Fig 2: ER stress and VIP induce a synergistic decrease in membrane NHE3 expression. Tm (5 μM) was applied to the apical side of Caco-2 cells for 24 h, followed by 1 μM VIP applied to the basolateral side for 1 h. Cell surface biotinylation was used to evaluate NHE3 expression at the apical membrane and in total lysates. The histogram represents the relative ratio of the surface over total NHE3 normalized to β-actin. Data are the mean ± SEM from three separate experiments. *** p < 0.001; (2-way ANOVA).

Fig 3: Secretomes of ER-stressed enterocytes induce VIP production in enteric neurons. (A) Xbp1s expression in control and Tm-treated Caco-2 cells. (B) HSP70 expression in the conditioned media of control and Tm-treated cells. (C) Mouse primary enteric neurons were co-cultured with Tm-treated Caco-2 cells for 24 h. The abundance of VIP peptide in the medium of neuronal culture was determined by ELISA. (D) rHSP70-treated mouse enteric neuronal cells upregulate VIP mRNA expression by RT-PCR and (E) VIP concentration by ELISA. Data are the mean ± SEM from three separate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; (two-way ANOVA).

Fig 4: Proposed mechanism of epithelial-enteric neuronal crosstalk in SARS-CoV-2 induced diarrhea. The SARS-CoV-2 infection causes ER stress and the production of DAMPs, which increase the expression and release of VIP by enteric neurons. The presence of ER stress, together with the produced VIP, restricts fluid absorption by downregulating the brush–border membrane expression of NHE3 in enterocytes, resulting in the luminal fluid buildup and increased intestinal motility, which contributes to the onset of diarrhea.