Abstract
BackgroundGuanylate 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.MethodsWe 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.ResultsRelative 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.ConclusionsThe 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.
Highlights
Ligand binding to transmembrane guanylate cyclase (GC) receptors initiates cyclic guanosine monophosphate production and activates a variety of cell-type specific signaling cascades [1,2]
Analysis of whole colonic tissue revealed that GC-C2/2 mice were highly responsive and strongly expressed cytokines, chemokines, and immunoregulatory genes, including IFNc, IL-17, IL-22, CXC motif-like (CXCL)5/9/10, and indoleamine 2,3-dioxygenase 1 to a greater degree than wildtype animals (Table 1)
While we have primarily focused on gene expression responses in the colon, we found that small bowel epithelial cell gene expression was affected by LPS challenge (CXCL5 WT 52.762.1 vs. GC-C2/2279.3694.4, p = 0.02; thymic stromal lymphopoietin (TSLP) WT 21.1366.7 vs. GC-C2/2120.9664.2, p = 0.03)
Summary
Ligand binding to transmembrane guanylate cyclase (GC) receptors initiates cyclic guanosine monophosphate (cGMP) production and activates a variety of cell-type specific signaling cascades [1,2]. The best characterized effector protein of GC-C-produced cGMP in intestinal epithelial cells is protein kinase G II (PKG II) which regulates the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ H+ exchanger 3 (NHE3) [6]. Signaling to these membrane channels through GC-C results in ion and water flow into the intestinal lumen and, GC-C is thought to be important for luminal hydration of intestinal contents. We investigated the impact of GC-C activity on mucosal immune responses
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