Gnotobiotic IL-10-/-;NF-kappa B(EGFP) mice reveal the critical role of TLR/NF-kappa B signaling in commensal bacteria-induced colitis

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-kappaB activation in a model of commensal bacteria-induced T cell-mediated colitis. A NF-kappaB reporter gene mouse (NF-kappaBEGFP) (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-kappaBEGFP and IL-10-/-;NF-kappaBEGFP 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-kappaBEGFP mice developed severe inflammation by 7 wk. Macroscopic analysis showed elevated EGFP expression throughout the colon of bacteria-associated IL-10-/-;NF-kappaBEGFP 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-kappaB inhibitor BAY 11-7085 attenuated E. faecalis/E. coli-induced EGFP expression and development of colitis. Additionally, E. faecalis/E. coli-induced NF-kappaB 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-kappaB signaling derived mostly from mucosal immune cells. Blocking TLR-induced NF-kappaB activity may represent an attractive strategy to treat immune-mediated intestinal inflammation.     

IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis

Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IEC expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10(-/-) IEC. Of note, TGF-beta1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappaB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-), but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.     

Bcl10 mediates LPS-induced activation of NF-kappaB and IL-8 in human intestinal epithelial cells

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bcl10 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Bcl10 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-kappaB in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2-6 h. Effects on Bcl10, phospho-IkappaBalpha, NF-kappaB, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bcl10 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bcl10 silencing, LPS-induced increases in NF-kappaB, IkappaBalpha, and IL-8 were significantly reduced (P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bcl10 protein when quantified by ELISA, and the association between LPS exposure and increased Bcl10 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bcl10, NF-kappaB, and IL-8. Identification of Bcl10 as a mediator of LPS-induced activation of NF-kappaB and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention.     

CD4+ T cells from IL-10-deficient mice transfer susceptibility to NSAID-induced Rag colitis

Products of arachidonic acid metabolism are important for mucosal homeostasis, because blockade of this pathway with an NSAID triggers rapid onset of severe colitis in the IL-10 knockout (IL-10(-/-)) model of IBD. Rag mice do not make T or B cells. This study determined whether reconstitution of Rag mice with T cells from IL-10(-/-) mice transferred NSAID colitis susceptibility. Rag mice were reconstituted by intraperitoneal injection with splenocytes from wild-type (WT) or IL-10(-/-) animals. Colitis was induced by using piroxicam and was graded histologically. Isolated lamina propria mononuclear cells (LPMC), lamina propria T cells, and LPMC depleted of T cells from reconstituted Rag mice were studied for cytokine production. Only animals reconstituted with IL-10(-/-) CD4(+) T cells and administered piroxicam developed severe colitis. LPMC from these colitic animals made IFN-gamma, whose production was dependent on T cells. Some IL-10 was produced but only from non-T cells. LPMC from the healthy Rag mice that were reconstituted with WT T cells and were piroxicam resistant made much more IL-10. This was mostly T cell dependent. In conclusion, only CD4(+) T cells from IL-10(-/-) animals leave Rag mice susceptible to NSAID-induced, Th1 colitis. Lamina propria T cells normally make large quantities of IL-10, suggesting that IL-10 from T cells may be protective.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.     

Induction of chronic colitis in IL-10 deficient mice requires IL-4

Th 1 cells activated by IL-12 and secreting IFN-gamma have been described as the main mediators for onset and maintenance of chronic colitis in IL-10 deficient mice. It was therefore surprising that mice deficient for IL-4 in addition to IL-10 showed intestinal pathology very rarely, whereas IL-10 KO mice developed rectal prolapse in most cases. To investigate the underlying mechanisms, we studied changes of ongoing inflammatory processes in mice deficient for IL-4, IL-10 or both cytokines. Levels of IFN-gamma, IL-12p40 and MHCII mRNA were elevated to a much higher degree in colonic tissue of IL-10 KO compared to IL-4/10 KO at the onset of colitis. Furthermore, the influx of eosinophils, a marker for Th2 responses, was investigated. Only IL-10 deficient mice displayed a significant increase of eosinophils in the lamina propria of the colon and rectum. In contrast, IL-4/10 deficient mice had eosinophil levels comparable to wildtype controls and IL-4 KO. Together these results indicate an important role of IL-4 for the onset of colitis in IL-10 KO mice by promoting a Th1 response and induction of a deleterious Th2 effector response.     

IL-12 induction of resistance to pulmonary blastomycosis

BACKGROUND: Why severity varies in blastomycosis outbreaks remains unresolved. In experimental pulmonary blastomycosis, susceptibility varied in mouse strains. In susceptible BALB/c the response is Th-2, in immunized, resistance is associated with Th-1. Can susceptibility be redirected by IL-12? Methods, results: BALB/c bronchoalveolar and peritoneal macrophages (PM) were shown deficient in IL-12 production in response to IFN-gamma+LPS. High dose IL-12 (1 microg, subcutaneously) treatment of BALB/c infected intranasally with Blastomyces resulted in enhanced survival (P<0.008). Since IL-12 was poorly tolerated, a new protocol for infected mice, IL-12 0.1 or 0.3 microg, every other day, resulted in minimal toxicity; almost all treated mice survived (P<0.002 vs. controls). When lungs of surviving mice were cultured, the 0.1 microg regimen resulted in fewer (P<0.02) cfu. For weeks after treatment, in vitro IFN-gamma treatment enabled PM Blastomyces killing. After infection spleen cells from IL-12 treated mice produced 4-fold more IFN-gamma and 3-fold less IL-10 in response to Blastomyces. IL-10 abrogated activation of macrophages by IFN-gamma for enhanced Blastomyces killing. Conclusions: A proper IL-12 treatment protocol induces resistance (survival and decreased growth in lungs), low toxicity, macrophage responsiveness to IFN-gamma for killing Blastomyces, up-regulation of IFN-gamma and down-regulation of IL-10 production.     

Intestinal epithelial cell proteome in IL-10 deficient mice and IL-10 receptor reconstituted epithelial cells: impact on chronic inflammation

The interaction of nonpathogenic enteric bacteria with intestinal epithelial cells (IEC) in the absence of host-derived Interleukin 10 (IL-10) may contribute to the development of chronic inflammation. Functional proteome analysis of primary IEC from Enterococcus faecalis-monoassociated WT and IL-10-/- mice as well as IL-10 receptor reconstituted IEC revealed expression changes of proteins that are involved in endoplasmic reticulum stress, energy metabolism, and apoptosis, suggesting a protective role for IL-10 at the epithelial cell level.     

IL-10 controls ultraviolet-induced carcinogenesis in mice

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.     

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.     

Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10-/- mice

A deficiency in understanding the steps responsible for colitis is the lack of comprehension for the role chemokines play in mucosal inflammation. IFN-gamma-inducible protein-10 (IP-10) and CXCR3 are highly expressed at sites of colitis. Our findings show that IP-10 significantly contributes to the development of Th1 and inflammatory responses. Specifically, IP-10 inhibition in IL-10(-/-) mice attenuates the associated increases in serum and/or local amyloid A, IL-2, IL-6, TNF-alpha, IFN-gamma, IL-1alpha, and IL-1beta with colitis as compared with IL-10(-/-) mice that develop colitis similar to human Crohn's disease. Correspondingly, the rate or intensity of inflammation in IL-10(-/-) mice treated with anti-IP-10 Abs showed improved scoring of inflammation, compared with control IL-10(-/-) mice. This study provides important and novel information regarding IP-10 as a target for the treatment of colitis.     

5-Lipoxygenase-derived lipid mediators are not required for the development of NSAID-induced inflammatory bowel disease in IL-10-/- mice

Leukotrienes are potent lipid mediators derived from the metabolism of arachidonic acid by the enzyme 5-lipoxygenase (5-LO). Elevated levels of the proinflammatory leukotriene LTB(4) have been found in preclinical models of inflammatory bowel disease (IBD) as well as in colon tissue from individuals with IBD. We therefore determined the extent to which absence of 5-LO-derived lipid mediators would alter the colitis in IL-10(-/-) mice, a model of human IBD. IL-10(-/-)/5-LO(-/-) mice were generated and were healthy. Absence of 5-LO did not alter the development of spontaneous colitis in IL-10-deficient mice. We then evaluated the extent to which absence of 5-LO would alter the development of NSAID-induced colitis in IL-10(-/-) mice. Absence of 5-LO did not delay the onset or alter the severity of inflammation in NSAID-treated IL-10(-/-) mice. At an early time point, 3 days after NSAID treatment was initiated, a qualitative increase in the number of dendritic cells and CD4(+) T cells was noted in the colons of IL-10(-/-)/5-LO(-/-); however, this difference was no longer present after 14 days of NSAID treatment. Absence of 5-LO did not alter the degree of neutrophil infiltration into the in this model. Absence of 5-LO does not alter the development of IFN-gamma producing Th1-type CD4(+) T cells or IL-17 producing CD4(+) T cells. Absence of 5-LO-derived mediators did not alter the expression of the adhesion molecules ICAM-1 and P-selectin. Development of colitis in IL-10(-/-) mice was associated with increased levels of the 5-LO-derived anti-inflammatory lipoxin LXA(4). These studies demonstrate that 5-LO-derived leukotrienes are not required for the development or maintenance of spontaneous or NSAID-induced colonic inflammation in IL-10(-/-) mice.     

Proinflammatory properties of IL-4 in the intestinal microenvironment

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.     

NHE3 modulates the severity of colitis in IL-10-deficient mice

NHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohn's disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation.     

Inhibition of Helicobacter hepaticus-induced colitis by IL-10 requires the p50/p105 subunit of NF-kappa B

Defects within the innate immune system sensitize NF-kappaB-deficient (p50(-/-); p65(+/-)) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-kappaB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2(-/-) (RAG) and p50(-/-); p65(+/-); RAG-2(-/-) (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50(-/-); RAG-2(-/-) (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-kappaB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.     

Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria

Disorders in enteric bacteria recognition by intestinal macrophages (Mphi) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mphi in intestinal inflammation by using an IL-10-deficient (IL-10-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mphi (GM-Mphi) and M-CSF-induced bone marrow-derived Mphi (M-Mphi) were generated from bone marrow CD11b+ cells. M-Mphi from IL-10-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mphi from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mphi was not significantly different between WT and IL-10-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mphi (CLPMphi) but not splenic Mphi from WT mice was similar to that of M-Mphi, and CLPMphi but not splenic Mphi from IL-10-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from M-Mphi from IL-10-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMphi and M-Mphi act as anti-inflammatory Mphi and suppress excess inflammation induced by bacteria in WT mice. In IL-10-/- mice, however, such Mphi subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mphi may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mphi to gut flora relationship in the development of colitis in IL-10-/- mice.     

Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ(-/-) mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ(+) Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti-IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ(+) Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.