Elucidating the Role of Cyclic GMP in Diarrhoea and Intestinal Inflammation

Abstract

Cyclic guanosine 3’,5’-monophosphate (cGMP) performs a wide range of functions in various cell types and tissues. The cellular levels of cGMP are maintained by the enzymatic conversion of guanosine 5’-triphosphate (GTP) to cGMP by guanylyl cyclases, which can also be membrane-associated receptors. Receptor guanylyl cyclase C (GC-C; gene GUCY2C) is predominantly expressed on the apical surface of the intestinal epithelial cells. GC-C is activated by peptide hormones guanylin and uroguanylin and the heat-stable enterotoxin (ST) produced by enterotoxigenic E.coli that causes traveller’s diarrhoea.

Several disease-causing mutations in GUCY2C have been reported. Patients with gain-of-function mutations show diarrhoea and inflammatory bowel disease (IBD). Several cases of paediatric IBD have also been associated with mutations in GUCY2C. This study addresses the physiological implications of increased cGMP using a transgenic mouse model harbouring the first-identified hyperactive mutation in human patients leading to familial GUCY2C diarrhoea syndrome (FGDS). Mice with hyperactive GC-C showed increased levels of cGMP in intestinal epithelial cells, which led to activation of Cftr and inhibition of Nhe3, resulting in diarrhoea-like symptoms and increased luminal pH and faecal sodium levels. Global transcriptome analysis of the distal colon revealed activation of the interferon signalling pathway, and transgenic mice showed greater susceptibility to DSS-induced colitis.

Histological analysis of the terminal ileum revealed a reduction in functional Paneth cells, goblet cells, and mucus barrier. The barrier integrity of the small intestine was compromised in these mice. Global transcriptome analysis of the terminal ileum revealed a Th1-type gene signature. Immune cell profiling across the gut-associated lymphoid tissue (GALT) showed reduced regulatory dendritic cells and an increased abundance of CD4+ Th cells. Increased levels of Stat1 were observed in the ileal epithelial cells of these mice, along with elevated expression of interferon-stimulated genes.

Small intestinal organoids were prepared from wild type and transgenic mice. The organoids from transgenic mice showed greater swelling in the presence of ST and uroguanylin due to increased fluid secretion into the lumen. Administration of cGMP increased Stat1 phosphorylation in the intestinal organoids. Our observations suggest that high cGMP levels have epithelial cell-extrinsic and cell-intrinsic roles in inducing intestinal inflammation. Furthermore, the administration of zinc inhibited the activity of GC-C and reduced diarrhoea and intestinal inflammation in the transgenic mice. Thus, the similarities observed in these transgenic mice with that of chronic diarrhoea and IBD patients indicate that they can be used as a pre-clinical model to understand the effects of chronically elevated cGMP on intestinal pathophysiology and for identifying novel therapeutic strategies for patients with hyperactivating mutations in GUCY2C.