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The Journal of Immunology, 2007, 178: 6522-6532.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Gnotobiotic IL-10–/–;NF-{kappa}BEGFP Mice Reveal the Critical Role of TLR/NF-{kappa}B Signaling in Commensal Bacteria-Induced Colitis1

Thomas Karrasch, Joo-Sung Kim2, Marcus Muhlbauer, Scott T. Magness and Christian Jobin3

Department of Medicine and Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Commensal bacteria and TLR signaling have been associated with the maintenance of intestinal homeostasis in dextran sodium sulfate-induced intestinal injury. The aim of this study was to determine the in vivo role of TLR/NF-{kappa}B activation in a model of commensal bacteria-induced T cell-mediated colitis. A NF-{kappa}B reporter gene mouse (NF-{kappa}BEGFP) (EGFP, enhanced GFP) was crossed to the colitogenic susceptible strain IL-10–/– and derived into germfree conditions using embryo-transfer technology. Germfree IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice (wt, wild type) were dual associated with the nonpathogenic commensal bacteria strains Enterococcus faecalis and Escherichia coli. EGFP was detected using macroimaging, confocal microscopy, and flow cytometry. IL-10–/–;MyD88–/– mice were used to assess E. faecalis/E. coli-induced TLR-dependent signaling and IL-23 gene expression. Dual-associated IL-10–/–;NF-{kappa}BEGFP mice developed severe inflammation by 7 wk. Macroscopic analysis showed elevated EGFP expression throughout the colon of bacteria-associated IL-10–/–;NF-{kappa}BEGFP mice. Confocal microscopy analysis revealed EGFP-positive enterocytes during the early phase of bacterial colonization (1 wk) in both IL-10wt/wt and IL-10–/– mice, while the signal shifted toward lamina propria T cells, dendritic cells, neutrophils, and macrophages in IL-10–/– mice during colitis (7 wk). The NF-{kappa}B inhibitor BAY 11-7085 attenuated E. faecalis/E. coli-induced EGFP expression and development of colitis. Additionally, E. faecalis/E. coli-induced NF-{kappa}B signaling and IL-23 gene expression were blocked in bone marrow-derived dendritic cells derived from IL-10–/–;MyD88–/– mice. We conclude that bacteria-induced experimental colitis involves the activation of TLR-induced NF-{kappa}B signaling derived mostly from mucosal immune cells. Blocking TLR-induced NF-{kappa}B activity may represent an attractive strategy to treat immune-mediated intestinal inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The intestine is a unique organ where a highly sophisticated lymphoid system and an abundant mucosal immune cell network coexist with a sea of commensal bacteria (>1012/g), a high load of bacterial products, potentially immunogenic dietary by-products (food Ags), and various toxins (1, 2). Despite this potentially explosive inflammatory environment, the intestine remains mostly tolerant to its bacterial neighbors and manages to maintain homeostasis. Numerous physical and cellular parameters contribute to the maintenance of intestinal homeostasis. Among the physical parameters, the presence of a tight epithelium consisting of a single intestinal epithelial cell layer is certainly at the forefront of protective mechanisms. A tight intestinal epithelium prevents the uncontrolled uptake of luminal products and translocation of microorganisms, thereby isolating the host’s mucosal immune system from the rich antigenic environment. At the cellular level, production of immunosuppressive molecules from specialized underlying immune cells helps maintain a relative state of tolerance toward the intestinal luminal content. For instance, production of immunoregulatory factors such as IL-10 and TGF-beta by various mucosal immune cells is essential in maintaining a state of tolerance toward the intestinal luminal content in a normal host (3, 4, 5, 6, 7, 8, 9, 10, 11).

TLR signaling cassettes are an essential component of the innate/adaptive immune response. These receptors sense the presence of pathogenic and/or invading microorganisms and mount a swift immunological response through the production of proinflammatory and immune molecules. This host response is central to the elimination of the damaging agents and to the re-establishment of homeostasis (12, 13, 14, 15, 16, 17, 18). An important TLR downstream effector target is the NF-{kappa}B transcriptional system, which controls the expression of numerous genes involved in both innate and adaptive responses (19, 20). Although beneficial, this response threatens the host integrity when directed toward self- and/or persisting luminal Ags, as exemplified in inflammatory bowel diseases. Although the etiology of inflammatory bowel diseases remains uncertain, numerous experimental models of colitis point to an aberrant innate/adaptive mucosal immune response to ubiquitous luminal nonpathogenic bacteria as a key event in the development of the disease (21). In a normal host, the sequential activation of regulatory immune cells producing immunosuppressive molecules and effector cells secreting inflammatory mediators ensures a proper and balanced response to intestinal microorganisms. Factors causing the disruption of this fragile equilibrium often lead to the development of chronic intestinal inflammation (22). The importance of a proper immunosuppressive response is illustrated clearly in IL-10-deficient mice. These mice spontaneously react to their endogenous luminal flora and develop chronic intestinal inflammation caused by T lymphocytes producing IL-17 (ThIL-17) and IL-6, both cytokines critical in mediating disease (23). Another essential component of this ThIL-17 response is the induction of IL-23 by APC such as dendritic cells (DC)4 (23).

Although the endogenous microflora plays an important role in the initiation of intestinal inflammation in IL-10–/– mice (24), molecular events associated with the host response to bacterial colonization and with disease development are defined poorly. Moreover, the biological impact of TLR/NF-{kappa}B signaling in disease and health is still unclear. A landmark report published a decade ago showed that blocking NF-{kappa}B activity with an antisense oligonucleotide directed against the p65 (RelA) subunit of NF-{kappa}B prevented chemical-induced colitis in mice (25). This finding documented for the first time the key role of the transcription factor NF-{kappa}B in promoting intestinal inflammation. Interestingly, other recent studies using genetically engineered mice indicate a protective function for TLR and/or NF-{kappa}B in experimental colitis. For example, deletion of I{kappa}B kinase beta gene in enterocytes failed to prevent dextran sodium sulfate (DSS)-induced experimental colitis and rather exacerbated the inflammatory response (26). Helicobacter hepaticus-induced typhlocolitis was increased in p50–/–;p65+/– mice compared with wild-type (wt) mice (27). Also, transgenic mice selectively expressing the I{kappa}B{alpha} superrepressor in enterocytes displayed enhanced DSS-induced colitis compared with wt mice (28). Importantly, DSS-induced acute colitis is exacerbated in mice deficient for various TLR signaling components (MyD88, TLR2, and TLR4) (29, 30). Altogether, these findings indicate that the TLR/NF-{kappa}B signaling system in addition to its proinflammatory effects participates in the induction of protective genes associated with the maintenance of intestinal homeostasis. Recently, a new report (31) demonstrated the critical role of TLR/MyD88 in promoting differentiation of Th1 cells and development of spontaneous colitis in IL-10–/– mice. However, the exact role TLR/NF-{kappa}B signaling in the context of bacterial-mediated chronic T cell-induced colitis remains to be defined. To specifically interrogate the host/bacteria relationship in disease and health, we used gnotobiotic IL-10–/–;NF-{kappa}BEGFP mice to selectively monitor the NF-{kappa}B response following bacterial colonization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The NF-{kappa}BEGFP knockin mice (C57BL6) were described previously (32). IL-10–/– mice (129/SvEv) were crossed to NF-{kappa}BEGFP mice to generate IL-10+/–;NF-{kappa}BEGFP (129/SvEv/C57BL6 background). Reproductive tracts of specific pathogen-free IL-10+/–;NF-{kappa}BEGFP females who had mated with IL-10+/–;NF-{kappa}BEGFP males were collected in M-2 medium (Chemicon International) and brought to the University of North Carolina Mutant Mouse Regional Resource Center lab for dissection. Embryos were released from the ampulla of the oviducts and placed in culture medium (potassium simplex optimized medium plus amino acids; Chemicon International) under oil (Sigma-Aldrich embryo tested oil) and cultured at 37°C overnight. Embryos were collected aseptically and transplanted into the oviducts of germfree pseudopregnant females. IL-10+/–;NF-{kappa}BEGFP offspring were further mated for six to seven generations in germfree conditions to generate IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice on the same genetic background (129/SvEv/C57BL6). These mice were used in our experiments. The genotype was confirmed by PCR analysis using primers specific for IL-10–/– and knockin NF-{kappa}BEGFP locus. IL-10–/– genotyping was performed following the protocol described by The Jackson Laboratory. The following oligonucleotide primers were used for NF-{kappa}BEGFP: primers for wt genotyping were (5') 5'-AAAGCGGTCTGAGGAGGAA-3' and (3') 5'-TCAGGCCCACCTAGTCAGAT-3' (amplicon size, 241 bp) and primers for NF-{kappa}BEGFP transgene were (5') 5'-GAGCTGAAGGGCATCGACTTCAAG-3' and (3') 5'-GGACTGGGTGCTCAGGTAGTGG-3' (amplicon size, 246 bp). Independent PCR were performed for each primer pair.

Bacterial colonization and LPS stimulation

IL-10–/–;NF-{kappa}BEGFP mice or control IL-10wt/wt;NF-{kappa}BEGFP mice were dual associated at 10–12 wk of age with a human oral isolate of Enterococcus faecalis (strain OG1RF; provided by Dr. M. Huycke, University of Oklahoma Health Science Center) and a murine strain of Escherichia coli (randomly isolated from wt mice raised in specific pathogen-free conditions) by gavage feeding and rectal swabbing with viable cultured bacteria. Dual-associated mice were maintained in the Gnotobiotic Animal Facility at the University of North Carolina (Chapel Hill, NC). Bacterial association and absence of contamination by other bacterial species were confirmed by periodic aerobic culture of stool samples. In a separate experiment, NF-{kappa}BEGFP mice were injected i.p. with LPS (25 mg/kg) for 4, 12, and 24 h. All animal protocols were approved by the Institutional Animal Care and Use Committee of University of North Carolina.

Enhanced GFP (EGFP) imaging

Dual-associated IL-10–/–;NF-{kappa}BEGFP and control IL-10wt/wt;NF-{kappa}BEGFP mice were sacrificed, the intestine dissected and immediately imaged using a charge-coupled device camera in a light-tight imaging box with a dual-filtered light source and emission filters specific for EGFP (LT-99D2 Illumatools; Lightools Research). Identical exposure times were used to capture images within each experiment. For confocal microscopy on living tissues, colonic segments were cut open longitudinally and placed on the stage of a Leica SP2 Upright Laser Scanning Confocal Microscope (Leica) lumen side facing the lens without further processing or fixation. EGFP was excited at 495 nm wavelength, and images were acquired using detection filters specific for the EGFP emission spectrum. Detecting transmitted light allowed for spacial orientation visualizing the shadows of crypts and interstitium. Images were analyzed with the Leica SP2 Laser Scanning Confocal Imaging Software (Leica).

Generation and stimulation of bone marrow-derived DC (BMDC)

BMDC were generated from the femur and tibia of IL-10wt/wt;NF-{kappa}BEGFP, IL-10–/–;NF-{kappa}BEGFP and IL-10–/–;MyD88–/– mice as described previously (33). Bone marrow cells were flushed and depleted of RBC using RBC lysing buffer (Sigma-Aldrich), and then cultured in ultralow-adherence 24-well plates (Costar) in complete medium (RPMI 1640) supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-ME, 10 ng/ml murine rGM-CSF, and 10 ng/ml murine rIL-4 (both from PeproTech). Half of complete medium was refreshed on the third and fifth day. On the seventh day of culturing, nonadherent cells were collected as BMDC. The resulting population was >93% CD11c+ and CD11b+, as determined by flow cytometry. Cells (2 x 106/6-well plates) were stimulated with E. faecalis/E. coli lysates (40 µg/ml) for various times to determine the expression of IL-12p40 and IL-23p19.

Immunohistochemical analysis and histological scoring

IL-10–/–;NF-{kappa}BEGFP mice and control IL-10wt/wt;NF-{kappa}BEGFP mice were dual associated for 7 wk. Sections of the cecum, proximal, and distal colon were fixed in 10% neutral-buffered Formalin. The fixed tissue was embedded in paraffin, and immunohistochemistry was performed using anti phospho-p65 (RelA) Ab (Cell Signaling Technology) as described previously (34). Histological evaluation of mucosal inflammation was performed by scoring (0–4) the degree of lamina propria mononuclear cell (LPMNC) infiltration, crypt hyperplasia, goblet cell depletion, and architectural distortion, as described previously (24).

Primary intestinal epithelial cells study

The small intestine of LPS-stimulated NF-{kappa}BEGFP mice was dissected, sliced open, and washed three times in PBS and twice in 3 mM EDTA and 0.5 mM DTT (solution A). The small intestine was incubated for 60 min at room temperature in solution A with shaking, and then the resulting supernatant was passed over nylon filter. The cellular suspension was centrifuged, washed, and resuspended in TRIzol for RNA analysis or in 1% formaldehyde for chromatin immunoprecipitation (ChIP) analysis. Vimentin and CD64, markers for myofibroblasts and myeloid cells, respectively, were not detected, whereas the epithelial marker cytokeratin was clearly expressed in our epithelial cell preparation as assayed by Western blot analysis (data not shown).

ChIP and PCR analysis

DNA was sheared from formaldehyde-fixed intestinal epithelial cells and ChIP performed using anti-RelA Ab and anti-RNApolymeraseII Abs (Santa Cruz Biotechnology) as described previously (34, 35, 36). PCR was performed with total DNA (2 µl, input control) and immunoprecipitated DNA (2 µl) using promoter-specific primers. The PCR products were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye (FMC). Fluorescent staining was captured using an AlphaImager 2000 (Alpha Innotech). The following promoter-specific oligos were used to amplify ChIP DNA: {kappa}B-EGFP (5') 5'-CGAATTCTGCAGGTCGACGGAAAG-3' and (3') 5'-CGGTGAACAGCTCCTCGCCCTTG-3'; and IL-6 (5') 5'-GACATGCTCAAGTGCTGAGTCAC-3' and (3') 5'-AGATTGCACAATGTGACGTCG-3'. The size of the amplified products is 200 and 125 bp for {kappa}B-EGFP and IL-6, respectively.

RNA extraction and amplification by RT-PCR

RNA was isolated from primary intestinal epithelial cells, colonic tissues, or BMDC using the TRIzol method (Invitrogen Life Technologies), reverse transcribed (1 µg of RNA), and amplified as previously described (37) using specific primers for murine IL-6, TNF-{alpha}, IL-12p40, and IL-23p19. The PCR products were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye (FMC). Fluorescence staining was captured using an Alpha Imager 2000 (Alpha Innotech). The following oligonucleotide primers were used: IL-6, 5'-ATGAAGTTCCTCTCTGCAAGAGACT-3', and 5'-CACTAGGTTTGCCGAGTAGATCTC-3'; IL-12p40, 5'-GGAAGCACGGCAGCAGAATA-3', and 5'-AACTTGAGGGAGAAGTAGGAATGG-3'; TNF-{alpha}, 5'-ATGAGCACAGAAAGCATGATC-3', and 5'-TACAGGCTTGTCACTCGAATT-3'; IL-23p19, 5'-GCGGGACATATGAATCTACTAAGAGA-3', and 5'-AGCCAGACCTTGGCGGATCCTTTG-3'; and actin, 5'-TGGAATCCTGTGGCATCCATGAAAC-3', and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'. The size of the amplified products was 590, 180, 175, 142, and 349 bp for IL-6, IL-12p40, TNF-{alpha}, IL-23p19, and actin, respectively.

Western blot analysis

Following stimulation, BMDC were collected, lysed in 1x Laemmli buffer, and the protein concentration was measured using Bio-Rad quantification assay (Bio-Rad). Protein extracts (20 µg) were subjected to electrophoresis on a 10% SDS-PAGE and transferred to nitrocellulose membranes. The Ab recognizing I{kappa}B{alpha} was from Santa Cruz Biotechnology. The Abs recognizing phospho-I{kappa}B{alpha} (Ser32) and phospho-RelA (Ser536) were from Cell Signaling (Cell Signaling Technology). All Abs were used at a 1/1000 dilution in a solution containing 5% milk in TBS-T. Immunoreactive proteins were detected using the ECL light detecting kit (Amersham Biosciences) as described previously (37).

Cytokine measurement

BMDC (5 x 105/well in a 24-well plate) were stimulated for 24 h with E. faecalis/E. coli lysate (40 µg/ml), supernatants were collected, and cytokine levels were measured using commercially available kits specific for IL-12p40 and IL-23p19 (BD Pharmingen/BD Biosciences), according to the manufacturer’s instructions. Cytokine levels were determined in triplicate culture supernatants in each separate experiment.

Isolation of intestinal lamina propria mononuclear cells (LPMNC) and flow cytometry analysis

Intestinal lymphocytes from both epithelial (IEL) and lamina propria (LPL) compartments of the large intestine of IL-10–/–;NF-{kappa}BEGFP mice and control IL-10wt/wt;NF-{kappa}BEGFP mice were isolated according to a modification of published procedures (38). In brief, the intestines were opened longitudinally, washed, and cut into small pieces. The intestinal epithelial layer was selectively detached from the mucosa by treatment with 1 mM DTT in a shaker water-bath at 37°C for 20 min, followed by vortexing for 4 min. The supernatant containing IEL and epithelial cells was collected. The residual tissues were then incubated twice in a shaker water-bath at 37°C for 30 min with a mixture of collagenase type II and IV (400 U/ml) (Sigma-Aldrich) and then disrupted mechanically. A stainless steel grid (60 mesh) was used to remove large particulate material. Single cells passing through the grid were harvested and IEL/LPL fractions were further purified by Percoll (Amersham Biosciences) density gradient centrifugation. Lymphocytes enriched in the interface between 70 and 40% Percoll were recovered. More than 90% of the gated cells were CD45+ (data not shown), demonstrating a high purity of immune cells achieved by our isolation procedure.

Freshly isolated intestinal IEL and LPL (2 x 105/100 µl) were then analyzed on a flow cytometer (CyAn; DakoCytomation) using the Summit version 4.3 software. All Abs including isotype controls were purchased from Caltag Laboratories. The following fluorochrome-labeled or unlabelled reagents were used: CD11c+ cells were identified using PE-conjugated anti-CD11c+ (clone N418) mAb; B220+ cells were identified using PE-conjugated anti-B220+ (clone RA3-6B2) mAb; CD45+ cells were identified using TC-conjugated anti-CD45+ (clone 30-F11) mAb; CD4+ cells were identified using PE-conjugated anti-CD4+ (clone CT-CD4) mAb; CD8+ cells were identified using TC-conjugated anti-CD8+ (clone CT-CD8a) mAb; and GR1+ cells were identified using PE-conjugated anti-GR1c+ (clone RB6-8C5) mAb. Cells incubated with PE- or TC-conjugated isotype standard from the same species at the same concentrations served as negative controls.

Statistical analysis

Statistical significance was evaluated by the Mann-Whitney test. A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NF-{kappa}B EGFP gene reporter mice (NF-{kappa}BEGFP) allow for real-time monitoring of LPS-induced NF-{kappa}B-dependent inflammatory responses in the intestine

We applied gnotobiotic technology to a NF-{kappa}B EGFP gene reporter mouse (NF-{kappa}BEGFP) to study the kinetics and cellular localization of NF-{kappa}B-derived transcriptional activity following bacteria-induced chronic intestinal inflammation. Gnotobiotic technology allows for the well-controlled introduction of a select subset of bacteria into a germfree host and a close and timely monitoring of the elicited host responses (39, 40). We first validated the NF-{kappa}BEGFP mice by monitoring EGFP expression following in vivo LPS stimulation. NF-{kappa}BEGFP mice were injected i.p. with LPS (25 µg/g body weight) for various times, the small intestine was dissected (stomach-duodenum), and EGFP expression was visualized using a charge-coupled device camera in a light-tight imaging box with a dual-filtered light source and emission filters specific for EGFP. Unstimulated NF-{kappa}BEGFP mice showed minimal signs of EGFP expression throughout the duodenum (Fig. 1A). Importantly, duodenum from LPS-injected NF-{kappa}BEGFP mice displayed a strong increase in EGFP expression (Fig. 1A; compare 4–24 h with PBS). To correlate enhanced EGFP expression with endogenous inflammatory genes, we isolated primary enterocytes from LPS-stimulated NF-{kappa}BEGFP mice and performed RT-PCR analysis. LPS enhanced mRNA accumulation of the NF-{kappa}B-dependent genes TNF and IL-6 compared with PBS control (Fig. 1B).


Figure 1
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  		FIGURE 1. Enhanced EGFP expression correlates with increased cytokine mRNA accumulation and RelA loading to NF-{kappa}B-dependent gene promoter in LPS-injected NF-{kappa}BEGFP mice. A, NF-{kappa}BEGFP mice were injected i.p. with LPS (40 µg/g body weight) for 4, 12, and 24 h, and the stomach down to the duodenum was resected. EGFP fluorescence was assessed using the Lightools Research macroimaging system with a dual output lighting. Data are representative of three independent experiments. B, RNA from the small bowel of LPS-injected NF-{kappa}BEGFP mice was isolated using the TRIzol procedure. One microgram of total RNA was reverse transcribed and amplified using specific primers for TNF, IL-6, and actin (loading control). The PCR products were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye. Data are representative of three independent experiments. C, NF-{kappa}BEGFP mice were injected i.p. with LPS (40 µg/g body weight) for 0, 30, 60, and 90 min, and enterocytes were isolated as described in Materials and Methods. ChIP assays were performed using anti-RelA or anti-RNA polymerase II Abs as described in Materials and Methods. PCR was performed using primers specific for the {kappa}B consensus site of the EGFP transgene and the IL-6 gene promoter. Data are representative of two independent experiments.

 
To directly demonstrate that NF-{kappa}B was activated following LPS stimulation, we performed ChIP analysis using enterocytes isolated from LPS-stimulated NF-{kappa}BEGFP mice. Results demonstrate that the NF-{kappa}B transcriptional subunit RelA is recruited to the {kappa}B-EGFP gene promoter following LPS stimulation (Fig. 1C). In addition, RNA polymerase II is also loaded onto the {kappa}B-EGFP gene promoter, indicating ongoing transcriptional activation (Fig. 1C). Similarly, LPS induced in vivo RelA and RNApolII recruitment to the IL-6 gene promoter in enterocytes. Altogether, these findings validate the usefulness of NF-{kappa}BEGFP mice to study the innate immune response in vivo.

IL-10–/–;NF-{kappa}BEGFP mice develop pancolitis following 7 wk of E. faecalis/E. coli dual association

To directly study the relationship between bacteria, TLR/NF-{kappa}B signaling and the development of colitis, we crossed the NF-{kappa}BEGFP mice to the IL-10–/– mice (IL-10–/–;NF-{kappa}BEGFP, 129SvEv/C57B6 background) and derived them into germfree condition. These mice remain disease free with no signs of immune activation when maintained in germfree condition (data not shown) (40). To study the relationship between commensal bacteria and NF-{kappa}B signaling, we dual associated IL-10–/–;NF-{kappa}BEGFP mice with E. faecalis/E. coli for 1 and 7 wk. These commensal bacteria have been shown to induce distal and cecal colitis in IL-10–/– mice (40). Histology indicated that E. faecalis/E. coli induced cecal, proximal, and distal inflammation after 7 wk of colonization in IL-10–/–;NF-{kappa}BEGFP mice (Fig. 2A). In contrast, no signs of inflammation were observed in E. faecalis/E. coli dual-associated wt mice. Representative histologic sections of the distal colons from wt mice dual associated with E. faecalis/E. coli showed normal physiologic cellularity (Fig. 2B). However, sections of the distal colon from dual-associated E. faecalis/E. coli IL-10–/–;NF-{kappa}BEGFP mice exhibited clear signs of inflammation with crypt hyperplasia, infiltration of predominantly mononuclear cells, and goblet cell depletion (Fig. 2B). These findings clearly demonstrate that IL-10–/–;NF-{kappa}BEGFP mice dual associated with commensal bacteria develop severe pancolitis.


Figure 2
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  		FIGURE 2. E. faecalis/E. coli dual-associated IL-10–/–;NF-{kappa}BEGFP mice develop severe pancolitis after 7 wk. A, Germfree IL-10–/–;NF-{kappa}BEGFP and IL-10wt/wt;NF-{kappa}BEGFP mice (seven mice per group) were dual associated with E. faecalis/E. coli for 7 wk. Mice were euthanized, and colonic histological sections of the cecum, proximal, and distal colons were scored as described in Materials and Methods (*, p < 0.05 vs control). B, Representative histologic sections of IL-10–/–;NF-{kappa}BEGFP mice dual associated with E. faecalis/E. coli showed evidence of severe inflammation compared with normal physiologic cellularity in IL-10wt/wt;NF-{kappa}BEGFP.

 
Temporal and spatial differences in the pattern of EGFP expression between 1- and 7-wk, dual-associated IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice

To monitor the state of NF-{kappa}B activation following bacterial colonization, we associated germfree IL-10–/–;NF-{kappa}BEGFP mice with E. faecalis/E. coli for 1 wk (no disease) and 7 wk (colitis). Germfree IL-10–/–;NF-{kappa}BEGFP mice, dual-associated IL-10–/–;NF-{kappa}BEGFP mice, and IL-10wt/wt;NF-{kappa}BEGFP mice were euthanized, their colons were dissected, and EGFP expression was macroscopically imaged. After 1 wk, macroscopic EGFP expression (NF-{kappa}B activation) was similarly low between germfree IL-10–/–;NF-{kappa}BEGFP mice, dual-associated IL-10wt/wt;NF-{kappa}BEGFP mice, and IL-10–/–;NF-{kappa}BEGFP mice (Fig. 3A). We next imaged EGFP expression using a sensitive confocal microscopy approach. Live colonic tissues obtained from 1-wk, dual-associated IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice were imaged, and Z-stack sections were computer generated to have a transversal view of the colon. Interestingly, EGFP expression was observed in the epithelium during the early phase of bacterial colonization (1 wk), with little evidence of activation in interstitial cells (Fig. 3B, upper panel). Imaging from the lumen (top-down) confirmed the similar distribution of EGFP-positive epithelium between IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice (Fig. 3B, lower panel). No clinical and histological sign of inflammation was visible after 1 wk of bacterial colonization (data not shown). In contrast to early colonization, IL-10–/–;NF-{kappa}BEGFP mice dual associated for 7 wk but not germfree or IL-10wt/wt mice displayed a strong increase in EGFP expression throughout the colon as determined by macroscopic imaging (Fig. 3C). Germfree IL-10–/–;NF-{kappa}BEGFP mice showed minimal signs of EGFP expression throughout the colon with positive "dots" likely representing NF-{kappa}B-activated cells located in the lymphoid follicles. Confocal microscopy showed that EGFP-positive cells are predominantly interstitial cells and thus likely represent LPMNC, whereas no signal was detected in IL-10wt/wt;NF-{kappa}BEGFP mice (Fig. 3D, upper panel). As opposed to the first week of colonization, minimal evidence of EGFP-positive epithelium was observed after 7 wk. Imaging from the lumen confirmed the different pattern of EGFP-positive cells between 7 wk (Fig. 3D, lower panel) and 1 wk of bacterial colonization (Fig. 3B, lower panel). These findings indicate that bacterial colonization initially triggers NF-{kappa}B activity in the epithelium (enterocytes) but that the signaling is mostly present in interstitial cells during chronic colitis.


Figure 3
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  		FIGURE 3. Enhanced EGFP expression in 7-wk but not 1-wk E. faecalis/E. coli dual-associated IL-10–/–;NF-{kappa}BEGFP mice. A, Germfree IL-10–/–;NF-{kappa}BEGFP and IL-10wt/wt;NF-{kappa}BEGFP mice (seven mice per group) were dual associated with E. faecalis/E. coli for 1 (A and B) or 7 wk (C and D). A, Mice were euthanized after 1 wk of bacterial colonization, and their colons were resected and EGFP fluorescence imaged as described in Fig. 1. B, Confocal imaging of colonic sections immediately after dissection was performed as described in Materials and Methods. Localization of EGFP-positive cells is mostly confined to the surface epithelium. Upper panels represent a transversal view (Z-stack), and lower panels represent a view of EGFP expression from the lumen down, showing epithelial cells. Left panel, A schematic representation of the Z-stack section (plane 1, longitudinal crypt axis) and the transverse section (plane 2, colonic surface axis). C, Mice were euthanized after 7 wk of bacterial colonization, and their colons were resected and EGFP fluorescence imaged as described in Fig. 1. D, Confocal imaging of colonic sections immediately after dissection was performed as described in Materials and Methods. Upper panels represent a transversal view (Z-stack), and lower panels represent a view of EGFP expression from the lumen down, which shows interstitial EGFP-positive cells. Left panel, A schematic representation as described in B.

 
To gain more insight into the profile of EGFP in lamina propria cells, we isolated LPMNC from 7-wk, dual-associated IL-10wt/wt;NF-{kappa}BEGFP and IL-10–/–;NF-{kappa}BEGFP mice and performed FACS. Interestingly, we found a relative increase in Th cells (CD4+, 2.0-fold increase), T cytotoxic cells (CD8+, 1.9-fold increase), DC (CD11c+, 3.3-fold increase), neutrophils (GR1+, 2.8-fold increase), and macrophages (MAC1+, 5.3-fold increase) in dual-associated IL-10–/–;NF-{kappa}BEGFP mice as compared with IL-10wt/wt;NF-{kappa}BEGFP mice in total EGFP+CD45+ LPMNC (Fig. 4A). Moreover, a relative decrease in the proportion of B cells (B220+) was observed between IL-10–/– and IL-10wt/wt animals (0.6-fold; Fig. 4A). Similar differences were found when total CD45+ LPMNC were examined (data not shown). These findings implicate that commensal bacteria-associated IL-10–/– mice demonstrate an increased ratio of activated lamina propria immune cells of the dendritic and T cell but not B cell fraction.


Figure 4
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  		FIGURE 4. Enhanced CD4+, CD8+, CD11c+, GR1+, and MAC1+ surface marker expression in the EGFP+ LPMNC in IL-10–/–;NF-{kappa}BEGFP as compared with IL-10wt/wt;NF-{kappa}BEGFP mice. A, Lamina propria immune cells were isolated from the colon of 7-wk dual-isolated IL-10–/–;NF-{kappa}BEGFP and IL-10wt/wt;NF-{kappa}BEGFP mice as described in Materials and Methods. Cells were stained for CD45+, CD4+, CD8+, CD11c+, GR1+, and MAC1+ surface markers and analyzed by flow cytometry as described in Materials and Methods. The graph shows the percentage of CD45+EGFP+ LPMNC additionally expressing the indicated surface markers in total CD45+EGFP+ LPMNC (R4/R6 in B). B, Representative flow cytometry scans for CD11c+ cell analysis. Data are representative of three independent experiments.

 
To document NF-{kappa}B signaling at the molecular level, we investigated the state of RelA phosphorylation following dual bacterial colonization. Immunohistochemical analysis of colonic tissue sections obtained from 7-wk, dual-associated IL-10–/–;NF-{kappa}BEGFP but not wt mice showed the presence of phosphorylated RelASer536 in invading immune mononuclear cells, as well as a few enterocytes (Fig. 5). Interestingly, RelA phosphorylation is stronger in LPMNC than enterocytes, indicating prolonged and sustained activation of NF-{kappa}B signaling in these immune cells. Isotype Ab controls demonstrated the specificity of the staining (Fig. 5, lower panel). Overall, these findings indicate that bacterial colonization triggers the sequential activation of NF-{kappa}B in enterocytes and LPMNC, which then is associated with the development of colitis.


Figure 5
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  		FIGURE 5. Increased RelA phosphorylation in LPMNC of 7-wk E. faecalis/E. coli dual-associated IL-10–/–;NF-{kappa}BEGFP mice compared with wt mice. IL-10–/–;NF-{kappa}BEGFP mice or control IL-10wt/wt;NF-{kappa}BEGFP were dual associated with E. faecalis/E. coli for 7 wk. Mice were euthanized, sections from the colon were fixed, and immunohistochemistry was performed on paraffin-embedded tissues using anti-phospho RelA (Ser536). Sections from IL-10–/–;NF-{kappa}BEGFP mice were stained with anti-phospho RelA (left panel) or isotype control (right panel). Sections were counterstained with hematoxylin.

 
E. faecalis/E. coli lysates induce I{kappa}B{alpha} and RelA phosphorylation, followed by NF-{kappa}B-dependent IL-12p40 and IL-23p19 mRNA accumulation in BMDC isolated from IL-10–/–; NF-{kappa}BEGFP mice

Bacterial-activated APC such as DC play a pivotal role in T cell-mediated colitis through the release of numerous immune mediators, including IL-12, IL-23, and TNF (41, 42, 43). Because of the limited numbers of intestinal DC, we studied the molecular mechanisms of E. faecalis/E. coli-induced gene expression using BMDC generated from IL-10–/–;NF-{kappa}BEGFP mice. I{kappa}B{alpha} and RelA are rapidly phosphorylated in BMDC following stimulation with E. faecalis/E. coli lysates (Fig. 6A). Similarly, EGFP expression is enhanced in E. faecalis/E. coli-stimulated BMDC (Fig. 6B).


Figure 6
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  		FIGURE 6. E. faecalis/E. coli lysate induced NF-{kappa}B signaling and EGFP expression in BMDC generated from IL-10–/–;NF-{kappa}BEGFP mice. A, BMDC from IL-10–/–;NF-{kappa}BEGFP mice were stimulated with E. faecalis/E. coli lysate (40 µg/ml) and harvested at 0, 0.5, 1, and 2 h. Western blot analysis was performed for phospho-I{kappa}B{alpha}, phospho-RelA, and actin. B, BMDC from IL-10–/–;NF-{kappa}BEGFP mice were stimulated with E. faecalis/E. coli lysates (40 µg/ml) for 24 h, and EGFP expression was assessed using fluorescence microscopy. The results are representative of four independent experiments.

 
Production of IL-23 is critical for the development of colitis in IL-10–/– mice (23). We next investigated the impact of E. faecalis/E. coli on IL-23 expression and examined the role of NF-{kappa}B in this process. E. faecalis/E. coli lysate led to the rapid induction of IL-12p40 and IL-23p19 mRNA in BMDC (Fig. 7). Interestingly, blocking NF-{kappa}B activity with the specific inhibitor Bay11-7085 prevented induction of IL-12p40 and IL-23p19 mRNA in E. faecalis/E. coli-stimulated BMDC (Fig. 7). These findings indicate that bacteria-induced IL-23 gene expression requires NF-{kappa}B signaling.


Figure 7
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  		FIGURE 7. E. faecalis/E. coli lysates induced IL-12p40 and IL-23p19 mRNA accumulation through a NF-{kappa}B-dependent pathway in IL-10–/–;NF-{kappa}BEGFP BMDC. BMDC from IL-10–/–;NF-{kappa}BEGFP mice were pretreated with Bay 11-7085 (10 µM) or vehicle control (DMSO 0.5%) for 45 min and then stimulated with E. faecalis/E. coli lysate (40 µg/ml) for 0, 2, 4, and 6 h. RNA was isolated using the TRIzol procedure, and 1 µg was reverse transcribed and amplified using specific primers for IL-12p40, IL-23p19, and actin (loading control). The PCR products were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye. Data are representative of three independent experiments.

 
E. faecalis/E. coli lysates induced I{kappa}B{alpha} degradation and IL-12p40 as well as IL-23p19 protein secretion in BMDC is dependent on MyD88 signaling

Recent findings demonstrated that spontaneous colitis is absent in IL-10–/–;MyD88–/– mice (31), indicating that commensal bacteria use the TLR signaling grid to induce inflammation. To better define the role of TLR in E. faecalis/E. coli-induced NF-{kappa}B signaling and gene expression, we generated BMDC from IL-10wt/wt, IL-10–/–, and IL-10–/–;MyD88–/– mice. E. faecalis/E. coli-induced I{kappa}B{alpha} degradation was inhibited in BMDC derived from IL-10–/–;MyD88–/– mice compared with IL-10–/– and wt mice (Fig. 8A). In addition, E. faecalis/E. coli lysates induced IL-12p40 and IL-23p19 secretion in both IL-10wt/wt and IL-10–/– mice, with higher induction in the latter (Fig. 8, B and C). This is consistent with a previous finding showing higher LPS-induced IL-12p40 secretion in IL-10–/– compared with IL-10wt/wt mice (33). Importantly, E. faecalis/E. coli lysate-induced IL-12p40 and IL-23p19 secretion was strongly inhibited in IL-10–/–;MyD88–/– mice compared with IL-10–/– and wt mice (Fig. 8, B and C). These findings indicate that E. faecalis/E. coli-induced NF-{kappa}B activity and IL-23 gene expression is dependent on TLR signaling.


Figure 8
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  		FIGURE 8. E. faecalis/E. coli lysates induced NF-{kappa}B signaling, and IL-23 gene expression is blocked in IL-10–/–;MyD88–/– BMDC compared with IL-10–/– BMDC. A, BMDC generated from IL-10–/–;MyD88–/–, IL-10–/–, and wt mice were stimulated with E. faecalis/E. coli lysate (40 µg/ml), harvested at 0 and 30 min, and Western blot analysis was performed for phospho-I{kappa}B{alpha} and actin. B, BMDC generated from IL-10–/–;MyD88–/–, IL-10–/–, and wt mice were stimulated with E. faecalis/E. coli lysate (40 µg/ml) for 24 h, and IL-12p40 secretion was measured in triplicate supernatants by ELISA. C, BMDC were treated as described above, and IL-23p19 secretion was measured in triplicate supernatants by ELISA. All results are representative of three independent experiments.

 
Pharmacological NF-{kappa}B-inhibition (Bay 11-7085) ameliorates dual association-induced colitis and reduces colonic EGFP expression, as well as IL-6, IL-12p40, and IL-17 mRNA accumulation in IL-10–/–;NF-{kappa}BEGFP mice

To further define the role of NF-{kappa}B signaling in the development of bacteria-induced colitis, NF-{kappa}B activation was pharmacologically inhibited in dual-associated IL-10–/–;NF-{kappa}BEGFP mice (Bay 11-7085 (5 mg/kg i.p.) or DMSO solvent control, three injections per week for 5 wk). At the macroscopic level, E. faecalis/E. coli dual-associated IL-10–/–;NF-{kappa}BEGFP mice displayed enhanced EGFP expression compared with wt E. faecalis/E. coli dual-associated mice (Fig. 9A). Importantly, colonic levels of EGFP expression were strongly reduced in Bay 11-7085-treated IL-10–/–;NF-{kappa}BEGFP mice (Fig. 9A). Confocal microscopic analysis of live colonic sections obtained from Bay 11-7085-treated IL-10–/–;NF-{kappa}BEGFP mice showed a strong reduction of EGFP expression in LPMNC compared with vehicle-treated mice (Fig. 9B).


Figure 9
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  		FIGURE 9. Blocking NF-{kappa}B activity prevented EGFP expression and colitis in E. faecalis/E. coli dual-associated IL-10–/–;NF-{kappa}BEGFP mice. A, Germfree IL-10–/–;NF-{kappa}BEGFP and IL-10wt/wt;NF-{kappa}BEGFP mice (seven mice per group) were dual associated with E. faecalis/E. coli and injected i.p. three times weekly with the NF-{kappa}B inhibitor Bay 11-7085 (5 mg/kg) or vehicle control (DMSO) for 5 wk. Mice were euthanized, and their colons were resected and EGFP fluorescence imaged as described in Fig. 1. B, Confocal imaging of colonic sections immediately after dissection was performed as described in Materials and Methods. EGFP-positive cells decreased in Bay 11-7085-treated mice compared with control. C, RNA from the colon of Bay 11-7085 and control-injected IL-10–/–;NF-{kappa}BEGFP mice was isolated using the TRIzol procedure. One microgram of total RNA was reverse transcribed and amplified using specific primers for IL-6, IL-12/IL-23p40, IL-17, and actin (loading control). The PCR products were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye. D, Colonic histological sections of the cecum, proximal, and distal colons were scored as described in Materials and Methods (*, p < 0.05 vs control).

 
To evaluate the correlation between EGFP expression and the extent of colitis in these mice, RNA from total colonic sections were isolated, and cytokine expression was evaluated by RT-PCR. IL-6, IL-17, and, to a lesser extent, IL-12p40 mRNA accumulation decreased in the colon of mice treated with Bay 11-7085 compared with control mice (Fig. 9C). In addition, mice treated with Bay 11-7085 showed a significant attenuation of bacteria-induced colitis in the cecum, proximal, and distal colon compared with control mice as evaluated by histological scoring (Fig. 9D; p < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we associated germfree IL-10–/–;NF-{kappa}BEGFP mice with two nonpathogenic commensal bacteria strains to specifically study the relationship between TLR/NF-{kappa}B signaling and the development of chronic intestinal inflammation. We present clear evidence that NF-{kappa}B (EGFP) is activated throughout the colon of E. faecalis/E. coli dual-associated mice. Histology showed the presence of inflammation in the cecum, proximal, and distal colon of 7-wk, but not in 1-wk, dual-associated mice, which correlated with the overall pattern of NF-{kappa}B activation. Moreover, the direct relationship between bacteria-induced inflammation and NF-{kappa}B activity was clearly demonstrated by pharmacological blockade, where the NF-{kappa}B-inhibitor Bay 11-7085 prevented E. faecalis/E. coli-induced colonic EGFP expression and significantly attenuated the development of colitis. Of critical importance was that, following pharmacologic inhibition of NF-{kappa}B, there was a strong reduction in EGFP expression specifically in the lamina propria (Fig. 9B), suggesting that bacteria-induced NF-{kappa}B activation in this cell compartment is responsible for the development of colitis in IL-10–/–;NF-{kappa}BEGFP mice.

Interestingly, we observed a temporally differential distribution of EGFP-positive cells (NF-{kappa}B activation) after bacterial colonization. During the first week of colonization, thus before any histological signs of inflammation (40), EGFP expression was mainly confined to the epithelium (enterocytes) with minimal evidence of activation in the lamina propria of IL-10–/–;NF-{kappa}BEGFP mice. Of note, IL-10wt/wt;NF-{kappa}BEGFP mice displayed a similar EGFP-positive epithelium following bacterial colonization, indicating that enterocytes are early responders of bacterial colonization and activate an innate response leading to NF-{kappa}B activity (EGFP expression). However, a clear shift in the EGFP expression pattern was observed during colitis (7-wk dual association) in IL-10–/–;NF-{kappa}BEGFP mice: in this chronic inflammatory environment, EGFP accumulation was present predominantly in the lamina propria with minimal evidence of NF-{kappa}B activation in the enterocytes; conversely, EGFP expression was barely detectable in the colon of 7-wk, dual-associated IL-10wt/wt;NF-{kappa}BEGFP mice. It should be stressed that, although the localization of EGFP in confocal microscopy (Figs. 3D and 9B), as well as immunohistochemical analysis of RelA-phosphorylation (Fig. 5), is consistent with NF-{kappa}B activation predominantly in lamina propria immune cells (LPMNC), at this point our findings do not exclude the possible contribution of other intestinal cells such as myofibroblasts, fibroblasts, and endothelial cells to the overall pattern of EGFP expression and in the development of colitis. Collectively, our findings indicate that bacterial colonization transiently activates NF-{kappa}B signaling in enterocytes (IL-10–/– and IL-10wt/wt). In the setting of a dysregulated immunosuppressive response (e.g., IL-10–/–), NF-{kappa}B is later persistently activated in LPMNC associated with the development of intestinal inflammation.

An attenuated response of colonic enterocytes to numerous commensal bacteria is a critical feature of intestinal homeostasis (14, 44, 45, 46, 47, 48, 49). The mechanism controlling the enterocytes’ tolerance to luminal bacteria is not fully elucidated but likely involves the action of negative regulators of TLR signaling and the presence of a complex network of immunosuppressive molecules (48, 50, 51, 52). Because initial EGFP expression (1 wk) is decreased later on in both IL-10–/– and IL-10wt/wt mice, it is unlikely that this event is mediated through an IL-10-dependent mechanism. The enterocytes’ tolerance to luminal bacteria and bacterial products may be a unique and intrinsic feature of the intestinal epithelium: indeed, fetal enterocytes are highly responsive to LPS stimulation whereas neonatal and adult enterocytes become unresponsive due to enhanced IRAK-1 degradation. The intestine of newborn mice is colonized rapidly by bacteria following their passage through the birth canal, and it is this event that likely initiates the rapid establishment of intestinal tolerance toward the luminal contents (49). It is possible that germfree mice in a similar manner rapidly respond to bacterial colonization by inducing TLR/NF-{kappa}B signaling but that the signal is down-regulated through activation of intrinsic negative feedback loops and extrinsic regulatory factors. Whether enhanced IRAK degradation is responsible for this acquired tolerance remains to be investigated.

EGFP-positive lymphocytes and DC, as well as neutrophils and macrophages, are likely to be a driving force in bacteria-induced colitis in IL-10–/– mice (24, 53, 54). For example, IL-23 secreted by APC such as DC plays a critical role in supporting the development of T lymphocytes producing IL-17 (ThIL-17), an essential step for the development of colitis in IL-10–/– mice (23). Interestingly, we found that E. faecalis/E. coli lysate induced NF-{kappa}B signaling, EGFP expression, and IL-23 mRNA accumulation in BMDC generated from IL-10–/–;NF-{kappa}BEGFP mice. Moreover, bacteria-induced IL-23p19 gene expression was higher in IL-10–/– mice compared with IL-10wt/wt control, indicating a dysregulated innate response in these mice. Importantly, blocking NF-{kappa}B activity with Bay 11-7085 prevented bacterial lysate-induced IL-23 mRNA accumulation. These in vitro findings correlated with the decrease of colonic EGFP, IL-6, and IL-17 expression and with the attenuation of colitis in Bay 11-7085-treated mice.

A recent study demonstrated the critical role of TLR/MyD88 in promoting differentiation of Th1 cells and development of spontaneous colitis in IL-10–/– mice (31). MyD88 is involved in both TLR2 and TLR4 signaling, and mice deficient for this adaptor molecule are expected to be defective for E. faecalis (gram +) and E. coli (gram –)-induced signaling (55, 56, 57). Indeed, we found that E. faecalis/E. coli-induced NF-{kappa}B signaling and IL-23 gene expression were blocked in IL-10–/–;MyD88–/– compared with IL-10–/– BMDC, showing the key role of TLR in mediating these events. Paradoxically, however, mice with defective TLR signaling (TLR2–/–, TLR4–/–, and MyD88–/–) have been shown to be highly susceptible to chemical-induced acute colitis (29, 30), and interestingly, TLR2 signaling enhances barrier integrity through translocation of ZO-1 (58). These studies illustrate that TLR signaling, apart from its proinflammatory effects, can have protective functions, which are missing in MyD88–/– animals and thus render the host more susceptible to intestinal injury. A similar bipartite effect can be seen in cyclooxygenase-2, which is induced rapidly following intestinal injury and participates in mucosal inflammation, restitution, and protection (35, 59, 60).

In summary, our results indicate that intestinal bacterial association induces NF-{kappa}B activation through TLR signaling mainly in mucosal immune cells and that these events participate in the development of colitis in a susceptible host (IL-10–/–). In this context, they outweigh both the protective effects of MyD88 signaling and NF-{kappa}B-regulatory feedback loops (e.g., A20 (61, 62)). Hence, blocking TLR-induced NF-{kappa}B activity may represent an attractive strategy to treat chronic immune-mediated intestinal inflammation.


   Acknowledgments
 
We are grateful for the expert work of the following individuals: Kathy Mohr (Mutant Mouse Regional Resource Center) for embryos dissection and transplantation; Maureen Bower and Silmara Camargo at the Gnotobiotic Core Facility of the Center for Gastrointestinal Biology and Disease for their expert help in conducting the mice dual-association studies; Dr. Sandra Kim (Department of Pediatrics, University of North Carolina (UNC), Chapel Hill, NC) for providing the bacteria strains E. coli and E. faecalis; Wendy Salmon and Dr. Michael Chua in the Michael Hooker Microscopy Facility at UNC for their expert advise on confocal microscopic imaging and for providing access to the Leica SP2 Upright Laser Scanning Confocal Microscope (Leica); Dr. Sue Tonkonogy and Bayley Crane at the College of Veterinary Medicine, North Carolina State University (Raleigh, NC), for their expert help with the ELISA studies on IL-23p19 secretion; Monica Mattmuller at the College of Veterinary Medicine, North Carolina State University, for her expert help with the immunohistochemical studies; Kathy Thompson from the Histology Core and Rosemary Link from the Immunoassay Core of the Center for Gastroenterology Biology and Disease at UNC for their expert assistance in histology and IL-12p40 and IL-23p19 quantification, respectively; and Dr. Larry W. Arnold in the Flow Cytometry Core at UNC for his expert advice on flow cytometry analysis. We thank Dr. Eyal Raz (University of California, San Diego, CA) for providing the IL-10–/–MyD88–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R0I DK 47700, Crohn’s and Colitis Foundation of America Grants, and a American Gastroenterological Association Fiterman Basic Research Award (to C.J.); the Crohn’s and Colitis Foundation of America Fellowship (to T.K.); National Institutes of Health Grant P30 DK03498; and National Gnotobiotic Rodent Research Center Grant P40 R018603. Back

2 Current address: Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, 110-744 Korea. Back

3 Address correspondence and reprint requests to Dr. Christian Jobin, University of North Carolina, CB 7032, Department of Medicine 7341B, Medical Biomolecular Research Building, Chapel Hill, NC 27599. E-mail address: Job{at}med.unc.edu Back

4 Abbreviations used in this paper: DC, dendritic cell; EGFP, enhanced GFP; BMDC, bone marrow-derived dendritic cell; ChIP, chromatin immunoprecipitation; LPMNC, lamina propria mononuclear cell; LPL, lamina propria lymphocyte; IEL, intestinal epithelial lymphocyte; wt, wild type. Back

Received for publication January 26, 2007. Accepted for publication March 9, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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