Human MUC2 mucin-producing colonic goblet-like cells secrete the chemokine CXCL8 by activating multiple pro-inflammatory pathways in response to Entamoeba histolytica.

Abstract

The mucus layer produced by highly stressed goblet cells forms a protective shield in the gut to protect the underlying mucosal epithelial cells from external threats. Hypersecretion and depletion of MUC2 mucin from goblet cells is characteristic of symptomatic Entamoeba histolytica (Eh) infections. We hypothesized that MUC2 depleted goblet cells could mount a second line of innate host defence by producing pro-inflammatory cytokines. To investigate this, we determined whether Eh could stimulate pro-inflammatory responses in wild type (WT) high MUC2 mucin-producing goblet-like cells and in CRISPR-Cas9 gene-edited MUC2KO cells. In response to live Eh and soluble Eh proteins, WT and to a lesser extent, MUC2KO cells, produced high levels of CXCL8. Eh temporally induced greater levels of CXCL8 mRNA expression and protein secretion in WT vs MUC2KO cells which was abrogated with alleviation of ER stress with the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). WT cells produced elevated ROS that induced longer half-lives of CXCL8 transcripts which was abrogated with DPI. Western blotting and proteomic analyses revealed that WT, but not MUC2KO cells, were basally primed to respond to external stressors and responded to Eh through rapid activation of the MAPK/ERK, MAPK/p38, and PI3K/Akt pathways, to induce CXCL8. These results suggest that colonic goblet-like cells defend against Eh infections by hypersecreting mucus and to produce the chemokine, CXCL8, to recruit neutrophils.