ABSTRACT
Introduction: Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium and can persist long after the respiratory infection has cleared. We previously observed that intestinal SARS-CoV-2 infection levels varied by individual donors and did not correlate positively with ACE2, the cognate SARS-CoV-2 receptor. Therefore we aimed to delineate host factors that influence viral infection in the intestine. Methods: Published dataset GSE75214 was downloaded and expression levels of select genes were querried. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn's disease (CD), were grown on 2D transwells until confluent. Cells were differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and GFP for 1h at a multiplicity of infection of ~0.5. Cells were harvested pre-infection and 24h after infection and expression of select genes was performed by qRT-PCR. Expression data were fit to a linear regression model to predict viral RNA levels. Results: Small intestine biopsy samples from CD patients demonstrated a reduction in ACE and an increase in CTSB and CTSL expression during active inflammation compared to healthy controls. Viral RNA expression did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). Expression of these proteases was higher in the pre-infection for the sample subset. Principle component analysis of uninfected expression data demonstrated these samples clustered separately from the others, with the difference driven by TMPRSS2, TMPRSS4, and CTSL. Modeling viral RNA levels based on gene expression revealed expression levels of these proteases are a predictive expression signature. Conclusions: Host protease expression can predict SARS-CoV-2 infection and represent potential therapeutic targets for COVID-19. This is consistent with the recent report showing that cathepsin inhibition reduces SARS-CoV-2 spike-mediated syncytia formation. High expression of these proteases in the intestine may also be a novel biomarker for the risk of intestinal complications associated with COVID-19.(Figure Presented)RNA data from dataset GSE75214 demonstrating reduced ACE2 and increased CTSB and CTSL in patients with Crohn's disease during active inflammation compared to healthy controls. (Figure Presented) Enteroids from healthy control donors and patients with Crohn's disease were grown in 2D transwells and expression of indicated genes was assessed in pre-infection (A) and after infection with VSV-SARS-CoV-2 (B)
ABSTRACT
Patients infected with the SARS-Cov2 virus have been severely affected [1]. The high rate damaged reported by the effects of this virus is due mainly to a storm pro-inflammatory cytokines production that is triggered by immune response overexpression [2]. Thus, research on this virus has become a high priority for public health [3]. However, biosafety regulations hinder rapid advancements on this research line. Therefore, it is essential to find an in vitro model that provides a close approximation to the actual SARS-Cov2 infection. The developed model is based on the detection of anti-inflammatory cytokines produced by Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) as a response of HCV-229E infection at a lung spheroid. For this purpose, a co-culture of lung fibroblast spheroid (MRC-5) constituted in human collagen type 1, infected with HCV229E, and human peripheral blood leukocytes were grown for 3 days, pursuing the generation of the pro-inflammatory signaling cascade. Subsequently, WJ-MSCs were co-cultured within the previous described environment. As a result, it was demonstrated that WJ-MSCs play an immunomodulatory role against HCV-229E infection. After 72 h of WJMSCs exposure a significant decrease in pro-inflammatory cytokines IL(1,2,6,7) and TNF α was observed. Additionally, a very marked increase in anti-inflammatory cytokines such as IL(4,10,11,13) and TGF-β was detected. We conclude that this model could be used for COVID-19 research and other similar respiratory syndromes without the manipulation of the actual virulent strand, this would aid in the understanding of mechanism behind the disease and the use WJMSCS such as therapy.