Guanylate cyclase C reduces invasion of intestinal epithelial cells by bacterial pathogens.

The guanylate cyclase C (GC-C) receptor regulates electrolyte and water secretion into the gut following activation by the E. coli enterotoxin STa, or by weaker endogenous agonists guanylin and uroguanylin. Our previous work has demonstrated that GC-C plays an important role in controlling initial infection as well as carrying load of non-invasive bacterial pathogens in the gut. Here, we use Salmonella enterica serovar Typhimurium to determine whether GC-C signaling is important in host defense against pathogens that actively invade enterocytes. In vitro studies indicated that GC-C signaling significantly reduces Salmonella invasion into Caco2-BBE monolayers. Relative to controls, GC-C knockout mice develop severe systemic illness following oral Salmonella infection, characterized by disrupted intestinal mucus layer, elevated cytokines and organ CFUs, and reduced animal survival. In Salmonella-infected wildtype mice, oral gavage of GC-C agonist peptide reduced host/pathogen physical interaction and diminished bacterial translocation to mesenteric lymph nodes. These studies suggest that early life susceptibility to STa-secreting enterotoxigenic E. coli may be counter-balanced by a critical role of GC-C in protecting the mucosa from non-STa producing, invasive bacterial pathogens.

Activation of TGF-ß activated kinase 1 promotes colon mucosal pathogenesis in inflammatory bowel disease.

The etiology and mechanisms for inflammatory bowel disease (IBD) are incompletely known. Determination of new, clinically important mechanisms for intestinal inflammation is imperative for developing effective therapies to treat IBD We sought to define a widespread mechanism for colon mucosal inflammation via the activation of TGF-ß activated Kinase 1 (TAK1), a central regulator of cellular inflammatory actions. Activation of TAK1 and the downstream inflammatory signaling mediators was determined in pediatric patients with ulcerative colitis (UC) or Crohn's disease (CD) as well as in DSS-induced and spontaneous IBD in mice. The role of TAK1 in facilitating intestinal inflammation in murine models of IBD was investigated by using (5Z)-7-Oxozeaenol, a highly selective pharmacological inhibitor of TAK1. We found hyper-activation of TAK1 in patients with UC or CD and in murine models of IBD Pharmacological inhibition of TAK1 prevented loss in body weight, disease activity, microscopic histopathology, infiltration of inflammatory cells in the colon mucosa, and elevated proinflammatory cytokine production in two murine models of IBD We demonstrated that at the early phase of the disease activation of TAK1 is restricted in the epithelial cells. However, at a more advanced stage of the disease, TAK1 activation predominantly occurs in nonepithelial cells, especially in macrophages. These findings elucidate the activation of TAK1 as crucial in promoting intestinal inflammation. Thus, the TAK1 activation pathway may represent a suitable target to design new therapies for treating IBD in humans.

Thrombin drives tumorigenesis in colitis-associated colon cancer.

The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. On the basis of evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/- mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacologic inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration, and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, precancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression.

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.

Guanylate cyclase C limits systemic dissemination of a murine enteric pathogen.

BACKGROUND: Guanylate 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. METHODS: GC-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. RESULTS: GC-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. CONCLUSIONS: This 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.

Murine guanylate cyclase C regulates colonic injury and inflammation.

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 and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine whether GC-C and its ligands direct the course of intestinal inflammation. In this article, we show that dextran sodium sulfate (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 wild-type controls, GC-C(-/-) and Gn(-/-) mice had reduced apoptosis and increased proliferation of intestinal epithelial cells during DSS colitis. Basal and DSS-induced production of resistin-like molecule ß (RELMß) was substantially diminished in GC-C(-/-) mice. RELMß is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNF-α and IFN-γ production was minimal in GC-C(-/-) animals. RELMß and cytokine levels were similar to wild-type in Gn(-/-) mice, however. Colonic instillation of recombinant RELMß 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 RELMß production.

Colitis-associated cancer is dependent on the interplay between the hemostatic and inflammatory systems and supported by integrin alpha(M)beta(2) engagement of fibrinogen.

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 used 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 alpha(M)beta(2) binding motif (Fibgamma(390-396A)), revealed that alpha(M)beta(2)-mediated engagement of fibrin(ogen) is mechanistically coupled to local inflammatory processes (e.g., interleukin-6 elaboration) and epithelial alterations that contribute to adenoma formation. Consistent with these findings, the majority of Fibgamma(390-396A) mice developed no discernable adenomas, whereas penetrance was 100% in controls. Furthermore, the adenomas harvested from Fibgamma(390-396A) 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 show, 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)-alpha(M)beta(2) interactions may be useful in preventing and/or treating this important subset of malignancies.

Loss of T cell and B cell quiescence precedes the onset of microbial flora-dependent wasting disease and intestinal inflammation in Gimap5-deficient mice.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

Differential sensitivity of CYP1A to 3,3',4',4-tetrachlorobiphenyl and benzo(a)pyrene in two Lepomis species.

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3',4'-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.

Loss of epithelial RelA results in deregulated intestinal proliferative/apoptotic homeostasis and susceptibility to inflammation.

NF-kappaB 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-kappaB 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. IkappaBalpha and IkappaBbeta 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, RegIIIgamma) and antiapoptotic, prorestitution genes (Bcl-x(L), 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.