Diarrhea Is a Hallmark of Inflammation in Pediatric COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogen with enteric tropism. We compared the clinical, biochemical and radiological features of children hospitalized for acute SARS-CoV-2 infection, classified in two groups based on the presence of diarrhea. Logistic regression analyses were used to investigate the variables associated with diarrhea. Overall, 407 children were included in the study (226 males, 55.5%, mean age 3.9 ± 5.0 years), of whom 77 (18.9%) presented with diarrhea, which was mild in most cases. Diarrhea prevalence was higher during the Alpha (23.6%) and Delta waves (21.9%), and in children aged 5-11 y (23.8%). Other gastrointestinal symptoms were most commonly reported in children with diarrhea (p < 0.05). Children with diarrhea showed an increased systemic inflammatory state (higher C-reactive protein, procalcitonin and ferritin levels, p < 0.005), higher local inflammation as judged by mesenteric fat hyperechogenicity (adjusted Odds Ratio 3.31, 95%CI 1.13-9.70) and a lower chance of previous immunosuppressive state (adjusted Odds Ratio 0.19, 95%CI 0.05-0.70). Diarrhea is a frequent feature of pediatric COVID-19 and is associated with increased systemic inflammation, which is related to the local mesenteric fat inflammatory response, confirming the implication of the gut not only in multisystem inflammatory syndrome but also in the acute phase of the infection.

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

Background and aims: The pathophysiology of SARS-CoV-2-associated diarrhea is unknown. Using an experimental model validated for rotavirus-induced diarrhea, we investigated the effects of SARS-CoV-2 on transepithelial ion fluxes and epithelial integrity of human intestinal cells. The effect of the antidiarrheal agent diosmectite on secretion was also evaluated following its inclusion in COVID-19 management protocols. Methods: We evaluated electrical parameters (intensity of short-circuit current [Isc] and transepithelial electrical resistance [TEER]) in polarized Caco-2 cells and in colonic specimens mounted in Ussing chambers after exposure to heat-inactivated (hi) SARS-CoV-2 and spike protein. Spectrofluorometry was used to measure reactive oxygen species (ROS), a marker of oxidative stress. Experiments were repeated after pretreatment with diosmectite, an antidiarrheal drug used in COVID-19 patients. Results: hiSARS-CoV-2 induced an increase in Isc when added to the mucosal (but not serosal) side of Caco-2 cells. The effect was inhibited in the absence of chloride and calcium and by the mucosal addition of the Ca2+-activated Cl- channel inhibitor A01, suggesting calcium-dependent chloride secretion. Spike protein had a lower, but similar, effect on Isc. The findings were consistent when repeated in human colonic mucosa specimens. Neither hiSARS-CoV-2 nor spike protein affected TEER, indicating epithelial integrity; both increased ROS production. Pretreatment with diosmectite inhibited the secretory effect and significantly reduced ROS of both hiSARS-CoV-2 and spike protein. Conclusions: SARS-CoV-2 induces calcium-dependent chloride secretion and oxidative stress without damaging intestinal epithelial structure. The effects are largely induced by the spike protein and are significantly reduced by diosmectite. SARS-CoV-2 should be added to the list of human enteric pathogens.

Spike S1 domain interactome in non-pulmonary systems: A role beyond the receptor recognition

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which, since 2019 in China, has rapidly become a worldwide pandemic. The aggressiveness and global spread were enhanced by the many SARS-CoV-2 variants that have been isolated up to now. These mutations affect mostly the viral glycoprotein Spike (S), the capsid protein mainly involved in the early stages of viral entry processes, through the recognition of specific receptors on the host cell surface. In particular, the subunit S1 of the Spike glycoprotein contains the Receptor Binding Domain (RBD) and it is responsible for the interaction with the angiotensin-converting enzyme 2 (ACE2). Although ACE2 is the primary Spike host receptor currently studied, it has been demonstrated that SARS-CoV-2 is also able to infect cells expressing low levels of ACE2, indicating that the virus may have alternative receptors on the host cells. The identification of the alternative receptors can better elucidate the pathogenicity and the tropism of SARS-CoV-2. Therefore, we investigated the Spike S1 interactomes, starting from host membrane proteins of non-pulmonary cell lines, such as human kidney (HK-2), normal colon (NCM460D), and colorectal adenocarcinoma (Caco-2). We employed an affinity purification-mass spectrometry (AP-MS) to pull down, from the membrane protein extracts of all cell lines, the protein partners of the recombinant form of the Spike S1 domain. The purified interactors were identified by a shotgun proteomics approach. The lists of S1 potential interacting proteins were then clusterized according to cellular localization, biological processes, and pathways, highlighting new possible S1 intracellular functions, crucial not only for the entrance mechanisms but also for viral replication and propagation processes.

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion.

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.