SARS-COV-2 induced Diarrhea is inflammatory, Ca²⁺Dependent and involves activation of calcium activated Cl channels

Abstract

Diarrhea occurs in 2-50% of cases of COVID-19 (~8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2 ) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike- D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS- D614G exposure to enteroids that were pretreated for ~24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by ~8 min . The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco- 2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of ~10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

SARS-CoV-2 causes secretory diarrhea with an enterotoxin-like mechanism, which is reduced by diosmectite

“…The SARS-CoV-2 spike protein has been shown to lead to the activation of TMEM16 proteins, part of the CaCC family that is essential in syncytia formation from infected pneumocytes [ 23 ]. Modulation of these transmembrane channels may also be involved in the development of secretory diarrhea [ 13 ]. Consistent with this, our results suggest that CaCC is implicated in SARS-CoV-2-induced anion secretion.…”

“…Recently, Donowitz et al. reported CaCC-dependent Cl – secretion induced by spike protein in a preliminary in vitro study using human enteroids from healthy individuals [ 13 ]. Their findings differ from our results in that they only observed a spike-protein-induced electrical response and secretion in the presence of preexisting inflammation (induced by IL-6 plus IL-8 pretreatment), which suggests that an inflammatory response may be a prerequisite for secretion.…”

“…It may be that there are two distinct pathogenic mechanisms involved in SARS-CoV-2-induced diarrhea, the first being Cl – -dependent secretory diarrhea (similar to that involved in rotavirus-associated diarrhea, which causes more mild or moderate symptoms in patients with COVID-19), and the second being characterized by inflammation, as described by Donowitz et al. [ 13 ] (which results in more severe symptoms).…”

“…In the absence of clear causal evidence of structural damage in patients with SARS-CoV-2-associated diarrhea, a functional pathology could be implicated. This hypothesis is supported by evidence of virus-induced dysfunction of ACE2 receptors, leading to an imbalance in membrane ion channels (i.e., dysfunction in Na + -dependent glucose transporter [ 8 ] and Na + /H + exchanger and neutral amino acid transporter [ 12 ] mechanisms, and in Ca 2+ -activated Cl – channels [CaCC] [ 13 ]). SARS-CoV-2 has also been shown to induce the production of reactive oxygen species (ROS) [ 14 ], suggesting a pathogenic mechanism potentially involving alteration of the intracellular redox state and promotion of chloride secretion in enterocytes, as observed for rotavirus [ 15 ].…”

Causal relationship of CA3 back-projection to the dentate gyrus and its role in CA1 fast ripple generation

“…Regarding the G-causality analysis, the two “non-canonical” trisynaptic back-projections caught our attention (CA1 to CA3 and CA3 to DG), in the case of the CA1 to CA3 connection, a recent work has physically mapped this connection, using multiple retrograde viral tracing approaches, several and significant “non-canonical” synaptic inputs to dorsal hippocampal CA3 from ventral CA1, perirhinal cortex, and the subicular complex have been found [ 71 ]. Functional role of this connection remains unclear but it could be related with the auto-associative function of CA3, even though, some of this “non-canonical” inputs appear to have a role in pathological conditions such as epilepsy, particularly, disrupting the local network rhythms and modulating the spike timing by a theta wave “reverse flow”, boosting the propagation of epileptiform events [ 71 – 73 ].…”