ABSTRACT
Background: Diarrhea is present in up to 36.6% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of Damage Associated Molecular Patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. The influence of ER stress and enteric neuronderived vasoactive intestinal peptide (VIP) on the expression of Na+/H+ exchanger 3 (NHE3), an important transporter that mediates intestinal Na+/fluid absorption, was further examined. Methods: SARS-CoV-2 propagated in Vero-E6 cells was used to infect Caco-2, a human colon epithelial cell line that expresses SARS-CoV-2 entry receptor ACE2. The expression of ER stress markers, phospho-PERK, Xbp1s, and DAMP proteins, was examined by Western blotting. Primary mouse enteric neurons were treated with a conditioned medium of Caco- 2 cells that were infected with SARS-CoV-2 or treated with tunicamycin. VIP expression by cultured enteric neurons was assessed by RT-qPCR, Western blotting, and ELISA. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s indicating increased ER stress. Infected Caco-2 cells secreted DAMP proteins, including HSP70 and calreticulin, as revealed by proteomic and Western analyses. The expression of VIP mRNA in enteric neurons was up-regulated after treatment with a conditioned medium of SARS-CoV-2- infected Caco-2 cells (Mock, 1 ± 0.0885;and SARS-CoV-2, 1.351 ± 0.020, P=.005). CD91, a receptor for HSP70 and calreticulin, is abundantly expressed in cultured mouse and human enteric neurons and was up-regulated by a conditioned medium of SARS-CoV-2-infected Caco-2 cells. Tunicamycin, an inducer of ER stress, also induced the secretion of HSP70 and calreticulin, mimicking SARS-CoV-2 infection. Moreover, co-culture of enteric neurons with tunicamycin-treated Caco-2 cells stimulated VIP production as determined by ELISA. Co-treatment of Caco-2 cells with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in the membrane expression of NHE3. Conclusions: Our findings demonstrate that SARS-CoV-2 infection of enterocytes leads to ER stress and the release of DAMPs that up-regulate the expression and release of VIP by enteric neurons. The presence of ER stress together with the secreted VIP, in turn, inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in the enterocytes. These data highlight epithelial-neuronal crosstalk in COVID-19 related diarrhea. (Figure Presented)
ABSTRACT
Background: Up to 36.6% of COVID-19 patients have diarrheal symptoms and 48.1% test positive for SARS-CoV-2 via stool test. The mechanism of SARS-CoV-2-associated diarrhea remains poorly understood. We hypothesize that crosstalk between enterocytes and the enteric nervous system (ENS) plays a critical role in the pathogenesis of COVID-19-associated diarrhea. We studied the effects of SARS-CoV-2 on induction of endoplasmic reticulum (ER) stress and release of Damage Associated Molecular Patterns (DAMPs), which act on enteric neurons and stimulate the production of neurotransmitters. The influence of ER stress and enteric neuron-derived vasoactive intestinal peptide (VIP) on the expression of electrolyte transporter Na+/H+ exchanger 3 (NHE3) was also examined. Methods: SARS-CoV-2 (2019-nCoV/USA-WA1/2020) was propagated in Vero-E6 cells. Caco-2, a human colon epithelial cell line, expresses the essential SARS-CoV-2 entry receptor ACE2 and was thus used for infection (MOI, ~0.01). We used Western blotting to assess the expression of ER stress (phospho-PERK and Xbp1s) and DAMP (HMGB1) markers at 48 hours post-infection. Primary mouse enteric neurons were co-cultured with Caco-2 cells, pre-treated for 24 hours with 2 μM tunicamycin to induce ER stress. Supernatants from enteric neurons were used to assess the expression of VIP by ELISA. Primary enteric neurons were treated with HMGB1 or ATP (another form of DAMPs), and the expression of c-FOS, a marker of neuronal activity, was determined by Western blotting and immunofluorescence staining. Results: We found that SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s. Compared to uninfected control, infected Caco-2 cells secreted HMGB1 into culture media, indicating epithelial production of DAMPs in response to SARS-CoV-2 infection. Tunicamycin was used to induce ER-stress and secretion of HMGB1 by Caco-2, mimicking SARS-CoV-2 infection. Importantly, enteric neurons co-cultured with tunicamycin-treated Caco-2 cells secreted significantly higher levels of VIP. Treating Caco-2 cells with tunicamycin or VIP on the basolateral side led to decreased surface NHE3 expression, suggesting a potential impairment of intestinal electrolyte/fluid absorption. More-over, HMGB1 and ATP both increased the expression of phospho-c-FOS in cultured enteric neurons, indicating DAMP-induced neuronal activation. Conclusions: Our findings demon-strate that enterocytes infected by SARS-CoV-2 release DAMPs with the capacity to induce VIP secretion by the enteric neurons, which in turn acts on enterocytes and inhibits apical localization of NHE3. These findings establish basic mechanisms relevant to diarrheal disease in COVID-19 patients and identify potential targets for the treatment of SARS-CoV-2 infection of the gastrointestinal tract.