Colonic Goblet Cells Produce Augmented Levels of Pro‐inflammatory Cytokines due to Metabolically Stressful MUC2 Mucin Biosynthesis

An intact mucus layer is vital in innate host defense in the gastrointestinal tract to protect the underlying mucosal epithelium from potential pathogens and toxins from the external environment whilst supporting the commensal microbiota. In the colon, goblet cells produce this essential mucus bilayer by secreting MUC2 mucin under a highly ER stressful mechanism. In conditions such as inflammatory bowel diseases and infectious colitis, goblet cells hypersecrete mucins resulting in depletion that disrupts the protective mucus layers. Goblet cells also produce pro-inflammatory cytokines that interact with and activate other arms of the innate immune system in host defence. In this study we hypothesize that ER stress induced by MUC2 biosynthesis and production augment pro-inflammatory responses in goblet cells. The aims of the study are: 1) to determine the pro-inflammatory responses in WT and CRISPR/Cas9 MUC2 KO LS174T human goblet cells and 2) to determine the MAPK signalling pathways by which the pro-inflammatory responses are altered in WT and MUC2 KO goblet cells. mRNA and protein expression of pro-inflammatory chemokines/cytokines were analyzed via RT-qPCR and 15-plex Luminex array, at basal conditions and in response to various mucus secretagogues, including phorbol myristate acetate (PMA, positive control), live Entamoeba histolytica(Eh), and lysed soluble amebic proteins (SAP). To determine the impact of ER stress, the expression of various stress proteins was quantified by Western blotting basally and following treatment with IL-22 to alleviate ER stress. To determine which MAPK signalling pathways were altered in the cell lines resulting in differing pro-inflammatory phenotypes, specific pharmacological inhibitors were used and analyzed by Western blotting. WT goblet cells expressed high levels of the ER stress proteins, ATF4 and GRP78, as compared to MUC2 KO cells, both basally and in response to live Eh. In response to the various mucus secretagogues, PMA, live Eh, and SAP, WT cells expressed higher levels of mRNA transcripts and secreted high amounts of the pro-inflammatory chemokines, interleukin-8 (IL-8), interleukin-13 (IL-13) and monocyte chemoattractant protein-1 (MCP-1) as compared to MUC2 KO cells. Specifically, IL-8 secretion was significantly elevated in both cell lines by mitigating ER stress with IL-22 pre-treatment. In particular, WT cells showed rapid and elevated phospho-ERK MAPK signalling responses, whereas the response in MUC2 KO cells was delayed. These results demonstrate that goblet cells under high metabolic ER stress triggered by MUC2 mucin biosynthesis activates the ERK MAPK signalling pathway basally and in response to inflammatory agonists to produce elevated levels of pro-inflammatory chemokines. In contrast, goblet cells that are genetically deleted of MUC2 showed dysregulated pro-inflammatory responses and altered MAPK signalling that could dysregulate innate host defences analogous to the pathology observed in Muc2-/- littermates.