). The chemotactic effect was specific because migration
did not occur when MCP1 or LTB4 was added to the upper compartment of the Boyden chamber (data not shown). In addition,
GM-CSF–induced increase in chemotaxis was also specific to the
stimulus because we did not find increased migration toward IL-8
(Fig. 3B). Integrins and CC chemokine receptors play a potential
role in monocyte trafficking into the mucosa in the context of
mucosal homeostasis at the intestinal epithelial barrier. These
molecules are also known to play a role in the pathogenesis of
human inflammatory bowel diseases. Expression of integrins and
CC chemokine receptors was analyzed by flow cytometry gated on
2430
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
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-like
monocyte/macrophage subsets in humans. (B) Microarray analysis data were confirmed by protein quantification (ELISA) for two selected chemokines
(CCL 18 and CCL 23). (C and D) IL-4– (C) and IFN-g–dependent (D) gene expression analysis (RT-PCR) in human monocytes at different time points.
Bars represent the relative n-fold regulation (mean 6 SEM) of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 compared with
untreated monocytes. (E) Effects of GM-CSF activation on already primed monocytes. Monocytes were primed for 24 h toward M1 or M2 and subsequently
treated for 24 h 6 GM-CSF. Expression (mean 6 SEM) of IL-1b, TNF-a, IL-10, and CD206 is shown gated on CD14+ cells of three independent
experiments. *p , 0.05, **p , 0.01, ***p , 0.001.
CD14+ cells, and expression is stated as mean fluorescence intensity
(MFI; geo-mean) 6 SEM of three independent experiments.
Expression of CCR2 and CCR6 was significantly increased in
GM-CSF–stimulated monocytes (CCR2 150.2 6 31.1; CCR6
24.5 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 of
CCR7 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 CX3CR1
was similar in GM-CSF–stimulated and untreated cells (data not
shown).
Production of reactive oxygen species is increased and
phagocytosis is unimpaired in GMaMs. Another important function of monocytes after being recruited, for example, to sites of
infection or defective barriers, is the phagocytosis and killing of
pathogens (e.g., via production of reactive oxygen species [ROS]).
Spontaneous and PMA-induced production of ROS was significantly enhanced in GMaMs (Fig. 3C). A few molecules involved
in phagocytosis/endocytosis were significantly downregulated by
GM-CSF stimulation (Table I). We therefore tested phagocytosis
of latex beads and of complement opsonized living L. major
parasites after activation of monocytes with GM-CSF. We detected
no significant difference in phagocytosis of latex beads by
GMaMs, and also phagocytosis of L. major promastigotes was not
altered compared with control monocytes (data not shown).
GM-CSF primes the monocyte response to a secondary microbial
stimulus. To test the hypothesis that GM-CSF specifically activates
monocyte functions by augmenting anti-infectious/antimicrobial
defense and bacterial clearance, we analyzed GMaMs for an increased response to a secondary microbial stimulus. Therefore, we
analyzed the influence of GM-CSF stimulation (24 h) on cytokine
production and gene expression in human monocytes after 4 h of
costimulation with bacterial endotoxin (LPS). Compared with control monocytes, GMaMs exposed to LPS produced significantly
more IL-1b and TNF-a (Fig. 3D). LPS stimulation of GMaMs
also resulted in a much more pronounced expression of inflammatory genes as revealed by qRT-PCR (Fig. 3E). This confirms
a GM-CSF–induced priming effect on monocytes, leading to an
increase in vitro response to other stimuli (42).
GMaM impact on epithelial healing and on adaptive immunity
GMaMs accelerate epithelial healing. In addition to their innate
phagocytic and killing activity in antimicrobial defense, monocytes
are also involved in wound repair. Because we observed phenotypic
The Journal of Immunology
2431
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
Chemokine (C-X-C motif) ligand 11
Chemokine (C-X-C motif) ligand 13
IFN, g
TNF a
TNF ligand superfamily
NO synthase 2, inducible
B7-1
IL 1 b
IL 6
IL 8
IL 12
IL 18
IL 23
IFN regulatory factor 5
PG-endoperoxidase synthase 2
Colony stimulating factor 2
Colony stimulating factor 3
Chemokine (C-X-C motif) ligand
Chemokine ligand 1
Chemokine ligand 13
Chemokine ligand 23
TGF b
Arginase
Mannose receptor C type 1 (MRC1)
CD163 molecule
DC-SIGN
IL 1 receptor, type 2
IL 10
IFN regulatory factor 4
CXCL10
CXCL11
CXCL13
IFNg
TNFa
TRAIL
iNOS
CD80
IL1b
IL6
IL8
IL12
IL18
IL23
IRF5
COX2
GM-CSF
G-CSF
CXCL5
CCL1
CCL13
CCL23
TGFb
ARG1
CD206
CD163
CD209
CD121b
IL10
IRF4
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M1
M2
M2
M2
M2
M2
M2
M2
M2
M2
M2
M2
M2
↓
↓
↓↓
↔
↑
↓
↔
↑
↑
↑
↔
↔
↔
↔
↔
↔
↓
↓
↑
↑↑
↑↑↑
↑↑
↔
↑
↑↑
↔
↑
↑
↔
↔
22.36
22.75
25.27
21.20
4.50
22.03
1.94
4.22
2.81
2.42
1.15
1.43
1.59
21.05
21.07
21.03
21.36
23.81
3.87
6.82
21.06
6.87
1.12
3.90
6.45
21.90
2.65
2.70
21.25
1.01
0.005
0.004
0.000
0.700
0.000
0.001
0.230
0.005
0.000
0.005
1.000
0.700
0.100
1.000
1.000
1.000
0.000
0.000
0.015
0.000
0.000
0.000
0.694
0.005
0.000
0.113
0.009
0.001
0.206
1.000
similarities between GMaMs and alternatively activated (M2-like)
macrophages, which have been originally described as “woundhealing macrophages,” we analyzed the influence of GMaMs on
epithelial healing. In epithelial cell (Caco-2) monolayers, preactivation of monocytes with GM-CSF significantly accelerated
wound closure compared with unstimulated monocytes (Fig. 3F).
GMaMs attract T cells and induce Tregs. Another important
function of monocytes is the cross talk to adaptive immunity.
Monocytes and macrophages serve as APCs, but they also
shape lymphocyte activation by a whole battery of different costimulatory molecules and cytokines. As shown in Fig. 2, gene
expression (Fig. 2A) and protein production (Fig. 2B) of chemokines CCL18 and CCL23 were strongly increased in GMaMs.
Because CCL18 and CCL23 have been shown to attract naive and
resting T cells (43, 44), we analyzed the capacity of GMaMs to
attract T cells by using a modified Boyden chamber. T cells were
allowed to migrate toward the culture supernatants of GMaMs or
untreated monocytes. We observed a significantly increased T cell
migration toward cell supernatants of GMaMs (Fig. 3G). In addition, we have addressed effects of GM-CSF on Treg differentiation. To this end, we stimulated human monocytes with GMCSF and cocultured monocytes and autologous T cells for 7 d, and
analyzed resulting T cells for Foxp3 expression to evaluate Treg
differentiation. CD25 and Foxp3 expression in T cells were already increased upon interaction with monocytes that had been
stimulated with GM-CSF for only 24 h. The induction of Foxp3
expression in T cells cocultured with GMaMs was further increased 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 confirmed
a specific activation pattern of human monocytes in response to
GM-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 the
context of CD. Because systematic analyses in the human system
are not feasible, we went on analyzing the effects of GMaMs in
the murine system. Analysis by qRT-PCR showed that murine
GMaMs had a similar gene regulation profile when compared
with the GM-CSF–dependent gene expression in human monocytes
(Fig. 4A). We chose the CD4+ T cell–dependent experimental colitis 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). The
onset of colitis is monitored clinically by weight loss. Untreated
monocytes (control) or ex vivo GMaMs were injected i.v. after
mice had lost weight on consecutive days (∼5–6 wk after eliciting
colitis by injection of CD4+CD252 T cells). Migration of injected
GMaMs to the inflamed gut was confirmed by in vivo imaging. In
agreement with the in vitro data on cell migration, we observed an
increased infiltration of GMaMs into MLNs compared with control monocytes in two independent experiments (Fig. 5). Mice that
had received GMaMs showed no weight loss at all over a period of
12 d after monocyte transfer (Fig. 4B). Normally, the inflamed
colon becomes shorter and presents with reduced length, and thus
shortening of the colon is a measure of inflammation. Also, in this
study, mice that received GMaMs did not show relevant shortening of the colon, whereas all other groups were not protected from
colitis (Fig. 4B, 4C). Control mice that had received untreated
monocytes or no monocytes showed progressive weight loss and
signs of intestinal inflammation, and had to be euthanized on day
12 (Fig. 4B, 4C). Animals that had received untreated monocytes
had significantly severe histopathologic alterations of the colon,
most evident in the distal part (Fig. 4D, 4E). In addition, we
performed high-resolution colonoscopy and graded inflammation
(MEICS-Score) (34). The colon of mice receiving no treatment
presented with a vulnerable and bleeding mucosa, rarefication of
2432
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
FIGURE 3. Functional properties of GMaMs and interaction of human GMaMs with T cells. (A) Human monocytes were activated with GM-CSF or left
untreated (control) for 24/48 h in Teflon bags and subsequently allowed to adhere to multiwell plates for 4 h. Adherent cells were stained with crystal violet,
and staining was quantified measuring the OD at 560 nm. (B) Monocytes were activated with GM-CSF or left untreated for 24 h in Teflon bags and placed
into the upper chamber of a Transwell filter. The lower chamber contained monocyte medium with the addition of LTB4, MCP-1/CCL2, IL-8/CXCL8, or no
attractants. After 4 h cells that had migrated into the lower compartment were counted, and numbers are presented as the percentage of cells, which
migrated in the absence of any chemotactic stimulus. The p values refer to the migration of untreated (control) cells in the absence of any chemotactic
stimuli (w/o). (C) Oxidative burst of GMaMs or untreated monocytes was initiated by the addition of PMA. Isoluminol chemiluminescence was measured in
PMA-treated cells and control cells after induction of oxidative burst. (D) Cytokine secretion was measured in supernatants of GMaMs (24 h) after exposure
to LPS (4 h) and compared with untreated monocytes exposed to LPS. **p , 0.01, ***p , 0.001 compared with LPS-treated control. (E) The gene
expression of GMaMs (24 h) after exposure to LPS (4 h) was assessed by qRT-PCR. (F) Shown is the extent of wound closure in scratch assays of Caco-2
monolayers at 24 and 48 h in the absence of monocytes (control; n = 30), the presence of untreated monocytes (monocytes; n = 45), or the presence of
monocytes preactivated for 48 h with GM-CSF (GMaM; n = 45). (G) T cell migration was analyzed. Monocytes were activated with GM-CSF or left
untreated for 24 h in Teflon bags. Fifty thousand T cells were added to top chamber, and cell culture supernatants of untreated monocytes or GMaMs, as
well as CCL18, CCL23, and medium (control), were added to the bottom chamber. T cells that had migrated into the lower compartment within 4 h were
counted. *p , 0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes. (H and I) Human autologous T cells were cocultured with untreated
monocytes (control) or GMaMs (24 and 48 h) at a ratio of 10:1 for 7 d. Cells were stained for CD4, CD25, and intracellular for Foxp3 expression and
analyzed by flow cytometry. (H) Representative dot plots are shown for 48 h. (I) Cells were gated on CD4+ cells and analyzed for CD25 and Foxp3
expression. Data shown are the means (6 SEM) of three independent experiments. (A–I) *p , 0.05, **p , 0.01, ***p , 0.001.
vascular pattern, presence of fibrin, and ulcerations. Mice treated
with GMaMs depicted a transparent colonic mucosa with a regular
vascular pattern resembling healthy animals (Fig. 4F). Taken together, treatment of an established CD4+ T cell–induced colitis
with GMaMs alleviates inflammation of the colon, resulting in
significantly improved clinical parameters and histology, suggesting that GMaMs potentially exert regulatory effects on T cells
in vivo.
GMaMs regulate T cell responses in vivo and in vitro. After termination of colitis experiments, LPMCs and MLNs were harvested.
Migration of injected GMaMs to gut tissue and MLNs was confirmed by in vivo imaging (Fig. 5). Single-cell suspensions were
restimulated with anti-CD3/anti-CD28 to explore how the capacity and strength of T cell cytokine production has changed in vivo
during colitis and treatment with GMaMs. After restimulation for
24 h, we tested supernatants for production of IFN-g (Th1 cell
response) and IL-4, IL-10, and IL-13 (Th2 cell response). As
shown in Fig. 6, GMaM transfer led to significantly reduced IFN-g
production in T cells from LPMCs (Fig. 6A) and MLNs (Fig. 6B).
The Th2 cytokines IL-4, IL-10, and IL-13, however, were slightly
increased in supernatants from LPMCs of animals treated with
GMaMs (Fig. 6A), and IL-4 and IL-13 were also increased in
supernatants from MLNs of animals treated with GMaMs (Fig.
6B). In summary, treatment of mice suffering from colitis with
GMaM results in a shift in cytokine production of T cells in vivo.
To confirm these in vivo data, we performed coculture experiments with anti-CD3e/anti-CD28–stimulated T cells in vitro.
Also, in this study, GMaMs skewed the T cell response and led
The Journal of Immunology
2433
FIGURE 4. Treatment with GMaMs protects from experimental colitis. (A) GM-CSF–dependent gene regulation in murine monocytes derived from bone
marrow of C57BL/6 mice and human peripheral blood monocytes compared with unstimulated monocytes. Shown is the mean relative n-fold regulation (6
SEM) of three independent experiments. *p , 0.05 compared with GM-CSF–treated human monocytes. (B) Rag12/2 mice were injected i.v. with
CD4+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 magnification 3100. (E) Intestinal inflammation scores of
the proximal and distal colon of mice with colitis injected with control monocytes or GMaM (0, no inflammation; 1, mild inflammation; 2, moderate
inflammation; 3, severe inflammation; 4, extreme inflammation). (F) MEICS score and representative pictures of high-resolution colonoscopy showing the
colon of a mouse receiving no treatment with a vulnerable and bleeding mucosa, rarefication of vascular pattern, fibrin, and ulcerations. Mice treated with
GMaMs depicted a transparent mucosa with a regular vascular pattern resembling healthy animals. Graphs in (B)–(E) show mean values (6 SEM) of 10
control mice and 14 mice injected with GMaMs from 3 independent experiments. *p , 0.05, **p , 0.01 compared with untreated monocytes; #p , 0.05
compared with T cells only.
to significant upregulation of Th2 cytokines IL-4, IL-13, and
IL-10, whereas the Th1 cytokine IFN-g was downregulated
(Fig. 6C). These data demonstrate that GMaM cross talk with
T cells results in a phenotype shift that attenuates classical Th1
responses, which may contribute to an immunomodulatory effect.
We did not observe an increase of Tregs, represented by Foxp3
expression in CD4+ T cells, in MLNs or LPMCs of mice with
experimental T cell transfer colitis after the transfer of GMaMs
(Fig. 6D, 6E). However, we were able to demonstrate that murine
GMaMs induce Tregs in vitro (Fig. 6F).
Peripheral blood monocytes from patients with CD behave like
GMaMs
We next studied phenotypic and functional features of untreated
versus GM-CSF–activated peripheral blood monocytes of 18
patients with quiescent CD by analyses of cell adherence, migration, chemotaxis, phagocytosis, oxidative burst, and cytokine
expression and secretion. Collectively, our data suggest that the
effects of GM-CSF activation of peripheral monocytes of patients
with CD (Fig. 7) are similar to the observed effects in GMaMs
from healthy donors (Figs. 1–3). This includes the GM-CSF–induced increase in adherence, migration, chemotaxis, and oxidative
burst, as well as the priming of monocytes to secondary microbial
stimuli (Fig. 7A–E). In addition, changes in GM-CSF–dependent
mRNA expression of selected key inflammatory cytokines were in
agreement with our transcriptomic data obtained from GMaMs of
healthy individuals (Fig. 7F). Importantly, there was no evidence
that GM-CSF activation had different effects on monocytes when
compared between individual patients.
Discussion
Despite the fact that the concept of CD as a chronic granulomatous
Th1-driven disease shifts toward a theory of CD as an immunodeficiency of macrophages, while we only begin to understand the
regulatory or suppressive functions of our immune system, our
general approach to chronic inflammatory diseases including CD is
still mainly based upon the paradigm of immunosuppression as the
primary therapeutic intervention. In line with that, phagocytes are
primarily seen as driving forces of inflammation that need to be
inhibited. This traditional view of immune interventions, however, is
in sharp contrast with our currently changing view of immunity. It is
now accepted that cells of the monocyte–macrophage lineage are
characterized by considerable diversity and plasticity that may encompass, as an example, classical M1-macrophage differentiation
(when stimulated by IFN-g) or alternative M2 differentiation (when
stimulated by IL-4/IL-13) as outer margins of a broad phenotypical
plasticity (2). Serving another example, the population resulting from
GM-CSF–stimulated human monocytes has been referred to as M1like 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 in
animal IBD models and in human patients with CD (47). Intraperitoneal administration of GM-CSF alleviated acute DSSinduced colitis in mice, resulting in decreased proinflammatory
cytokine release, improved clinical and histologic parameters, as
well as more rapid ulcer healing, and facilitated epithelial regeneration (15, 16). Importantly, transfer of splenic GM-CSF–
induced CD11b+ myeloid cells into DSS-exposed mice improved
2434
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
FIGURE 5. GMaMs rapidly infiltrate the intestine. (A) Mesenteric lymph node single-cell suspensions of congenic CD45.2 mice, suffering from colitis
(and treated as indicated), were analyzed for infiltration of injected donor monocytes (CD45.1+) using CD45.1 Ab by flow cytometry. (B) Results of two
independent experiments are shown as percent infiltrated donor CD45.1+ cells. *p , 0.05 compared with untreated monocytes. (C) GMaMs were labeled
with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, and 2 3 106 cells were injected according to the standard treatment regimen. Monocyte
infiltration in the intestine was visualized after 24 h by a planar small-animal fluorescence-mediated tomography system, and representative pictures are
depicted.
colitis, and GM-CSF–expanded CD11b+ splenocytes were shown
to promote in vitro wound repair (16). Furthermore, it has been
shown that: 1) neutralization of GM-CSF increases intestinal
permeability and bacterial translocation in mice; and 2) increased
levels of GM-CSF autoantibodies are associated with an increase
in bowel permeability, disease relapse, stricturing ileal disease,
and surgery in patients with CD (48–50). As a therapy that could
help in overcoming insufficient macrophage functions, GM-CSF
had strikingly beneficial effects in subgroups of CD patients (17).
Seemingly, these beneficial effects were rather unexpected in light
of the previously reported proinflammatory polarization of macrophages upon stimulation with GM-CSF (41, 46).
We speculated that GM-CSF exerts its beneficial effects in intestinal inflammation in vivo by specific activation of monocytes
that combines innate immune activation, thus facilitating antiinfectious defense, with a simultaneous regulatory function serving to limit adaptive immunity and excessive inflammation. We
thus set off in an unbiased systems biology approach to comprehensively study the many facets of monocyte activation in vitro,
ranging from gene expression to innate immune functions and the
interplay with adaptive immunity. All these aspects have previously been studied on monocytes but separately and independently
from each other (41, 46, 51–54). Our findings suggest that the
early imprinting of monocytes after activation with GM-CSF is
of crucial importance, because monocytes play an important role
during the recruitment phase of the innate immune response and
have the potential to regulate adaptive immune mechanisms. GMCSF has been shown to have a pleiotropic role in inflammation
and autoimmunity (23). Data from other groups suggest that cell
culture conditions, concentration, time point, and duration chosen
for GM-CSF stimulation of human monocytes may determine
transcriptional outcomes relating to M1/M2 polarity (53–55). In
this respect, it has been described that different biologic responses
induced by GM-CSF depend on its concentration (53), and that the
time point chosen for the CSF treatment of human monocytes can
markedly determine the relative expression of cytokine genes (41).
Collectively, it is conceivable that the described population of
GMaMs in this study represents an intermediate cell type, combining cell-surface expression characteristics and functional features of different M2 macrophage subsets including CD206 and
CD209 cell-surface expression (M2a), Th2 responses/activation
(M2a/b), killing, and type II inflammation (M2a) and immunoregulation (M2b). In contrast with proinflammatory and antimicrobial responses of classically activated monocytes, M2-like
phenotypes are broadly anti-inflammatory and play important
roles in wound healing (54). GMaMs combine: 1) features of
augmented host defense functions; 2) the ability to facilitate epithelial healing; and 3) the regulatory potential on adaptive immunity. Specifically, we found a GMaM-dependent accelerated
wound closure in Caco-2 monolayers using an in vitro scratch
closure assay and in addition an upregulation of genes involved in
cell proliferation (e.g., FGF13, CDKN1C, TGF-b). Furthermore,
we found that the expression of a number of genes that are associated with M2 polarization is increased in human monocytes
after activation with GM-CSF. In particular, we found a significant
regulation of chemokines and chemokine receptors in human
monocytes. In this study, we found a GM-CSF–dependent downregulation of the M1 chemokines CXCL9, CXCL10, CXCL11, CXCL13,
and CXCL16 in monocytes. At the same time, GM-CSF significantly
induced expression of chemokines CXCL3, CCL1, CCL13, CCL17,
CCL18, CCL23, and CCL24 in monocytes, which are characteristic for
M2 macrophages (39, 54, 56). In addition, we showed that a number
of other M2a/b macrophage markers were significantly upregulated
in human monocytes after GM-CSF activation (e.g., CD206,
CD209, CD121b, ARG1). As mentioned earlier, we also found an
enhanced production of proinflammatory cytokines (IL-1b, TNF-a,
The Journal of Immunology
2435
FIGURE 6. GMaMs alter T cell cytokine
responses in vivo and in vitro. LPMCs and MLNs
were isolated from the colon of mice that were
treated as indicated. Expression of cytokines after
24-h stimulation with anti-CD3e/anti-CD28 Abs is
shown for LPMCs (A) and after 96 h for MLNs (B).
Graphs show mean values (6 SEM) from 3 independent experiments and n = 6–8 per group. (C)
Naive pan T cells were cocultured for 4 d with
GMaM, control monocytes, or left alone. T cells
were stimulated with anti-CD3e/anti-CD28 Abs.
Ratio of respective monocytes to T cells was 1:10,
and cytokines were measured in supernatants. Data
shown are the means (6 SEM) of nine independent
experiments. *p , 0.05, **p , 0.01, ***p , 0.001
compared with untreated monocytes. Cell populations from the lamina propria (D) and MLNs (E)
were stained for CD4 and Foxp3 expression and
analyzed by flow cytometry. Bars refer to mean 6
SEM of three independent experiments. (F) Murine
T cells were cocultured with GMaMs or control
monocytes at a ratio of 10:1 (T cells/monocytes). Cells
were stained for CD4 and Foxp3 expression and analyzed by flow cytometry. Bars refer to mean 6 SEM
of three independent experiments. **p , 0.01 compared with untreated monocytes (control).
and IL-6) in GMaMs, a feature also seen in M2b macrophages
upon exposure to immune complexes and LPS (56). In addition to
these strengthened innate immune functions, GMaMs simultaneously indeed have a regulatory potential on adaptive immunity.
Overall, our data indicate that GMaMs represent a distinctive cell
population 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 published
that GM-CSF increases the adherence of purified peripheral blood
monocytes to plastic surfaces and to monolayers of HUVECs (55),
and that GM-CSF can prime monocytes for increased transendothelial migration (57). Furthermore, reported results on GMCSF effects on other functions such as oxidative metabolism,
cytotoxicity, phagocytosis, and the in vitro response to other
stimuli are conflicting to some extent (23, 52, 58, 59). We confirm
in this study that short-term treatment (24 h) of human monocytes
with GM-CSF promotes: 1) cell adherence and migration, 2)
production of ROS, and 3) response to a second microbial stimulus (LPS). Thus, GM-CSF enhances selective effector functions
of monocytes, an effect of GM-CSF previously described for tissue-derived macrophages (60). In addition, our data indicate that
GM-CSF–activated peripheral blood monocytes from patients
with CD behave the same way as GMaMs (Fig. 7) and that GMCSF may regulate the homing molecules CCR2 and CCR6, which
are involved in regulating several aspects of mucosal immunity,
including the ability to mediate the recruitment of innate immune
cells to the sites of epithelial inflammation (61, 62).
Monocytes may also significantly regulate the immune context
by interaction with other cells. In particular, chemokines and
chemokine receptors have a key role in intestinal epithelial barrier repair and maintenance (63, 64). We found a significant regulation of chemokines and chemokine receptors with GM-CSF–dependent downregulation of the chemokines CXCL9, CXCL10, and
CXCL11 in monocytes. These factors are known to be increased
in IBD and to attract Th1 and NK cells (65). At the same time,
GM-CSF significantly induced a short-termed expression of the
chemokines CCL13, CCL17, CCL18, CCL23, and CCL24 in
monocytes, which are known to attract naive T cells, Th2 cells,
and/or Tregs (43, 44, 63, 65). Because of the observed upregulation of costimulatory molecule CD80 and the chemotactic factors
for naive and quiescent T cells CCL18 and CCL23 (43, 44), we
next analyzed the interaction of GMaMs with T cells. Indeed,
migration of naive, autologous T cells toward GMaMs was accelerated. Our data suggest that particularly CCL18 and CCL23
might be responsible for the increased T cell migration. However,
our transcriptomic data suggest that other GM-CSF–induced
chemokines (e.g., CCL13, CCL17, CCL24) might also be responsible for the increased T cell migration because they are
known to attract T cells. Studies to address this question more
specifically are beyond the scope of this study. It has been shown
that treatment of human monocytes with GM-CSF generates a
subtype of cells that regulate CD4+ T cell proliferation partially
via production of IL-10 (52), and that GM-CSF may sustain Treg
homeostasis and enhance their suppressive functions (47), indi-
2436
GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
FIGURE 7. Features of GM-CSF–activated peripheral blood monocytes of patients with quiescent CD (n = 18). (A) Adhesion of untreated (w/o) versus
GM-CSF–activated patient monocytes (24 h) to fibronectin-coated plastic surface. Adhering cells were stained with 0.5% crystal violet, and staining was
quantified measuring the OD at 560 nm. (B) Migration and chemotaxis studies of untreated (w/o) versus GMaMs (24 h) using a modified Boyden chamber
and LTB4 (100 nM) as an additional chemoattractant. After 4 h, cells that had migrated into the lower compartment were counted, and numbers are
presented as the percentage of untreated cells, which migrated in the absence of any chemotactic stimuli. (C) Phagocytosis of fluorescein-labeled E. coli by
untreated (w/o) versus GMaMs (24 h). E. coli phagocytosis was analyzed by flow cytometry, and phagocytic internalization was confirmed by fluorescence
microscopy. (D) Production of ROS by untreated (w/o) versus GMaMs (24 h) with and without further LPS stimulation for 2 h in the presence of rhodamine
for the final 15 min. Oxidative burst was analyzed by flow cytometry. (E) Cytokine secretion of untreated (w/o) versus GMaMs (24 h) with and without
further LPS stimulation for 2 h. (F) Gene expression (relative n-fold regulation) in untreated (w/o) versus GMaMs (24 h). Bars refer to mean 6 SEM. *p ,
0.05, **p , 0.01, ***p , 0.001 compared with untreated monocytes; ###p , 0.001 compared with controls without LTB4 or LPS.
cating the regulatory potential of GMaMs toward CD4+ T cells
and Tregs. When we cocultured GMaMs with syngeneic CD4+
T cells, we observed that GMaMs shape T cell response toward
a Th2 phenotype and induce Tregs.
After having analyzed the programming of monocytes with
GM-CSF in vitro, we next aimed at analyzing the therapeutic
effects of this cell population in a model of CD. We found that T
and B cell–deficient (Rag12/2) mice in which Crohn-like colitis was
induced with the CD4+CD252 T cell transfer model (66, 67) were
protected from disease progression when they received GMaMs
(but not untreated monocytes). Interestingly, Rag12/2 mice that
did not receive GMaMs but i.p. GM-CSF injections after the
transfer of T cells were not completely protected from colitis but
showed reduced disease severity. This is in agreement with earlier
work demonstrating positive effects of GM-CSF administration
(i.p.) in DSS-induced colitis in BALB/c, and more importantly, in
Rag12/2 mice (15, 16). We postulate that the alleviating effects of
GM-CSF in experimental colitis are due to direct modulation of
monocyte/macrophage functions including accelerated epithelial
healing. Our data demonstrate that the protective effect of
monocytes depends on their GM-CSF prestimulation in a T cell–
dependent model of colitis. The therapeutic mechanism of action
of 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 functions
regarding polarization and expansion of lymphocytes and may
also contribute to shaping T cell responses (22). The in vivo
effects of GMaMs showed increased levels of Th2 cytokines in
LPMCs (IL-4, IL-10, IL-13) and MLNs (IL-4, IL-13), but this
trend was not statistically significant. However, together with the
observed significantly reduced production of IFN-g in T cells
from LMPCs and MLNs, we concluded that treatment of mice
suffering from colitis with GMaMs results in a shift toward Th2
cytokine production of T cells in vivo. Our in vitro experiments
confirmed that GMaMs skew the T cell response and lead to significant upregulation of Th2 cytokines (IL-4, IL-10, IL-13), whereas
the Th1 cytokine IFN-g was significantly downregulated. This
might be explained by our observation that GMaMs display characteristics 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 the
T cell transfer colitis model also resulted in a strongly downregulated release of IFN-g by T cells from LPMCs and MLNs. The
production of IL-4 and IL-13 was not influenced in single-cell
suspensions from LPMCs and MLNs after treatment of mice
The Journal of Immunology
with injection of GCsMs, which is also in contrast with the in vitro
system, where cytokine production of IFN-g, IL-4, and IL-13 by
T cells was significantly regulated in cocultures with GCsMs (68).
The same study also showed that in the T cell transfer colitis
model, CD4+Foxp3+ Tregs accumulate locally in the colon after
treatment with GCsMs and that repetitive stimulation of naive
splenic T cells with GCsMs induces Tregs in vitro. However,
Tregs also did not expand in draining MLNs and LPMCs of animals 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-mediated
protection from colitis is facilitated via expansion of mucosal
Tregs in vivo.
Limitations of our study include the lack of in vivo data exploring specifically GMaMs in human CD, which may become
feasible in the future. Although we address the tissue context by
analyzing LPMCs and MLN, the further differentiation of ex vivo–
activated monocytes after transfer in vivo, especially after infiltrating the inflamed tissue, is certainly a complex issue. The circulating monocyte population is not homogenous but consists of
both inflammatory and regulatory populations that counterbalance
each other. We speculate that GM-CSF exerts its beneficial effects in intestinal inflammation in vivo by specific activation of
monocytes that combines innate immune activation, facilitating
anti-infectious defense and a simultaneous regulatory function
serving to limit adaptive immunity and excessive inflammation
rather short term. The pleiotropic GM-CSF functions on monocyte
activation and their consequences for innate and adaptive immunity range from activation of M2-like monocytes, chemotactic
migration, and antimicrobial response to mucosal healing, but also
encompasses regulation of adaptive immunity by attraction of, for
example, T cells with the possibility to differentiate Th cells and
subsequently limit inflammation induced by adaptive immunity.
However, it remains a question for further studies whether and
how monocytes differentiate long term at sites of inflammation
and within damaged tissue. It is also important to investigate the
immunomodulatory properties of monocytes in other animal
models of experimental colitis, for example, in DSS-induced colitis. Recent work by Kurmaeva et al. (69) has demonstrated that
immunosuppressive monocytes (CD11b+Ly6GnegLy6Chigh cells)
accumulate in the spleen and inflamed intestine during experimental colitis not only in T cell–induced colitis but also in an
acute 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 findings
support the exploration of stimulating rather than suppressive
therapies with the potential to more specifically reprogram monocytes toward immunomodulatory functions to alleviate chronic
inflammatory bowel disease.
Acknowledgments
We thank Melanie Saers, Susanne Schleifenbaum, Andrea Dick, Claudia
Solé, Eva Nattkemper, Andrea Stadtbäumer, and Sonja Dufentester for
excellent technical work. We also thank Dr. Tilmann Spieker (Department
of Pathology, University Hospital M€
unster, Germany, and St. FranziskusHospital, Institute of Pathology, M€
unster, Germany), who helped with
histopathology. We also thank the American Gastroenterological Association Research Foundation for the Moti L and Kamla Rustgi Awards, which
supported the presentation of the research at the 50th and 51st Digestive
Disease Week annual meetings.
Disclosures
The authors have no financial conflicts of interest.
2437
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T. Hussell, M. Feldmann, and I. A. Udalova. 2011. IRF5 promotes inflammatory
macrophage polarization and TH1-TH17 responses. Nat. Immunol. 12: 231–238.
Däbritz, J. 2014. Granulocyte macrophage colony-stimulating factor and the
intestinal innate immune cell homeostasis in Crohn’s disease. Am. J. Physiol.
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GM-CSF–ACTIVATED MONOCYTES REGULATE COLITIS
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Däbritz, J., E. Bonkowski, C. Chalk, B. C. Trapnell, J. Langhorst, L. A. Denson,
and D. Foell. 2013. Granulocyte macrophage colony-stimulating factor autoantibodies and disease relapse in inflammatory bowel disease. Am. J. Gastroenterol. 108: 1901–1910.
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skews macrophage polarization by promoting a proinflammatory phenotype and
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factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary
switch. EMBO J. 25: 479–489.
Murray, P. J., and T. A. Wynn. 2011. Protective and pathogenic functions of
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CCR6 as a mediator of immunity in the lung and gut. Exp. Cell Res. 317: 613–619.
Thomas, S., and D. C. Baumgart. 2012. Targeting leukocyte migration and adhesion in Crohn’s disease and ulcerative colitis. Inflammopharmacology 20: 1–18.
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Chemokines and chemokine receptors in mucosal homeostasis at the intestinal
epithelial barrier in inflammatory bowel disease. Inflamm. Bowel Dis. 14: 1000–
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R. S. Blumberg, and K. Kakimi. 2005. The simultaneous blockade of chemokine
receptors CCR2, CCR5 and CXCR3 by a non-peptide chemokine receptor antagonist protects mice from dextran sodium sulfate-mediated colitis. Int.
Immunol. 17: 1023–1034.
Koelink, P. J., S. A. Overbeek, S. Braber, P. de Kruijf, G. Folkerts, M. J. Smit,
and A. D. Kraneveld. 2012. Targeting chemokine receptors in chronic inflammatory diseases: an extensive review. Pharmacol. Ther. 133: 1–18.
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critical role for transforming growth factor-beta but not interleukin 4 in the
suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells.
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chronic colitis in SCID mice induced by adoptive transfer of CD62L+ CD4+
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A. Tsianakas, M. Ross, D. Bettenworth, T. Spieker, et al. 2014. Immune suppression via glucocorticoid-stimulated monocytes: a novel mechanism to cope
with inflammation. J. Immunol. 193: 1090–1099.
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
SUPPLEMENTARY MATERIAL
SUPPLEMENTARY TABLES
Table S1. Computational Ascertainment of Regulatory Relationships (Inferred from
Expression)
Pattern name
P
# Pattern
Up-regulated by GM-CSF
STAT 5b
STAT 5a
c-Ets-2 binding sites
cut-like homeodomain protein
CCAAT/enhancer binding protein beta
androgen receptor
PEA3
C/EBPbeta
9.63 x 10-11
3.44 x 10-8
3.38 x 10-7
3.46 x 10-3
4.84 x 10-2
5.86 x 10-4
2.29 x 10-3
4.89 x 10-2
M00459
M00457
M00340
M00104
M00109
M00447
M00655
M00621
Down-regulated by GM-CSF
LBP-1
NF-kappaB (p65)
c-Rel
NF-kappaB
activator protein 4
E2F
NF-kappaB (p50)
serum response factor
LEF1
1.54 x 10-2
7.19 x 10-6
1.94 x 10-5
5.41 x 10-5
3.93 x 10-5
1.12 x 10-4
4.20 x 10-3
2.76 x 10-2
6.32 x 10-5
M00644
M00052
M00053
M00774
M00175
M00803
M00051
M00186
M00805
2
SUPPLEMENTARY MATERIAL
Table S2. Complete list of up- and down-regulated genes.
Affymetrix identifier
Gene symbol
p-value
N-fold
205582_s_at
213800_at
218717_s_at
209909_s_at
206932_at
1553311_at
201904_s_at
205110_s_at
205743_at
205987_at
206407_s_at
225987_at
220407_s_at
206749_at
235171_at
223374_s_at
204529_s_at
221019_s_at
1555600_s_at
217504_at
213906_at
207819_s_at
201906_s_at
220187_at
230323_s_at
206637_at
215784_at
216375_s_at
213265_at
202565_s_at
223939_at
240232_at
205404_at
1554519_at
210549_s_at
238439_at
210548_at
229568_at
204044_at
211379_x_at
229566_at
225646_at
203650_at
214146_s_at
202566_s_at
226844_at
231234_at
GGTLA1
CFH
LEPREL1
TGFB2
CH25H
C20orf197
CTDSPL
FGF13
STAC
CD1C
CCL13
STEAP4
TGFB2
CD1B
--B3GALNT1
TOX
COLEC12
APOL4
ABCA6
MYBL1
ABCB4
CTDSPL
STEAP4
TMEM45B
P2RY14
CD1E
ETV5
PGA3 /// PGA4 /// PGA5
SVIL
SUCNR1
--HSD11B1
CD80
CCL23
ANKRD22
CCL23
MOBKL2B
QPRT
B3GALNT1
LOC645638
CTSC
PROCR
PPBP
SVIL
MOBKL2B
CTSC
0.0001693
0.01343855
0.00119232
0.00633007
0.00570894
0.04907555
0.00068324
0.01835098
4.2567E-05
8.1715E-05
0.00013093
0.00599794
0.00332232
0.00825223
1.0164E-05
0.00616719
0.00012043
0.00037242
0.00946476
0.00196413
3.3347E-05
0.0149746
0.02083693
0.00566497
0.00368398
0.00047201
0.00036557
0.0173255
0.00541377
3.1281E-05
0.00069322
0.00422387
0.00033605
0.00016535
0.00221019
0.00058313
0.02167974
8.2151E-07
0.00550087
0.00157536
0.00379492
1.6407E-05
3.0279E-05
0.00149519
0.00073725
8.6574E-05
0.00062037
50.50649347
44.90593032
31.26424011
26.84033465
25.42228437
24.09862336
20.2349671
19.81260534
18.5919308
18.39333098
16.68245451
15.99445019
15.54808562
15.48920616
14.64152207
14.21784293
14.18535443
14.12670214
14.04685127
13.78119273
13.60347906
12.31540717
12.01831432
11.88489592
11.74804279
11.24085437
9.842376733
9.690221163
9.501098446
8.967745921
8.966488497
8.593775661
8.415616905
8.351698565
8.328243908
7.987773267
7.849443119
7.821421311
7.615064549
7.570312236
7.570145619
7.545684655
7.481681815
7.231768627
6.975562537
6.820100758
6.764636072
3
SUPPLEMENTARY MATERIAL
228121_at
226226_at
210325_at
209795_at
220144_s_at
242541_at
215101_s_at
219265_at
223961_s_at
1555689_at
231698_at
207900_at
223377_x_at
219667_s_at
225647_s_at
210375_at
207008_at
203680_at
242714_at
206181_at
222915_s_at
205798_at
205220_at
215388_s_at
213189_at
227856_at
219890_at
221223_x_at
239196_at
210233_at
228450_at
206983_at
201577_at
209994_s_at
207826_s_at
39248_at
204174_at
229733_s_at
224480_s_at
227620_at
224596_at
1568964_x_at
226218_at
228485_s_at
242417_at
222364_at
1558662_s_at
216913_s_at
207339_s_at
1559065_a_at
TGFB2
TMEM45B
CD1A
CD69
ANKRD5
ABCA9
CXCL5
MOBKL2B
CISH
CD80
FLJ36848
CCL17
CISH
BANK1
CTSC
PTGER3
IL8RB
PRKAR2B
--SLAMF1
BANK1
IL7R
GPR109B
CFH /// CFHR1
MINA
C4orf32
CLEC5A
CISH
ANKRD22
IL1RAP
PLEKHA7
CCR6
NME1
ABCB1 /// ABCB4
ID3
AQP3
ALOX5AP
--MAG1
SLC44A1
SLC44A1
SPN
IL7R
SLC44A1
LOC283278
SLC44A1
BANK1
RRP12
LTB
CLEC4G
0.00162072
0.01204346
0.00073108
0.00247926
0.00079353
0.00175565
0.02476789
3.0354E-05
0.00060494
0.00077794
0.0031729
0.00336947
0.00026023
0.01092215
0.00016548
0.03294313
0.00226392
0.00150321
0.00218649
0.04087152
0.02367735
0.00144021
0.00788387
0.00996691
0.00032132
2.1238E-05
0.01078711
0.00054497
0.00348723
0.00674947
0.0008014
0.00064013
0.00034558
0.00030352
4.0282E-05
0.00402339
0.00329028
0.01119593
0.00061558
0.00065128
0.0004088
0.00713234
0.00313406
0.00126762
0.0022643
0.01630046
0.00768653
0.02004205
0.00193252
0.02320381
4
6.508429981
6.45310024
6.410251988
6.394661483
6.077013983
5.840155931
5.752733193
5.718559619
5.684694152
5.653435999
5.570477754
5.533766308
5.229202004
5.212358558
5.176559771
5.140865608
5.083832786
5.058741774
5.03093753
4.964063377
4.859928302
4.839081662
4.672372409
4.654094368
4.537621679
4.534985676
4.518367139
4.506457024
4.492226897
4.373316171
4.333857776
4.329658646
4.293962423
4.27137836
4.1913959
4.17568041
4.131184206
4.119293143
4.073673324
4.019685784
3.845367812
3.838904836
3.718450837
3.718450837
3.702809602
3.683552983
3.677144378
3.677144378
3.568058872
3.552553229
SUPPLEMENTARY MATERIAL
203349_s_at
213188_s_at
220581_at
224595_at
222457_s_at
207113_s_at
206682_at
228486_at
222500_at
205153_s_at
203348_s_at
211668_s_at
209567_at
205786_s_at
205128_x_at
205479_s_at
215813_s_at
244439_at
215346_at
238846_at
205659_at
203373_at
209679_s_at
203372_s_at
208029_s_at
221830_at
217892_s_at
220865_s_at
230102_at
205282_at
217097_s_at
210839_s_at
202613_at
226837_at
213268_at
227607_at
209803_s_at
221724_s_at
221053_s_at
204829_s_at
211864_s_at
229132_at
202314_at
214039_s_at
205831_at
218984_at
204601_at
208433_s_at
201798_s_at
212110_at
ETV5
MINA
C6orf97
SLC44A1
LIMA1
TNF
CLEC10A
SLC44A1
PPIL1
CD40
ETV5
PLAU
RRS1
ITGAM
PTGS1
PLAU
PTGS1
SPRED1
CD40
TNFRSF11A
HDAC9
SOCS2
LOC57228
SOCS2
LAPTM4B
RAP2A
LIMA1
PDSS1
ETV5
LRP8
PHTF2
ENPP2
CTPS
SPRED1
CAMTA1
STAMBPL1
PHLDA2
CLEC4A
TDRKH
FOLR2
FER1L3
MINA
CYP51A1
LAPTM4B
CD2
PUS7
N4BP1
LRP8
FER1L3
SLC39A14
0.00106768
0.00047061
0.0007264
0.00037046
0.01282761
0.0029579
0.00289933
0.00040979
1.521E-05
0.02377785
0.00028432
0.00074962
0.02196573
0.0002406
0.00328404
0.00015452
0.00802737
0.00542101
0.01173246
0.00061909
0.0058376
0.00485244
0.01483812
0.03734608
0.01760473
0.00202405
0.02583751
0.0217345
0.00299339
0.00157745
0.01550453
0.03517238
0.00083074
0.00054464
0.00738793
0.00259953
0.01473078
0.00339806
0.02137839
0.01145529
0.01082703
0.00479289
0.03331124
0.0075295
0.00342397
0.00058368
0.00060159
0.00166523
0.00681365
0.00410054
5
3.552553229
3.546338607
3.520186364
3.504545129
3.499339724
3.449986705
3.436876459
3.430897019
3.410050379
3.405097868
3.35914557
3.347587115
3.298850568
3.260271937
3.254214991
3.186546321
3.186546321
3.170576976
3.155814315
3.155814315
3.119293715
3.114020837
3.096905317
3.092846087
3.074502127
3.064500222
3.064500222
3.046352168
3.036289948
3.020320603
2.978804177
2.937010699
2.931805294
2.931805294
2.919895179
2.914243848
2.901368672
2.900627363
2.885727437
2.868611917
2.846575179
2.841718364
2.838231571
2.838231571
2.802259969
2.786754325
2.783438078
2.783438078
2.740327878
2.735471064
SUPPLEMENTARY MATERIAL
209499_x_at
239648_at
207419_s_at
205227_at
211495_x_at
202047_s_at
218195_at
206148_at
205016_at
203234_at
227333_at
241937_s_at
220578_at
201487_at
205505_at
225438_at
224634_at
209392_at
239761_at
228955_at
202551_s_at
204393_s_at
214487_s_at
201700_at
211974_x_at
32069_at
201797_s_at
203119_at
228372_at
218681_s_at
209500_x_at
210314_x_at
228231_at
1558517_s_at
212295_s_at
217809_at
223533_at
226923_at
204744_s_at
219551_at
225173_at
235085_at
224516_s_at
207275_s_at
208918_s_at
221731_x_at
225598_at
204043_at
212658_at
219367_s_at
TNFSF12-TNFSF13 /// TNFSF13
DCUN1D3
RAC2
IL1RAP
TNFSF12-TNFSF13 /// TNFSF13
CBX6
C6orf211
IL3RA
TGFA
UPP1
--WDR4
ADAMTSL4
CTSC
GCNT1
NUDCD1
GPATCH4
ENPP2
GCNT1
--CRIM1
ACPP
RAP2A /// RAP2B
CCND3
RBPJ
N4BP1
VARS
CCDC86
C10orf128
SDF2L1
TNFSF12-TNFSF13 /// TNFSF13
TNFSF12-TNFSF13 /// TNFSF13
--LRRC8C
SLC7A1
BZW2
LRRC8C
SCFD2
IARS
EAF2
ARHGAP18
DKFZp761P0423
CXXC5
ACSL1
NADK
VCAN
SLC45A4
TCN2
LHFPL2
---
0.00352244
0.00083273
0.01000203
0.00047687
0.00435195
0.00210421
0.00078258
0.03245599
0.0282456
0.00170427
0.00055681
0.00043505
0.03093454
0.00107415
0.01054675
0.00112599
0.0064642
0.00658615
0.00758671
0.00395472
0.01264565
0.04665685
0.00202447
0.00077809
6.6175E-05
0.00226608
0.00074228
0.0022327
0.0079918
0.0001793
0.00045191
0.00106288
0.00190143
0.00477875
3.6157E-05
0.00038262
0.00786109
0.00024058
0.00094695
0.0061163
0.00555202
0.00417984
0.0237143
0.02708995
0.00039223
0.03341251
0.00050444
0.02674388
0.00319037
0.00319825
6
2.706381025
2.706381025
2.673075272
2.669565041
2.655948555
2.643243924
2.614601002
2.604665293
2.600133725
2.588279885
2.580106823
2.542075518
2.542075518
2.536996624
2.536996624
2.536996624
2.529370887
2.474406848
2.474406848
2.474406848
2.473859523
2.473859523
2.473859523
2.414950524
2.414950524
2.411269747
2.407324788
2.407324788
2.371840325
2.359986486
2.308703224
2.308703224
2.308703224
2.301077487
2.249794226
2.249794226
2.249794226
2.246113448
2.194830187
2.194830187
2.194830187
2.194830187
-2.143546925
-2.194830187
-2.194830187
-2.194830187
-2.194830187
-2.249794226
-2.249794226
-2.249794226
SUPPLEMENTARY MATERIAL
204647_at
208981_at
235457_at
232231_at
204620_s_at
206472_s_at
208161_s_at
207574_s_at
208982_at
225956_at
215211_at
233955_x_at
225755_at
207167_at
222996_s_at
200897_s_at
208983_s_at
209298_s_at
202391_at
235556_at
213397_x_at
240137_at
200907_s_at
208919_s_at
224817_at
214581_x_at
204526_s_at
212390_at
219622_at
213241_at
208626_s_at
215646_s_at
205382_s_at
213109_at
214129_at
216705_s_at
225631_at
221563_at
228340_at
33304_at
204639_at
221081_s_at
235146_at
200766_at
203505_at
217889_s_at
225762_x_at
241742_at
209122_at
209831_x_at
HOMER3
PECAM1
MAML2
RUNX2
VCAN
TLE3
ABCC3
GADD45B
PECAM1
LOC153222
LOC730092
CXXC5
KLHDC8B
IGSF2
CXXC5
PALLD
PECAM1
ITSN1
BASP1
--RNASE4
--PALLD
NADK
SH3PXD2A
TNFRSF21
TBC1D8
LOC727893 /// PDE4DIP
RAB20
PLXNC1
VAT1
VCAN
CFD
TNIK
LOC727942
ADA
KIAA1706
DUSP10
TLE3
ISG20
ADA
DENND2D
--CTSD
ABCA1
CYBRD1
LOC284801
PRAM1
ADFP
DNASE2
0.00536832
2.2395E-05
0.00120286
0.00344245
0.03173514
0.01436113
0.01987907
0.00088207
0.00578303
0.00166546
0.00928518
0.01714169
0.00224667
0.00028724
0.00922852
0.00126053
0.0004589
0.0315577
0.00307102
0.00478104
0.03852373
0.00445196
0.00047184
0.00671747
0.00113436
0.04706226
0.00046135
0.00189182
0.00129368
0.00077876
0.00087169
0.04737497
0.00781013
0.01434917
0.01629392
0.00096118
4.9247E-05
0.00354435
0.00588266
0.00106415
0.00111826
0.00595754
0.00477377
0.00318172
0.00193347
0.02209698
0.00161705
0.00486406
5.9025E-05
0.00033775
7
-2.301077487
-2.301077487
-2.301077487
-2.308703224
-2.352360749
-2.352360749
-2.356041526
-2.359986486
-2.359986486
-2.359986486
-2.371840325
-2.407324788
-2.411269747
-2.414950524
-2.423123587
-2.439508994
-2.462288827
-2.466233786
-2.474406848
-2.478087626
-2.525142784
-2.529370887
-2.536996624
-2.536996624
-2.588279885
-2.589765368
-2.639015822
-2.643243924
-2.643243924
-2.651416986
-2.667802394
-2.667802394
-2.669565041
-2.669565041
-2.669565041
-2.702152923
-2.702152923
-2.706381025
-2.706381025
-2.706381025
-2.710912594
-2.710912594
-2.710912594
-2.719085656
-2.719085656
-2.719085656
-2.728474039
-2.74729593
-2.765290024
-2.765290024
SUPPLEMENTARY MATERIAL
218729_at
213388_at
227969_at
41660_at
228933_at
218856_at
215111_s_at
239287_at
213006_at
221246_x_at
213061_s_at
1552540_s_at
207704_s_at
209263_x_at
213222_at
219371_s_at
228325_at
232530_at
213902_at
205801_s_at
221042_s_at
222857_s_at
215223_s_at
209264_s_at
216894_x_at
214099_s_at
230233_at
211571_s_at
1552701_a_at
222838_at
213062_at
1553787_at
1555419_a_at
213258_at
211067_s_at
201212_at
218999_at
228570_at
227220_at
205466_s_at
221022_s_at
226534_at
203504_s_at
225757_s_at
213839_at
209969_s_at
230139_at
226869_at
1552846_s_at
229296_at
LXN
LOC727942
LOC400960
CELSR1
NHS
TNFRSF21
TSC22D1
--CEBPD
TNS1
NTAN1
IQCD
GAS7
TSPAN4
PLCB1
KLF2
KIAA0146
PLD1
ASAH1
RASGRP3
CLMN
KCNMB4
SOD2
TSPAN4
CDKN1C
LOC727927 /// PDE4DIP
--VCAN
COP1
SLAMF7
NTAN1
C11orf45
ASAH1
TFPI
GAS7
LGMN
TMEM140
BTBD11
NFXL1
HS3ST1
PMFBP1
KITLG
ABCA1
CLMN
KIAA0500
STAT1
--MEGF6
RAB42
---
0.03165556
0.0036434
0.00964696
0.00257308
0.00893871
0.034557
0.00113387
0.01385525
0.00376504
0.00562485
0.00041726
0.0172296
0.01181464
0.00354284
4.0846E-05
0.01766364
0.00328205
0.00626143
0.000186
0.00778405
0.00078339
0.0339815
0.02198291
0.01110871
0.02188963
0.00261202
0.00923389
0.02136148
0.00277034
0.00908658
0.00216046
0.00304653
0.00243214
0.00197907
0.00573692
4.5658E-05
0.00051421
0.03948206
0.00212586
0.00591447
0.00053595
0.00180129
0.00505662
0.01857931
0.00197276
0.03399577
0.01517874
0.04222651
0.00010919
0.0013362
8
-2.769821592
-2.774049695
-2.779322573
-2.786754325
-2.79161114
-2.79161114
-2.832958693
-2.838231571
-2.842347076
-2.842347076
-2.846575179
-2.868611917
-2.900627363
-2.900627363
-2.900627363
-2.901368672
-2.927520916
-2.937010699
-2.968621278
-2.983215066
-3.009536183
-3.033768216
-3.03598381
-3.036289948
-3.041941278
-3.046352168
-3.050883736
-3.064500222
-3.075284642
-3.086355967
-3.092677214
-3.103958617
-3.103958617
-3.103958617
-3.108815432
-3.114020837
-3.12340922
-3.12340922
-3.129466166
-3.13085217
-3.13419364
-3.137639228
-3.186546321
-3.18892249
-3.201140109
-3.239658426
-3.251512265
-3.256591159
-3.26427721
-3.284450014
SUPPLEMENTARY MATERIAL
228274_at
203510_at
202191_s_at
225102_at
210664_s_at
221747_at
205888_s_at
211026_s_at
222877_at
229686_at
204533_at
219534_x_at
202748_at
219403_s_at
201911_s_at
200665_s_at
202192_s_at
203979_at
204972_at
213624_at
213348_at
213182_x_at
204961_s_at
202074_s_at
1558397_at
214992_s_at
222881_at
204146_at
205552_s_at
206701_x_at
227618_at
214084_x_at
229620_at
203920_at
209540_at
202086_at
219895_at
209960_at
228170_at
212912_at
206545_at
221748_s_at
208268_at
206756_at
210997_at
201185_at
222173_s_at
201141_at
202464_s_at
212226_s_at
SDSL
MET
GAS7
MGLL
TFPI
TNS1
JAKMIP2
MGLL
--P2RY8
CXCL10
CDKN1C
GBP2
HPSE
FARP1
SPARC
GAS7
CYP27A1
OAS2
SMPDL3A
CDKN1C
CDKN1C
NCF1 /// NCF1B /// NCF1C
OPTN
--DNASE2
HPSE
RAD51AP1
OAS1
EDNRB
--NCF1C
SEPP1
NR1H3
IGF1
MX1
FAM70A
HGF
OLIG1
RPS6KA2
CD28
TNS1
ADAM28
CHST7
HGF
HTRA1
TBC1D2
GPNMB
PFKFB3
PPAP2B
0.00098447
0.02533322
0.00582831
0.00323484
0.00529179
0.0014137
0.00030406
0.0004981
0.00032681
9.4761E-05
0.00675986
0.04093356
0.00222671
0.00289568
0.01115936
0.00275603
0.00096308
0.01277469
0.0431925
0.00021902
0.00351901
0.00418554
0.00286032
0.00442087
0.00210786
0.00229182
0.00118532
0.00658881
0.04871288
0.00522793
0.00110585
0.00046392
0.02556105
0.00048322
0.0117016
0.02266035
0.00463366
0.00197154
0.00325159
0.00092461
0.00808069
0.00091278
0.00282439
0.01187277
0.01408447
0.01263869
4.8979E-05
0.00048491
0.00060661
0.00374695
9
-3.286620086
-3.337524895
-3.337524895
-3.337524895
-3.354078789
-3.35914557
-3.404471364
-3.404471364
-3.449986705
-3.482202253
-3.493838214
-3.513094833
-3.531675536
-3.552553229
-3.552747213
-3.557410043
-3.595037674
-3.654801502
-3.678280105
-3.683552983
-3.702809602
-3.775335086
-3.79562216
-3.799749906
-3.833809356
-3.833809356
-3.858644991
-3.923098701
-3.978792852
-4.019685784
-4.081034879
-4.121363174
-4.124566021
-4.129613862
-4.135412309
-4.175051698
-4.183601402
-4.192167217
-4.204673079
-4.218903207
-4.225234306
-4.256126886
-4.28972232
-4.361874194
-4.42052809
-4.426010548
-4.432419153
-4.462995405
-4.500219085
-4.577949974
SUPPLEMENTARY MATERIAL
217028_at
209030_s_at
202007_at
214770_at
238780_s_at
219452_at
1569403_at
219863_at
204271_s_at
226301_at
226702_at
204273_at
226189_at
209541_at
204747_at
238581_at
224694_at
212230_at
208450_at
205568_at
206134_at
205997_at
225207_at
211919_s_at
209355_s_at
205844_at
219697_at
217897_at
209160_at
1554018_at
221061_at
226560_at
239675_at
205695_at
209201_x_at
221584_s_at
205242_at
201005_at
238727_at
203066_at
210163_at
219525_at
216950_s_at
206392_s_at
209031_at
221872_at
214511_x_at
207092_at
210998_s_at
204698_at
CXCR4
CADM1
NID1
MSR1
--DPEP2
--HERC5
EDNRB
C6orf192
LOC129607
EDNRB
ITGB8
IGF1
IFIT3
GBP5
ANTXR1
PPAP2B
LGALS2
AQP9
ADAMDEC1
ADAM28
PDK4
CXCR4
PPAP2B
VNN1
HS3ST2
FXYD6
AKR1C3
GPNMB
PKD2L1
--LOC283143
SDS
CXCR4
KCNMA1
CXCL13
CD9
LOC440934
GALNAC4S-6ST
CXCL11
SLC47A1
FCGR1A
RARRES1
CADM1
RARRES1
FCGR1B
LEP
HGF
ISG20
1.8921E-06
0.00178361
0.00027978
0.0407896
0.00350254
0.00030538
0.00119758
0.01161955
0.00333527
0.00014043
0.04907369
0.00179274
0.00641868
0.0008278
0.03743987
0.00937433
0.00228002
0.00037949
0.031045
0.00420534
0.00524947
0.00417381
0.00456378
0.00122355
0.01837133
0.00233577
0.00275271
0.00116334
0.02049248
0.00010869
0.00666769
0.00426252
0.00164347
0.00051795
0.00112863
0.02349066
0.01926834
0.01166181
0.00218154
0.00026469
0.04645725
0.0059081
0.02233428
0.01824988
0.00102294
0.01653019
0.01078419
0.0002238
0.03097396
0.00039274
10
-4.602154975
-4.610147163
-4.687878052
-4.693308494
-4.734201426
-4.771187956
-4.815535587
-4.818009986
-4.860477301
-4.956175251
-4.957194803
-4.978295298
-4.994433515
-5.000122456
-5.012158842
-5.038506992
-5.124914039
-5.168669852
-5.244043989
-5.318670262
-5.324856039
-5.347695897
-5.412762051
-5.539643184
-5.652960981
-5.665917386
-5.707039394
-5.713018834
-5.848356173
-5.927528927
-6.007580849
-6.058693564
-6.207917234
-6.219219895
-6.24681844
-6.251570778
-6.37784498
-6.389936088
-6.394661483
-6.853628479
-7.105225164
-7.435807023
-7.679580716
-8.105591263
-8.483319219
-8.779320747
-8.990724541
-9.069971353
-11.43966736
-12.26609988
SUPPLEMENTARY MATERIAL
205922_at
209687_at
205960_at
224215_s_at
VNN2
CXCL12
PDK4
DLL1
0.00516081
0.00874068
0.04772079
0.00908821
11
-14.62125494
-16.90131603
-19.67973809
-25.91147956
SUPPLEMENTARY MATERIAL
Table S3. Primer sequences for RT-PCR
Species
Gene
Forward Primer Sequence (5’-3’)
Reverse Primer Sequence (5’-3’)
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
ARG1
CCL1
CD163
CD1a
CD1c
CD1e
CD206
CD209
COX2
CXCL11
CXCL13
CXCL5
G-CSF
GMCSF
IFNα
IFNβ
IFNγ
IL12
IL18
IL1R2
IL23
iNOS
IRF4
IRF5
MRC1
TRAIL
CCL13
CCL23
CD1c
CD80
CFH
CXCL10
CXCL12
GGTLA
IL10
IL1b
IL4
IL6
IL8
LTB
TGFb
TNFa
RPL
CD80
CXCL10
CXCL11
CXCL12
CXCL13
GMCSF
LTB
TGFb
IL1β
ARG1
MRC1
TNFα
RPL
GTG GAA ACT TGC ATG GAC AAC
CTC ATT TGC GGA GCA AGA GAT
TCA GTG CAG AAA TGG CCA ACA GA
TCA TCT TGG CGG TGA TAG TG
TGA ATT GGA TTG CCT TGG TAG
AGT TAC CCT GGT CAT ATT GGT TG
AGG GGG AAG AGT CTC ACA GG
CAC CTG GAT GGG ACT TTC AG
GGG TTG CTG GTG GTA GGA ATG
CAG AAT TCC ACT GCC CAA AGG
CTT CCC TTA TCC CTG CTC TGG A
GAT CCA GAA GCC CCT TTT CTA AAG
ACA AGC AGA GGT GGC CAG AG
GTC ATC TCA GAA ATG TTT GAC CTC C
GCC TCG CCC TTT GCT TTA CT
ATG ACC AAC AAG TGT CTC CTC C
TCG GTA ACT GAC TTG AAT GTC CA
ATG GCC CTG TGC CTT AGT AGT
TTC AAC TCT CTC CTG TGA GAA CA
TCC TGA CAT TTG CCC ATG AAG
GGA CAA CAG TCA GTT CTG CTT
CCT ACC AAC TGA CGG GAG ATG
GCT GAT CGA CCA GAT CGA CAG
TTC TCT CCT GGG CTG TCT CTG
AAG GCG GTG ACC TCA CAA G
CCG TCA GCT CGT TAG AAA GAC TCC A
ATC TCC TTG CAG AGG CTG AA
TTT GAA ACG AAC AGC GAG TG
TGA ATT GGA TTG CCT TGG TAG
CTG CTT TGC CCC AAG ATG C
AAG CGC AGA CCA CAG TTA CA
GCA AGC CAA TTT TGT CCA CG
CCA ACG TCA AGC ATC TCA AA
CAG GGG TCG AAG CTA GTG AT
GCT GAG AAC CAA GAC CCA GAC A
GCG GCC AGG ATA TAA CTG ACT TC
CCA ACT GCT TCC CCC TCT G
CAA GAA GGG TTT TTG TGA CTG AAT C
CTT GTT CCA CTG TGC CTT GGT T
CCA GAA ACA GAT CTC AGC CCC
ATG GTG TGT GAG ACG TTG ACT GA
CTT CTC GAA CCC CGA GTG AC
AGG TAT GCT GCC CCA CAA AAC
AAA TAT GGA GAT GCT CAC GTG TCA G
TTC ACC ATG TGC CAT GCC
AAA ATG GCA GAG ATC GAG AAA GC
ATC CTC AAC ACT CCA AAC TGT G
CAT AGA TCG GAT TCA AGT TAC GCC
TGC TTT TGT GCC TGC GTA ATG
AAC ACT TCC CCT CGA GC
GGA CCC TGC CCC TAT ATT TGG
TGT CTT GGC CGA GGA CTA AGG
CTC CAA GCC AAA GTC CTT AGA G
AGA CGA AAT CCC TGC TAC TGA A
AGA AAC ACA AGA TGC TGG GAC AGT
TGG TCC CTG CTG CTC TCA AG
CCT GGC ACA TCG GGA ATC TTT
GGA GCT GGT ATT TCT GTA ACA CA
CGA CGA AAA TGG CCA ACA GA
GAG GAG GCT CAT GGT GTG TC
AGG GGG AAG AGT CTC ACA GG
GGC TCC CAT GAG AAA GAC AG
AAA GTC CAA TTC CTC GAT GGT G
TGT TGG GCT CTC CTC TGT TC
AGC ATA AAG CGT TTG CGG TAC TC
GTA AAC TCC GAT GGT AAC CAG CC
CCA TCA GCT CCT GCA AGG TTA TT
AGA GAC CTC CAG AAA ACT TCT CTG C
CAA ACC ATG TCC CAA AAG TCT TAA G
GTG CTG TTT GTA GTG GCT GGC
CTG TGG GTC TCA GGG AGA TCA
GGA ATC CAA GCA AGT TGT AGC TC
TCG CTT CCC TGT TTT AGC TGC
AGC TTT GCA TTC ATG GTC TTG A
ATG TCC TGG GAC ACT TCT CTG
GGA AAT GAT CAC AGG AAT GGT CTC
CAC AGG GCT ATC AGG GAG C
ATG GCC GAC CTG ATG TTG C
CGG TTG TAG TCC TGC TTG C
CTA TAC AGC TAG GCC CCA GGG
AAA GTC CAA TTC CTC GAT GGT G
GCC CAC TCC TTG ATG ATT CCC AGG
CTT CTC CTT TGG GTC AGC AC
CAG CAT TCT CAC GCA AAC C
AGG GGG AAG AGT CTC ACA GG
CAG ATC TTT TCA GCC CCT TGC
TCA AGC TGG AGA GGG ATG AC
ACA TTT CCT TGC TAA CTG CTT TCA G
TAG CTT CGG GTC AAT GCA C
CTC TCA GGT CAA AGC CAA GC
CGG CCT TGC TCT TGT TTT CA
TCC ACA TTC AGC ACA GGA CTC TC
TCT GTT ACG GTC AAC TCG GTG
TCC TTG TTT TGC TCC AAC ACT AAT C
GCT TCC ACA TGT CCT CAC AAC AT
AAC GCC TGT TCC TTC GTC G
CGA GAG CCT GTC CAG ATG CT
TGA GGT ACA GGC CCT CTG ATG
TGT AGG CTT CAG ACG CAC GAC
CTG TTA TTA CTG CGC CGA ATC C
GAA CTG ACG AGC CTG AGC TAG G
CAG GCA CCT TTG TCG TTT ATG AG
TTT CTC CAG GTA CTC TTG GAT C
TCT TGG TCC AGA TCA CAA CTT CA
TCC AAG CTG AGT CAG CGT TTT C
ATG GCC AGC AGT AGC ATT GC
TGT TGC AGG TCA TTT AAC CAA GTG
TGG GCT GGA CTG TTT CTA ATG C
GGA GCT GTC ATT AGG GAC ATC A
TAG AAA GGA ATC CAC GCA GTC T
CCT TTGCAG AAC TCA GGAATG G
GGC CTT TTC CTT CCG TTT CTC
12
SUPPLEMENTARY MATERIAL
Table S4. Antibodies for flow cytometry
Species
Molecule
Clone
Manufacturer
Human
CD9
MEM-61
Immunotools, Hamburg, Germany
Human
CD80
2D10
BD Pharmingen, Heidelberg, Germany
Human
CD4
RPA-T4
Biolegend, San Diego, CA, USA
Human
CD25
BC96
Biolegend, San Diego, CA, USA
Human
Il1β
CRM56
eBioscience, San Diego, CA, USA
Human
TNFα
MAb11
eBioscience, San Diego, CA, USA
Human
IL10
JES3-9D7
Biolegend, San Diego, CA, USA
Human
CD206
eBioscience, San Diego, CA, USA
Human
Beta7
19.2
FIB504
Human
CCR1
5F10B29
Biolegend, San Diego, CA, USA
Human
CCR2
K036C2
Biolegend, San Diego, CA, USA
Human
CCR4
L291H4
Biolegend, San Diego, CA, USA
Human
CCR6
G034E3
Biolegend, San Diego, CA, USA
Human
CCR7
G043H7
Biolegend, San Diego, CA, USA
Human
CCR9
L053E8
Biolegend, San Diego, CA, USA
Human
CX3CR1
528728
R&D Systems, Minneapolis, MN, USA
Human
Foxp3
Biolegend, San Diego, CA, USA
Mouse
CD3e
259D
145-2C11
Mouse
CD28
37.51
BD Pharmingen, Heidelberg, Germany
Mouse
CD4
RM4-5
Biolegend, San Diego, CA, USA
Mouse
CD11b
M1/70
Biolegend, San Diego, CA, USA
Mouse
CD45.1
A20
Biolegend, San Diego, CA, USA
Mouse
Foxp3
FJK-16s
eBioscience, San Diego, CA, USA
13
Biolegend, San Diego, CA, USA
BD Pharmingen, Heidelberg, Germany