G Protein-coupled Estrogen Receptor:Effects On Intestinal Mucosal Barrier And Immune Inflammation

Objective Inflammatory bowel disease(IBD)is a chronic inflammatory disease including ulcerative colitis(UC)and Crohn’s disease(CD).The incidence of IBD has been rising around the globe,but the etiology remains unclear.It is generally believed that multiple factors such as environment,genetics and intestinal microecology may interact and lead to intestinal imbalance and intestinal mucosal barrier injury.In recent years,accumulating evidence highlights the potential role of estrogens in many autoimmune diseases and intestinal barrier,and a lower prevalence of IBD has been reported in women than in men in Asian countries.Estrogens act via interacting with nuclear receptors ERα and ERβ or G protein-coupled estrogen receptor(GPER).Although the expression of GPER intestinal tissues has been reported,the exact localization and the function of GPER in the gut remain poorly understood.Therefore,this study aims to elucidate the exact distribution of GPER in the intestine,and to further study the potential role of GPER in the regulation of intestinal inflammation and mucosal barrier function.Methods The distribution of GPER in the intestinal tissues of mice,rats and IBD patients was explored by immunofluorescence and by using GPER reporter mice(GPER-cre-tdTomato or GPER-cre-ZsGreen).In addition,GPER protein level in the intestinal tissues of CD patients with different degrees of inflammation was detected by Western blot.The types of GPER positive immune cells were detected by using double fluorescent reporter mice(CX3CR1-GFP/GPER-cre-tdTomato),macrophage/dendritic cell fluorescent reporter mice(CX3CR1-GFP)and immune cell markers(F4/80 or CD 11c).Finally,we compared the severity of DSS-induced colitis and intestinal permeability(FITC-dextran test)between wild-type and GPER-/-mice to explore the effect of GPER on intestinal inflammation and barrier.Results GPER immunofluorescence was detected in intestinal lamina propria and the enteric ganglion in rats.Consistent with the immunohistochemistry,Tomato or zsGreen-labeled cells were also detected in the submucosal and myenteric plexus and in the lamina propria.Interestingly,within the mucosal layer,GPER+cells were seen immediately below the epithelial cells,with higher cell numbers in the colorectum than in small intestine.Immunofluorescence staining showed GPER+cells in the lamina propria were immune-positive for macrophage and dendritic cell marker Iba-1,F4/80 and CD 11c,suggesting that they were macrophages or dendritic cells with antigen presenting functions.In the 3%DSS-induced acute colitis model,GPER-/-mice showed greater weight loss,higher disease activity index,mortality rate and pathological scores.Immunofluorescence staining showed that the distribution pattern of GPER+cells in intestinal mucosa of IBD patients was similar to that of mice and rats,and Western blot showed that the expression of GPER protein was increased in inflammatory tissues.Conclusions GPER is uniquely distributed in the intestine:GPER is not only expressed in certain types of intestinal ganglia,but also expressed in macrophages or dendritic cells in the lamina propria of intestinal mucosa.The expression of GPER in the lamina propria presents a gradient increase from small intestine to large intestine.The protein expression of GPER was positively correlated with severity of inflammation of the intestine in IBD patients,suggesting that GPER plays a role in regulation of mucosal immunity and inflammation.GPER deficient mice showed more severe inflammatory phenotype than wild-type mice in the DSS-indcued colitis model,suggesting that GPER on macrophages or DCs has important anti-inflammatory effect and is a potential intervention target for inflammatory bowel disease.