The Journal of Immunology
Colonic Eosinophilic Inflammation in Experimental Colitis Is
Mediated by Ly6ChighCCR2+Inflammatory
Amanda Waddell,* Richard Ahrens,* Kris Steinbrecher,†Burke Donovan,*
Marc E. Rothenberg,* Ariel Munitz,*,‡and Simon P. Hogan*
Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the
eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we
employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic
intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoi-
etic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate
(DSS) treatment promotes the recruitment of F4/80+CD11b+CCR2+Ly6Chighinflammatory monocytes into the colon. F4/80+
CD11b+CCR2+Ly6Chighmonocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflam-
mation. Phenotypic analysis of purified Ly6Chighintestinal inflammatory macrophages revealed that these cells express both M1-
and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80+
CD11b+CCR2+Ly6Chighmonocyte recruitment to the colon in CCR22/2mice was associated with decreased colonic CCL11
expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced
colonic eosinophilia mediated by Ly6ChighCCR2+inflammatory monocyte/macrophage-derived CCL11.
nology, 2011, 186: 5993–6003.
substantial morbidity and decreased quality of life. Over 30 years
ago, Rutgeerts and colleagues (1) observed that apthous ulcers
containing an eosinophilc infiltrate and blunted villi were the ear-
liest endoscopic signs of recurrence in the neoterminal ileum
and anastomosis following surgical resection for CD. Eosinophils
usually represent only a small percentage of the infiltrating leu-
kocytes (2, 3), but their level has been proposed to be a negative
The Journal of Immu-
he inflammatory bowel diseases (IBD) Crohn’s disease
(CD) and ulcerative colitis (UC) are chronic relapsing
gastrointestinal (GI) inflammatory diseases that cause
prognostic indicator (3, 4). Notably, elevated fecal concentrations
of the eosinophil-derived granule proteins ECP and EPO were
associated with clinical relapse within 3 mo in CD (5). Since these
initial studies, there have been a number of studies suggesting
eosinophil involvement in IBD. Elevated levels of eosinophils
have been observed in colonic biopsy samples from UC patients,
and increased numbers of this cell and eosinophil-derived granule
proteins MBP, ECP, EPO, and EDN have been shown to correlate
with morphological changes to the GI tract, disease severity, and
GI dysfunction (5–10). Increased numbers of tissue eosinophils
with ultrastructural evidence of activation has also been observed
in patients with CD (11–13). Consistent with this clinical obser-
vation, dextran sodium sulfate (DSS)-induced histopathology is
attenuated in mice deficient in eosinophils (14–16).
Genome-wide association studies of pediatric and adult IBD
haverevealed a number of IBDsusceptibility genes associated with
innate (CARD15, ATG16L1, and IRGM) and adaptive (IL23R,
IL10, IL12B, and STAT3) immunity. Furthermore, a recent in-
vestigation identified a significant association between the C-C
motif chemokine cluster on 17q12 loci, which contains the
eosinophil-specific chemokine gene CCL11, and early-onset CD
(17). CCL11 is a member of the CC chemokine family (18) and is
a relatively potent and specific eosinophil chemoattractant (19–
21). CCL11 is constitutively expressed in a variety of tissues that
contain eosinophils such as the GI tract and thymus (22–25).
Genetic deletion of CCL11 abrogates eosinophil recruitment dur-
ing eosinophil-associated pulmonary and GI disease, suggesting
an important role for CCL11 in eosinophil trafficking during
disease (26–29). Consistent with this, clinical investigations by us
and others demonstrate increased CCL11 mRNA levels in sputum
and intestinal biopsy samples from asthmatic and eosinophilic GI
disorder patients. Importantly, the levels of CCL11 positively
correlated with tissue eosinophilia (14, 25, 26).
*Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Cen-
ter, Cincinnati, OH 45229;†Division of Gastroenterology, Hepatology and Nutri-
tion, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and
‡Department of Microbiology and Clinical Immunology, Sackler Faculty of Medi-
cine, Tel Aviv University, Ramat Aviv 69978, Israel
Received for publication November 23, 2010. Accepted for publication March 12,
This work was supported by a Crohn’s and Colitis Foundation of America Career
Development Award (to S.P.H.), National Institutes of Health Grant R01 AI073553
(to S.P.H.), National Institutes of Health Grant R01 AI45898 (to M.E.R.), and an
American Gastroenterological Association Foundation Graduate Student Research
Fellowship Award (to A.W.).
A.W. and S.P.H. designed and performed experiments, analyzed and interpreted data,
and wrote the manuscript. B.D. performed experiments and analyzed and interpreted
data. M.E.R. provided mice. K.S., R.A., and A.M. discussed experimental design and
Address correspondence and reprint requests to Dr. Simon P. Hogan, Division of
Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Bur-
net Avenue, Cincinnati, OH 45229. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; CD, Crohn’s disease; DAI,
disease activity index; DSS, dextran sodium sulfate; eGFP, enhanced GFP; GI, gas-
trointestinal; hpf, high-power field; IBD, inflammatory bowel diseases; MF, macro-
phages; PDL1, programmed death ligand 1; PHIL, mice deficient in eosinophils; SC,
stromal compartment; UC, ulcerative colitis; WT, wild-type.
established, the cellular source of CCL11 and molecular regulation
of CCL11 expression in experimental colitis is not yet delineated.
In the current study, we employed a model of DSS-induced coli-
tis, which features a pronounced CCL11-dependent eosinophilic
inflammation, to decipher the molecular regulation of CCL11 in
experimental colitis. Performing bone marrow (BM) chimera ex-
periments, we show that hematopoietic cell-derived CCL11 is
required for DSS-induced colonic eosinophilic inflammation. We
show that DSS exposure promotes the recruitment of F4/80+
CD11b+CCR2+Ly6Chighinflammatory monocytes to the colon and
that F4/80+CD11b+CCR2+Ly6Chighcolonic monocytes/macrophages
(MFs) positively correlate with colonic eosinophilic inflammation.
Ablation of DSS-induced F4/80+CD11b+CCR2+Ly6Chighmonocyte
recruitment was associated with decreased intestinal CCL11 ex-
pression, colonic eosinophilic inflammation, and DSS-induced his-
topathology. These studies demonstrate that inflammatory monocyte/
MF-derived CCL11 drives colonic eosinophilic inflammation in
Materials and Methods
Male and female, 6–8-wk-old strain-, age-, and weight-matched CCR22/2
(C57BL/6), CCL22/2(C57BL/6) (The Jackson Laboratory, Bar Harbor,
ME), C57BL/6, BALB/c, CCL112/2(BALB/c) (30), CX3CR1GFP/+(The
Jackson Laboratory), and Nzeg-enhanced GFP (eGFP) (31) mice were
used. All mice were housed under specific pathogen-free conditions and
treated according to institutional guidelines.
DSS-induced colonic injury and histopathologic examination
DSS (ICN Chemicals; 40–45 kDa) was administered in the drinking water
as a 2.5–5% (w/v) solution for up to 8 d. Disease monitoring and histo-
pathologic changes in the colon were scored as previously described (14).
Immunofluorescence analysis was performed as previously described (14,
32). In brief, frozen sections were fixed in 10% acetone for 10 min, rinsed
in PBS, blocked with 3% goat serum/PBS for 2 h at room temperature, and
incubated with primary Ab rat anti-mouse F4/80 (5 mg/ml; eBioscience,
San Diego, CA) in 3% normal goat serum/PBS. Sections were incubated
with isotype control alone in place of primary Ab as a negative control.
After an overnight incubation at 4˚C, sections were washed with 0.1%
BSA and 0.05% Tween/PBS and incubated with goat anti-rat Alexa Fluor
594 (Invitrogen, Carlsbad, CA) for 2 h at room temperature. Slides
were washed in PBS and counterstained with DAPI/Supermount G solu-
tion (Southern Biotechnology Associates, Birmingham, AL). Images were
captured using a Zeiss microscope fitted with Zeiss UPlanApo lenses
(310, 320, and 340 magnification) and an AxioCam MRc camera and
analyzed with Axioviewer version 3.1 image analysis software (Carl Zeiss,
Jena, Germany). Postacquisition processing (brightness, opacity, contrast,
and color balance) was applied to the entire image and accurately reflects
that of the original.
CCL11, IL-6, and TNF-a levels were measured in the supernatants using
the ELISA Duo-Set kit according to the manufacturer’s instructions (R&D
Systems, Minneapolis, MN).
Eosinophil levels were quantified by anti-MBP immunohistochemistry as
previously described (33).
Colons were excised, flushed with PBS with gentamicin (20 mg/ml), and
opened along a longitudinal axis. Thereafter, 3-mm2punch biopsies were
excised and incubated for 24 h in a 24-well plate with RPMI 1640 sup-
plemented with 10% FCS and antibiotics. Supernatants were collected and
kept at 220˚C until assessed for cytokines/chemokines by ELISA.
Real-time PCR analysis
Mouse Hprt, Ccl11, Retnla, Chi3l3, Arg1, Trem1, Ccl22, Ccl17, Cxcl2,
Ccl4, Cxcl10, Pdgfb, Il1b, Tnf, Il6, Ccl3, and Il10 mRNA were quantified
by real-time PCR as previously described (34). In brief, the RNA samples
(1 mg) were subjected to reverse transcription analysis using SuperScript II
reverse transcriptase (Invitrogen) according to the manufacturer’s
instructions and quantified using the iQ5 multicolor real-time PCR de-
tection system (Bio-Rad, Hercules, CA) with iQ5 software V2.0 and
LightCycler FastStart DNA Master SYBR Green I (Bio-Rad). Primer sets
are listed in Supplemental Table I. Gene expression was determined as
relative expression on a linear curve based on a gel-extracted standard and
was normalized to Hprt amplified from the same cDNA mix. Results were
expressed as gene of interest/Hprt ratio.
Intestinal MF purification
MF populations from the colons of CX3CR1eGFP/+(C57BL/6) were iso-
lated as previously described (14). In brief, the colon segment of the GI
tract was removed and flushed with 20 ml Ca2+- and Mg2+-free HBSS. The
colon was cut longitudinally, placed in Ca2+- and Mg2+-free HBSS con-
taining 10% FBS/5 mM EDTA/25 mM HEPES, and shaken vigorously at
37˚C for 30 min. The tissue was cut into 1-cm segments and incubated in
digestion buffer containing 2.4 mg/ml collagenase A (Roche Diagnostics,
Indianapolis, IN) and 0.2 mg/ml DNase I (Roche Diagnostics) in RPMI
1640 for 45 min on a shaker at 37˚C. Following incubation, the cell
aggregates were dissociated by filtering thorough a 19-gauge needle and
70-mm filter and centrifuged at 1200 rpm for 20 min at 4˚C. The super-
natant was decanted and the cell pellet resuspended in 1% FBS/5 mM
EDTA/PBS, and cells were incubated for 30 min with biotinylated rat anti-
mouse CD11b (1 mg/1 3 106cells; BD Pharmingen, San Jose, CA) at 4˚C.
Cells were subsequently incubated with anti-biotin microbeads (Miltenyi
Biotec, Auburn, CA) for 15 min at 10˚C and purified by LS MACS column
by positive selection as described by the manufacturer. In brief, 1 ml of cell
suspension was added to the LS column, and the column was washed three
times with 3 ml of 5 mM EDTA/1% FBS/PBS. CD11b+cells were re-
moved from the column using a plunger. After washing, CD11b+-selected
cells were labeled with rat anti-mouse Ly6C-Alexa 647 (AbD Serotec,
Raleigh, NC) and immediately sorted using an FACSAria cell sorter
(BD Biosciences, San Jose, CA) for CX3CR1-eGFP and Ly6C. Purity of
CX3CR1lowLy6Chighcells was .95% as assessed by flow cytometry. RNA
was isolated using the Qiagen RNeasy micro kit for cDNA synthesis
(Qiagen) and RT-PCR analysis was performed as described above.
Peripheral blood was collected in K2EDTA tubes, and RBCs were lysed
using RBC Lysing Buffer (Sigma-Aldrich, St. Louis, MO). Monocytes
were enriched using the StemCell Technologies monocyte enrichment kit
following the manufacturer’s protocol (StemCell Technologies). Purity
was assessed by flow cytometry at .80% F4/80+CD11b+. RNA was iso-
lated using the Qiagen RNeasy micro kit (Qiagen), and cDNA was gen-
erated for RT-PCR analysis as described above.
Single-cell suspensions were washed with FACS buffer (PBS/1% FCS) and
incubated with combinations of the following Abs: PE anti-mouse F4/80
(clone CI:A3-1; AbD Serotec), PE-Cy7 anti-mouse CD11b (clone M1/
70; BD Pharmingen), Alexa Fluor 647 anti-mouse Ly6C (clone ER-
MP20; AbD Serotec), FITC anti-mouse CD206 (MR5D3; Biolegend,
San Diego, CA), allophycocyanin anti-mouse CD11c (clone HL3; BD
Pharmingen), FITC anti-mouse programmed death ligand 1 (PDL1) (clone
MIH6; AbD Serotec), and goat anti-mouse CCR2 (polyclonal; GeneTex,
Irvine, CA) followed by donkey anti-goat FITC (Jackson ImmunoResearch
Laboratories, West Grove, PA), and biotinylated anti-mouse TLR2 (clone
mT2.7; eBioscience), followed by streptavidin-FITC (BD Pharmingen),
allophycocyanin anti-mouse CCR3 (clone 83101; R&D Systems), and PE
anti-mouse Siglec F (clone E50-2440; BD Pharmingen). The following
Abs were used as appropriate isotype controls: FITC rat IgG2a (clone B39-
4; BD Pharmingen), PE rat IgG2a (clone 53-6.7; BD Pharmingen), PE-Cy7
rat IgG2b (clone DTA-1; BD Pharmingen), and Alexa Fluor 647 rat IgG2a
(clone R35-95; BD Pharmingen). Cells were analyzed on an FACSCalibur
(BD Immunocytometry Systems, San Jose, CA), and analysis was per-
formed using FlowJo software (Tree Star, Ashland, OR).
Generation of BM chimeras
BM was isolated from Nzeg-eGFP (BALB/c) (31) and CCL112/2mice
(BALB/c). Lethally irradiated (two doses of [137Cs] [475 and 475 rad, 3 h
5994LY6ChighMONOCYTE/MACROPHAGES REGULATE COLONIC INFLAMMATION
apart]) wild-type (WT) or CCL112/2BALB/c recipients were injected i.v.
with 5–10 3 106BM cells/mouse. Engraftment was checked by eGFP+
(donor)/eGFP2(recipient) cells from the peripheral blood, mesenteric
lymph node, and colon by flow cytometry. Seven to 8 wk postirradiation,
the mice were administered 5% DSS for 7 d, and colonic eosinophil ac-
cumulation was assessed.
Data were analyzed by means of ANOVA, followed by the Tukey post hoc
test, and correlative analysis was performed using a Spearman rank order
correlation coefficient analysis with GraphPad Prism 5 (GraphPad, San
Diego, CA). Data are presented as the mean 6 SE. The p values ,0.05
were considered statistically significant.
BM-derived CCL11 is sufficient to reconstitute eosinophil
recruitment to the colon of CCL112/2mice during
We have previously reported a pathological role for eosinophils in
DSS-induced colonic injury and that eosinophil recruitment into
the colon following DSS treatment was dependent on CCL11 (14).
With the emerging experimental and clinical data demonstrating
an important function for CCL11/eosinophils in IBD, we were
interested in defining the cellular source of CCL11 in experi-
mental colitis. To assess if hematopoietic or stromal compartment
(SC) expression of CCL11 is sufficient for DSS-induced colonic
eosinophilic inflammation, we restricted CCL11 expression to
either the BM or SC using BM chimeric mice on the BALB/c
background. To restrict CCL11 expression to the BM compart-
ment, we irradiated recipient CCL112/2mice and reconstituted
them with WT BM. In these mice, referred to as SC2BM+, only
cells derived from transferred WT BM are CCL11 sufficient (+),
whereas the radioresistant MFs and SC native to the recipient
CCL112/2mice do not express functional CCL11 (2). Con-
versely, in SC+BM2mice, we restricted CCL11 signaling to only
the SC compartment by irradiating WT mice and reconstituting
with CCL112/2BM. As controls, WT or CCL112/2mice were
irradiated and reconstituted with their own BM type (SC+BM+and
SC2BM2, respectively). Seven to 8 wk following BM re-
constitution, chimeric mice were exposed to 5% DSS, and eo-
sinophilic inflammation was evaluated. To facilitate analysis of
chimerism in BALB/c mice, we transferred BM from BALB/c
Nzeg-eGFP mice to WT and CCL112/2BALB/c mice. All
cells from the Nzeg-eGFP mice are constitutively GFP positive
(31) and can be detected by flow cytometric analysis of auto-
fluorescence. In chimeric mice, eGFP+cells derived from the
donor BM are distinguished from any remaining recipient eGFP2
cells. The degree of chimerism at 7 wk as determined by flow
cytometry was 98.2 6 0.2% and 95.4 6 2.1% for peripheral blood
monocytes and eosinophils, respectively, and 72.0 6 0.6% for
mesenteric lymph nodes (CD4+T cells) (mean 6 SEM; n = 3 to 4
mice per group) (Supplemental Fig. 1A–C). Percent reconstitution
of colonic MFs at baseline and following DSS was 66.9 6 2.8%
and 93.3 6 1.2%, respectively, as determined by flow cytometry
(Supplemental Fig. 1D, 1E). Chimerism was also determined for
baseline colonic CD4+T cells, B cells, and eosinophils (Supple-
mental Fig. 1D, 1E). Following the verification of reconstitution,
mice were exposed to DSS for 7 d, and colonic eosinophil in-
flammation was quantitated. DSS treatment of SC+BM+and SC2
BM+mice induced a significant increase in colonic eosinophil
levels compared with control-treated mice (Fig. 1A, 1B: SC+BM+
baseline 8.0 6 1.0 versus SC+BM+DSS 17.2 6 2.9 eosinophils/
high-power field (hpf), p , 0.05; SC2BM+baseline 3.4 6 0.1
versus SC2BM+13.2 6 1.2 eosinophils/hpf, p , 0.05; n = 3 to 5
mice baseline; n = 7 to 8 mice DSS). Similarly, DSS treatment of
CCL112/2mice reconstituted with WT BM (SC2BM+) induced
a 3-fold increase in eosinophil recruitment in the distal colon
compared with SC2BM2mice (Fig. 1A, 1B: SC2BM+12.4 6 1.6
eosinophils/hpf versus SC2BM23.8 6 1.3 eosinophils/hpf; n = 3
to 4 mice per group). Eosinophils were also significantly increased
in SC+BM2mice (Fig. 1B). Importantly, DSS-induced colonic
eosinophilic inflammation was attenuated in CCL112/2mice
reconstituted with CCL112/2BM (SC2BM2; Fig. 1B). These
data indicate that BM-derived CCL11 is sufficient to drive eo-
sinophilic recruitment into the colon during DSS-induced colonic
DSS-induced colonic injury promotes the specific recruitment
Following our demonstration that BM-derived CCL11 expression
was sufficient to reconstitute DSS-induced colonic eosinophilic
inflammation, we were next interested in identifying the hema-
topoietic source of CCL11 that drove DSS-induced colonic eo-
sinophilic inflammation. We have previously demonstrated CCL11
expression in F4/80+myeloid cells within the lamina propria of
colonic sections from day 7 DSS-treated mice. B, Eosinophil quantification/hpf in the distal sections of the colon of baseline and DSS-treated mice. Data
represent the mean 6 SEM of n = 3 to 4 mice per group from duplicate experiments. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001)
between groups. Original magnification 3100.
BM chimeras SC2BM+have increased eosinophil levels compared with SC2BM2. A, Representative photomicrographs of MBP-stained
The Journal of Immunology5995
the colon of mice following 7 d of exposure to 2.5% DSS (14).
We therefore assessed the relationship between myeloid cell and
DSS-induced eosinophilic inflammation. First, we performed flow
cytometry analysis on peripheral blood monocytes and colonic
MFs at baseline and following DSS exposure (Fig. 2A). Under
homeostatic conditions, the peripheral blood was predominantly
composed of F4/80+CD11b+Ly6Chighmyeloid cells, whereas the
colon consisted of F4/80+CD11b+Ly6Clowand F4/80+CD11b+
Ly6Chighmyeloid cells (Fig. 2A). The predominant F4/80+CD11b+
Ly6Clowmyeloid population (.80%) within the colon was
CX3CR1highPDL1+TLR-22CD206+(Fig. 2B), consistent with the
resident intestinal MF phenotype (35). DSS exposure (5 d) in-
duced a significant influx of F4/80+CD11b+Ly6Chighmonocytes
(Fig. 2A). Notably, the increase in colonic F4/80+CD11b+Ly6Chigh
monocyte/MF cell numbers (control 7,497 6 1,565 versus DSS
39,996 6 8,708, p , 0.01; mean 6 SEM; n = 5 to 6 per group)
occurred in the absence of any change in F4/80+CD11b+Ly6Clow
MF levels (control 55,979 6 12,490 versus 42,818 6 7,190;
mean 6 SEM; n = 5 to 6 per group) (Fig. 2A). The infiltrating
F4/80+CD11b+Ly6Chighmyeloid population was predominantly
CCR2+CX3CR1loTLR-2+CD2062CD11c2PDL12(Fig. 2B, Sup-
plemental Fig. 2). We next assessed the colonic eosinophil pop-
ulation following DSS exposure. We show that eosinophils were
a distinct population characterized by forward light scatterlowand
side scatterhigh. Eosinophil lineage was confirmed as the cells
were Siglec F+CCR3+double positive. Notably, the colonic eo-
sinophil population was CD11b+, F4/80+, and CCR22(Fig. 2C).
Colonic Ly6ChighMFs express Ccl11
To directly assess if Ly6Chighcolonic MFs were a source of
CCL11, we purified F4/80+CD11b+Ly6ChighMFs from the co-
lon of DSS-treated mice by using CX3CR1eGFP/+mice. Previous
studies have demonstrated that inflammatory (Ly6ChighCCR2+
CX3CR1low) and noninflammatory (Ly6ClowCCR22CX3CR1hi)
tissue MFs can be distinguished based upon the level of CX3CR1
expression (35). Consistent with this, the resident noninflammatory
F4/80+CD11b+Ly6ClowMF population of CX3CR1eGFP/+mice was
CX3CR1high, whereas the DSS-induced colonic F4/80+CD11b+
Ly6ChighMF population was CX3CR1low(Fig. 2B). In conjunction,
the CX3CR1lowcells within the colon of DSS-treated mice were
found to be F4/80+CD11b+Ly6Chigh, consistent with the infiltrating
monocyte population (Fig. 3A).
Following our confirmation that CX3CR1 and Ly6C expres-
sion could distinguish between the two intestinal myeloid sub-
populations, we purified CX3CR1lowLy6Chighcells from the
colons of CX3CR1eGFP/+mice following DSS exposure using flow
sorting (Fig. 3B). Purity of CX3CR1lowLy6Chighcells was .95%
as assessed by flow cytometry (data not shown). For compara-
CD11b+Ly6Chighmonocyte/MF populations from the peripheral blood (top panel), colon at baseline (middle panel), and colon following DSS exposure
(bottom panel). MF were initially gated by side scatter (SSC) versus forward light scatter (FSC) followed by F4/80+CD11b+. The double-positive cell
populations from baseline and DSS-treated colons were assessed for Ly6C. B, Ly6Chighand Ly6Clowcolonic MF expression of CCR2, CX3CR1, TLR2,
PDL1, and CD206 by flow cytometry at baseline and following DSS. C, Characterization of F4/80, CD11b, Siglec F, CCR3, and CCR2 on colonic
eosinophils from DSS-treated mice. Data represent the mean 6 SEM of n = 5 to 6 mice per group from duplicate experiments. Significant differences (*p ,
0.05) between groups.
DSS exposure induces the recruitment of F4/80+CD11b+Ly6Chighmonocyte population. A, Representative flow cytometry plots of the F4/80+
5996LY6ChighMONOCYTE/MACROPHAGES REGULATE COLONIC INFLAMMATION
tive analyses, we also purified blood CX3CR1lowF4/80+CD11b+
Ly6Chighmonocytes from CX3CR1eGFP/+mice at baseline and on
day 5 of DSS (Fig. 4A, 4B). PCR analyses revealed no detectable
Ccl11 mRNA expression in CX3CR1lowLy6Chighperipheral blood
monocytes at baseline or following DSS (Fig. 3C). Ccl11 mRNA
expression was induced in the colonic CX3CR1lowLy6Chighcells
following infiltration into the colon during DSS-induced colitis
(Fig. 3C). Assessment of other genes expressed by the Ly6Chigh
myeloid population revealed that recruited F4/80+CD11b+Ly6Chigh
MFs had a similar phenotype to the peripheral blood popula-
tion, as they were positive for Chi3l3, Trem1, Cxcl10, Tnfa, and
Pdgfb mRNA transcripts with no detectable Ccl17 expression
(Fig. 3C and data not shown). Interestingly, DSS exposure did not
significantly influence the Ly6Chighperipheral blood population,
as we observed no significant difference in the gene profile be-
tween control and DSS-treated Ly6Chighperipheral blood cells
(Fig. 3C). However, we detected increased mRNA transcripts for
Arg1, Il10, Ccl4, Il1b, Il6, Cxcl2, Retnla, and Ccl22 in the
Ly6Chighcolonic population compared with the Ly6Chighperiph-
eral blood population (Fig. 3C).
To determine the relationship between the intestinal inflam-
matory F4/80+CD11b+Ly6Chighmonocytes/MFs and eosinophil
recruitment in DSS colitis, we quantified colonic F4/80+CD11b+
Ly6ChighMF and eosinophil levels following DSS-induced co-
lonic injury. We found a positive correlation between numbers
of colonic F4/80+CD11b+Ly6ChighMFs and eosinophils (Fig. 4;
p , 0.005). Notably, levels of colonic F4/80+CD11b+Ly6Chigh
MFs did not correlate with colonic neutrophil (F4/802CD11b+
Ly6G+) levels (Fig. 4), indicating a specific link between F4/80+
CD11b+Ly6Chighmonocyte recruitment and colonic eosinophilic
inflammation. For F4/80+CD11b+Ly6Chighmonocytes/MFs to
drive eosinophil recruitment into the colon would require the in-
flux of F4/80+CD11b+Ly6Chighmonocyte/MF cells prior to eo-
sinophil infiltration. We have previously reported that eosinophil
infiltration of the colon occurs at day 5 of DSS exposure (14).
Assessment of F4/80+CD11b+Ly6Chighmonocyte/MF and eosin-
ophil levels in the colon revealed a significant increase in F4/80+
CD11b+Ly6Chighmonocytes/MFs on days 3 and 4 of DSS ex-
posure in the absence of increased eosinophil numbers (Supple-
mental Fig. 3). These data indicate that F4/80+CD11b+Ly6Chigh
monocyte/MF recruitment into the colon precedes eosinophil
MFs express CCL11 and are a mixed M1/M2
phenotype in DSS-induced colonic injury. A,
CX3CR1-eGFP expression in F4/80+CD11b+
Ly6Chighcolonic monocytes/MFs. B, Repre-
sentative FACS plot of CX3CR1+Ly6Chighco-
lonic monocyte/MF flow sorted on day 5 of
DSS. C, cDNAwas made, and gene expression
of Ccl11, Il1b, Tnf, Il6, Ccl4, Cxcl2, Arg1, Il10,
Retnla, and Ccl22 was analyzed by RT-PCR.
Three to four mice were pooled per sample.
Peripheral blood baseline (6 samples), peri-
pheral blood day 5 DSS (3 samples), and co-
lon day 5 DSS (7–10 samples) monocyte/MF
expression was analyzed. Data represent the
mean 6 SEM. Significant differences (*p ,
0.05, **p , 0.01) between groups. ND, below
limit of detection.
els positively correlated with colonic eosinophilic inflammation. Corre-
lative analyses of eosinophils, neutrophils, and F4/80+CD11b+Ly6Chigh
monocyte/MF levels in the colon following DSS exposure. Cells were
gated as described in Fig. 1.
DSS-induced F4/80+CD11b+Ly6Chighmonocyte/MF lev-
The Journal of Immunology 5997
DSS-induced colonic injury recruitment of F4/80+CD11b+
Ly6Chighmonocytes is CCR2-dependent
We next assessed the relative contribution of F4/80+CD11b+
Ly6Chighblood monocytes to DSS-induced colonic eosinophilic
inflammation and colitis in CCR22/2mice, as CCR2 is important
for Ly6Chighmonocyte mobilization from the BM to the periph-
eral circulation and tissue inflammatory sites (36). Consistent
with this, basal homeostatic levels of peripheral blood Ly6Chigh
monocytes were 6-fold lower in CCR22/2mice compared with
WT mice (Fig. 5A, 5B). DSS-induced recruitment of F4/80+
CD11b+Ly6ChighMFs into the colon was attenuated in CCR22/2
mice (Fig. 5C, 5D). Immunofluorescence analyses of F4/80+cells
in the colonic lamina propria of DSS-treated WT mice and
CCR22/2mice revealed a loss of the large F4/80+infiltrate in the
CCR22/2mice (Fig. 5E). This reduction was specific for Ly6Chigh
monocytes, as recruitment of F4/802CD11b+cells (neutrophils)
was not impaired (Fig. 5F, 5G). The reduction in intestinal MF
levels was not due to decreased levels of homeostatic intestinal
MFs, as the levels of resident F4/80+CD11b+Ly6Clowcolonic
MFs were comparable between WTand CCR22/2mice (Fig. 5H,
5I). Collectively, these data indicate that homeostatic resident
F4/80+CD11b+Ly6Clowcolonic MF levels are independent of
CCR2, whereas F4/80+CD11b+Ly6Chighmonocyte recruitment to
the colon during DSS-induced colitis is CCR2 dependent.
DSS-induced colonic inflammation and colitis is attenuated in
To assess the contribution of F4/80+CD11b+Ly6Chighmonocytes to
DSS-induced colonic CCL11 expression and eosinophilia, we
quantitated CCL11 and eosinophil levels in the colon of CCR22/2
mice following DSS exposure. Importantly, colonic eosinophil
levels and distribution at baseline were comparable between WT
and CCR22/2mice, indicating no role for CCR2 in basal colonic
eosinophil recruitment (Fig. 6A, 6B and data not shown). DSS
treatment of WT mice induced a significant increase in colonic
eosinophil levels (Fig. 6A, 6B). In contrast, there was no signifi-
cant increase in eosinophil levels in DSS-treated CCR22/2mice
(Fig. 6A, 6B). Notably, the reduction in colonic eosinophil levels
was associated with no significant increase in colonic CCL11
levels in colonic punch biopsies from DSS-treated CCR22/2mice
(Fig. 6C: WT baseline 8.7 6 1.2 pg/ml versus WT DSS 42.4 6
9.4 pg/ml, p , 0.05; CCR22/2baseline: 12.6 6 3.1 pg/ml;
Ly6Chighperipheral blood monocytes in WT and CCR22/2mice. B, Quantification of percent Ly6Chighperipheral blood monocytes in WT and CCR22/2
mice. C, Flow cytometric analysis of F4/80+CD11b+Ly6Chighcolonic monocytes/MFs at baseline and following DSS in WT and CCR22/2mice. D,
Quantification of Ly6Chighcolonic MF numbers based on flow cytometry analysis. E, Colonic sections from WT and CCR22/2mice treated with vehicle
and 2.5% DSS for 5 d were stained with either anti-F4/80 or isotype Ig and goat anti-rat Alexa Fluor 594. Slides were counterstained with nuclei stain
DAPI. Images represent overlay DAPI/F480. Original magnification 3100. F, Representative flow cytometry plots of F4/802CD11b+cells (neutrophils) in
colon at baseline and following DSS in WT and CCR22/2mice. G, Percent F4/802CD11b+in colon at baseline and following DSS in WT and CCR22/2.
H, Colonic lamina propria cells were stained for flow cytometry with F4/80, CD11b, and Ly6C, and the number of triple-positive cells was examined. I,
Quantification of percent of F4/80+CD11b+and F4/80+CD11b+Ly6ChighWT and CCR22/2colonic MFs at baseline. Data represent the mean 6 SEM of
n = 3 to 4 mice per group from duplicate experiments. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001) between groups.
DSS-induced recruitment of F4/80+CD11b+Ly6Chighmonocytes in WT and CCR22/2mice. A, Flow cytometric analysis of F4/80+CD11b+
5998LY6ChighMONOCYTE/MACROPHAGES REGULATE COLONIC INFLAMMATION
CCR22/2DSS: 19.27 6 4.4 pg/ml; mean 6 SEM; n = 9 to 10
mice per group). These data directly implicate F4/80+CD11b+
Ly6Chighmonocyte/MF-derived CCL11 in the regulation of co-
lonic eosinophilic inflammation in DSS-induced colonic injury.
To assess the effect of ablation of Ly6Chighmonocyte re-
cruitment and colonic eosinophils to DSS-induced colonic injury,
we performed histopathological assessment of the colon in WT
and CCR22/2mice. DSS treatment of WT mice induced crypt
loss, epithelial erosion, and a large inflammatory infiltrate (Fig.
7A). The DSS-induced epithelial damage was significantly re-
duced in CCR22/2mice compared with WT mice (Fig. 7A, 7B;
histological score of WT 15.7 6 0.84 versus CCR22/27.67 6
0.62, p , 0.001; mean 6 SEM; n = 10 per group). Consistent with
this observation, CCR22/2mice displayed less weight loss and
delayed development of diarrhea and rectal bleeding (disease
activity index [DAI]) resulting in decreased DAI score (Fig. 7C;
DAI of WT 5.5 6 0.65 versus CCR22/22.2 6 0.32, p , 0.05;
mean 6 SEM; n = 4 per group). Attenuation of DSS-induced
colitis and recruitment of F4/80+CD11b+Ly6Chighmonocytes to
the colon by CCR2 deficiency was associated with decreased
production of proinflammatory cytokines IL-6 and TNF-a (Fig.
7D). Collectively, these studies identify that Ly6Chighcolonic
monocytes/MFs have a pathogenic role in DSS-induced proin-
flammatory cytokine production and histopathology and that re-
cruitment of this cell population is mediated by CCR2-dependent
CCL2 is a CC chemokine that binds to the CCR2 receptor, and
experimental data indicate that this chemokine is important in the
recruitment of monocytes and MFs into inflamed tissues (37–39).
Notably, DSS exposure induced a significant increase in colonic
CCL2 protein levels (Supplemental Fig. 4A). To evaluate the
relative contribution of CCL2 to DSS-induced Ly6Chighmonocyte
recruitment into the colon and disease pathology, we examined
CCL22/2mice. Surprisingly, the levels of colonic F4/80+CD11b+
Ly6Chighmonocytes in the colon of DSS-treated CCL22/2mice
were comparable to that of strain- and weight-matched DSS-
treated WT mice (Supplemental Fig. 4B, 4C). Consistent with
our data, recruitment of F4/80+CD11b+Ly6Chighmonocytes into
the colon of CCL22/2mice was associated with DSS-induced
weight loss and disease activity (Supplemental Fig. 4D), dis-
ease pathology (Supplemental Fig. 4E, 4F), and colonic eosino-
phil inflammation (Supplemental Fig. 4G, 4H). Assessment of
Ly6Chighperipheral blood monocytes and colonic MFs at base-
line revealed comparable levels between WT and CCL22/2mice,
indicating that CCL2 does not contribute to either homeosta-
tic resident intestinal F4/80+CD11b+Ly6ClowMF levels or DSS-
induced recruitment of F4/80+CD11b+Ly6Chighmonocytes into
the colon (Supplemental Fig. 5).
In the current study, we have investigated the molecular regulation
of CCL11 and colonic eosinophilic inflammation in an experi-
mental mouse model of DSS-induced colitis. We demonstrate
that reconstitution of CCL112/2mice with BM-derived CCL11 is
sufficient for DSS-induced colonic eosinophil inflammation. We
show that DSS exposure promotes the influx of F4/80+CD11b+
Ly6Chighmonocytes into the colon and that recruitment of this
cell population positively correlated with colonic eosinophilic in-
flammation. Purification of the F4/80+CD11b+Ly6Chighpopula-
tion revealed that these cells express a mixture of M1- and
M2-associated genes, including Chi3l3, Retnla, Il10, Il6, Il1b,
Trem1, Cxcl2, and Ccl11. Abrogation of F4/80+CD11b+Ly6Chigh
monocyte recruitment by genetic deletion of CCR2 was associated
with decreased DSS-induced histopathology, CCL11 expression,
and eosinophil recruitment. These studies indicate that colonic
eosinophilic inflammation in experimental colitis is mediated by
Ly6ChighCCR2+inflammatory monocyte/MF-derived CCL11.
Flow cytometry analyses identified the presence of two distinct
monocyte populations in the peripheral blood of mice charac-
terized by F4/80+CD11b+Ly6Chighand F4/80+CD11b+Ly6Clow
phenotype. We show that the F4/80+CD11b+Ly6Chighpopulation
was CCR2+and CX3CR1low, whereas the F4/80+CD11b+Ly6Clow
monocytes were CCR22and CX3CR1high. The F4/80+CD11b+
Ly6Clowblood monocytes are a precursor to resident homeo-
static tissue MFs (35). Consistent with this, under homoeostatic
conditions, the colon predominantly consisted of F4/80+CD11b+
Ly6Clowmyeloid cells. Intestinal dendritic cells under homeostatic
conditions also express CX3CR1 and F4/80. Analysis of CD11c
expression revealed that F4/80+CD11b+Ly6Clowcells consisted
of 80% CD11c2and 20% CD11c+cells, indicating the presence
of homeostatic tissue MFs and dendritic cells within the F4/80+
CD11b+Ly6Clowpopulation (Supplemental Fig. 2). The presence
of an F4/80+CD11b+Ly6ClowCD11c+dendritic cell population
may have contributed to the gene expression heterogeneity seen
within this cell population. The F4/80+CD11b+Ly6Chighmono-
cytes are an inflammatory monocyte population and rapidly re-
cruited into tissues following inflammatory insult. Large infil-
trates of Ly6ChighCX3CR1lowCCR2+blood monocytes have been
observed in the peritoneum and injured myocardium follow-
ing immune stimulation (35, 40). Similarly, we observed the se-
lective increase in the numbers of F4/80+CD11b+Ly6Chighcells
within the colon following DSS exposure. Importantly, the F4/80+
CD11b+Ly6Chighcells within the colon were CD11c2, indicating
that this population did not contain a contaminating dendritic cell
population (Supplemental Fig. 2). Consistent with previous in-
vestigations, we show that the recruitment of the inflammatory
F4/80+CD11b+Ly6Chighmonocytes/MFs into inflamed tissues
graphs of anti-MBP–stained colonic sections from baseline and DSS-treated mice (day 7, 2.5% DSS). Original magnification 3100. Colonic eosinophil
levels (B) and CCL11 levels (C) in supernatants from punch biopsies from WTand CCR22/2mice at baseline and following 5 d of DSS. Data represent the
mean 6 SEM of n = 9 to 10 mice per group from duplicate experiments. Significant differences (*p , 0.05, **p , 0.01) between groups.
Reduced CCL11 and eosinophil levels in CCR22/2mice during DSS-induced colitis compared with WT. A, Representative photomicro-
The Journal of Immunology5999
was dependent on CCR2 (35). Previous investigations have in-
dicated that CCR2-dependent recruitment of this monocytic pop-
ulation is mediated by CCL2 (37, 41); however, the CCR2-
mediated recruitment of F4/80+CD11b+Ly6Chighmonocytes/MFs
into the colon was not dependent on CCL2. CCR2 is a pro-
miscuous chemokine receptor binding multiple ligands, includ-
ing CCL2, CCL8, CCL7, and CCL13 (42). Additionally, CCL2,
CCL8, and CCL7 have been shown to be increased in the colon in
inflamed tissue from IBD patients as well as in the DSS model of
colitis (43, 44).
The demonstration that ablation of F4/80+CD11b+Ly6Chigh
monocytes/MFs into the colon in CCR22/2mice was associated
with a reduction in proinflammatory cytokines (IL-6 and TNF-a)
indicates that this cell population is an important source of cyto-
kines that drive DSS-induced intestinal inflammation and colitic
disease. Consistent with this, PCR analyses of purified Ly6Chigh
monocytes/MFs revealed that these cells expressed high levels
of proinflammatory markers Trem1 and TLR2, but not the anti-
inflammatory markers CD206 or PDL1. Similar to our obser-
vations, Platt and colleagues (45) identified a TLR2+CCR2+
CX3CR1IntLy6ChiTNF-a+monocyte/MF population in the colon
following DSS exposure and that this cell type drives DSS-
induced pathology. We have extended these observations, identi-
fying a new cellular function for the F4/80+CD11b+Ly6Chigh
TLR2+population in driving colonic eosinophilic inflammation
and histopathology associated with DSS-induced colitis. Further-
more, we have developed a protocol that permits purification of
this cell population and demonstrated that the Ly6Chighcolonic
monocyte/MFs express Arg1, Retnla, Chi3l3, Ccl22 (MDC), and
Il10. The Arg1 and Chi3l3 (YM1) mRNA expression of the in-
filtrating Ly6Chighcell population is consistent with the previously
reported DSS-induced total increase in tissue ARG1 and YM1
protein (46). The expression of Arg1 and Chi3l3 (YM1) suggests
that the monocytes/MFs are alternatively activated; however,
we also observed evidence of classical activation as the cells
expressed the proinflammatory cytokines/chemokines Il1b, Il6,
Ccl4, and Cxcl2. Surprisingly, Tnfa mRNA expression was not
increased in Ly6Chighcolonic monocytes compared with peri-
pheral blood monocytes, although protein expression correlated
with Ly6Chighmonocyte recruitment. However, it has previously
been shown that Tnf mRNA stability and translation regulate its
bioreactivity (47–50). Our results suggest that Ly6Chighcolonic
monocytes/MFs have a mixed classically/alternatively activated
phenotype in the acute phase of DSS-induced colitis, further in-
dicating that classical/alternatively activated phenotypes are part
of a spectrum of activation as opposed to the strongly polarized
phenotypes that are often seen in vitro (51, 52). Alternatively, the
mixed phenotype could suggest the presence of subpopulations of
Ly6Chighcolonic monocytes/MFs within the colon. DSS-induced
colitis has been linked with increased expression of the classical/
alternative activation cytokines IL-4/IFN-g (53, 54). In a mouse
model of kidney injury, Chi3l3 was expressed in Ly6C+MFs in
the kidney; however, other M2 genes, including Ccl17 and Ccl22,
were only expressed at very low levels, whereas high levels of
of DSS. Original magnification 3100. B, Histological score. C, Percent weight loss and diarrhea/rectal bleeding score during the course of DSS exposure.
D, Cytokine analysis of punch biopsy supernatants from day 6 DSS in WT and CCR22/2mice. Data represent the mean 6 SEM of n = 9 to 10 mice per
group from duplicate experiments. Significant differences (*p , 0.05, ***p , 0.001) between groups.
DSS-induced disease pathology in WTand CCR22/2mice. A, Representative photomicrographs of H&E-stained colonic sections from day 7
6000 LY6ChighMONOCYTE/MACROPHAGES REGULATE COLONIC INFLAMMATION
Cxcl2 and Il1b were expressed, indicating an M1-biased pattern
(55). Further investigations are required to determine whether the
Ly6Chighmonocyte/MF population consists of multiple polarized
monocyte/MF subsets of different phenotypes or whether the
Ly6Chighmonocyte/MF population is simply plastic and adapts to
exogenous and endogenous stimuli within the microenvironment.
The role of monocytes/MFs in eosinophil recruitment during
chronic inflammatory responses and the relative contribution of
CCL11 to this response are not yet fully delineated. Previous
studies have demonstrated Ccl11 mRNA expression in MFs in
allergen-induced cutaneous biopsies in atopic patients (56) and in
bronchial biopsies from asthmatic patients that possess eosino-
phils (57). Furthermore, a recent study also demonstrated CCL11
expression in lung MFs from rhinovirus-infected allergic mice
with pulmonary eosinophilic inflammation (58). Experimental an-
alyses employing a Nippostrongylus brasiliensis infestation
model have demonstrated a role for monocyte/MF populations
in eosinophil recruitment into peritoneum. Similar to our obser-
vations, eosinophil recruitment was associated with Retnla and
Chi3l3 monocyte/MF expression but was not associated with
Ccl11 expression (59). We show that during colonic inflammation,
CX3CR1+Ly6Chighcolonic monocytes/MFs are the primary cel-
lular source of CCL11 and that this cell population is sufficient
to mediate colonic eosinophilic inflammation. These observations
suggest that DSS exposure stimulates CX3CR1+Ly6Chighcolonic
monocyte/MF recruitment in the colon and that the recruited in-
flammatory monocytes/MFs subsequently drive eosinophil in-
filtration via a CCL11-dependent pathway. We have previously
characterized eosinophil infiltration of the colon following DSS
exposure and reported that eosinophil levels begin to increase
on day 5 of DSS exposure (14). Assessment of F4/80+CD11b+
Ly6Chighand eosinophil numbers in the colon of mice 3 and
4 d following DSS exposure revealed a significant influx of F4/80+
CD11b+Ly6Chighmonocytes/MFs prior to commencement of eo-
sinophil recruitment (Supplemental Fig. 3). These kinetic data
indicate that F4/80+CD11b+Ly6Chighmonocytes/MFs are strate-
gically positioned in the colon to regulate DSS-induced eosinophil
recruitment. These studies strongly suggest that CCL11 is pre-
dominantly monocyte/MF-derived and is important in colonic
eosinophilic recruitment in experimental colitis. We have pre-
viously reported a critical role for CCL11 and not the other
eotaxin family members CCL24 and CCL26 in the regulation of
eosinophilic inflammation in experimental and human IBD (14).
Moreover, eosinophil recruitment into the colon during experi-
mental colitis was attenuated in CCL112/2and not CCL242/2
mice, and elevated Ccl11 mRNA levels in lesional biopsy samples
from IBD patients positively correlated with eosinophil numbers
(14). Furthermore, we have previously identified CCL11+CD68+
monocytes/MFs in colonic biopsies of pediatric UC patients. The
demonstration of a link between colonic eosinophilic inflamma-
tion and DSS-induced histopathology suggests a role for eosino-
phils in the DSS-induced colonic injury. This is consistent with
previous demonstration by us and others of a partial role for
eosinophils in DSS-induced epithelial histopathology (14–16, 33).
Furthermore, this is supported by significant clinical evidence
demonstrating increased eosinophils and eosinophil-derived
granule proteins in adult UC and CD and a positive correlation
between levels of eosinophils and histological score in recto-
sigmoid colonic biopsy samples from pediatric UC patients (4, 9,
14, 60, 61). We have previously published that mice deficient in
eosinophils (PHIL) are partially protected from DSS-induced co-
litis (33). Moreover, we observed a 48% reduction in histological
score between DSS-treated WT versus PHIL mice (14). Notably,
in the current study, we observed a 52% reduction in histological
score between DSS-treated WT versus CCR22/2mice (histolog-
ical score WT DSS: 15.7 6 0.8 versus PHIL DSS: 7.7 6 0.6; n = 9
to 10 per group; mean 6 SEM; p = 0.001; Fig. 7). The reduction
in histopathology in CCR22/2mice was associated with reduced
F4/80+CD11b+Ly6Chighmonocyte/MF recruitment and reduced
CCL11 expression and eosinophil infiltration. These data suggest
that a significant component of monocyte/MF-mediated DSS-
induced histopathology is mediated via regulation of eosinophil
BM chimera experiments in mice indicate that BM cell-derived
CCL11 is sufficient for DSS-induced colonic eosinophilic in-
flammation. Moreover, CCL112/2mice reconstituted with WT
BM restored eosinophil recruitment to the colon during DSS-
induced colonic injury. As BM reconstitution is not selective for
Ly6Chighmonocyte-derived CCL11, we cannot rule out the con-
tribution of other BM-derived cell populations; however, we have
previously demonstrated that DSS-induced CCL11 expression in
the colon was restricted to F4/80+myeloid cells and not F4/802
cells (14). Surprisingly, we observed a significant eosinophilic
infiltrate in the colon following DSS exposure in WT mice
reconstituted with BM from CCL112/2mice, suggesting a role
for structural cell-derived CCL11 in colonic eosinophilic in-
flammation. We postulate that this paradoxical observation is due
to the radioresistance of monocytes/MFs and the effects of radi-
ation on monocyte/MF function. Many studies have demonstrated
that tissue MFs are radioresistant (62–64). For example, in the
lung, it takes .90 d for alveolar MFs to reach ∼80% re-
constitution (64). Similarly, we found that Ly6Clowcolonic MFs
only reached ∼60% reconstitution at ∼50 d postirradiation and
that ∼5% of monocytes/MFs in the colon of DSS-treated mice
were recipient-derived cells. Furthermore, irradiation has been
shown to induce MF oxidative injury (65) as well as alter
MF activation, which may lead to homeostatic resident MF in-
volvement in eosinophil recruitment (66). We currently cannot
rule out a contribution for non-BM cell-derived CCL11 to eo-
sinophil recruitment during DSS-induced colitis; however, our
previous data demonstrating CCL11 expression restricted to F4/
80+cells in the lamina propria of the colon on day 7 of DSS
exposure (14) and our observations in CCR22/2mice indicate that
the CCR2-dependent Ly6Chighmonocytes are sufficient to drive
eosinophil recruitment in DSS-induced colonic injury.
Recently, a number of unique loci were identified to be asso-
ciated with early-onset IBD susceptibility including the C-C motif
chemokine cluster on 17q12 loci, which contains the eosinophil-
specific chemokine gene CCL11 (17). Clinical and experimental
data indicate: 1) a strong relationship between eosinophils and the
exacerbation and severity to IBD; and 2) a pivotal role for MFs in
the augmentation of the intestinal inflammatory response associ-
ated with IBD (67). We provide evidence of a direct pathway
involving Ly6Chighcolonic monocyte/MF-derived CCL11 in co-
lonic eosinophilic inflammation and histopathology in experi-
mental colitis. These studies provide significant rationale for the
assessment of monocyte/MF-derived CCL11 in human IBD and
the targeting of the monocyte/MF/CCL11 pathway as a thera-
peutic modality for the treatment and prevention of IBD.
We thank Drs. Patricia Fulkerson and Debroski Herbert and members of the
Division of Allergy and Immunology and the Division of Gastroenterology,
Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical Center
for critical review of the manuscript and insightful conversations. We thank
Dr. Nives Zimmermann, Victoria Summey, and Jeff Bailey from the Cin-
cinnati Children’s Hospital Medical Center Comprehensive Mouse and
Cancer Core facility for expertise and assistance with BM transplantation.
The Journal of Immunology 6001
We thank Jamie and Nancy Lee for the generous provision of anti-MBPAb
and Dr. Klaus Matthaei for the Nzeg-eGFP mice. We also thank Emily
Stucke and Heather Osterfeld for animal husbandry and Shawna Hottinger
for editorial assistance and manuscript preparation.
The authors have no financial conflicts of interest.
1. Rutgeerts, P., K. Geboes, G. Vantrappen, R. Kerremans, J. L. Coenegrachts, and
G. Coremans. 1984. Natural history of recurrent Crohn’s disease at the ileoco-
lonic anastomosis after curative surgery. Gut 25: 665–672.
2. Walsh, R. E., and T. S. Gaginella. 1991. The eosinophil in inflammatory bowel
disease. Scand. J. Gastroenterol. 26: 1217–1224.
3. Desreumaux, P., S. Nutten, and J. F. Colombel. 1999. Activated eosinophils in
inflammatory bowel disease: do they matter? Am. J. Gastroenterol. 94: 3396–
4. Nishitani, H., M. Okabayashi, M. Satomi, T. Shimoyama, and Y. Dohi. 1998.
Infiltration of peroxidase-producing eosinophils into the lamina propria of
patients with ulcerative colitis. J. Gastroenterol. 33: 189–195.
5. Saitoh, O., K. Kojima, K. Sugi, R. Matsuse, K. Uchida, K. Tabata, K. Nakagawa,
M. Kayazawa, I. Hirata, and K. Katsu. 1999. Fecal eosinophil granule-derived
proteins reflect disease activity in inflammatory bowel disease. Am. J. Gastro-
enterol. 94: 3513–3520.
6. Bischoff, S. C., J. Mayer, Q. T. Nguyen, M. Stolte, and M. P. Manns. 1999.
Immunnohistological assessment of intestinal eosinophil activation in patients
with eosinophilic gastroenteritis and inflammatory bowel disease. Am. J. Gas-
troenterol. 94: 3521–3529.
7. Bischoff, S. C., J. Wedemeyer, A. Herrmann, P. N. Meier, C. Trautwein, Y. Cetin,
H. Maschek, M. Stolte, M. Gebel, and M. P. Manns. 1996. Quantitative as-
sessment of intestinal eosinophils and mast cells in inflammatory bowel disease.
Histopathology 28: 1–13.
8. Raab, Y., K. Fredens, B. Gerdin, and R. Ha ¨llgren. 1998. Eosinophil activation in
ulcerative colitis: studies on mucosal release and localization of eosinophil
granule constituents. Dig. Dis. Sci. 43: 1061–1070.
9. Carlson, M., Y. Raab, C. Peterson, R. Ha ¨llgren, and P. Venge. 1999. Increased
intraluminal release of eosinophil granule proteins EPO, ECP, EPX, and cyto-
kines in ulcerative colitis and proctitis in segmental perfusion. Am. J. Gastro-
enterol. 94: 1876–1883.
10. Carvalho, A. T., C. C. Elia, H. S. de Souza, P. R. Elias, E. L. Pontes,
H. P. Lukashok, F. C. de Freitas, and J. R. Lapa e Silva. 2003. Immunohisto-
chemical study of intestinal eosinophils in inflammatory bowel disease. J. Clin.
Gastroenterol. 36: 120–125.
11. Dubucquoi, S., A. Janin, O. Klein, P. Desreumaux, P. Quandalle, A. Cortot,
M. Capron, and J.-F. Colombel. 1995. Activated eosinophils and interleukin 5
expression in early recurrence of Crohn’s disease. Gut 37: 242–246.
12. Ha ¨llgren, R., J. F. Colombel, R. Dahl, K. Fredens, A. Kruse, N. O. Jacobsen,
P. Venge, and J. C. Rambaud. 1989. Neutrophil and eosinophil involvement of
the small bowel in patients with celiac disease and Crohn’s disease: studies on
the secretion rate and immunohistochemical localization of granulocyte granule
constituents. Am. J. Med. 86: 56–64.
13. Dvorak, A. M. 1980. Ultrastructural evidence for release of major basic protein-
containing crystalline cores of eosinophil granules in vivo: cytotoxic potential in
Crohn’s disease. J. Immunol. 125: 460–462.
14. Ahrens, R., A. Waddell, L. Seidu, C. Blanchard, R. Carey, E. Forbes, M. Lampinen,
T. Wilson, E. Cohen, K. Stringer, et al. 2008. Intestinal macrophage/epithelial cell-
derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric
ulcerative colitis. J. Immunol. 181: 7390–7399.
15. Maltby, S., C. Wohlfarth, M. Gold, L. Zbytnuik, M. R. Hughes, and
K. M. McNagny. 2010. CD34 is required for infiltration of eosinophils into
the colon and pathology associated with DSS-induced ulcerative colitis. Am.
J. Pathol. 177: 1244–1254.
16. Vieira, A. T., C. T. Fagundes, A. L. Alessandri, M. G. Castor, R. Guabiraba,
V. O. Borges, K. D. Silveira, E. L. Vieira, J. L. Gonc ¸alves, T. A. Silva, et al.
2009. Treatment with a novel chemokine-binding protein or eosinophil lineage-
ablation protects mice from experimental colitis. Am. J. Pathol. 175: 2382–2391.
17. Imielinski, M., R. N. Baldassano, A. Griffiths, R. K. Russell, V. Annese,
M. Dubinsky, S. Kugathasan, J. P. Bradfield, T. D. Walters, P. Sleiman, et al;
Western Regional Alliance for Pediatric IBD; International IBD Genetics Con-
sortium; NIDDK IBD Genetics Consortium; Belgian-French IBD Consortium;
Wellcome Trust Case Control Consortium. 2009. Common variants at five new
loci associated with early-onset inflammatory bowel disease. Nat. Genet. 41:
18. Zimmermann, N., G. K. Hershey, P. S. Foster, and M. E. Rothenberg. 2003.
Chemokines in asthma: cooperative interaction between chemokines and IL-13.
J. Allergy Clin. Immunol. 111: 227–242, quiz 243.
19. Jose, P. J., D. A. Griffiths-Johnson, P. D. Collins, D. T. Walsh, R. Moqbel,
N. F. Totty, O. Truong, J. J. Hsuan, and T. J. Williams. 1994. Eotaxin: a potent
eosinophil chemoattractant cytokine detected in a guinea pig model of allergic
airways inflammation. J. Exp. Med. 179: 881–887.
20. Rothenberg, M. E., R. Ownbey, P. D. Mehlhop, P. M. Loiselle, M. van de Rijn,
J. V. Bonventre, H. C. Oettgen, P. Leder, and A. D. Luster. 1996. Eotaxin triggers
eosinophil-selective chemotaxis and calcium flux via a distinct receptor and
induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Mol.
Med. 2: 334–348.
21. Ganzalo, J. A., G. Q. Jia, V. Aguirre, D. Friend, A. J. Coyle, N. A. Jenkins,
G. S. Lin, H. Katz, A. Lichtman, N. Copeland, et al. 1996. Mouse Eotaxin ex-
pression parallels eosinophil accumulation during lung allergic inflammation but
it is not restricted to a Th2-type response. Immunity 4: 1–14.
22. Mishra, A., S. P. Hogan, J. J. Lee, P. S. Foster, and M. E. Rothenberg. 1999.
Fundamental signals that regulate eosinophil homing to the gastrointestinal tract.
J. Clin. Invest. 103: 1719–1727.
23. Zimmermann, N., S. P. Hogan, A. Mishra, E. B. Brandt, T. R. Bodette,
S. M. Pope, F. D. Finkelman, and M. E. Rothenberg. 2000. Murine eotaxin-2:
a constitutive eosinophil chemokine induced by allergen challenge and IL-4
overexpression. J. Immunol. 165: 5839–5846.
24. Rothenberg, M. E., A. D. Luster, C. M. Lilly, J. M. Drazen, and P. Leder. 1995.
Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig
lung. J. Exp. Med. 181: 1211–1216.
25. Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, and
A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil
cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2:
26. Hogan, S. P., A. Mishra, E. B. Brandt, P. S. Foster, and M. E. Rothenberg. 2000.
A critical role for eotaxin in experimental oral antigen-induced eosinophilic
gastrointestinal allergy. Proc. Natl. Acad. Sci. USA 97: 6681–6686.
27. Matthews, A. N., D. S. Friend, N. Zimmermann, M. N. Sarafi, A. D. Luster,
E. Pearlman, S. E. Wert, and M. E. Rothenberg. 1998. Eotaxin is required for the
baseline level of tissue eosinophils. Proc. Natl. Acad. Sci. USA 95: 6273–6278.
28. Mishra, A., S. P. Hogan, E. B. Brandt, N. Wagner, M. W. Crossman, P. S. Foster,
and M. E. Rothenberg. 2002. Enterocyte expression of the eotaxin and
interleukin-5 transgenes induces compartmentalized dysregulation of eosinophil
trafficking. J. Biol. Chem. 277: 4406–4412.
29. Hogan, S. P., A. Mishra, E. B. Brandt, M. P. Royalty, S. M. Pope,
N. Zimmermann, P. S. Foster, and M. E. Rothenberg. 2001. A pathological
function for eotaxin and eosinophils in eosinophilic gastrointestinal in-
flammation. Nat. Immunol. 2: 353–360.
30. Rothenberg, M. E., J. A. MacLean, E. Pearlman, A. D. Luster, and P. Leder.
1997. Targeted disruption of the chemokine eotaxin partially reduces antigen-
induced tissue eosinophilia. J. Exp. Med. 185: 785–790.
31. Quah, B. J., V. P. Barlow, V. McPhun, K. I. Matthaei, M. D. Hulett, and
C. R. Parish. 2008. Bystander B cells rapidly acquire antigen receptors from
activated B cells by membrane transfer. Proc. Natl. Acad. Sci. USA 105: 4259–
32. Munitz, A., E. T. Cole, A. Beichler, K. Groschwitz, R. Ahrens, K. Steinbrecher,
T. Willson, X. Han, L. Denson, M. E. Rothenberg, and S. P. Hogan. 2010. Paired
immunoglobulin-like receptor B (PIR-B) negatively regulates macrophage ac-
tivation in experimental colitis. Gastroenterology 139: 530–541.
33. Forbes, E., T. Murase, M. Yang, K. I. Matthaei, J. J. Lee, N. A. Lee, P. S. Foster,
and S. P. Hogan. 2004. Immunopathogenesis of experimental ulcerative colitis is
mediated by eosinophil peroxidase. J. Immunol. 172: 5664–5675.
34. Blanchard, C., N. Wang, K. F. Stringer, A. Mishra, P. C. Fulkerson, J. P. Abonia,
S. C. Jameson, C. Kirby, M. R. Konikoff, M. H. Collins, et al. 2006. Eotaxin-3
and a uniquely conserved gene-expression profile in eosinophilic esophagitis. J.
Clin. Invest. 116: 536–547.
35. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes consist of
two principal subsets with distinct migratory properties. Immunity 19: 71–82.
36. Serbina, N. V., and E. G. Pamer. 2006. Monocyte emigration from bone marrow
during bacterial infection requires signals mediated by chemokine receptor
CCR2. Nat. Immunol. 7: 311–317.
37. Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North,
C. Gerard, and B. J. Rollins. 1998. Abnormalities in monocyte recruitment and
cytokine expression in monocyte chemoattractant protein 1-deficient mice. J.
Exp. Med. 187: 601–608.
38. Gosling, J., S. Slaymaker, L. Gu, S. Tseng, C. H. Zlot, S. G. Young, B. J. Rollins,
and I. F. Charo. 1999. MCP-1 deficiency reduces susceptibility to atherosclerosis
in mice that overexpress human apolipoprotein B. J. Clin. Invest. 103: 773–778.
39. Huang, D. R., J. Wang, P. Kivisakk, B. J. Rollins, and R. M. Ransohoff. 2001.
Absence of monocyte chemoattractant protein 1 in mice leads to decreased local
macrophage recruitment and antigen-specific T helper cell type 1 immune re-
sponse in experimental autoimmune encephalomyelitis. J. Exp. Med. 193: 713–
40. Nahrendorf, M., F. K. Swirski, E. Aikawa, L. Stangenberg, T. Wurdinger,
J. L. Figueiredo, P. Libby, R. Weissleder, and M. J. Pittet. 2007. The healing
myocardium sequentially mobilizes two monocyte subsets with divergent and
complementary functions. J. Exp. Med. 204: 3037–3047.
41. Winter, C., W. Herbold, R. Maus, F. La ¨nger, D. E. Briles, J. C. Paton, T. Welte,
and U. A. Maus. 2009. Important role for CC chemokine ligand 2-dependent
lung mononuclear phagocyte recruitment to inhibit sepsis in mice infected with
Streptococcus pneumoniae. J. Immunol. 182: 4931–4937.
42. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and
their role in immunity. Immunity 12: 121–127.
43. Wedemeyer, J., A. Lorentz, M. Go ¨ke, P. N. Meier, P. Flemming, C. A. Dahinden,
M. P. Manns, and S. C. Bischoff. 1999. Enhanced production of monocyte
chemotactic protein 3 in inflammatory bowel disease mucosa. Gut 44: 629–635.
44. te Velde, A. A., F. de Kort, E. Sterrenburg, I. Pronk, F. J. ten Kate,
D. W. Hommes, and S. J. van Deventer. 2007. Comparative analysis of colonic
gene expression of three experimental colitis models mimicking inflammatory
bowel disease. Inflamm. Bowel Dis. 13: 325–330.
6002LY6ChighMONOCYTE/MACROPHAGES REGULATE COLONIC INFLAMMATION
45. Platt, A. M., C. C. Bain, Y. Bordon, D. P. Sester, and A. M. Mowat. 2010. An Download full-text
independent subset of TLR expressing CCR2-dependent macrophages promotes
colonic inflammation. J. Immunol. 184: 6843–6854.
46. Munitz, A, A Waddell, L Seidu, ET Cole, R Ahrens, SP Hogan, and
ME Rothenberg. 2008. Resistin-like molecule alpha enhances myeloid cell
activation and promotes colitis. J. Allergy Clin. Immunol. 122: 1200–1207.
47. Kontoyiannis, D., M. Pasparakis, T. T. Pizarro, F. Cominelli, and G. Kollias.
1999. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-
rich elements: implications for joint and gut-associated immunopathologies.
Immunity 10: 387–398.
48. McDonald, P. P., V. A. Fadok, D. Bratton, and P. M. Henson. 1999. Transcrip-
tional and translational regulation of inflammatory mediator production by en-
dogenous TGF-beta in macrophages that have ingested apoptotic cells. J.
Immunol. 163: 6164–6172.
49. Schook, L. B., H. Albrecht, P. Gallay, and C. V. Jongeneel. 1994. Cytokine
regulation of TNF-alpha mRNA and protein production by unprimed macro-
phages from C57Bl/6 and NZW mice. J. Leukoc. Biol. 56: 514–520.
50. Swantek, J. L., M. H. Cobb, and T. D. Geppert. 1997. Jun N-terminal kinase/
stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide
stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorti-
coids inhibit TNF-alpha translation by blocking JNK/SAPK. Mol. Cell. Biol. 17:
51. Benoit, M., B. Desnues, and J. L. Mege. 2008. Macrophage polarization in
bacterial infections. J. Immunol. 181: 3733–3739.
52. Mantovani, A., A. Sica, S. Sozzani, P. Allavena, A. Vecchi, and M. Locati. 2004.
The chemokine system in diverse forms of macrophage activation and polari-
zation. Trends Immunol. 25: 677–686.
53. Ito, R., M. Shin-Ya, T. Kishida, A. Urano, R. Takada, J. Sakagami, J. Imanishi,
M. Kita, Y. Ueda, Y. Iwakura, et al. 2006. Interferon-gamma is causatively in-
volved in experimental inflammatory bowel disease in mice. Clin. Exp. Immunol.
54. Stevceva, L., P. Pavli, A. Husband, A. Ramsay, and W. F. Doe. 2001. Dextran
sulphate sodium-induced colitis is ameliorated in interleukin 4 deficient mice.
Genes Immun. 2: 309–316.
55. Lin, S. L., A. P. Castan ˜o, B. T. Nowlin, M. L. Lupher, Jr., and J. S. Duffield.
2009. Bone marrow Ly6Chigh monocytes are selectively recruited to injured
kidney and differentiate into functionally distinct populations. J. Immunol. 183:
56. Ying, S., D. S. Robinson, Q. Meng, L. T. Barata, A. R. McEuen, M. G. Buckley,
A. F. Walls, P. W. Askenase, and A. B. Kay. 1999. C-C chemokines in allergen-
induced late-phase cutaneous responses in atopic subjects: association of eotaxin
with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant
protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils
and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES).
J. Immunol. 163: 3976–3984.
57. Ying, S., Q. Meng, K. Zeibecoglou, D. S. Robinson, A. Macfarlane, M. Humbert,
and A. B. Kay. 1999. Eosinophil chemotactic chemokines (eotaxin, eotaxin-2,
RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C
chemokine receptor 3 expression in bronchial biopsies from atopic and non-
atopic (Intrinsic) asthmatics. J. Immunol. 163: 6321–6329.
58. Nagarkar, D. R., E. R. Bowman, D. Schneider, Q. Wang, J. Shim, Y. Zhao,
M. J. Linn, C. L. McHenry, B. Gosangi, J. K. Bentley, et al. 2010. Rhinovirus
infection of allergen-sensitized and -challenged mice induces eotaxin release
from functionally polarized macrophages. J. Immunol. 185: 2525–2535.
59. Voehringer, D., N. van Rooijen, and R. M. Locksley. 2007. Eosinophils develop
in distinct stages and are recruited to peripheral sites by alternatively activated
macrophages. J. Leukoc. Biol. 81: 1434–1444.
60. Jeziorska, M., N. Haboubi, P. Schofield, and D. E. Woolley. 2001. Distribution
and activation of eosinophils in inflammatory bowel disease using an improved
immunohistochemical technique. J. Pathol. 194: 484–492.
61. Venge, P., J. Bystro ¨m, M. Carlson, L. Ha ˆkansson, M. Karawacjzyk, C. Peterson,
L. Seve ´us, and A. Trulson. 1999. Eosinophil cationic protein (ECP): molecular
and biological properties and the use of ECP as a marker of eosinophil activation
in disease. Clin. Exp. Allergy 29: 1172–1186.
62. Lambert, L. E., and D. M. Paulnock. 1987. Modulation of macrophage function
by gamma-irradiation. Acquisition of the primed cell intermediate stage of the
macrophage tumoricidal activation pathway. J. Immunol. 139: 2834–2841.
63. Lorimore, S. A., P. J. Coates, G. E. Scobie, G. Milne, and E. G. Wright. 2001.
Inflammatory-type responses after exposure to ionizing radiation in vivo:
a mechanism for radiation-induced bystander effects? Oncogene 20: 7085–7095.
64. Matute-Bello, G., J. S. Lee, C. W. Frevert, W. C. Liles, S. Sutlief, K. Ballman,
V. Wong, A. Selk, and T. R. Martin. 2004. Optimal timing to repopulation of
resident alveolar macrophages with donor cells following total body irradiation
and bone marrow transplantation in mice. J. Immunol. Methods 292: 25–34.
65. Coates, P. J., J. I. Robinson, S. A. Lorimore, and E. G. Wright. 2008. Ongoing
activation of p53 pathway responses is a long-term consequence of radiation
exposure in vivo and associates with altered macrophage activities. J. Pathol.
66. Coates, P. J., J. K. Rundle, S. A. Lorimore, and E. G. Wright. 2008. Indirect
macrophage responses to ionizing radiation: implications for genotype-
dependent bystander signaling. Cancer Res. 68: 450–456.
67. Heinsbroek, S. E. M., and S. Gordon. 2009. The role of macrophages in in-
flammatory bowel diseases. Expert Rev. Mol. Med. 11: e14.
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