key: cord-275199-y7b12vml
authors: Suárez-Fariñas, Mayte; Tokuyama, Minami; Wei, Gabrielle; Huang, Ruiqi; Livanos, Alexandra; Jha, Divya; Levescot, Anais; Irizar, Haritz; Kosoy, Roman; Cording, Sascha; Wang, Wenhui; Losic, Bojan; Ungaro, Ryan; Di’Narzo, Antonio; Martinez-Delgado, Gustavo; Suprun, Maria; Corley, Michael J.; Stojmirovic, Aleksandar; Houten, Sander M.; Peters, Lauren; Curran, Mark; Brodmerkel, Carrie; Perrigoue, Jacqueline; Friedman, Joshua R.; Hao, Ke; Schadt, Eric E.; Zhu, Jun; Ko, Huaibin M.; Cho, Judy; Dubinsky, Marla C.; Sands, Bruce E.; Ndhlovu, Lishomwa; Cerf-Bensusan, Nadine; Kasarskis, Andrew; Colombel, Jean Frederic; Harpaz, Noam; Argmann, Carmen; Mehandru, Saurabh
title: Intestinal inflammation modulates the expression of ACE2 and TMPRSS2 and potentially overlaps with the pathogenesis of SARS-CoV-2 related disease
date: 2020-09-25
journal: Gastroenterology
DOI: 10.1053/j.gastro.2020.09.029
sha: 
doc_id: 275199
cord_uid: y7b12vml

Background and Aims The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication within enterocytes. Methods Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation and IBD treatment. Results A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and non-biologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. Additionally, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. Conclusions These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease 2019 (COVID-19) 1 , have evolved into a global pandemic of unprecedented proportions 2 .

Angiotensin converting enzyme-2 (ACE2) is a carboxypeptidase that catalyzes the conversion of angiotensin I into angiotensin 1-9, and angiotensin II into angiotensin 1-7 [3] [4] [5] [6] . ACE2 can also be cleaved by serine proteases such as transmembrane serine protease (TMPRSS) 2, TMPRSS11D and TMPRSS1.

Early events in the pathogenesis of SARS-CoV-2 infection include attachment of the receptor binding domain of the viral spike (S) protein to epithelial ACE2 [7] [8] [9] [10] . The S protein is then cleaved by TMPRSS2, which facilitates viral entry into the cytoplasm of the host cell 11 . Following infection with SARS-CoV, ACE2 is downregulated in the lungs, resulting in unopposed renin-angiotensin-aldosterone system (RAAS) and contributing to disease severity 12, 13 .

Inflammatory bowel diseases (IBD) encompassing Crohn's disease (CD) and ulcerative colitis (UC) are chronic, inflammatory disorders of the gastrointestinal (GI) tract that are treated with conventional immunosuppressive drugs such as corticosteroids, biologic therapies and immunomodulatory drugs 14, 15 . Given that SARS-CoV-2 co-opts receptors expressed by intestinal epithelial cells, COVID-19 has the potential to intersect with the pathogenesis of IBD and by extension, its treatment, at a number of points 16, 17 . For example, ACE2 expression may be potentially be altered during gut inflammation or by IBD medications. Further, immunomodulatory drugs used in IBD therapeutics 14, 15 could potentially be used in COVID-19 patients to manage the "cytokine storm" associated with severe disease. Therefore, in this study we systematically examined potential areas of intersection between the uninflamed and inflamed GI tract and COVID-19 disease. The results of this study may improve our molecular understanding of how COVID-19 intersects IBD and may provide a rationale for further investigation of drugs used in IBD therapeutics for use in patients with COVID-19.

Specimens were obtained via clinical endoscopy during routine care (Table S1, S2). Tissue was formalin fixed and paraffin embedded by the clinical pathology core at our institution. Primary antibodies used included ACE2 (abcam-ab15348, 1:1000), EPCAM (abcam-ab228023, prediluted) and mouse anti- ii. The RISK cohort 18 : ACE2 and TMPRSS2 in treatment-free pediatric CD (<17 years of age) patients was studied using RNA-seq expression profiles from GSE57945, which includes ileal biopsies from endoscopically defined inflamed samples (n=160), non-inflamed (n=53) and non-IBD controls (n=42).

i) The GSE100833 series which includes expression profiles from the gut of 80 anti-TNFα refractory CD patients and the blood from 226 patients enrolled in a phase 2b crossover trial (CERTIFI trial) with ustekinumab 19 (detailed in supplementary methods).

ii)The GSE73661 series includes gene expression profiles (Affymetrix Human Gene 1.0 ST arrays) from colonic biopsies from moderate-to-severe UC patients enrolled in two Vedolizumab efficacy trials (GEMINI-I and GEMINI LTS) 20 (detailed in supplementary methods). GSE73661 series also included 12 non-IBD colonic biopsies and colonic biopsies from 23 UC patients before and 4-6 weeks after first infliximab treatment. Response was defined as endoscopic mucosal healing.

BGRNs can capture fundamental properties of complex systems in states that give rise to complex (diseased) phenotypes 19 . BGRNs were generated from intestinal biopsy RNA sequence data (MSCCR, using intestinal expression QTL information (eQTLs) as priors). The BGRNs were region-(ileum or colon/rectum) and disease-(CD, UC, and control) specific and included both inflamed and uninflamed biopsies and were constructed using RIMBAnet software 19 and visualized using Cytoscape 3.7 21 . We also used two publicly available BGRNs from the RISK and the CERTIFI cohort 19 (supplementary methods).

ACE2 and TMPRSS2 subnetworks: Gene-centric subnetworks were generated by selecting either ACE2 or TMPRSS2 from various BGRNs and expanding out three to five layers (undirected) to obtain the nearest J o u r n a l P r e -p r o o f 7 ACE2 or TMPRSS2 neighbors. The connected subnetworks obtained were generally between 200-500 genes in total.

We curated RNA-seq based IBD and COVID-19 response signatures by identifying differentially expressed genes (DEGs, supplementary methods). Genes differentially expressed in blood 22 , lung NHBE/A549 23 or human small intestinal organoids 24 (hSIO) following SARS-CoV-2 infection; IBD inflammation; or response to medications were separately projected onto various BGRNs allowing for 1 or 2 nearest neighbors depending on the signature sizes. The most connected subnetworks were then extracted to generate model-specific SARS-CoV-2 infection-; IBD inflammation-; or drug-responseassociated subnetworks (supplementary methods).

Gene subnetworks were tested for functional enrichment using a Fisher's exact test with Benjamini-Hochberg (BH) multiple test correction on a collection of genesets. The collection of genesets included i) Reactome pathways sourced from Enrichr 25 , i) gene sets from Smillie et al 26 , ii) Huang et al 27 , iii) various macrophage perturbations (e.g. cytokines) 28 , iv) ACE2 co-expressed genes 29 and v) reported IBD GWAS genes (see supplementary methods). Pathway and geneset enrichment, as well as intersection between networks were tested using a Fisher's exact test and p-values were adjusted for multiple hypothesis with BH correction.

Key driver analysis (KDA) identifies key or "master" driver genes for a given gene set in a given BGRN 37 (supplementary methods). Genesets for KDA included those associated with NHBE-COVID-19 infection or IBD inflammation. Key driver genes (KDGs) were summarized by frequency across the networks.

COVID-19 response gene expression (from whole blood or epithelial models) was evaluated in the context of IBD related inflammation using gene set variation analysis (GSVA). For each COVID-19 response signature, a sample-wise enrichment score was quantified from each transcriptomic profile in the MSCCR and CERTIFI cohorts using GSVA. COVID-19 response GSVA scores were then modeled to test the association with patient-derived phenotypic information.

Healthy gut segments express ACE2 and TMPRSS2 proteins J o u r n a l P r e -p r o o f 8 To define the localization and distribution of ACE2, immunofluorescence (IF) microscopy was performed on histologically normal GI tissue in 20 adults (9 males, 11 females) and 11 children (7 males, 4 females) ( Figure 1 and Table S1 ). ACE2 expression was observed on the small intestinal surface epithelium in all subjects in a continuous distribution with the exception of occasional breaks representing the mucin from goblet cells (Figure 1a-b) . In all examined small intestinal segments, ACE2 could be detected on the crypt epithelium, though to a lesser extent than on the surface epithelium. TMPRSS2 expression was less abundant in the small bowel, and when detectable, was exclusively found on crypt epithelium ( Figure   S1a -b). There was no observable age or sex dependence of ACE2 or TMPRSS2 protein expression in the small bowel.

In the colon, ACE2 expression was patchy and could not be identified in every subject, in contrast to the small bowel (Figure 1c) . This inconsistency across different donors could not be readily associated with age, sex, ACE inhibitor treatment, nor the colonic segment being examined. In contrast, TMPRSS2 expression was more robust in the colon and was readily detectable on both surface and crypt epithelia ( Figure S1c) . Thus, ACE2 in the healthy gut is higher in the small bowel than the large bowel and inversely, expresses more TMPRSS2 protein in the colon compared to the small bowel.

Next, we examined ACE2 and TMPRSS2 mRNA levels in the intestine of non-IBD controls and IBD patients with active and inactive disease enrolled in MSCCR ( Table S3) . As the number of samples for the colon non-rectum locations was low (Figure S3a /b) and no discernable differences were observed, colon non-rectum biopsies were grouped together to increase statistical power. Consistent with the protein data, ACE2 gene expression was higher in the uninflamed ileum compared to the uninflamed colon or rectum. With inflammation, ileal ACE2 mRNA expression was significantly decreased compared to either uninflamed biopsies from IBD patients or non-IBD controls. In contrast, in the rectum ACE2 mRNA expression was increased with inflammation when compared to the uninflamed IBD patients or non-IBD controls (Figure 2a, upper panels) . There were no significant differences by disease location or between patients with UC versus CD ( Figure S4) . We further validated these results using the pediatric IBD RISK cohort 18 where ACE2 mRNA was significantly decreased in the ileum of patients with active IBD as well (Figure 2b) .

The expression of TMPRSS2 in the MSCCR cohort was moderately higher in the colon compared to ileum. In both ileum and colon biopsies TMPRSS2 expression was found significantly increased in the inflamed relative to non-inflamed samples, although the effect sizes were small (Figure 2a (Table S2) , we could not appreciate differences in ACE2 expression in the ileum, which possibly stemmed from the elevated physiological expression of ACE2. In the colon, patchy epithelial ACE2 expression from control non-IBD controls increased in IBD patients with inflammation and this increase was mostly evident on the crypt epithelium ( Figure S2a ).

In the ileum, low intensity TMPRSS2 expression on the crypt epithelium was also comparable in inflamed or uninflamed mucosal segments from IBD patients and in non-inflamed mucosal segments from non-IBD controls. In the colon, TMPRSS2 expression appeared comparable in IBD patients and non-IBD controls. In the rectum, TMPRSS2 expression was enhanced by inflammation ( Figure S2a ).

Age and gender but not smoking increases ACE2 mRNA in the colon ACE2 mRNA was higher with age in the uninflamed rectum samples. However, these effects were essentially nullified in the presence of inflammation. A positive association with age in inflamed CD ileum and a negative association in inflamed UC rectum biopsies was observed. ACE2 mRNA in the uninflamed rectum was significantly lower in males versus females but no gender associations in TMPRSS2 mRNA levels were found (Figure S4b-c) . The expression of ACE2 and TMPRSS2 was similar when comparing active smokers to non-smokers, either between healthy controls or IBD patients (data not shown) and no significant interactions with inflammation status, region or other covariates were found. Thus, age and gender, but not smoking modulates ACE2 but not TMPRSS2 mRNA expression in the IBD colon.

We further evaluated the impact of non-biologic and biologic medication use (self-reported) on the expression of ACE2 and TMPRSS2 mRNA (Table S3) In the ileum of IBD (CD) patients, corticosteroid, thiopurine or 5-aminosalicylate use had no impact on ACE2 mRNA expression in either inflamed or uninflamed biopsies (Figure 3a-c) . In the rectum, however, a significant decrease in ACE2 mRNA expression was observed with corticosteroid use in inflamed biopsies. A similar decrease of ACE2 mRNA was noticed in thiopurine-treated non-inflamed samples from the rectum. The use of corticosteroids, thiopurine or 5-aminosalicylate did not significantly affect TMPRSS2 mRNA expression in ileum samples. However, each of these three medications significantly decreased TMPRSS2 mRNA expression in inflamed rectum or colon samples. Thus, corticosteroid, thiopurine or 5-aminosalicylate attenuated ACE2 and TMPRSS2 mRNA expression in inflamed colon and rectum.

We also defined the effect of current anti-TNF therapy (either adalimumab or infliximab) use on ACE2 and TMPRSS2 (Figure 3d ) expression. In the ileum, patients taking anti-TNF biologics did not show significantly different ACE2 or TMPRSS2 mRNA expression compared to those not on anti-TNF medication. In the large intestine, anti-TNF users showed increased ACE2 and TMPRSS expression, particularly in the inflamed rectum. Since the use cross-sectional cohort to study treatment effect has its limitations, we utilized published datasets from clinical trial cohorts, where longitudinal patient sampling was performed and information on endoscopic responses to treatment was available.

The results from a patient cohort treated with infliximab or vedolizumab on the GEMINI LTS trial 20 are summarized (Figure 4a and S5). Compared to baseline ACE2 expression, post-infliximab (week 6) colonic ACE2 gene expression was significantly lower and this decrease was observed predominantly in endoscopic responders ( Figure S5 ). In contrast, post-vedolizumab (week 6) ACE2 gene expression did not significantly change, although a nominally significant decrease was observed in endoscopic responders (Figure 4a and S5) . Finally, neither vedolizumab nor infliximab modified TMPRSS2 expression.

To study the impact of IL-12/IL-23-targeting ustekinumab on ACE2 and TMPRSS2 mRNA levels in IBD, we used the CERTIFI trial cohort 19, 30 . We first confirmed that, as in our MSCCR cohort, ACE2 gene expression was higher in uninflamed ileum compared to the colon regions ( Figure S6a ).

Next, we observed that ileal ACE2 gene expression was significantly decreased with inflammation as was observed in the ileal samples from both MSCCR and RISK cohorts ( Figure S6a ). In colonic and rectal samples, a trend to increase ACE2 expression in inflamed biopsies as compared with non-inflamed was observed ( Figure S6a ). To identify potential functions associated with ACE2 and TMPRSS2 in the gut, we studied these genes in the context of Bayesian gene regulatory networks (BGRN). These probabilistic graphical models consider all available trait data (gene expression and genotype) simultaneously in order to derive gene:gene casual relationships amongst thousands of intermediate molecular traits 31 .

The nearest neighbors of ACE2 were extracted including genes within either 3 to 5 path lengths in each BGRN network (5 networks total) keeping the subnetwork sizes relatively similar (~200-300 genes, Figure 5a , 5d, Table S7 ). Immediate neighbors of ACE2 in the ileum CD network included SLC6A19 (a known interacting partner of ACE2 32 ), and other SLC transporters but also other viralassociated receptor proteins, like DPP4 and EZR 33 . A summary of the recurring genes across subnetworks is shown (Figure 5b, 5e) .

Functional enrichment of ACE2-associated subnetwork was interrogated in the ileum (Figure 5c) or colon (Figure 5f ) using the Reactome database. Identified metabolic pathways included SLC-mediated transport; xenobiotic metabolism; vitamins and cofactors; and hexose transport. Additional ileumassociated pathways included amino acid and oligopeptide SLC transporters, while colon-associated networks included interferon and immune signaling.

To support our network approaches we verified a significant overlap was observed between the colonic ACE2-associated subnetworks and genes reported to be correlated with ACE2 expression in colonocytes 29 ( Figure S7a ). In addition, ACE2-subnetworks were significantly enriched in expression profiles associated with epithelial cell types, including enterocytes and absorptive cells 26, 27 (Figure S7b c). Colonic ACE2-associated subnetworks also co-enriched in immune cell types as well as macrophage gene signatures following various cytokine perturbations including IFNγ/β 28 ( Figure S7d) . In summary our ACE2-subnetworks are a novel source of insight into the regulation and function of ACE2.

To address the function of TMRPSS2, the subnetworks associated with TMPRSS2 were extracted from the BGRNs (Figure S8a ) allowing three or four layers to obtain similar subnetworks sizes (~200-300 genes). TMPRSS2 was not found in the BGRN from the ileum of controls, likely due to a low expression variance, which is a filter used before BGRN network construction (Table S13) (Figure S8c) . Consistent with these functions, we observed enrichment of the TMPRSS2 subnetworks in genesets associated with enterocytes, goblet and secretory cells ( Figure   S9 a-b) .

COVID-19 is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. Therefore, apart from alterations in the receptor expression, we investigated additional areas of overlap between COVID-19 responsive pathways and pathways associated with IBD. As the first step, we examined a host molecular response signature generated following SARS-CoV-2 infection of a primary human lung epithelium (NHBE) cell and a transformed lung alveolar cell line (A549) 23 . Using GSVA, we generated a per-sample score summarizing expression of either up or down-regulated COVID-19-responsive genes, and evaluated differences in these scores according to intestinal region, disease and inflammation status (Figure 6a and S10 a-b) .

as compared to uninflamed regions or non-IBD control subjects, an observation confirmed in the CERTIFI CD cohort (Figure S10c-d) . We directly compared the genes associated with response to lung cell SARS-CoV2 infection and various IBD-centric genesets generated in our MSCCR cohort 34 or IBD GWAS genes. We observed that genes: up-regulated with inflammation, or positively associated with macroscopic or microscopic measures of disease, or associated with the risk of IBD, were significantly enriched with genes up-regulated by SARS-CoV2 infection of lung epithelial cells ( Figure S10e ).

Next, we examined for the congruence of COVID-19 related peripheral blood gene responses 32 and active IBD inflammation. We observed that genes up-regulated in the blood of COVID-19 infected patients have significantly higher expression in the blood of IBD patients as compared to healthy control blood (Figure 6b and Figure S11a ) as well as in patients with active IBD versus quiescent IBD (Figure   6b and Figure S11b ).

To further interrogate the molecular congruence between SARS-CoV-2 infection and IBD inflammatory responses, we generated NHBE-COVID-19 associated subnetworks (NHBE-COVID_subnet) and IBD inflammation associated subnetworks (IBD_Inf_subnet) from three IBD BGRNs (Table S17) . We then determined the overlap between them, with the rationale that common gene membership implies similar molecular pathobiologies. We observed a significant overlap between IBD-Inf and NHBE-COVID_subnets across all three independent networks tested (Figure 6c ).

J o u r n a l P r e -p r o o f 13 Interestingly, the common genes (148 in ileum CD, 213 in Colon UC and 170 in Colon CD networks) were significantly enriched in candidate IBD GWAS genes (Table S18) .

To determine the underlying shared patho-biological mechanisms we evaluated enrichment of the three intersecting subnetworks against Reactome pathways. Interestingly, although subnetworks were generated by projecting a COVID-19 response signature generated in an epithelial-based model the majority of the pathways and cell type enrichments were immune oriented with the most striking related to innate immune signaling via interferon and interleukin-6 (Figure 6d, Figure S12 , Table S19 ). This is consistent with the fact that while SARS-CoV-2 infection initiates within the epithelium, COVID-19 is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. Our COVID-19 associated gut subnetwork findings are consistent with a large body of recent data that describe a dramatic up-regulation of pro-inflammatory cytokines in patients with COVID-19 35 including induction of IFN-stimulated genes 23 .

We confirmed these observations using the recent dataset where SARS-CoV-2 was shown to infect hSIOs 24 (Figure S13a-b) . Direct overlap of genesets from the SARS-CoV-2 infected hSIOs and various IBD-centric genesets showed significant gene enrichments, similar to those observed between the lung-COVID-19 model and IBD (Figure S13c) . Finally, subnetworks generated in the ileum CD using hSIO-COVID-19 responsive genesets (Table S20 ) overlapped with IBD-Inf associated subnetworks. The intersecting genes were similar to those found between IBD-Inf and NHBE-COVID19-associated ileum CD subnetworks, containing many interferon-stimulated genes (Figure S13d-e) .

Next, as a data reduction approach, we evaluated each of the NHBE-COVID-19 and IBD-Inflammation molecular response subnetworks for key driver genes (KDGs). We then summarized the KDGs across the three BGRNs and determined those shared by IBD inflammation and NHBE-COVID-19 molecular responses ( Figure S14 , Table S21 ). The shared KDGs identified were interferon-stimulated genes such as IRF1, GBP1/2, PARP14/9 and several of these were replicated in BGRNs from the RISK and CERTIFI cohort including CXCL1, GBP4, PARP9 amongst others (data not shown).

We also compared the intersections between COVID-19 associated genes and those associated with three different murine models of intestinal injury or inflammation. We observed a significant overlap 

14 Finally, we generated colonic or ileal gene subnetworks associated with response to infliximab or ustekinumab (Table S23 ) and tested their enrichment in the tissue and IBD-type specific subnetworks and observed significant overlap between many of the genes associated with IBD therapy and either IBDinflammation (used as positive control) or COVID-infection (Figure 6c) indicative of commonality between COVID-19 and response to IBD treatment. We also evaluated changes in the activity of the blood COVID-19 response signature in the blood of CERTIFI CD patients treated with ustekinumab and observed that COVID-19 up-regulated genes significantly decreased after 4 weeks of ustekinumab treatment (Figure 6f) .

Altogether, through analyses of multiple genesets, we observed a significant overlap between COVID-19 response genes and genes associated with IBD response.

The objective of this study was to systematically determine the molecular intersections between COVID-19-associated inflammation, IBD and immunomodulatory drugs. Our data provides mRNA-and proteinlevel evidence of the regional distribution of ACE2 and TMPRSS2 across different parts of the GI tract and the impact of the commonly used IBD medications on ACE2 and TMPRSS2 expression in the inflamed and uninflamed intestines. Additionally, our data highlight an overlap between COVIDresponsive pathways and pathways associated with IBD inflammation. These findings generate the possibility that some of the current and emerging therapies in IBD may be of benefit in patients with COVID-19.

In exploring the intersections between COVID-19 and IBD, we initially considered a) impact of inflammation on ACE2 expression and TMPRSS2 expression in the intestines; b) impact of IBD medications on ACE2 and TMPRSS2 expression. ACE2 has a less appreciated RAAS-independent role in the intestine by promoting amino acid absorption. Consistent with this function we observed high levels of ACE2 expression in the small bowel brush border [39] [40] [41] supportive of the role of ACE2 in mucosal homeostasis 40 . Remarkably, the analyses of gene:regulatory networks empirically derived from the terminal ileum, showed that ACE2 was co-regulated with SLC6A19, an amino acid transporter that physically interacts with ACE2 42 . The colon-derived ACE2 subnetwork would also suggest, a potential metabolic role for ACE2 in the colon including solute carrier dependent processes.

While the physiological function of TMPRSS2 remains largely elusive, it has been linked to epithelial sodium channel (ENaC) regulation 43 We have observed a reduction in ACE2 expression in the inflamed ileum. Since ACE2 appears to be a brush-border enzyme, its reduction with inflammation in the ileum is consistent with loss of expression of other brush border enzymes during enteritis 48 . That said, even during inflammation, the expression of ACE2 remains significant in the small intestines. In contrast, inflammation associated with IBD enhances the expression of ACE2 and TMPRSS2 in the rectum. Overall, modulation of ACE2 and TMPRSS2 by IBD-associated inflammation is complex and appears to be region-specific in the intestines.

Akin to the impact of inflammation on intestinal ACE2 and TMPRSS2 expression, the effect of IBD medications was complex. Corticosteroids, thiopurines or 5-aminosalicylates did not significantly affect TMPRSS2 mRNA expression in the ileum. However, each of these three medications significantly decreased TMPRSS2 mRNA expression in inflamed rectum or colon samples. Additionally, the impact of IBD medications like TNF inhibitors may vary by the stage of treatment. One may speculate that in early stages of therapy, anti-TNF drugs, similar to anti-IL-12/23 agents, may increase the expression of ACE2 in the intestines and potentially have a detrimental effect. However, these medications could have a beneficial effect in the long run due to their ability to reduce inflammation.

Importantly, while SARS-CoV-2 infection initiates with the viral attachment to ACE2 and its cleavage by TMPRSS2 within the epithelial surfaces 7-10 , COVID-19 is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. Therefore, apart from alterations in the receptor expression, we investigated additional areas of overlap between COVID-19 responsive pathways and pathways associated with IBD.

A number of such intersections appeared. IL6, CXCL1/2/5, PDPN, S100A8/A9 which were upregulated following SARS-CoV-2 infection of primary human lung epithelium (NHBE) 23 were also significantly upregulated in the inflamed intestines of patients in our IBD cohort (MSCCR). Similarly, a peripheral blood gene signature from COVID-19 patients 22 which included the upregulated genes, CLEC4D, S100A8/A9 and FCAR, were also significantly upregulated in the blood of MSCCR IBD patients (as compared to controls) and in patients with active IBD (as compared to patients with quiescent disease). Additionally, a number of SARS-CoV-2-associated genes were upregulated in murine models of intestinal injury with DSS 36 (DAPP1, PDPN, IL1RN, DUOX2, IL1B , S100A9, CXCL2, CXCL3) or TNBS 37 (MARCKSL1, IFITIM3, IFITIM1, C3, AGR2, REG4 ) or adoptive T cell transfer colitis model 38 (TAP2, MARCKSL1, SLP1, PARP9, IFITIM3, MMP13, IL1B, S100A8) . These genes relate to a number of IBD-relevant pathways including those associated with inflammatory cytokine signaling (including IL-6, J o u r n a l P r e -p r o o f 16 IL-1, IFN-G), chemokine signaling, but also with interferon-associated pathways, regulation of complement cascade, G protein coupled receptor (GPCR) signaling as well as collagen degradation.

Having observed significant molecular intersections between COVID-19-and IBD-associated pathways, we next examined the impact of biologic medications used in IBD therapeutics where pre-and post-treatment transcriptomic data was available. Interestingly, our network analyses had identified a number of shared COVID-19 and IBD-associated 'key driver genes', including CXCL1, GBP4, SOCS3, PARP14 and PARP9. Importantly, we observed that these KDGs which were up-regulated with COVID-19 and IBD inflammation, were all down-regulated following infliximab treatment. Thus, the impact of IBD medications on attenuating some of the key inflammatory genes and pathways would be independent of their complex effects on the expression of ACE2 and TMPRSS2 on enterocytes.

Given the unregulated inflammatory responses in patients with severe COVID-19, it has been argued that targeted use of anti-inflammatory medications be considered as a therapeutic option 49 . This approach has been met with variable success. While the use of dexamethasone has provided a striking mortality benefit 50 , the results of trials using anti-IL-6 have been equivocal (NCT04320615) and

NCT04372186. An anti-TNF trial is currently underway in the UK (NCT04425538). Our data suggest that anti-IL-12/23 therapy could also be considered in the therapeutic armamentarium in patients with COVID-19. Reassuringly, real-time data from a registry of IBD-COVID patients 51 did not report significant adverse outcomes associated with the use of biologic medications including anti-TNF and anti-IL-12/23 inhibitors. To the contrary, the risk of severe COVID-19 was found to be reduced in IBD patients on anti-TNF inhibitor medication 51 . This is consistent with the observation that individuals treated with cytokine inhibitors had lower rates of SARS-CoV-2 seroprevalence compared to controls. 52 In summary, through detailed analyses of intestinal tissues in health and IBD, we conclude that high expression of ACE2 and TMPRSS2 potentially supports local, GI-associated replication of SARS-COV-2. Further, a number of overlapping inflammatory pathways between COVID-19 and IBD are noted. These data support the use of specific anti-inflammatory agents in the treatment of patients with COVID-19. Table S3 and S4. Under the model, we estimated the change in ACE2 or TMPRSS2 gene expression between the medicated and non-medicated group according to disease subtype (CD, UC) region (ileum, colon, rectum) and tissue type (inflamed, non-inflamed). Samples sizes are in Table S5 . (Table S8 for full results). (Table S17 and S18). The three ileum-associated CD networks are shown for example. Venn diagrams of the overlap in subnetwork genes including the fold enrichment (FE) and a p-value of the enrichment test are shown.

Using the BGRNs drug response subnetworks were generated (See supplementary methods) for infliximab and ustekinumab response genes. The bar graph summarizes the FE for the overlaps between the drug response and either the i) COVID-19 subnetworks; ii) IBD inflammation subnetworks or iii) the intersecting nodes between COVID-19 and IBD-Inf subnetworks. A full table of network nodes and enrichment results can be found in Table S23 and S24 (D) The 3 sets of genes in the intersecting subnetworks from C were interrogated for enrichment in the Reactome database. Heatmap depicting the FE in pathways (BH adj p<0.1 and minimum 5-fold enrichment, Table S19 ). anti-TNF Inf n/a n/a n/a n/a CERTIFI RISK n/a n/a Rectum ACE2 TMPRSS2 Inf Inf Non.I n/a n/a n/a n/a n/a n/a Tissue location and in ammation state BEND7  CCT8P1  CD82  CHRNA7  CLCN5  EGFR-AS1  ENSG00000245958  ESPN  FADS6  GNA11  HAAO  HNRNPA1P9  JDP2  LRRC75A  MS4A8  MUC17  NBPF7  NSMF  NUCKS1  OIT3  PLB1  RHOU  SERPINA1  SLC15A1  SLC36A1  SLC7A7  SLC9A3R1  TCN2  TMEM117  TMEM150B   ACSL5  ACY3  ADGRG6  ANPEP  CA13  CNDP2  DGAT1  ENTPD7  MEP1B  MGAM2  PLEKHS1  SAT2  SEC16B  SLC46A3  SLC5A1  TKFC  TMEM92 d. e. f.

Adj P Value * = 0.1 -0.05, **=0.05-0.01, ***>0.01 ACE2 associated Subnetworks ACE2 associated Subnetworks

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A functional genomics predictive network model identifies regulators of inflammatory bowel disease

Effect of vedolizumab (anti-α4β7-integrin) therapy on histological healing and mucosal gene expression in patients with UC

Cytoscape: a software environment for integrated models of biomolecular interaction networks

Transcriptomic Similarities and Differences in Host Response between SARS-CoV-2 and Other Viral Infections

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

SARS-CoV-2 productively infects human gut enterocytes

Enrichr: a comprehensive gene set enrichment analysis web server 2016 update

Intra-and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis

Mucosal Profiling of Pediatric-Onset Colitis and IBD Reveals Common Pathogenics and Therapeutic Pathways

Transcriptome-based network analysis reveals a spectrum model of human macrophage activation

ACE2 expression by colonic epithelial cells is associated with viral infection, immunity and energy metabolism

Ustekinumab Induction and Maintenance Therapy in Refractory Crohn's Disease

An integrative genomics approach to infer causal associations between gene expression and disease

Tissue-Specific Amino Acid Transporter Partners ACE2 and Collectrin Differentially Interact With Hartnup Mutations

Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

OP17 A molecular measure of inflammation in IBD patients based on transcriptional profiles from 2495 intestinal biopsies

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Conserved transcriptomic profile between mouse and human colitis allows unsupervised patient stratification

Temporal colonic gene expression profiling in the recurrent colitis model identifies early and chronic inflammatory processes

Temporal genome expression profile analysis during tcell-mediated colitis: identification of novel targets and pathways

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Distribution of brush-border membrane peptidases along the rat intestine

A protein complex in the brush-border membrane explains a Hartnup disorder allele

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

TMPRSS2, a serine protease expressed in the prostate on the apical surface of luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells

Endothelial JAM-A promotes reovirus viremia and bloodstream dissemination

The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination

IRTKS negatively regulates antiviral immunity through PCBP2 sumoylation-mediated MAVS degradation

Early biochemical responses of the small intestine of coeliac patients to wheat gluten

Trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report

SECURE-IBD Database Public Data Update

Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion