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Originally published online as doi:10.1189/jlb.1205734 on August 3, 2006

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(Journal of Leukocyte Biology. 2006;80:802-815.)
© 2006 by Society for Leukocyte Biology

Suppression of experimental colitis by intestinal mononuclear phagocytes

Joseph E. Qualls*, Alan M. Kaplan*, Nico van Rooijen{dagger} and Donald A. Cohen*,1

* Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, College of Medicine, Lexington, Kentucky; and
{dagger} Vrije Universiteit, VUMC, Department of Molecular Cell Biology, Amsterdam, The Netherlands

1 Correspondence: Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, College of Medicine, Lexington, KY 40536-0084. E-mail: dcohen{at}uky.edu

ABSTRACT

The contribution of innate immunity to inflammatory bowel disease (IBD) remains an area of intense interest. Macrophages (MØ) and dendritic cells (DC) are considered important factors in regulating the onset of IBD. The goal of this study was to determine if intestinal mononuclear phagocytes (iMNP) serve a pathological or protective role in dextran sulfate sodium (DSS)-induced colitis in mice. Using a conditional MØ/DC depletion transgenic mouse line—MØ Fas-induced apoptosis—to systemically deplete iMNP, DSS colitis histopathology was shown to be more severe in MØ/DC-depleted compared with MØ/DC-intact mice. Similarly, localized iMNP depletion by clodronate-encapsulated liposomes into C57BL/6, BALB/c, and CB.17/SCID mice also increased DSS colitis severity, as indicated by increased histopathology, weight loss, rectal bleeding, decreased stool consistency, and colon length compared with MØ/DC-intact, DSS-treated mice. Histology revealed that iMNP depletion during DSS treatment led to increased neutrophilic inflammation, increased epithelial injury, and enhanced mucin depletion from Goblet cells. iMNP depletion did not further elevate DSS-induced expression of TNF-{alpha} and IFN-{gamma} mRNA but significantly increased expression of CXCL1 chemokine mRNA. Myeloperoxidase activity was increased in colons of MØ/DC-depleted, DSS-treated mice, compared with DSS alone, coincident with increased neutrophil infiltration in diseased colons. Neutrophil depletion combined with MØ/DC depletion prevented the increase in DSS colitis severity compared with MØ/DC depletion alone. This study demonstrates that iMNP can serve a protective role during development of acute colitis and that protection is associated with MØ/DC-mediated down-regulation of neutrophil infiltration.

Key Words: dextran sodium sulfate • dendritic cells • monocytes/macrophages • mice

INTRODUCTION

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is associated with severe and sometimes chronic inflammation in the lower gastroinstestinal (GI) tract, affecting more than 0.1% of the population in developed countries [1 ]. Although the incidence of IBD has been linked to a number of possible environmental [1 , 2 ], genetic [1 , 3 4 5 ], and immunological [6 ] causes, specific factors that contribute to the onset and severity of IBD have not yet been defined clearly. Evidence points toward contributions from aberrant immune responses against commensal bacteria in the intestinal tract. Although experimental colitis can be induced in germ-free mice [7 ], a variety of studies support a role for commensal bacteria in IBD [8 ], and treatment with broad-spectrum antibiotics has been shown to reduce inflammatory symptoms in some IBD patients [9 10 11 ]. During the disease process of IBD, the colonic epithelium becomes disrupted by an overt, inflammatory response directed against an unknown source, allowing the invasion of commensal microorganisms from the intestinal lumen into the mucosa. The disruption of the colonic epithelium coincides with an intense influx of leukocytes into affected tissue sites [12 ], presumably aimed at the invading microorganisms and subsequent repair of the mucosal epithelium.

Several studies have suggested a pathogenic role for innate immunity in the onset and severity of IBD. For example, experimental colitis has been induced in lymphocyte-deficient SCID mice by the oral administration of dextran sulfate sodium (DSS) [13 , 14 ], and depletion of macrophages (MØ)/dendritic cells (DC) by clodronate-encapsulated liposomes in an IL-10 knockout (KO) model of colitis in mice resulted in the amelioration of disease symptoms [15 ]. The MØ has long been considered part of the destructive force behind IBD, as a result of its involvement in regulating inflammation through the production of inflammatory cytokines and chemokines. MØ-derived inflammatory cytokines, such as IL-1ß, IL-6, and TNF-{alpha} as well as the neutrophil chemoattractant IL-8 are increased in IBD [16 17 18 ] and animal models of colitis [19 , 20 ]. However, MØ can exist in a range of activation states, some of which also produce factors that are immunosuppressive and/or can aid in the repair of damaged mucosal tissues [21 , 22 ]. Recent studies by Araki et al. [23 ] and Katakura et al. [24 ] have demonstrated a protective role for TLR signaling in experimental colitis; however, whether protection is mediated by MØ or other intestinal cells remains to be determined. DC have been shown to have an immunostimulatory role in activating T cells in experimental models of IBD [25 , 26 ]; however, whether their function as an innate immune cell also plays a role is presently unresolved. New evidence is emerging that DC can play an important role in innate immunity. Recent studies have shown that activation of TLRs on DC can result in the release of a variety of proinflammatory cytokines and chemokines, which promote the infiltration of neutrophils and the activation of NK cells [27 28 29 ]. The goal of the current study was to evaluate the pathophysiologic role of intestinal mononuclear phagocytes (iMNP) in the development of intestinal inflammation in the DSS-induced murine model of colitis. Using two different methods of MØ/DC depletion in colonic tissue, we demonstrate that iMNP can serve a protective role, which limits the severity of intestinal inflammation.

MATERIALS AND METHODS

Mice
MØ Fas-induced apoptosis (MaFIA) transgenic mice (C57BL/6 background) were developed in our laboratory at the University of Kentucky (Lexington) [30 ] from the enhanced GFP (EGFP) reporter driven by the c-fms promoter of Sasmono et al. [31 ] and the FK506-binding protein-Fas suicide construct described by Thomis et al. [32 ]. MaFIA mice were bred at the University of Kentucky’s Division of Laboratory Animal Resources. Female C57BL/6 and BALB/c mice (5–8 weeks old) were purchased from Harlan (Indianapolis, IN), and 5- to 8-week-old female CB.17/SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). These mice were housed according to the policies of the Animal Welfare Act, and the Institutional Animal Care and Use Committee at the University of Kentucky approved all studies. Mice were housed four to a cage in sterile, microisolator cages and allowed unrestricted access to food and water.

Induction of colitis
DSS (3%; molecular weight 36,000–50,000, MP Biomedicals, LLC, Aurora, OH) was dissolved in water (w/v) and given to mice in place of normal drinking water. DSS water was provided ad libitum up to 7 days; all mice were killed on Day 7, except CB.17/SCID mice, which were killed on Day 5. Measurements of the water volume were taken daily to determine the amount of DSS consumed per mouse. The amount of DSS consumed per mouse was comparable between MØ/DC-intact and MØ/DC-depleted mice.

Clinical scoring of colitis
The clinical scoring of a disease activity index (DAI) for DSS-induced colitis was based on weight loss, stool consistency, and bleeding, as described previously by Murthy et al. [33 ]. The DAI was scored 0–4 for each parameter and then averaged for each mouse and each group. Weight-loss scores were determined as 0 = no weight loss; 1 = 1–5% weight loss; 2 = 6–10% weight loss; 3 = 11–15% weight loss; and 4 = >15% weight loss. Stool scores were determined as 0 = normal stools; 2 = loose stools; 4 = diarrhea. Bleeding scores were determined as 0 = no bleeding; 1 = + Guiac occult blood test (minimal color change to green); 2 = + Guiac occult blood test (maximal color change to blue); 3 = blood visibly present in the stool and no clotting on the anus; and 4 = gross bleeding from the anus with clotting present.

Depletion of MØ
MaFIA transgenic mice
MØ, monocytes, and DC in MaFIA mice constitutively express an inactive, Fas-based suicide gene under the control of the c-fms promoter. The protein encoded by the suicide gene is incorporated into the cytosolic side of the plasma membrane and can be activated by chemical dimerization using a cell-permeable dimerizer, AP20187, developed by Ariad Pharmaceuticals (Cambridge, MA). AP20187 was injected once daily i.v. at a dose of 10 mg/kg for 5 consecutive days for initial cell depletion, and this depletion protocol has been shown by us to cause 80–95% depletion of monocytes, MØ, and DC in many tissues [30 ]. Depletion could be prolonged for up to 5 weeks by subsequent injections of AP20187 once weekly at a reduced dose of 1 mg/kg. As a mock-treated, negative control, MaFIA mice were similarly injected with diluent only, which had no effect on the percentage of MØ, monocytes, or DC in any tissues.

Clodronate liposomes
iMNP were depleted from lower large intestine (descending colon and rectum) of C57BL/6, BALB/c, or CB.17/SCID mice by intrarectal inoculation with clodronate-encapsulated liposomes. Minimal depletion was observed in more proximal regions of the colon by this technique (not shown). The liposomes were prepared as described previously [34 ]. Clodronate was a gift of Roche Diagnostics GmbH (Mannheim, Germany). Phosphatidylcholine was obtained from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was purchased from Sigma Chemical Co. (St. Louis, MO). Briefly, mice were anesthetized by i.p. injection of 200 µL 2.5% Avertin per mouse. Once anesthetized, mice were injected with 100 µL clodronate liposomes intrarectally using a micropipette. This was performed on Days –1, 1, 3, and 5 of DSS administration. Note that PBS-encapsulated liposomes were not used as a negative control, as preliminary studies indicated that uptake of these liposomes by colonic MØ caused a partial reduction in the percentage of MØ in the colon and could affect the physiology of the remaining colonic MØ (data not shown and ClodronateLiposomes.org, Amsterdam, The Netherlands).

Determining MØ/DC depletion
The efficiency of the MØ/DC depletion protocols was determined in preliminary studies on MaFIA mice by flow cytometry. MØ and DC in MaFIA mice constitutively coexpress the suicide gene and an EGFP reporter gene. Dissected colons were minced and cultured for 2 h at 37°C plus 5% CO2 in RPMI-1640 medium containing 5% FCS plus collagenase/DNase (400 U/mL and 160 U/mL, respectively) and Dispase (100 µg/mL). Free cells were separated from the mucosa by gentle vortexing. If necessary, RBCs were lysed by treatment with Tris-buffered ammonium chloride. Cells were passed through a 70-µm filter, washed, and stained with appropriate antibodies in PBS/BSA/Azide. Stained cells were washed in PBS and then analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). MØ/DC depletion was indicated by a reduction of EGFP-expressing and/or CD11b+/Ly6G– cells as compared with nondepleted mice.

Systemic depletion of neutrophils
Anti-Gr1 mAb was prepared from B cell hybridoma line RB6-8C5 (obtained from DNAX Research Institute, Palo Alto, CA) and partially purified using ammonium sulfate precipitation. The concentration of mAb was determined by electrophoresis, compared with reference standards. Rat IgG was obtained from Sigma Chemical Co. as a control. Anti-Gr1 mAb or rat IgG was injected i.v. via the retro-orbital plexus at 40 µg Ig/mouse on Days –1, +1, +3, and +5 of DSS administration. Neutrophil depletion was determined by flow cytometric analysis, as indicated by a loss of CD11b+/Ly6G+ cells from the bone marrow and peripheral blood compared with nondepleted mice.

Tissue preparation for histology
After mouse sacrifice, the length of the colon was measured, and intestine sections were prepared for histology. Sections were placed in cassettes and stored in 10%-buffered formalin until being embedded in paraffin. Cross-sections were mounted on slides and stained with H&E by the University of Kentucky histology services.

Microscopic scoring of colon pathology
Histological scoring was based on the method described previously by Berg et al. [35 ]. In brief, H&E-stained cross-sections of colon tissue were scored on a 0–4 scale, based on the following criteria for severity of histopathology: 0, No change from normal tissue; Grade 1, mild inflammation present in the mucosa, comprised mainly of mononuclear cells, with little epithelial damage; Grade 2, multifocal inflammation greater than a Grade 1 score, including mononuclear and few polymorphonuclear cells (neutrophils), crypt glands pulled away from the basement membrane, mucin depletion from Goblet cells, and the epithelium occasionally pulled away from the mucosa into the lumen; Grade 3, mutlifocal inflammation greater than a Grade 2 score, including mononuclear and neutrophils progressing into the submucosa, crypt abscesses present with increased mucin depletion, and presence of epithelial disruption, including some ulceration; Grade 4, crypts no longer present, severe mucosal inflammation mainly composed of neutrophils, and epithelium no longer present or completely detached. An average of four fields of view per colon was determined for each mouse. These scores were averaged per group and recorded as the histopathology score ± SE.

Screening for bacterial translocation
MØ/DC-depleted and MØ/DC-intact C57BL/6 and MaFIA mice were killed before DSS treatment and on Day 3 or Day 7 of 3% DSS treatment. Spleen and mesenteric lymph nodes (MLNs) were removed under sterile conditions, homogenized in sterile distilled water, and plated onto blood agar plates. Bacterial growth was evaluated 48 h after incubation at 37°C.

Tissue preparation for RT-PCR and myeloperoxidase (MPO) assays
After sacrifice, 1.5 cm of the descending colon and rectum from each mouse was cut longitudinally. Each section was snap-frozen in liquid nitrogen and subsequently stored at –80°C. Frozen tissues for RT-PCR were homogenized in 1 mL Trizol reagent (Invitrogen, Carlsbad, CA), followed by phenol/chloroform RNA extraction. RNA was dissolved in nuclease-free water and stored at –80°C until use. RNA was reverse-transcribed for 1 h at 42°C using oligo-dT primers (Promega, Madison, WI). cDNA samples were then amplified in a DNA thermal cycler (PerkinElmer Inc., Boston, MA) using primers specific for ß-actin (sense: 5' AAG TCA TCA CTA TTG GCA ACG AGC 3'; antisense: 5' GTC AAA GAA AGG GTG TAA AAC GCA 3'), IL-10 (sense: 5' GTG AAG ACT TTC TTT CAA ACA AAG 3'; antisense: 5' CTG CTC CAC TGC CTT GCT CTT ATT 3'), IFN-{gamma} (sense: 5' AGC GGC TGA CTG AAC TCA GAT TGT AG 3'; antisense: 5' GTC ACA GTT TTC AGC TGT ATA GGG 3'), TNF-{alpha} (sense: 5' ATG AGC ACA GAA AGC ATG ATC 3'; antisense: 5' TAC AGG CTT GTC ACT CGA ATT 3'), and CXCL1 (sense: 5' CTG GGA TTC ACC TCA AGA AC 3'; antisense 5' GGC ATT CCC ACT AGG AGA 3'). Samples were amplified at subsaturating conditions for 35 cycles for all primers except for ß-actin, which were amplified for only 25 cycles, and cycles for all primers were set at a denaturing temperature of 95°C, annealing temperature of 56°C, and elongation temperature of 72°C.

Frozen tissues for the MPO activity assay were homogenized in 1 mL Hepes buffer (1 M, pH 8.0). The homogenate was centrifuged at 10,000 g for 30 min, and the pellet was rehomogenized in 350 µL 0.5% cetyltrimethylammonium chloride. This homogenate was centrifuged again at 10,000 g for 30 min, and the supernatant was collected as the tissue extract. The tissue extract was diluted 1:1 in citrate buffer, pH 5.0, and 75 µL was added to wells of a 96-well culture plate. Citrate buffer alone was added as a background negative control. Tetramethylbenzidine/H2O2 substrate solution (75 µL, BD/PharMingen, San Diego, CA) was added to each well. After 30 min, the reaction was stopped with 75 µL 2 N H2SO4. The plate was read at 450 nm, minus the background at 570 nm, on a microplate reader.

RESULTS

MØ/DC offer a protective rather than destructive role in DSS-induced colitis
As previous studies in the IL-10 KO murine colitis model suggested a destructive role for MØ/DC in experimental colitis, we predicted that the depletion of iMNP from immunologically normal mice would result in less severe disease in DSS-induced colitis. When MaFIA mice were injected i.v. with AP20187 for 5 consecutive days, colonic MØ/DC were reduced by 86% compared with mock-treated MaFIA mice (Fig. 1A ), which was comparable with the level of depletion that we have seen previously in other peripheral tissues [30 ]. Immediately following the 5-day depletion protocol, mice were given 3% DSS in drinking water for an additional 7 days. At Day 7 of colitis induction, the percentage of MØ/DC in AP20187-treated MaFIA mice was still reduced 62% compared with mock-treated MaFIA mice (data not shown).


Figure 1
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  		Figure 1. MØ/DC depletion in MaFIA mice leads to increased severity of DSS-induced colitis. MaFIA mice were treated i.v. with diluent (Mock Depleted) or 10 mg/kg AP20187 dimerizer to deplete MØ/DC (MØ/DC Depleted). Total colon cells were analyzed for GFP expression by flow cytometric analysis (A). After MØ/DC depletion, mice were administered 3% DSS for 7 days, and the colon was prepared for histology and stained with H&E (B). Areas of interest are the epithelium (black arrows), Goblet cells (white arrows), and basement membrane (green arrowheads). Data are representative of two separate studies with four mice per group. Histology sections were scored for disease activity ± SE. (C). Data are representative of two separate studies. P ≤ 0.05 compared with DSS treatment alone.

In contrast to MØ/DC depletion studies in IL-10 KO mice, the histopathology in the colons of DSS-treated mice was much more severe in MØ/DC-depleted compared with MØ/DC-intact MaFIA mice (Fig. 1B) . The histology of the colon was normal in MaFIA mice allowed normal drinking water (0% DSS), regardless of whether iMNP were depleted. In both cases, the colons of mice on normal drinking water had normal crypt architecture, which was tightly associated with the basement membrane (green arrowheads), displayed intact epithelium (black arrows), and typical Goblet cell morphology (white arrows). When given 3% DSS for 7 days, MØ/DC-intact mice showed histological signs of colitis. Mixed mononuclear and granulocytic inflammation was present within the mucosa, and the epithelium showed ulceration and was being sloughed off in the inflamed areas. Goblet cells were abnormal in shape and showed evidence of mucin depletion. Although the crypts were atypical in structure, for the most part, they were still associated with the basement membrane. The colons of MØ/DC-depleted MaFIA mice given 3% DSS displayed pathology, which was much more severe than in MØ/DC-intact, DSS-treated MaFIA mice. The epithelium was usually disrupted in the areas of inflammation and almost completely separated from the lamina propria. Affected regions of the colon were usually devoid of crypts and most of the goblet cells. It is most notable that inflammation involved the mucosa and submucosa in MØ/DC-depleted, DSS-treated MaFIA mice; however, the submucosa remained relatively unaffected in the MØ/DC-intact mice after DSS treatment. Results of histological scoring (Fig. 1C) showed that iMNP depletion alone caused little or no change in colon histology. DSS-treated, MØ/DC-intact mice displayed significant histopathology (2.1±0.5) compared with untreated, MØ/DC-intact mice (0.0 score). However, MØ/DC depletion prior to DSS treatment led to a significant increase in histopathology (3.8±0.2) compared with DSS-treated, MØ/DC-intact mice. Overall, these results suggested that the net function of iMNP during the onset and progression of DSS-induced colitis was a protective rather than destructive role.

MØ/DC protection in DSS-induced colitis occurs in multiple mouse strains
To confirm that the effects of MØ/DC depletion on the severity of colitis was not a result of features, which were unique to the MaFIA transgenic mouse, and/or was strain-dependent, additional laboratory strains of mice were depleted locally of colonic MØ/DC by intrarectal administration of clodronate-encapsulated liposomes and then placed on DSS-containing drinking water. A single intrarectal treatment with clodronate liposomes was shown to deplete 92% of the MØ/DC from the descending colon and rectum, as shown by a drop from 2.4% of total cells in normal colon to 0.2% 24 h after the intrarectal injection of 100 uL clodronate liposomes (Fig. 2 ). Depletion also was observed to a lesser extent in the transverse and ascending colons (data not shown); however, depletion was localized to the colon, as the percentage of MØ in the peritoneum and bone marrow was unaffected by intrarectal clodronate/liposome administration (Fig. 2) . C57BL/6, BALB/c, and CB.17/SCID mice were injected with 100 uL clodronate liposomes on Days –1, +1, +3, and +5 of DSS administration (began on Day 0). Clodronate-treated and untreated mice were then killed 7 days after the addition of DSS to the drinking water. CB.17/SCID mice were killed on Day 5 before the last clodronate treatment as a result of disease severity. At the time of sacrifice, there were still significantly fewer MØ/DC present in the colon of depleted mice (24% of total CD45+ cells) compared with MØ/DC-intact mice (44% of total CD45+ cells), as determined by flow cytometry of CD11b+/Ly6G– cells. All mice were observed for weight loss, stool consistency, and rectal bleeding during DSS treatment. MØ/DC-intact C57BL/6 mice displayed a significant loss in body weight, beginning at Day 4 of DSS treatment (Fig. 3A ). When MØ/DC were depleted in addition to DSS administration, the weight loss was significantly greater on Days 4, 6, and 7. BALB/c and CB.17/SCID mice did not lose weight during the 7-day DSS treatment; however, significant weight loss was observed when these strains of mice were MØ/DC-depleted and placed on DSS (Fig. 3B and 3C , respectively).


Figure 2
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  		Figure 2. Localized depletion of colonic MØ/DC following intrarectal administration of clodronate-encapsulated liposomes. MaFIA mice were injected intrarectally (IR) with 100 uL clodronate-encapsulated liposomes under 2.5% Avertin anesthesia or left untreated. Total colon cells as well as peritoneal and bone marrow cells were analyzed for GFP expression by flow cytometry 24 h after clodronate treatment. D. Colon, Descending colon.


Figure 3
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  		Figure 3. MØ/DC depletion in C57BL/6, BALB/c, and CB.17/SCID mice by clodronate liposomes correlates with increased weight loss in DSS-induced colitis. Groups of four mice were administered 3% DSS in drinking water at Day 0 or allowed normal drinking water. MØ/DC were depleted by intrarectal injection of 100 µL clodronate-encapsulated liposomes (+) on Days –1, +1, +3, and +5 of DSS treatment. All mice were weighed daily, and data are presented as mean body weight ± SE. (A) C57BL/6 and (B) BALB/c mice were killed on Day 7 of DSS treatment. (C) CB.17/SCID mice were sacrificed on Day 5, prior to the last clodronate treatment, as a result of disease severity. Groups: 3% DSS-treated plus MØ/DC depletion ({square}); 3% DSS treated only ({blacksquare}); untreated ({diamondsuit}). Data are representative of two separate studies. +, Clodronate treatment. *, P ≤ 0.05, compared with DSS treatment alone.

The disease severity of colitis was calculated as a DAI by combining the weight loss with observed stool consistency and rectal bleeding. C57BL/6, BALB/c, and CB.17/SCID mice (Fig. 4A 4B 4C , respectively) each began to show signs of colitis by Day 3 of DSS treatment. In each strain, the depletion of MØ/DC in the colon led to a significant increase in DAI during DSS-induced colitis. The DAI was significantly greater on Days 3–7 for C57BL/6, Days 4–7 for BALB/c, and Days 1–5 for CB.17/SCID. An additional indicator of disease severity in colitis—colon shortening—also showed in all three strains of mice that depleting MØ/DC in the colon during DSS treatment led to more severe disease than DSS treatment alone (Fig. 5 ). DAI scores in MØ/DC-depleted and MØ/DC-intact mice were negligible in the absence of DSS treatment (data not shown).


Figure 4
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  		Figure 4. Disease activity is more severe in C57BL/6, BALB/c, and CB.17/SCID mice when MØ/DC are depleted during DSS-induced colitis. Groups of four mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. The DAI, based on weight loss, stool consistency, and rectal bleeding, was determined for each mouse. Data are presented as mean DAI ± SE. (A) C57BL/6 and (B) BALB/c mice were killed on Day 7 of DSS treatment. (C) CB.17/SCID mice were sacrificed on Day 5, prior to the last clodronate treatment, as a result of disease severity. Groups: 3% DSS-treated plus MØ/DC depletion (hatched bars); 3% DSS-treated only (shaded bars); untreated (solid bars). Data are representative of two separate studies. +, Clodronate treatment. *, P ≤ 0.05, compared with DSS treatment alone.


Figure 5
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  		Figure 5. MØ/DC-depleted C57BL/6, BALB/c, and CB.17/SCID mice have shorter colons after DSS-induced colitis compared with normal mice with colitis. Groups of four mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. Colons were removed at sacrifice, and their outstretched length was measured from the cecal/ascending colon junction to the rectum. Data are presented as mean colon length (cm) ± SE. C57BL/6 and BALB/c mice were killed on Day 7 of DSS treatment. CB.17/SCID mice were killed on Day 5, prior to the last clodronate treatment, as a result of disease severity. Groups: 3% DSS-treated plus MØ/DC depletion (hatched bars); 3% DSS-treated only (shaded bars); untreated (solid bars). Data are representative of two separate studies. *, P ≤ 0.05, compared with DSS treatment alone.

Inflammation, crypt destruction, and epithelial disruption increase in DSS-induced colitis when MØ/DC are depleted
Histological evaluation of H&E-stained sections from the rectum/descending colon was performed to delineate the effects of iMNP depletion on the histopathology of DSS colitis (Fig. 6A ). In MØ/DC-intact C57BL/6 mice treated with 3% DSS for 7 days, multiple foci of inflammation were present throughout the lamina propria and the submucosa, which consisted of mononuclear and granulocytic infiltration. Most of the crypts were absent, and those that remained had separated from the basement membrane. The epithelium was separated from the mucosa at multiple sites, and the goblet cells usually appeared depleted of mucin. The colons of MØ/DC-intact BALB/c mice, following DSS treatment, were less affected than colons from C57BL/6 mice, although mononuclear and granulocytic inflammation was still observed in the lamina propria and mucosal layers. Crypts were present and remained in contact with the basement membrane. Goblet cells were present but enlarged. The epithelium appeared generally intact with few areas of ulceration. MØ/DC-intact CB.17/SCID mice, when treated with 3% DSS, showed granulocytic inflammation, which was primarily present in the lamina propria. The epithelium remained largely intact with few ulcers, but the crypts were severely disrupted and were rarely in contact with the basement membrane. Goblet cells were much fewer in number than in the control animals.


Figure 6
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  		Figure 6. Colon pathology shows enhanced injury in MØ/DC-depleted DSS colitis mice compared with MØ/DC-intact DSS colitis mice. Groups of four mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. Colons were removed at sacrifice (Day 7 for C57BL/6 and BALB/c mice; Day 5 for CB.17/SCID mice), and tissue sections were prepared for histology by H&E staining (A). Arrows indicate the epithelium (black arrows), Goblet cells (white arrows), and the basement membrane (green arrowheads). Groups: 3% DSS-treated plus MØ/DC depletion; 3% DSS-treated only; untreated (0% DSS). Data are representative of two separate studies. Histology sections were scored for disease activity ± SE (B). Data are representative of two separate studies. *, P ≤ 0.05, compared with DSS treatment alone.

In all strains of mice, MØ/DC depletion during the onset of colitis resulted in an increase in histological disease. The affected areas of colons from MØ/DC-depleted, DSS-treated C57BL/6 mice were devoid of Goblet cells, crypts, and epithelium. A striking increase in the level of inflammation was observed throughout the colon, and many mononuclear and granulocytic inflammatory cells were evident within the lumen. MØ-depleted BALB/c mice with colitis also showed more intense inflammation within the lamina propria and a greater loss of crypts and epithelial separation than in DSS-treated mice with intact MØ/DC. CB.17/SCID mice also showed more intense granulocytic infiltration in the colons of mice depleted of MØ/DC compared with those with intact MØ/DC. Where the epithelium was present, it was often separated from the underlying tissue. The increase in histological severity in DSS-treated, MØ/DC-depleted mice was significant in BALB/c and CB.17/SCID mice (Fig. 6B) . Although the histological score was not increased significantly in MØ/DC-depleted C57BL/6 mice at Day 7, colon section scores from MØ/DC-depleted C57BL/6 mice killed at Day 4 of colitis induction approached significance (P=0.08) compared with MØ/DC-intact colitis mice (data not shown).

MØ/DC depletion during DSS treatment leads to increased CXCL1 expression and neutrophil influx without increased bacterial translocation
It was possible that the increase in colitis severity in MØ/DC-depleted mice was a result of increased bacterial translocation from the colon to draining lymph nodes and peripheral circulation, as bacterial clearance by resident MØ/DC would be eliminated. To address this possibility, the spleen and MLNs were removed from MØ/DC-depleted and MØ/DC-intact mice, which had no DSS treatment or were treated with 3% DSS for 3 or 7 days. Homogenized tissues were assayed for bacterial content by culture on blood agar plates. Neither MaFIA nor C57BL/6 mice had culturable bacteria in either tissue following the 5-day MØ/DC depletion protocol, indicating that the intestinal barrier remained intact following depletion of iMNP. Moreover, there was no bacterial growth in MLNs or spleen from MØ/DC-intact or depleted mice, which were treated with 3% DSS for 3 days. At Day 7 of DSS treatment in C57BL/6 mice, two of four MØ/DC-depleted and zero of four MØ/DC-intact mice had culture plates containing fewer than 10 colonies from the MLNs. Bacteria were not detected in the spleen of any mice (data not shown). In DSS-treated MaFIA mice, two of four MØ/DC-intact and two of four MØ/DC-depleted mice had fewer than 10 colonies in the MLNs, and only a single MØ/DC-intact mouse produced one colony from the spleen (data not shown). Taken together, these data indicate that the increase in colitis severity in MØ/DC-depleted mice was not a result of an increase in release of bacteria from the colon.

To determine if the increased severity of colitis, which occurred in MØ/DC-depleted mice, was associated with changes in proinflammatory and anti-inflammatory cytokine expression, mRNA levels for IFN-{gamma}, TNF-{alpha}, and IL-10 were evaluated in total RNA from colons of untreated, DSS-treated, and MØ/DC-depleted, DSS-treated C57BL/6 mice (Fig. 7 ). As enhanced neutrophil accumulation was also observed in all MØ/DC-depleted strains of mice with colitis histologically and by flow cytometry (data not shown), the expression of CXCL1 chemokine mRNA was also evaluated. mRNA for the proinflammatory cytokines, IFN-{gamma}, and TNF-{alpha} was elevated significantly in DSS-treated mice compared with untreated mice. It is unexpected that MØ/DC depletion did not further increase the steady-state levels of IFN-{gamma} and TNF-{alpha}. IL-10 mRNA was decreased significantly in DSS colitis mice following MØ/DC depletion; however, although a similar trend was observed in repeated studies, differences were not significant. In contrast, expression of CXCL1 mRNA was increased routinely and significantly in DSS colitis mice following MØ/DC depletion compared with DSS treatment alone. Furthermore, the increased CXCL1 expression in MØ/DC-depleted, DSS-treated mice was observed in C57BL/6, BALB/c, and CB.17/SCID mice (Fig. 8A ).


Figure 7
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  		Figure 7. MØ/DC depletion is associated with an increase in CXCL1 chemokine mRNA expression in colons of DSS-treated mice. Groups of four C57BL/6 mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. Colons were removed at sacrifice, and descending colon samples were snap-frozen in liquid nitrogen and processed to isolate RNA for expression of ß-actin, IL-10, IFN-{gamma}, TNF-{alpha}, and CXCL1 chemokine mRNA. Total RNA from each mouse was amplified by RT-PCR, and data are presented as mean ± SE of the integrated density, normalized to ß-actin. Groups: 3% DSS-treated plus MØ/DC depletion (hatched bars); 3% DSS-treated only (shaded bars); untreated (solid bars). Data are representative of two separate studies. *, P ≤ 0.05, compared with DSS treatment alone.


Figure 8
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  		Figure 8. MØ/DC depletion correlates with increased neutrophil influx and increased expression of neutrophil chemoattractants in the colon of DSS-induced colitis mice. Groups of four mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. Colons were removed at sacrifice (Day 7 for C57BL/6 and BALB/c mice; Day 5 for CB.17/SCID mice), and descending colon samples were snap-frozen in liquid nitrogen and processed to isolate RNA for expression of CXCL1 chemokine mRNA (A). Total RNA from each mouse was amplified by RT-PCR for ß-actin and CXCL1. Data are presented as mean ± SE of the integrated density of CXCL1 normalized to ß-actin. Additional frozen tissue samples were processed to isolate the tissue extract to detect MPO activity (B). Data are presented as mean ± SE of MPO activity. Groups: 3% DSS-treated plus MØ/DC depletion (hatched bars); 3% DSS-treated only (shaded bars); untreated (solid bars). C57BL/6 data are representative of two separate studies (BALB/c and CB.17/SCID data are from a single study). *, P < 0.05, compared with DSS treatment alone.

To quantify the increase in neutrophil infiltration, which was observed histologically in DSS colitis following depletion of iMNP, tissue extracts from colons of C57BL/6, BALB/c, and CB.17/SCID mice were analyzed for MPO activity 7 days after DSS treatment (Fig. 8B) . MØ/DC depletion during DSS treatment led to a significantly greater influx of neutrophils into diseased colons of C57BL/6 and CB.17/SCID mice, as indicated by MPO activity, compared with DSS treatment of MØ/DC intact mice. Thus, the increase in colitis severity following depletion of iMNP was not associated with changes in the levels of proinflammatory or anti-inflammatory cytokines; however, a significant increase in CXCL1 chemokine expression and neutrophil infiltration was observed consistently in DSS-treated mice following depletion of MØ/DC from the colon.

Depletion of neutrophils from MØ/DC-depleted mice prevents the increased colitis severity as a result of MØ/DC depletion
To determine if increased neutrophil influx and/or activity were responsible for the increase in colitis severity in MØ/DC-depleted mice, C57BL/6 mice were treated with clodronate liposomes intrarectally for local MØ/DC depletion. In addition, mice were treated i.v. with 40 µg anti-Gr1 mAb on Days –1, +1, +3, and +5 for systemic neutrophil depletion. Rat IgG was injected i.v. as a negative control. These studies were performed in wild-type C57BL/6 mice rather than MaFIA mice, as MØ/DC depletion in normal MaFIA mice has been shown to promote a noninflammatory neutrophilia [30 ]. Neither anti-Gr1 nor rat IgG had any effect on mice given 0% DSS in drinking water.

As shown in Figure 9A , MØ/DC-depleted mice additionally treated with control rat Ig developed more severe colitis, compared with those treated with DSS alone. However, when neutrophils were also depleted by simultaneous injection of anti-Gr1, MØ/DC-depleted mice failed to develop an increase in colitis severity. Neutrophil depletion alone in DSS-treated, MØ/DC-intact mice caused a reduction in disease severity, which approached significance at Day 5 of DSS treatment compared with DSS-treated, MØ/DC-intact mice treated with rat IgG, but this protective effect was lost on Day 7 (Fig. 9A) . A similar observation was seen in colitis-associated shortening of colon length after sacrifice on Day 7 of DSS treatment (Fig. 9B) . Mice in which neutrophils, MØ, and DC were depleted also failed to develop the degree of colon shortening, which was observed in MØ/DC-depleted mice, and their colon length remained the same as DSS-treated, MØ/DC-intact mice. Neutrophil depletion alone in DSS-treated, MØ/DC-intact mice failed to affect colon shortening in DSS-treated mice. Neutrophil depletion alone in the absence of DSS treatment had no demonstrable effect on mice during the period of study (data not shown). The above data suggest that the increase in colitis severity in MØ/DC-depleted mice is in large part a result of increased neutrophil numbers/activity.


Figure 9
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  		Figure 9. Neutrophil depletion, in combination with MØ/DC depletion, decreases disease severity to levels seen in MØ/DC-intact mice. Groups of four C57BL/6 mice were administered 3% DSS in drinking water or allowed normal drinking water for 7 days. MØ and DC were depleted by intrarectal clodronate liposome injections. Neutrophils were depleted by i.v. injections of {alpha}-Gr1 mAb. Injections for cellular depletion were performed on Days –1, +1, +3, and +5 of DSS administration. DAI scores were determined throughout the study (A). Data are presented as mean DAI ± SE. On Day 7, colons were removed at sacrifice, and their outstretched length was measured from the cecal/ascending colon junction to the rectum (B). Data are presented as mean colon length (cm) ± SE. Groups: 0% DSS-treated plus rat IgG control antibodies (solid bars); 3% DSS-treated plus rat IgG antibodies (shaded bars); 3% DSS-treated plus {alpha}-Gr1 mAb (thin, hatched bars); 3% DSS-treated plus MØ/DC depletion and rat IgG control antibodies (thick, hatched bars); 3% DSS-treated plus MØ/DC depletion and {alpha}-Gr1 mAb (dotted bars). Data are representative of two separate studies. ns, Not significant; *, P ≤ 0.05.

DISCUSSION

In the present study, we have shown in four different mouse strains with two different MØ/DC depletion techniques that the presence of iMNP plays a protective role in experimental colitis induced by DSS. The absence of MØ/DC in the colon during DSS treatment led to an increased loss of body weight, higher DAI scores, greater shortening of the colon, increased inflammation, and histopathology in colon tissue. These results are in contrast to those of Watanabe et al. [15 ], which showed that depletion of colonic MØ/DC by intrarectal injections of clodronate liposomes prevented chronic colitis in an IL-10 KO model of colitis. The basis of this discrepancy is unclear, as in both studies, local MØ/DC depletion by clodronate liposomes was used. However, there are distinct differences in IL-10 KO mice, which may contribute to these different results. IL-10-deficient mice are prone to the spontaneous development of colitis [36 ] as a result of the lack of IL-10-mediated regulation from birth. It is likely that chronic exposure to commensal bacteria throughout the life of these mice leads to chronic immune stimulation, which becomes manifest in colitis later in life. In contrast, all strains of mice in our study were immunologically normal until the MØ/DC depletion procedure was initiated. Thus, the resident population of MØ/DC in the colon was likely of a more resting phenotype at the initiation of DSS treatment, and it may be this resident phenotype that expressed the protective function seen in this study.

Resident, intestinal MØ have been shown to have more of an anti-inflammatory phenotype than the monocytes, which would be elicited into intestinal tissue during the development of colitis [37 ]. That resident intestinal MØ may serve an immunosuppressive role has been observed in several studies. Work by Smythies et al. [38 ] has shown that intestinal MØ have a profound deficiency in the ability to produce inflammatory cytokines; however, they retain the ability to phagocytize and kill microbes. In a separate study by Hausmann et al. [39 ], it was found that MØ of the intestine do not express mRNA for the TLRs recognizing LPS and lipotechoic acid, TLR4 and TLR2, respectively. Araki et al. [23 ] showed that MyD88–/– mice, which fail to signal through several TLRs, develop a more severe colitis in response to DSS treatment, compared with normal mice. These findings suggested there may be some basal level of MyD88 signaling through TLRs in normal intestine, which may prevent adverse immune responses toward commensal bacteria of the gut and could reduce the severity of colitis [40 ]. Similarly, studies by Katakura et al. [24 ] demonstrated that stimulation by TLR9 agonists resulted in protection against DSS-induced colitis. Although these studies by Araki and Katakura did not determine which cells were necessary for colitis protection by MyD88 or TLR9 signaling, they do suggest a possible pathway for the protection observed in this study.

The reason that normal intestinal MØ respond so poorly to inflammatory stimuli is not known. However, constitutive sources of IL-10 and TGF-ß have been observed in normal intestinal tissues [41 , 42 ], and both cytokines display potent immunosuppressive activity toward MØ. Tissue MØ themselves can produce immunosuppressive factors in some normal tissues, including IL-10, TGF-ß, PGE2, and NO [43 , 44 ]. So, it might not be unexpected that normal intestinal MØ may also suppress the immune reactivity of other cell types within the interstitial tissues.

Normal colonic DC have also been shown to be hyporesponsive to microbial stimuli in rodents and humans [45 46 47 48 ] and can be a source of immunosuppressive cytokines. Although DC were lost in both depletion methods used in this study, it is not likely that their role in adaptive immune responses plays a major part in the increased colitis pathology in this study. This would suggest a protective role for DC but would appear to be at the innate level. CB.17/SCID mice lack the ability to generate adaptive immunity but still displayed more severe DSS colitis when iMNP were depleted. This suggests that if DC participate in the protective response observed in this study, it is likely that involves their function as an innate immune cell. Like intestinal MØ, colon lamina propria DC express less TLR2 and TLR4 than blood DC, suggesting a lack of inflammatory response to commensals in the colon [49 ]. In addition, Iwasaki and Kelsall [50 ] demonstrated that DC in mouse Peyer’s patches up-regulate IL-10 rather than immunostimulating cytokines, supporting a possible role for DC in regulating inflammation in the gut. Differentiating the roles of DC versus MØ in experimental colitis is currently being addressed in our lab by studies in conditional DC ablation, transgenic mice.

In addition to an immunosuppressive role for intestinal MØ, these resident cells have been shown to play an important role in the regeneration of damaged epithelium after DSS-induced colitis [22 ]. Treatment of mice with DSS leads to an erosive form of colitis, in which the epithelial barrier is acutely injured. MØ have long been known to participate in the repair of injured tissues through the release of a variety of cytokines and growth factors [51 52 53 54 ], and it is likely that MØ contribute to intestinal repair through similar mechanisms. It should be noted that exposure of MØ to IL-10 and/or TGF-ß has been shown to alter the physiology of MØ toward a MØ type, which is effective in tissue repair [21 ], suggesting that constitutive exposure of resident intestinal MØ to IL-10 and TGF-ß would not only inhibit their proinflammatory functions but would also promote their ability to maintain and/or repair the intestinal mucosa. The elimination of resident MØ/DC in the colons of DSS-treated mice may have increased the time required to repair the epithelial injury caused by DSS, as growth-promoting cytokines released by these MØ/DC would have been reduced. In related studies, Rakoff-Nahoum et al. [55 ] and Fukata et al. [56 ] showed in MyD88–/– and TLR4–/– mice that TLR signaling caused by commensal bacteria is important for maintenance of intestinal epithelial homeostasis and for effective protection and repair from epithelial injury. Although these studies did not identify whether the protective mechanisms were mediated directly via TLRs on epithelial cells or indirectly via stimulation of innate immune cells in the subepithelium, that TLR ligands induce MØ, and DC to produce a variety of cytokines known to be protective to epithelial cells suggest that homeostatic interactions between intestinal epithelium and MØ/DC are critical to maintain epithelial integrity and to promote effective repair following epithelial injury.

Another potential mechanism for increased colitis severity in MØ/DC-depleted mice could be an increase in the migration of inflammatory monocytes into the colons of DSS-treated mice, as MØ, which are recent emigrants from the blood, have a potent ability to produce inflammatory cytokines and to contribute to inflammatory reactions [57 , 58 ]. Although it remains unknown whether inflammatory monocyte influx is the cause of increased colitis severity in this study, flow cytometric analysis of colonic tissues during DSS treatment indicated that MØ/DC-like cells remained depleted throughout the 7-day development of colitis. However, it should be noted that depletion was never complete, and it remains possible that the residual cells were recently arrived inflammatory monocytes, which could contribute to the colitis.

The influx and activity of neutrophils are increased during colitis. Studies in human IBD have shown an increase in human neutrophil lipocalin and MPO in patients with colitis [59 ], and separate studies have demonstrated that neutrophil involvement in colitis correlated with an increase in local IL-8 production [18 , 60 ]. Of note are observations from several labs that CXCL1 is expressed by GI epithelial cells in response to injury and/or proinflammatory cytokines [61 62 63 ]. It is interesting that recent studies by Dovi et al. [64 ] have shown that a robust influx of neutrophils into experimental wounds delays the rate of healing and that depletion of neutrophils from mice promotes healing. Thus, regulation of neutrophil influx by resident MØ/DC in the intestines may be advantageous under homeostatic conditions in maintaining an effective, intestinal barrier and during intestinal epithelial injury in enhancing the rate of tissue repair.

Colonic MØ/DC depletion could also have led to an increase in the number of commensal bacteria that remain viable upon gaining entry into the mucosal tissue. The intestines are constantly exposed to commensal bacteria, and iMNP are exposed frequently to bacterial components. To control the potential inflammatory responses as a result of this frequent exposure, resident iMNP are highly phagocytic and bacteriocidal but are in a functional state of "innate immune anergy" in that they are poor at inducing proinflammatory cytokines in response to bacteria [38 ]. Depletion of the resident intestinal MØ/DC would reduce this innate bacteriocidal activity in intestinal tissues and could lead to an increase in bacterial load in the lamina propria following acute DSS-mediated injury to the epithelial barrier. Although we do not yet know if bacterial entry and/or survival in intestinal mucosa are increased in MØ/DC-depleted mice, it was clear that MØ/DC depletion in DSS-treated mice did not increase escape of bacteria to draining lymph nodes or spleen. However, it remains a possibility that an increase in bacteria within the lamina propria could directly result in an increase in neutrophil infiltration elicited by bacteria-derived chemotactic agents.

In conclusion, depletion of colonic MØ/DC during the development of DSS colitis led to a more severe disease, including greater inflammation and disruption of tissue architecture in the colon. The depletion of MØ/DC was associated with increased expression of the chemokine CXCL1 and with a significant increase in the infiltration of neutrophils into affected tissue sites. Depletion of neutrophils in addition to MØ/DC depletion resulted in a reduction of colitis severity to levels seen in MØ/DC-intact mice. These results indicate that iMNP may serve a protective role, which helps protect and/or recover from intestinal injury, and the protection is in part to regulate neutrophil migration into injured colonic tissue.

ACKNOWLEDGEMENTS

Studies were funded in part by the following grants: NIH Grants HL57399 and HL69459 and a grant from the Crohn’s and Colitis Foundation of America.

Received December 14, 2005; revised June 22, 2006; accepted June 23, 2006.

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