Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function

Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.     

Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice

Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.     

Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in Celiac disease

Recent studies have demonstrated the importance of TLR signaling in intestinal homeostasis. Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. In this study, we sought to test the hypothesis that gliadin initiates this response by stimulating the innate immune response to increase intestinal permeability and by up-regulating macrophage proinflammatory gene expression and cytokine production. To this end, intestinal permeability and the release of zonulin (an endogenous mediator of gut permeability) in vitro, as well as proinflammatory gene expression and cytokine release by primary murine macrophage cultures, were measured. Gliadin and its peptide derivatives, 33-mer and p31-43, were found to be potent inducers of both a zonulin-dependent increase in intestinal permeability and macrophage proinflammatory gene expression and cytokine secretion. Gliadin-induced zonulin release, increased intestinal permeability, and cytokine production were dependent on myeloid differentiation factor 88 (MyD88), a key adapter molecule in the TLR/IL-1R signaling pathways, but were neither TLR2- nor TLR4-dependent. Our data support the following model for the innate immune response to gliadin in the initiation of CD. Gliadin interaction with the intestinal epithelium increases intestinal permeability through the MyD88-dependent release of zonulin that, in turn, enables paracellular translocation of gliadin and its subsequent interaction with macrophages within the intestinal submucosa. There, the interaction of gliadin with macrophages elicits a MyD88-dependent proinflammatory cytokine milieu that facilitates the interaction of T cells with APCs, leading ultimately to the Ag-specific adaptive immune response seen in patients with CD.     

Estrogen receptor-β signaling modulates epithelial barrier function

Impaired epithelial barrier function and estrogens are recognized as factors influencing inflammatory bowel disease (IBD) pathology and disease course. Estrogen receptor-β (ERβ) is the most abundant estrogen receptor in the colon and a complete absence of ERβ expression is associated with disrupted tight-junction formation and abnormal colonic architecture. The aim of this study was to determine whether ERβ signaling has a role in the maintenance of epithelial permeability in the colon. ERβ mRNA levels and colonic permeability were assessed in IL-10-deficient mice and HLA-B27 rats by RT-PCR and Ussing chambers. ERβ expression and monolayer resistance were measured in HT-29 and T84 colonic epithelial monolayers by RT-PCR and electric cell-substrate impedance sensing. The effect of 17β-estradiol and an estrogen agonist [diarylpropionitrile (DPN)] and antagonist (ICI 182780) on epithelial resistance in T84 cells was measured. Expression of ERβ and proinflammatory cytokines was investigated in colonic biopsies from IBD patients. Levels of ERβ mRNA were decreased, whereas colonic permeability was increased, in IL-10-deficient mice and HLA-B27 transgenic rats prior to the onset of colitis. T84 cells demonstrated higher resistance and increased levels of ERβ mRNA compared with HT-29 cells. 17β-estradiol and DPN induced increased epithelial resistance in T84 cells, whereas an ERβ blocker prevented the increased resistance. Decreased ERβ mRNA levels were observed in colonic biopsies from IBD patients. This study suggests a potential role for ERβ signaling in the modulation of epithelial permeability and demonstrates reduced ERβ mRNA in animal models of colitis and colon of patients with inflammatory bowel disease.     

Metabolomic Profiles Are Gender,Disease and Time Specific in the Interleukin-10 Gene-Deficient Mouse Model of Inflammatory Bowel Disease

Metabolomic profiling can be used to study disease-induced changes in inflammatory bowel diseases (IBD). The aim of this study was to investigate the difference in the metabolomic profile of males and females as they developed IBD. Using the IL-10 gene-deficient mouse model of IBD and wild-type mice, urine at age 4, 6, 8, 12, 16, and 20 weeks was collected and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Multivariate data analysis was employed to assess differences in metabolomic profiles that occurred as a consequence of IBD development and severity (at week 20). These changes were contrasted to those that occurred as a consequence of gender. Our results demonstrate that both IL-10 gene-deficient and wild-type mice exhibit gender-related changes in urinary metabolomic profile over time. Some male-female separating metabolites are common to both IL-10 gene-deficient and control wild-type mice and, therefore, appear to be related predominantly to gender maturation. In addition, we were able to identify gender-separating metabolites that are unique for IL-10 gene-deficient and wild-type mice and, therefore, may be indicative of a gender-specific involvement in the development and severity of the intestinal inflammation. The comparison of the gender-separating metabolomic profile from IL-10 gene-deficient mice and wild-type mice during the development of IBD allowed us to identify changes in profile patterns that appear to be imperative in the development of intestinal inflammation, but yet central to gender-related differences in IBD development. The knowledge of metabolomic profile differences by gender and by disease severity has potential clinical implications in the design of both biomarkers of disease as well as the development of optimal therapies.     

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.     

A protective role for human IL-10-expressing CD4+ T cells in colitis

IL-10 is an immunoregulatory cytokine expressed by numerous cell types. Studies in mice confirm that different IL-10-expressing cell subsets contribute differentially to disease phenotypes. However, little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. In this study, we used the previously described human (h)IL10BAC transgenic model to examine the role of hIL-10 in maintaining intestinal homeostasis. Genomically controlled hIL-10 expression rescued Il10(-/-) mice from Helicobacter-induced colitis and was associated with control of proinflammatory cytokine expression and Th17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4(+)Foxp3(+) regulatory T cells specifically within the lamina propria but not other secondary lymphoid tissues. Cotransfer of CD4(+)CD45RB(lo) cells from Il10(-/-)/hIL10BAC mice rescued Rag1(-/-) mice from colitis, further suggesting that CD4(+) T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4(+)CD44(+) T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark acetylated histone H3 in both the human and mouse IL10 locus compared with the spleen. These results provide experimental evidence verifying the importance of T cell-derived hIL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.     

Localized colonic stem cell transplantation enhances tissue regeneration in murine colitis

Many patients suffer from chronic gastrointestinal diseases characterized by chronic inflammation, increased intestinal permeability and visceral pain in which there is no definitive treatment. Adult stem cells have recently been used in various disease states to contribute wound-healing processes. In the current study we investigated the ability of intra-colonic adult stem cells application to heal colonic inflammation in IL-10(-/-) mice with active colitis. The aims of this study were to determine whether intra-colonic infusion of adult colonic stem cells (CSCs) (local stem cell transplantation): (i) restores intestinal permeability; (ii) attenuates visceral hypersensitivity; (iii) heals murine colitis. IL-10(-/-) mice with active colitis were transplanted with adult stem cells. Mice received either a single intracolonic infusion of CSCs or colonic epithelial cells. Two weeks after transplantation, we measured visceral hypersensitivity and intestinal permeability and correlated these with histological improvement of colitis. IL-10(-/-) mice that received stem cell transplantation showed histopathologic evidence of recovery from colitis. Improvement in colitis as graded by pathology scores correlated with restoration of intestinal permeability and decreased visceral hypersensitivity. Intra-colonic administration of CSCs is a potential therapeutic method for treating refractory symptoms in patients with chronic gastrointestinal diseases associated with chronic inflammation and visceral hypersensitivity. This method may be safer and should have far fewer side effects than systemic stem cell administration.     

IL-1 receptor type 1 gene-deficient mice demonstrate an impaired host defense against pneumococcal meningitis

The fatality rate associated with Streptococcus pneumoniae meningitis remains high despite adequate antibiotic treatment. IL-1 is an important proinflammatory cytokine, which is up-regulated in brain tissue after the induction of meningitis. To determine the role of IL-1 in pneumococcal meningitis we induced meningitis by intranasal inoculation with 8 x 10(4) CFU of S. pneumoniae and 180 U of hyaluronidase in IL-1R type I gene-deficient (IL-1R(-/-)) mice and wild-type mice. Meningitis resulted in elevated IL-1alpha and IL-1beta mRNA and protein levels in the brain. The absence of an intact IL-1 signal was associated with a higher susceptibility to develop meningitis. Furthermore, the lack of IL-1 impaired bacterial clearance, as reflected by an increased number of CFU in cerebrospinal fluid of IL-1R(-/-) mice. The characteristic pleocytosis of meningitis was not significantly altered in IL-1R(-/-) mice, but meningitis was associated with lower brain levels of cytokines. The mortality was significantly higher and earlier in the course of the disease in IL-1R(-/-) mice. These results demonstrate that endogenous IL-1 is required for an adequate host defense in pneumococcal meningitis.     

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

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.     

Colonic Inflammation in Mice Is Improved by Cigarette Smoke through iNKT Cells Recruitment

Cigarette smoke (CS) protects against intestinal inflammation during ulcerative colitis. Immunoregulatory mechanisms sustaining this effect remain unknown. The aim of this study was to assess the effects of CS on experimental colitis and to characterize the intestinal inflammatory response at the cellular and molecular levels. Using the InExpose® System, a smoking device accurately reproducing human smoking habit, we pre-exposed C57BL/6 mice for 2 weeks to CS, and then we induced colitis by administration of dextran sodium sulfate (DSS). This system allowed us to demonstrate that CS exposure improved colonic inflammation (significant decrease in clinical score, body weight loss and weight/length colonic ratio). This improvement was associated with a significant decrease in colonic proinflammatory Th1/Th17 cytokine expression, as compared to unexposed mice (TNF (p = 0.0169), IFNγ (p<0.0001), and IL-17 (p = 0.0008)). Smoke exposure also induced an increased expression of IL-10 mRNA (p = 0.0035) and a marked recruitment of iNKT (invariant Natural Killer T; CD45+ TCRβ+ CD1d tetramer+) cells in the colon of DSS-untreated mice. Demonstration of the role of iNKT cells in CS-dependent colitis improvement was performed using two different strains of NKT cells deficient mice. Indeed, in Jα18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Thus, our study demonstrates that iNKT cells are pivotal actors in the CS-dependent protection of the colon. These results highlight the role of intestinal iNKT lymphocytes and their responsiveness to environmental stimuli. Targeting iNKT cells would represent a new therapeutic way for inflammatory bowel diseases.     

Elastin receptor (spliced galactosidase) occupancy by elastin peptides counteracts proinflammatory cytokine expression in lipopolysaccharide-stimulated human monocytes through NF-kappaB down-regulation

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.     

Signal transducer and activator of transcription 3 is the dominant mediator of the anti-inflammatory effects of IL-10 in human macrophages

The signaling mechanism by which the anti-inflammatory cytokine IL-10 mediates suppression of proinflammatory cytokine synthesis remains largely unknown. Macrophage-specific STAT3-null mice have demonstrated that STAT3 plays a critical role in the suppression of LPS-induced TNF-alpha release, although the mechanism by which STAT3 mediates this inhibition is still not clear. Using an adenoviral system, we have expressed a dominant negative (DN) STAT3 in human macrophages to broaden the investigation to determine the role of STAT3 in IL-10-mediated anti-inflammatory signaling and gene expression. Overexpression of STAT3 DN completely inhibited IL-10-induced suppressor of cytokine signaling 3, tissue inhibitor of MMP-1, TNF receptor expression, and the recently identified IL-10-inducible genes, T cell protein tyrosine phosphatase and signaling lymphocyte activation molecule. STAT3 DN also blocked IL-10-mediated inhibition of MHC class II and COX2 expression. In agreement with the studies in STAT3-null mice, overexpression of the STAT3 DN completely reversed the ability of IL-10 to inhibit LPS-mediated TNF-alpha and IL-6 production. However, real-time PCR analysis showed that STAT3 DN expression did not affect immediate suppression of TNF-alpha mRNA, but did reverse the suppression observed at later time points, suggesting a biphasic regulation of TNF-alpha mRNA levels by IL-10. In conclusion, although STAT3 does appear to be the dominant mediator of the majority of IL-10 functions, there are elements of its anti-inflammatory activity that are STAT3 independent.     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

Cytokine and endothelial cell adhesion molecule expression in interleukin-10-deficient mice

The objectives of this study were to quantify cytokine mRNA levels and endothelial cell adhesion molecule message and protein expression in healthy wild-type and interleukin-10-deficient (IL-10(-/-)) mice that develop spontaneous and chronic colitis. We found that colonic message levels of IL-1, IL-6, tumor necrosis factor-alpha, interferon-gamma, lymphotoxin-beta, and transforming growth factor-beta were elevated in colitic mice 10- to 35-fold compared with their healthy wild-type controls. In addition, colonic message levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) were found to be increased 10-, 5-, and 23-fold, respectively, in colitic IL-10(-/-) mice compared with their wild-type controls. Immunoradiolabeling as well as immunohistochemistry revealed large and significant increases in vascular surface expression of colonic ICAM-1, VCAM-1, and MAdCAM-1 in the mucosa as well as the submucosa of the colons of colitic mice. These data are consistent with the hypothesis that deletion of IL-10 results in the sustained production of proinflammatory cytokines, leading to the upregulation of adhesion molecules and infiltration of mononuclear and polymorphonuclear leukocytes into the cecal and colonic interstitium.     

Helicobacter pylori-secreted factors inhibit dendritic cell IL-12 secretion: a mechanism of ineffective host defense

Helicobacter pylori evades host immune defenses and causes chronic gastritis. Immunity against intestinal pathogens is largely mediated by dendritic cells, yet the role of dendritic cells in acute H. pylori infection is largely unknown. We observed the recruitment of dendritic cells to the gastric mucosa of H. pylori-infected mice. Bone marrow-derived dendritic cells from mice responded to live H. pylori by upregulating the expression of proinflammatory cytokine mRNA (i.e., IL-1alpha, IL-1beta, and IL-6). The supernatant from dendritic cells stimulated with H. pylori for 18 h contained twofold higher levels of IL-12p70 than IL-10 and induced the proliferation of syngeneic splenocytes and type 1 T helper cell cytokine release (IFN-gamma and TNF-alpha). These responses were significantly lower compared with those induced by Acinetobacter lwoffi, another gastritis-causing pathogen more susceptible to host defenses. Analysis of whole H. pylori sonicate revealed the presence of a heat-stable factor secreted from H. pylori that specifically inhibited IL-12 but not IL-10 release from dendritic cells activated by A. lwoffi. Our findings suggest that dendritic cells participate in the host immune response against H. pylori and that their suppression by H. pylori may explain why infected hosts fail to prevent bacterial colonization.