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
Introduction Coronavirus Disease 2019 (COVID-19) is an ongoing publichealth crisis that has sickened or precipitated death in millions.The etiologic agent of COVID-19, Severe Acute RespiratorySyndrome Coronavirus 2 (SARS-CoV-2), infects the intestinalepithelium, with viral RNA shed in the stool, and can induce GIsymptoms similar to the human inflammatory bowel diseases(IBD). An international surveillance epidemiology study,SECURE-IBD, reported that the standardized mortality ratiotrends higher in IBD patients (1.5-1.8) and that 5-aminosalicylicacid (5-ASA) therapy correlates with poor outcome. Togetherthese data indicate patients with IBD may represent aparticularly vulnerable population during this COVID-19pandemic. Methods Published datasets GSE75214 and GSE16879 were downloadedand expression levels of select genes were querried usingRStudio. Primary human ileal spheroids (enteroids), derivedfrom healthy donors and patients with Crohn's disease (CD),were grown on 2D transwells until confluent. Cells were thendifferentiated for 3d before infection with a modified vesicularstomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and green fluorescent protein (GFP) for 1 h at amultiplicity of infection (MOI) of ∼0.5. Healthy enteroids weretreated with 10 ng/ml of human Tumor Necrosis Factor alpha (TNF-α) for 24h before infection via the basolateral reservoir or5-ASA 5h before infection via the apical reservoir. 24h afterinfection, cells were processed for immunofluorescence or RNAexpression of select genes by qRT-PCR. Results VSV-SARS-CoV-2 was able to infect both healthy and CDenteroids as determined by co-staining of GFP, indicative ofvirus infection, and the viral receptor ACE2. However, levels ofGFP fluorescence did not correlate with ACE2 expression in CDenteroids. A subset of CD enteroids exhibited enhanced proteaseexpression (TMPRSS2, TMPRSS4, CTSL), each of whichcorrelated with higher viral RNA levels (P=0.04, P=0.002,P=0.006, respectively). In Vero E6 cells, 5-ASA inhibited thereplication of a clinical isolate of SARS-CoV-2 in aconcentration-dependent manner. Treating healthy enteroidswith 5-ASA did not have any effect on viral proliferation, whileTNF-α pretreatment reduced viral RNA. 5-ASA treatment causeda reduction of ACE2 and an increase in CTSL expression. Conclusions Host proteases, particularly the lysosomal protease CTSL,contribute to the infection of CD enteroids and may representnovel therapeutic targets in patients with IBD and COVID-19. 5-ASA modulates the expression of several epithelial genesrelevant to SARS-CoV-2 infection, yet does not alter viralreplication in healthy enteroids.
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, with viral RNA shed in the stool, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study, SECURE-IBD, reported that the standardized mortality ratio trends higher in IBD patients (1.5–1.8) and that 5-aminosalicylic acid (5-ASA) therapy correlates with poor outcome. Together these data indicate patients with IBD may represent a particularly vulnerable population during this COVID-19 pandemic. Methods: Published datasets GSE75214 and GSE16879 were downloaded and expression levels of select genes were querried using RStudio. 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 then differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and green fluorescent protein (GFP) for 1 h at a multiplicity of infection (MOI) of ∼0.5. Healthy enteroids were treated with 10 ng/ml of human Tumor Necrosis Factor alpha (TNF-α) for 24h before infection via the basolateral reservoir or 5-ASA 5h before infection via the apical reservoir. 24h after infection, cells were processed for immunofluorescence or RNA expression of select genes by qRT-PCR. Results: VSV-SARS-CoV-2 was able to infect both healthy and CD enteroids as determined by co-staining of GFP, indicative of virus infection, and the viral receptor ACE2. However, levels of GFP fluorescence 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). In Vero E6 cells, 5-ASA inhibited the replication of a clinical isolate of SARS-CoV-2 in a concentration-dependent manner. Treating healthy enteroids with 5-ASA did not have any effect on viral proliferation, while TNF-α pretreatment reduced viral RNA. 5-ASA treatment caused a reduction of ACE2 and an increase in CTSL expression. Conclusions: Host proteases, particularly the lysosomal protease CTSL, contribute to the infection of CD enteroids and may represent novel therapeutic targets in patients with IBD and COVID-19. 5-ASA modulates the expression of several epithelial genes relevant to SARS-CoV-2 infection, yet does not alter viral replication in healthy enteroids. [Formula presented] [Formula presented]