NF-κB plays a central, proinflammatory role in chronic intestinal inflammation, yet recent work suggests a predominantly protective function for this transcription factor group in some cell types of the intestine. We herein describe the conditional deletion of the NF-κB RelA gene in murine intestinal epithelia and determine its function in homeostatic control of enterocyte proliferation/apoptosis and susceptibility to colonic inflammation. Mice lacking RelA in ileal and colonic enterocytes were born in expected Mendelian ratios, and RelA-null epithelia differentiated normally. Spontaneous intestinal disease and death occurred with low penetrance in neonates lacking epithelial RelA. IκBα and IκBβ were significantly diminished in RelA-null epithelia, and endotoxin challenge revealed elevated p50 and c-Rel DNA binding activity as compared with controls. Deletion of RelA resulted in diminished expression of antimicrobial (defensin-related cryptdin 4, defensin-related cryptdin 5, RegIIIγ) and antiapoptotic, prorestitution genes (Bcl-xL, RegIV, IL-11, IL-18), and basal rates of epithelial apoptosis and proliferation were elevated. Mice lacking colonic RelA were sensitive to dextran sodium sulfate-induced colitis. Although experimental colitis enhanced proliferation in cells lacking RelA, sustained epithelial cell apoptosis precluded mucosal healing and decreased animal survival. We conclude that activation of RelA is required for homeostatic regulation of cell death and division in intestinal epithelia, as well as for protection from development of severe, acute inflammation of the intestine.

A link between colitis and colon cancer is well established, but the mechanisms regulating inflammation in this context are not fully defined. Given substantial evidence that hemostatic system components are powerful modulators of both inflammation and tumor progression, we employed gene-targeted mice to directly test the hypothesis that the coagulation factor fibrinogen contributes to colitis-associated colon cancer in mice. This fundamental provisional matrix protein was found to be an important determinant of colon cancer. Fibrinogen deficiency resulted in a dramatic diminution in the number of colonic adenomas formed following azoxymethane/dextran sodium sulfate challenge. More detailed analyses in mice expressing a mutant form of fibrinogen that retains clotting function, but lacks the leukocyte integrin receptor αMβ2 binding motif (Fibγ390-396A) revealed αMβ2-mediated engagement of fibrin(ogen) is mechanistically coupled to local inflammatory processes (e.g., IL-6 elaboration) and epithelial alterations which contribute to adenoma formation. Consistent with these findings, the majority of Fibγ390-396A mice developed no discernable adenomas, whereas penetrance was 100% in controls. Furthermore, the adenomas harvested from Fibγ390-396A mice were significantly smaller than those from control mice and less proliferative based on quantitative analyses of mitotic indices, suggesting an additional role for fibrin(ogen) in the growth of established adenomas. These studies demonstrate, for the first time, a unique link between fibrin(ogen) and the development of inflammation-driven malignancy. Given the importance of antecedent inflammation in the progression of numerous cancers, these studies suggest that therapies targeting fibrin(ogen)-αMβ2 interactions may be useful in preventing and/or treating this important subset of malignancies.

Guanylate cyclase C (GUCY2C or GC-C) and its ligands, guanylin (GUCA2A or Gn) and uroguanylin (GUCA2B or Ugn), are expressed in intestinal epithelial cells (IECs) and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine if GC-C and its ligands direct the course of intestinal inflammation. Here, we show that DSS-induced clinical disease and histological damage to the colonic mucosa were significantly less severe in GC-C−/− mice and moderately reduced in Gn−/− animals. Relative to wildtype controls, GC-C−/− and Gn−/− mice had reduced apoptosis and increased proliferation of IECs during DSS colitis. Basal and DSS-induced production of resistin-like molecule β (RELMβ) was substantially diminished in GC-C−/− mice. RELMβ is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNFα and IFNγ production was minimal in GC-C−/− animals. RELMβ and cytokine levels were similar to wildtype in Gn−/− mice, however. Colonic instillation of recombinant RELMβ by enema into GC-C−/− mice restores sensitivity to DSS-mediated mucosal injury. These findings demonstrate a novel role for GC-C signaling in facilitating mucosal wounding and inflammation and further suggest that this may be mediated, in part, through control of RELMβ production.

BackgroundGuanylate Cyclase C (GC-C) is an apically-oriented transmembrane receptor that is expressed on epithelial cells of the intestine. Activation of GC-C by the endogenous ligands guanylin or uroguanylin elevates intracellular cGMP and is implicated in intestinal ion secretion, cell proliferation, apoptosis, intestinal barrier function, as well as the susceptibility of the intestine to inflammation. Our aim was to determine if GC-C is required for host defense during infection by the murine enteric pathogen Citrobacter rodentium of the family Enterobacteriacea.MethodsGC-C+/+ control mice or those having GC-C genetically ablated (GC-C−/−) were administered C. rodentium by orogastric gavage and analyzed at multiple time points up to post-infection day 20. Commensal bacteria were characterized in uninfected GC-C+/+ and GC-C−/− mice using 16S rRNA PCR analysis.ResultsGC-C−/− mice had an increase in C. rodentium bacterial load in stool relative to GC-C+/+. C. rodentium infection strongly decreased guanylin expression in GC-C+/+ mice and, to an even greater degree, in GC-C−/− animals. Fluorescent tracer studies indicated that mice lacking GC-C, unlike GC-C+/+ animals, had a substantial loss of intestinal barrier function early in the course of infection. Epithelial cell apoptosis was significantly increased in GC-C−/− mice following 10 days of infection and this was associated with increased frequency and numbers of C. rodentium translocation out of the intestine. Infection led to significant liver histopathology in GC-C−/− mice as well as lymphocyte infiltration and elevated cytokine and chemokine expression. Relative to naïve GC-C+/+ mice, the commensal microflora load in uninfected GC-C−/− mice was decreased and bacterial composition was imbalanced and included outgrowth of the Enterobacteriacea family.ConclusionsThis work demonstrates the novel finding that GC-C signaling is an essential component of host defense during murine enteric infection by reducing bacterial load and preventing systemic dissemination of attaching/effacing-lesion forming bacterial pathogens such as C. rodentium.