High MUC2 Biosynthesis and Production Augment Pro-Inflammatory Responses in Colonic Goblet Cells
Date
2023-09-11
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Abstract
Goblet cells are specialized secretory epithelial cells whose primary function is to secrete mucins into the lumen of the gastrointestinal tract to form the mucus layer, which acts as a physical barrier separating luminal contents from the underlying epithelium. During amoebiasis, caused by the human intestinal protozoan parasite, Entamoeba histolytica, goblet cells hypersecrete and become depleted of MUC2 mucin, impairing the protective mucus barrier to allow the parasite to contact the colonic epithelium and induce a robust pro-inflammatory cytokine response. While colonic absorptive epithelial and immune cells have been shown to produce pro-inflammatory cytokines, it is not known if goblet cells do so as well in addition to mucin production. In this study, I investigated if the pro-inflammatory chemokine, CXCL8, was altered in WT and CRISPR/Cas9 MUC2KO human colonic goblet cells in response to E. histolytica. CXCL8 (IL-8) is a potent chemokine that recruits neutrophils to sites of inflammation, and symptomatic E. histolytica infections are marked by robust production of CXCL8 and subsequent neutrophil tissue infiltration. RT-qPCR and ELISA were used to analyze mRNA and protein expression in response to various stimuli. Specific pro-inflammatory pathway pharmacological inhibitors were used to enumerate defects in intracellular signaling, actinomycin D to inhibit transcription, and cycloheximide to inhibit translation. Diphenyleneiodonium chloride (DPI) was used to alleviate reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress and ROS production was measured by the 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) assay. WT goblet cells produced more CXCL8 and ROS than MUC2KO cells in response to amoebic stimulation. Whereas CXCL8 mRNA was reduced by inhibiting multiple pro-inflammatory signaling pathways in WT, only the MAPK/ERK pathway reduced transcription in MUC2KO cells. CXCL8 mRNA from WT was more stable than MUC2KO cells, and this stability was reduced when ER stress was alleviated. This study identified that metabolically stressed goblet cells regulate the expression of CXCL8 in response to E. histolytica by a ROS-dependent mechanism, activating multiple signalling pathways to stabilize transcripts and maximize pro-inflammatory responses against E. histolytica. Importantly, these results unraveled a previously uncharacterized dualistic role for goblet cells- mucus secretion and pro-inflammatory chemokine production- in innate host defence against E. histolytica.
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Kim, A. J. (2023). High MUC2 biosynthesis and production augment pro-inflammatory responses in colonic goblet cells (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.