ABSTRACT
Introduction: Obesity is a major risk factor for severe coronavirus disease, and clinical evidence now supports the GI tract, in addition to the respiratory system, as a potential route for SARS-CoV-2 infection. Expression of viral entry factors ACE2, TMPRSS2, and CTSL have been detected along the human GI tract including gastric, ileal and colonic mucosa. It is unclear whether obesity confers increased susceptibility to initial SARS-CoV-2 infection, or what gut mechanisms in obesity predispose to vulnerability to SARS-CoV-2. Thus, we aimed to investigate, by single cell RNA-sequencing (scRNA-Seq) of human colonic mucosa, whether patients with obesity may be more susceptible to SARS-CoV-2 infection, by virtue of enhanced expression of SARS-CoV2 entry cofactors followed protein assessment in colon biopsies. Methods: We studied 19 patients: 10 lean (age 33±3y, BMI 23±1kg/m2, 90% female), and 9 with obesity (age 43±3y, BMI 36±1kg/m2, 89% female). Human colonic biopsies from lean (n=4 scRNA-Seq;n=6 validation) and obesity (n=6 scRNASeq;n=3 validation) participants were obtained by sigmoidoscopy. Biopsies were dissociated, and viable cells were FACS-isolated. Chromium-10X Genomics was used for scRNA-Seq library prep, followed by Illumina HiSeq4000 sequencing. COVID-19 entry factors displaying significant differential expression between lean and obesity were then validated for gene, and protein expression in the validation cohort using Illumina TruSeq, and quantitative immunofluorescence confocal microscopy, respectively. Results: The initial dataset analysis revealed sequencing of 59,653 cells, 705 million reads, at 127,000 reads per cell. The human colonic mucosa partitioned into 20 cell subsets (Fig1A,B), and 15 of the 20 clusters displayed detectable expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2 (Fig1C,D,E). Goblet cell expression of TMPRSS2 was increased 4.6-fold (p<0.05), stromal cell expression of CTSL was increased 1.2-fold (p<0.0001), and ACE2 expression was increased 1.27-fold (p<0.001) in crypt-top (CT) colonocytes of obesity compared to lean controls (Fig2A). Colonic overexpression of TMPRSS2 mRNA (p<0.05) and protein (p<0.05), and CTSL (p<0.05) mRNA, but not ACE2 mRNA, in obesity was further validated in a second validation cohort (Fig2B-F). Conclusions: scRNA-Seq analysis of human colonic epithelium in obesity compared to healthy controls revealed multiple epithelial cell subsets (goblet cell, stromal, and colonocytes) with overexpression of COVID-19 entry factors TMPRSS2, CTSL, and ACE2, confirming the digestive system as a portal for infection by SARS-CoV-2.Furthermore goblet, stromal, and colonocyte-specific overexpression of TMPRSS2, CTSL, and ACE2 in obesity may play a significant role in increased initial susceptibility to COVID-19, and worse disease outcomes in human obesity.(Figure Presented) Single-Cell RNA-Seq Profiling of Human Colonic Epithelium in Obesity. A) t-SNE plot of single-cell RNA-seq profiles of native human colonic epithelial cells, colored by cluster number and identity, listed by largest to smallest population, and annotated by cluster identity, determined by highest ranking gene marker for the colonic cells clustered and profiled between lean and obesity, where dotted blue, red, and green circles represents goblet cells, stromal cells, and CT colonocytes, respectively. B) Proposed cluster identities based on conserved expression of known markers for annotated cell types. Clusters identified displayed expression of at least one of the COVID-19 entry factors: TMPRSS2, CTSL, or ACE2. The average proportion of cells in annotated clusters expressing, C) TMPRSS2, D) CTSL, and E) ACE2 among all studied participants.