Giardia‐Induced Alterations to Intestinal Mucus Production Involve Protease‐Activated Receptor‐2‐Mediated Activation of MAPK and Calcium Release

Abstract

IntroductionDisruption of the intestinal mucus layers is associated with increased rates of infection and inflammation and has been implicated in a wide variety of intestinal disorders. Giardia duodenalis has been shown to disrupt mucus production and secretion [1] and may therefore be used as a model system to study regulation of these processes in health and disease.AimsDetermine the mechanisms involved in Giardia mediated disruptions to mucus production by intestinal goblet cells. The roles of protease‐activated receptor‐2 (PAR2) and Giardia cysteine proteases were investigated.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR).Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using fluorescein‐coupled wheat germ agglutinin, and qPCR was performed for Muc2 and Muc5ac.ResultsGiardia isolates NF, S2, and WB, but not GSM, increased MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or MAPK signaling in LS174T cells.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsResults demonstrate that PAR2 plays a significant role in the regulation of mucin gene expression in mice and a human colonic cell line, and indicate a potential pathway in which Giardia cysteine proteases cleave PAR2 at the N‐terminus, activating canonical PAR2 signaling which involves both calcium release and MAPK activation. These pathways in turn contribute to altered mucin gene expression.Support or Funding InformationCrohn's Colitis Canada (CCC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.