ABSTRACT
SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, are increasingly recognised. Through immunohistochemistry against the SARS-CoV-2 nucleocapsid protein (NP), we aimed to characterise the multisystem viral tropism of SARS-CoV-2. FFPE tissue was obtained from 16 PCR-confirmed post-mortem COVID cases. Of these cases, 10 were full-body, 5 were brain only and 1 was a brain biopsy. Brain regions studied included frontal cortex, medulla, cerebellum, pons and olfactory bulb. Neurological symptoms featured in the cohort included brainstem encephalitis, acute disseminated encephalomyelitis (ADEM) and brain infarction. Immunohistochemistry of digestive system tissues revealed presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE-2 expression, the entry receptor for SARS-CoV-2 and one of the earliest affected cells in Parkinson's disease (PD). Within the brain, staining was widespread in all sampled regions but limited to endothelial cells only (including in the olfactory bulb). Furthermore, in the full-body post-mortem cases, positivity in brain endothelia was restricted to cases exhibiting multiorgan tissue positivity (3/9 cases). The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days vs mean = 20.3 days, p = 0.0416) suggesting NP detection was confined to the infectious period. Together, our findings provide evidence for enteric nervous system but not brain neuroinvasion of SARS-CoV-2 as well as potential insights into long-term complications of COVID-19 and PD pathogenesis.
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.