key: cord-0813799-ixz6t7gb authors: Krzysztof, Nowak Jan; Christoffer, Lindstrøm Jonas; Rahul, Kalla; Ricanek, Petr; Jonas, Halfvarson; Jack, Satsangi title: Age, inflammation and disease location are critical determinants of intestinal expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in inflammatory bowel disease. date: 2020-05-12 journal: Gastroenterology DOI: 10.1053/j.gastro.2020.05.030 sha: b3eb240255977b2090a3fa3507ed36f2bf6d6c74 doc_id: 813799 cord_uid: ixz6t7gb nan Funding: EU FP7 grant IBD-Character (2858546); the Polish National Science Centre (2017/25/B/NZ5/02783). Author contributions: JKN, RK -data analysis and writing manuscript; JCL -analysis, drafting manuscript; MHV -patient recruitment, consortium management, revision of manuscript, JH -consortium management, patient recruitment and sample collection, revision of manuscript, JS -project design, consortium management, patient recruitment, drafting and revising the manuscript. Although the respiratory tract is implicated as the primary portal of entry of the SARS-CoV-2 coronavirus, gastrointestinal involvement is well-reported, associated with nausea, vomiting, diarrhoea and highly persistent viral particle shedding in faeces 1,2 . There is critical need to determine factors determining susceptibility to COVID-19 in inflammatory bowel disease (IBD) patients. Age, co-morbidity, disease activity and exposure to immuno-modulatory and biological therapies provide the basis for new guidelines for risk stratification and shielding 3 . We hypothesise that expression levels of the SARS-CoV-2 spike protein receptor, angiotensin-converting enzyme 2 (ACE2) 4 may also determine susceptibility to SARS-CoV-2-inflicted damage. Transmembrane serine protease 2 (TMPRSS2) primes the viral spike protein 5 , allowing for the potent binding of ACE2. Both are known to be highly expressed in healthy ileal epithelium, with lower levels in epithelial cells in the colon. We report dysregulated mucosal ACE2 and TMPRSS2 expression in the colon and ileum in IBD, and identify the critical determinants of altered expression. We compared RNA expression of ACE2 and TMPRSS2 in blood (paired-end sequencing), ileal and colonic mucosal biopsies (microarray) from 138 treatmentnaïve IBD patients (cases) and 154 controls, predominantly with functional gastrointestinal disorders. They were recruited at six European centres, between 2012-2015, as part of the IBD Character program (EU Character reference no. ACE2 expression in the terminal ileum in controls was 25-fold higher than in the colon (p=7.0×10 -14 ; Supplementary Table 2) , consistent with previous reports. In IBD, expression in the terminal ileum was increased 10-fold compared with the colon (p=7.9×10 -14 ). In contrast, TMPRSS2 expression in the terminal ileum was lower than in the colon, both in controls (p=3.6×10 -16 ), and in IBD overall (p=6.0×10 - ). Dysregulated ileal gene expression. The expression of ACE2 in inflamed CD ileum was 60% lower (p=0.0175) than in controls (Fig 1A) . Ileal TMPRSS2 was higher in CD non-inflamed tissue than in controls (by 70%, p=0.023, Fig 1B) . Ileal ACE2 did not differ between UC patients and controls, but ileal TMPRSS2 was 30% higher (p=0.023, Fig 1B) . In CD, colonic ACE2 expression was increased by 30% relative to control (p=0.006; Fig 1C) . TMPRSS2 expression in CD colon was similar to controls ( Fig 1D) . In UC, the inflamed colonic mucosa expressed 70% more (p=2.1×10 -11 ) ACE2 transcript copies. UC mucosal ACE2 was 50% higher in inflamed vs non-inflamed sites (p=6.3×10 -5 ). Colonic ACE2 levels associated with Montreal disease extent ( Fig 1E) and the Mayo endoscopic subscore (rho=0.43, p=3.2×10 -5 , Fig 1F) . Colonic TMPRSS2 was upregulated by inflammation (p=0.0179, Fig 1D) , and extent (E1 vs E3: 150%, p=0.0002, Fig 1G) , and was greater by 20% in men (p=0.03). Among IBD patients, colonic ACE2 expression correlated weakly with hsCRP (rho=0.23, p=0.0043), age (rho=0.19, p=0.014, Fig 1H) and serum albumin (rho=-0.17, p=0.037). In controls, colonic ACE2 expression correlated with fecal calprotectin (n=136, rho=0.39, p=2.7×10 -6 ), hsCRP (n=180, rho=0.25, p=0.00083), and age (rho=0.20, p=0.0066). In the control ileum tissue, ACE2 increased with age (rho=0.64, p=0.0099) and was 130% greater in men (p=0.0256). The colonic expression of TMPRSS2, but not ACE2, was 20% higher in smokers from the control group (p=0.0034). We found no important differences in ACE2 and TMPRSS2 expression with regard to recruitment centres. We examined the relationship between mucosal ACE2 or TMPRSS2 and blood expression of TNF, OSM, IL10, TGFB1, GATA3, and STAT6 in IBD. Colonic (and also ileal) ACE2 correlated with blood OSM in patients with UC (rho=0.35, p=0.00076, Fig 1I) . Mucosal angiotensinogen correlated with tissue inflammation (p=4.7×10 -11 in UC colon, p=2.0×10 -5 in CD colon) and disease severity (Mayo sub-score rho=0.58, p=5.3×10 -22 ; Froslie score rho=0.39, p=3.4×10 -9 ). ACE expression in the blood associated negatively with IBD status (in UC p=2.4×10 -6 , in CD p=8.2×10 -5 ) but ACE expression was greater in inflamed UC colon (p=0.019). Renin was detectable in biopsies only, where it was reduced in colonic IBD compared with controls (UC p=1.4×10 -5 , CD p=0.0034). No other factors were implicated. We demonstrate that age, the presence of inflammation and anatomical location are key determinants of expression of ACE2 and TMPRSS2 in patients presenting with IBD. These findings have potential implications for disease management, as well for mechanistic studies. Thus, the inflammation-related increase in ACE2 expression in the colon is consistent with recent mechanistic data highlighting the influence of cytokines on ACE2 expression in the respiratory epithelium 6 . These data raise the possibility that active IBD may enhance viral particle production and uptake in the colon; and furthermore that infection and consequent inflammatory activation may exacerbate colitis. The apparent reduction in ACE2 in the ileum in active Crohn's disease also bears further investigation -this alteration may relate to the loss of epithelial surface in active ulceration, reduced ACE2 production by maturating epithelium, and consequent sampling effects; or may indeed be directly relevant to CD pathogenesis. In summary, we identify age, smoking, and active disease as potential additional risk factors of vulnerability to COVID-19 in IBD patients, through alterations of receptor expression. Our findings lend support to registry initiatives such as SECURE-IBD, which are necessary to monitor the possible impact of COVID-19 on IBD; and to the on-going translational research program characterizing sites for therapeutic intervention in the molecular pathways of SARS-CoV-2 recognition. Evidence for gastrointestinal infection of SARS-CoV-2 Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms Management of IBD during the COVID-19 outbreak: resetting clinical priorities Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor SARS-CoV-2 Receptor ACE2 is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Enriched in Specific Cell Subsets Across Tissues