Since the novel betacoronavirus SARS-CoV-2 was first reported in the province of Wuhan, China, at the 54 end of 2019, the virus has spread over more than 200 countries, causing more than 8.9 million infections, 55 including almost 470.000 death globally. 1 Despite being primarily a respiratory virus, coronavirus disease 56 2019 can also present with non-respiratory signs, including digestive symptoms as diarrhea, 57 nausea and ageusia. 2, 3, 4 58 59 Although it is thought that SARS-CoV-2 primarily infects the lungs with transmission via the respiratory 60 route, the gastro-intestinal tract can be an alternative viral target organ. Indeed, the SARS-CoV-2 receptor 61 angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes, with strong 62 induction of generic viral response programs upon viral binding. 5, 6 The cellular entry of coronaviruses 63 depends on the binding of the spike (S) protein to a specific receptor, followed by an S protein priming by 64 proteases, with key players ACE2 (receptor for the S protein) and TMPRSS2 (protease) in case of 7, 8 Furthermore, based on protein crystal structures, data predicted that the Middle East respiratory 66 syndrome (MERS)-CoV receptor dipeptidyl peptidase 4 (DDP4) might act as a candidate binding target or 67 co-receptor of SARS-CoV-2. 9, 10 In line, proteomic studies in COVID-19 patients suggested a prognostic 68 role for DDP4. 11 Upon cellular entry in nasal goblet secretory cells, lung type II pneumocytes and ileal 69 absorptive enterocytes, an interferon-driven mechanism is initiated, including the upregulation of ACE2 70 which further enhances infection. 7 71 72 Why ACE2, the S protein receptor, is abundantly expressed on intestinal epithelium, is not entirely 73 understood. Recent studies have addressed the homeostatic role of ACE2 on intestinal epithelial cells 74 demonstrating defective intestinal amino acid absorption in ACE2 deficient mice. 12 Mechanistically, ACE2 75 independently of its role on the renin angiotensin system (RAS), is essential for regulating epithelial 76 tryptophan absorption, expression of antimicrobial peptides, and consequently regulating the ecology of the 77 gut microbiome promoting homeostasis and preventing intestinal inflammation. 13 Thus, ACE2 regulation 78 could be link to the pathogenesis of IBD, playing a role as modulator of epithelial immune homeostatic 79 functions. 80 81 82 Individual susceptibility to COVID-19 may correlate with the expression of these designated (co)receptors. 83
In this respect, it is unknown how inflammation affects ACE2, TMPRSS2 and/or DDP4 expression in ileum 84 and colon. Inflammatory Bowel Disease (IBD) is a prototype gastrointestinal disease characterized by a 85 chronic relapsing inflammatory infiltrate of the small and/or large bowel. So far, data on COVID-19 in 86 patients with IBD are rather limited, 14, 15, 16, 17, 18 although suggest that increasing age, a diagnosis of 87 ulcerative colitis [UC] (as opposed to Crohn's disease [CD] ) and increasing disease activity are linked with 88 a more severe course of COVID-19. In contrast, anti-inflammatory IBD therapy has not yet been associated 89 with COVID-19 risk. Using a combination of bulk and single cell transcriptomics and organoid cultures, we 90 studied the intestinal expression of several SARS-CoV-2 co-receptors in the healthy gut and in IBD and 91 investigated whether inflammation alters co-receptor expression. 92
Patients 95
This study was carried out at the University Hospitals Leuven (Leuven, Belgium Mucosal biopsies from both uninflamed and macroscopically inflamed colon segments (UC only) were 106 processed as reported earlier. 19 In brief, crypts were isolated and cultured as organoids for at least four 107 weeks. Inflammation was then re-induced using an inflammatory mix (100 ng/ml TNF-α, 20 ng/ml IL-1β, 108 1μg/ml Flagellin) during 24 hours. 19 109
Inflamed biopsies were taken at the most affected site at the edge of an ulcerative surface, whereas 111 uninflamed biopsies were taken randomly in macroscopic unaffected areas. All were stored in RNALater 112 buffer (Ambion, Austin, TX, USA) and preserved at -80°C. As described previously, 20 RNA from biopsies 113 was isolated using the AllPrep DNA/RNA Mini kit (Qiagen, Hilden, Germany), and RNA libraries were 114 prepared using the TruSeq Stranded mRNA protocol (Illumina, San Diego, USA). RNA from organoids was 115 extracted using the RNeasy Mini Kit (Qiagen) and libraries were constructed by the Lexogen QuantSeq 3' 116 mRNA-Seq Library Kit FWD (Lexogen, Vienna, Austria). 19 All RNA libraries were sequenced by the Illumina 117 HiSeq4000 (Illumina, San Diego, CA). Raw sequencing data were aligned to the reference genome 118 (GRCh37) using Hisat2 (version 2.1.0) 21 and absolute counts generated using HTSeq. 22 Counts were 119 normalized and protein coding genes selected (Ensemble hg 19 reference build) 23 using the DESeq2 120 package. 24. A weighted gene co-expression network (WGCNA) was generated, 25 as described earlier. (HBSS), and 1mM EDTA in HBSS at 37°C for 30 minutes, respectively. Then, tissue was transferred into a 132 sterile gentleMACS C tube (Miltenyi Biotec), and digested with 5.4 U/mL collagenase D (Roche applied 133 science), 100 U/mL DNase I (Sigma) and 39.6 U/mL dispase II (Gibco) with the gentleMACS™ Dissociator 134 (program human_tumor_02.01). Samples were incubated for 30 minutes at 37°C at 250 rpm. Dissociated 135 samples were filtered with 70 µm cell strainers, and treated with red blood cell lysis buffer (11814389001, 136 Roche). After centrifugation, single-cell suspensions were re-suspended in 0.4% BSA in PBS, and were 137 immediately processed with 10x 3' v3 GEM kit, and loaded on a 10x chromium controller to create Single 138 Cell Gel beads in Emulsion (GEM). A cDNA library was created and assessed using a 10x 3' v3 library kit, 139 and was then sequenced on a NovaSeq 6000 system (Illumina). Pre-processing of the samples including 140 alignment, and counting was performed using Cell Ranger Software from 10x (Version: 3.0.2). 141
Publicly available colonic single cell RNA sequencing data (sc-RNA seq) (Single Cell Portal, SCP 259) 143 were downloaded and visualized using the SCP data browser. 31 For colonic epithelial single cell data, tSNE 144 coordinates and publicly available annotation with the data was used for visualization and analysis. 145 146 Annotation of the ileal data was performed using SingleR R package, with inbuilt Human Cell Atlas data as 147 reference. Quality control, clustering and dimensionality reduction of sc-RNA seq data was performed using 148
Seurat R package (Version 3.1.5). 32, 33 Data from each 10x run were integrated after performing 149 SCTransform on each dataset, with percentage of mitochondrial genes set as a parameter to be regressed. After deparaffinization, antigen retrieval was done in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA 158 solution, 0.05% Tween 20, pH 9.0) at 95°C for 30 minutes. 1% BSA in PBST (0.1% tween-20 and 0.5% 159 sodium azide) was used to block non-specific binding of detection antibodies and gently permeabilize 160 before ACE2 and Cytokeratin AE1/AE3 staining. In brief, ACE2 (Polyclonal, Cell Signaling Technology) and 161 cytokeratin (IgG1-kappa, clone AE1/AE3, Dako) were applied in 1% BSA, followed by donkey anti-rabbit 162 Cy3 (Jackson Immuno Research) and donkey anti-mouse Alexa fluor 488 (Invitrogen). Slides were mounted 163 in SlowFade™ Diamond Antifade Mountant (Invitrogen), and stored at 4 °C before imaging. Images were 164 acquired using a Zeiss LSM 780 at the Cell and Tissue Imaging Cluster (CIC) at KU Leuven. 165 166 Genetics 167
All samples were genotyped using the Illumina GSA array. All SNPs and samples with more than 10% 168 missingness rate were removed, as were SNPs with minor allele frequency (MAF)<0.001. Genotypes for 169 rs6017342 (HNF4A) were extracted. All steps were performed using PLINK (v1.90b4.9). 35 170 171
Statistical analysis was performed using R 3.6.2 (The R foundation, Vienna, Austria). Pearson correlation 173 coefficients were computed to assess the correlation between individual genes. Multivariate analysis was 174 performed using the R package "lm.beta". Continuous variables on graphs were expressed as median and 175 interquartile range (IQR). ACE2, DPP4 and TMPRSS2 comparisons were done using two sample t-tests or 176
Wilcoxon tests, as appropriate. In case of hypothesis-free comparisons, ie. genome-wide differential gene 177 expression analyses, multiple testing correction was applied (adjusted p [adj. p], Benjamini-Hochberg 178 method). 179
First, we studied the expression patterns of ACE2, DPP4 and TMPRSS2 in ileum and colon biopsies from 183 351 IBD patients (193 CD, 158 UC) and 51 non-IBD controls based on bulk RNA sequencing. 184
In non-IBD controls, ACE2 and DPP4 expression levels were strongly increased in ileum compared to colon 186 (fold change (FC) =32.0, p=6.3E-13; FC=16.5, p=6.3E-13) ( Figure 1A-B) . In contrast, ileal TMPRSS2 was 187 lower compared to colon (FC=-2.9, p=6.3E-13) ( Figure 1C) . 188 189 When turning to tissue from IBD patients, ACE2 and DPP4 levels in uninflamed IBD ileum were similar to 190 those observed in matched control ileum (p=1.6E-01; p=8.0E-01) ( Figure 1A-B) . TMPRSS2 however, was 191 significantly upregulated compared to control ileum (FC=1.2, p=3.4E-02) ( Figure 1C) . In uninflamed IBD 192 colon, expression levels of ACE2, DDP4 and TMPRSS2 did not differ from control colon (p=2. 0E-01; 193 p=3.3E-01; 2.2E-01) ( Figure 1A (Figure 2A) . Moreover, ACE2 also seemed to have a central role in this co-expression 217 network with a correlation value of r=0.86 (p=4.6E-45) (Figure 2A) . Pathway analysis of the ACE2/DPP4-218 module found enrichment for epithelium-related metabolic pathways such as Xenobiotic Metabolism 219
Signaling, Nicotine Degradation II and Melatonin Degradation (p<1.0E-08). Predicted upstream analysis 220 (using curated datasets in IPA) highlighted the transcription regulator HNF4A, an IBD susceptibility gene, 221 as the most likely upstream regulator of the ACE2/DPP4-module (p=1.2E-11). 222 TMPRSS2 belonged to a separate module "yellow" (1126 genes) with hub gene COA3 (r=0.92, p=4.7E-61) 223 ( As the expression of ACE2-modules was found to be driven by the IBD susceptibility locus, HNF4A, we 241 next studied the genetic variability in rs6017342 (i.e. the causal IBD variant in this locus, 36 ), and its 242 relationship with ACE2 and HNF4A expression, both in inflamed ileum and colon. Ileal ACE2 levels were 243 lower in patients carrying the HNF4A-AA genotype, compared to patients carrying the C-allele, i.e. HNF4A-244 AC or HNF4A-CC genotypes (p=2.8E-02) (Figure 3) . Colonic ACE2 expression was independent of the 245 HNF4A genotype (p=6.7E-01). 246 247 248
ACE2 expression in the gastrointestinal tract is primarily found in absorptive enterocytes, 7, 37 which could 250 indirectly be confirmed through the significant correlation (p<2.2E-16) between mucosal ACE2 and several 251 epithelial marker genes (APOA1, SI, FABP6, ENPEP) (Supplementary Figure S2) . To further examine 252 the expression of genes associated with risk of SARS-CoV-2 infection in IBD patients, we employed sc-253 RNA seq to profile transmural biopsies of (un)inflamed regions of resected tissue from six CD patients 254 undergoing ileocaecal resection. Unaffected ileal tissue from five patients with CRC undergoing right 255 hemicolectomy was used as control. A total of 78,722 cells were used for downstream analyses containing 256 a similar number of cells from each type of tissue (inflamed CD, uninflamed CD and healthy tissue) 257 (Supplementary Figure S3B) . Sixty-one cell clusters belonging to epithelial, immune and stromal cells 258
were obtained using unsupervised clustering ( Figure 4A, Supplementary Figure S3A ). Cell clusters were 259 annotated by correlating the cluster gene expression profiles with Human Cell Atlas using SingleR, as 260 previously described. 38 ACE2 expression was found exclusively in epithelial cell clusters (Figure 4B-C) , 261 which could also be confirmed using immunofluorescence staining (Figure 5) . To define the epithelial cell 262 subtypes expressing ACE2 at deeper resolution, clusters annotated as epithelial cells by SingleR were 263 extracted and re-clustered ( Figure 4D) . The re-clustered epithelial cell subtypes were annotated using a 264 marker panel designed based on previous reports (Supplementary Figure S3C) . 39 Three enterocyte 265 clusters were identified, out of which two clusters co-expressed ACE2, TMPRSS2 and DPP4. Most 266 prominent ACE2 expression was observed in the ACE2/TMPRSS2 Enterocytes 1 cluster (Figure 4G -I, 267
Supplementary Figure S3D) . 268 269 Next, we asked whether ACE2 expression varied across ileal tissue with inflammatory state, as observed 270 in our bulk transcriptomic data ( Figure 1A) . ACE2 expression and frequency of ACE2 positive cells were 271 clearly reduced in ileum of patients with active CD, compared to uninflamed or healthy tissue ( Figure 4E-272   F, Supplementary Figure S3E) . A similar reduction of DPP4 expression was observed in the inflamed 273 samples in the ACE2/TMPRSS2 Enterocytes 1 and ACE2/TMPRSS2 Enterocytes 2 clusters ( Figure 4E) . 274
In line, reduction of ACE2 expression in inflamed ileum compared to healthy tissue was also confirmed with 275 confocal imaging (Figure 5) . 276
To define ACE2 expression in healthy and inflamed colon, we visualized publicly available colonic sc-RNA 278 seq data containing 366,650 cells from colonic mucosa obtained in 18 (in)active UC patients and 12 healthy 279 individuals (Single Cell Portal, SCP 259) (Supplementary Figure S4A-C) . 31 As for the ileum, ACE2 was 280 solely expressed in colonic epithelium, mainly in a subset of enterocyte (Figure 6A -B, Supplementary 281 Figure S4D ). As in the ileum, the ACE2 positive colonic enterocyte cluster co-expressed TMPRSS2 and 282 DPP4 (Figure 6B, 6E-G) . However, in contrast to ileum, colonic ACE2 expression was mainly restricted to 283 enterocytes isolated form patient with active UC, while undetectable in colonic enterocytes isolated from 284 the mucosa of healthy subjects. (Figure 6C-D, Supplementary Figure S4E to identify specific transcription programs in ACE2 expressing enterocytes, both in ileum and colon. As 292 demonstrated using bulk RNA analysis, we found a relatively higher HNF4A regulon activation in ileal ACE2 293 positive cells, compared to colonic ACE2 enterocytes ( Figure 6I) . Differently, colonic ACE2 expressing 294 enterocytes were found to have increased regulon activity of interferon responsive factors, such as IRF6 295 and IRF7, when compared to ileum ( Figure 6I) . 296 297 298
We then asked whether particular expression patterns within ACE2 positive cells depend on the tissue 300 and/or inflammatory state, and studied which upstream regulators were linked to these changes. When 301 comparing expression profiles of ACE2 positive cells from inflamed CD ileum with control ileum, we found 302 56 differentially expressed genes (adj. p<0.05, FC>2.0). Predicted upstream regulators of these genes were 303 HNF4A (inhibited, p=2.3E-04) and IFNγ (activated, p=5.2E-05). At the colonic level, we identified 54 304 differentially expressed genes in ACE2 positive cells from inflamed colon, as compared to control tissue. Given the ex vivo model clearly confirmed the effect of a pro-inflammatory mix, including TNF, on epithelial 322 ACE2 expression, we subsequently studied the effect of neutralizing TNF (through administration of 323 infliximab) on intestinal ACE2 expression in IBD patients with active endoscopic disease. Paired 324 transcriptomic data, generated prior to first infliximab administration and 4-6 weeks after treatment initiation, 325 confirmed a significant downregulation of colonic ACE2 in endoscopic remitters, but not in non-remitters 326 (p=1.8E-04, p=6.5E-01 respectively) (Supplementary Figure S6) . In contrast, infliximab therapy did not 327 significantly affect ileal ACE2 expression in remitters and non-remitters (p=7.8E-02, p=2.25E-01 328 respectively). 329
Many patients with IBD have long-term exposure to corticosteroids, thiopurines, methotrexate, small 332 molecules and/or biological agents, classifying them as high-risk patients because of their 333
immunosuppression. In addition, intestinal inflammation has shown to be an important risk factor for SARS-334
CoV-2 infection and prognosis in IBD. 14, 15, 16, 17, 18 However, emerging evidence now suggests that IBD 335 patients do not seem more vulnerable for COVID-19. To reconcile these observations, we investigated the Being an IBD susceptibility locus, 46 epithelial HNF4A plays a protective role in IBD by consolidating the 354 epithelial barrier, 47 especially in small intestine. 48 As it appears to be a transcriptional sensor of 355 inflammation, 49 and because of its key role as transcription factor in the regulation of angiotensinogen 356 metabolism, 50 the decrease in ACE2 in inflamed ileum does therefore not come as a surprise. In individuals 357 carrying the minor AA genotype at the IBD HNF4A susceptibility locus, ileal ACE2 expression was even 358 further downregulated, without any effect on colonic ACE2. Of note, our sc-RNAseq data showing ACE2 359 downregulation in enterocytes from inflamed CD ileum further suggest an intrinsic regulation of ACE2. In 360 addition, as we observed significant correlations between enterocyte markers and ileal ACE2 as well as an 361 overall decrease in number of cells expressing ACE2 in inflamed CD ileum, a loss of enterocytes might also 362 explain lower ACE2 levels. 363 364 Remarkably, a very recent GWAS study identified 3p21.31 as a genetic locus being associated with COVID-365 19-induced respiratory failure. 51 This locus covers a cluster of 6 genes (SLC6A20, LZTFL1, CCR9, FYCO1, 366 CXCR6, and XCR1), with the identified risk allele (i.e. worse COVID-19 outcome) being associated with 367 increased SCL6A20 expression. Strikingly, SCL6A20 is known to be regulated by HNF4A. 52 368 369 Although the colonic ACE2 co-expression cluster in bulk tissue was also enriched for HNF4A as upstream 370
transcriptional regulator, single cell data revealed that colonic ACE2 expression seems primarily driven by 371 interferon regulator factors. Upstream regulating analysis further supported that pro-inflammatory 372 cytokines, including TNF, IFNγ and IL-1β contribute to colonic ACE2 upregulation. Elevated colonic ACE2 373 levels in patients with active inflammation may thus promote viral entry and, in theory, could promote 374 COVID-19 disease severity. One can question this hypothesis as downregulated ACE2 in inflamed ileum 375 remains much higher than in normal and IBD colon. However, ACE2 expression is the most abundant in 376 the small intestine, followed by the large intestine, whereas its expression is limited in the respiratory 377 system. 53, 54 While there is yet no direct evidence that altered expression of intestinal ACE2 directly impacts 378 SARS-CoV-2 intestinal entry and tropisms to different intestinal sites, 55 using ex vivo organoid models we 379 confirmed that pro-inflammatory cytokines can upregulate colonic epithelial ACE2 expression in IBD 380 patients, but not in healthy individuals. Different genetic susceptibility and/or microbial composition may be 381 responsible for difference in response to inflammatory stimuli observed in controls and IBD. Indeed, it has 382 already been demonstrated that organoids from UC patients maintain some inherent differences as 383 compared to non-IBD tissue, 19, 56 presumably reflecting inherent genetic factors which could result in a more 384 sensitive epithelium. 385 386 Being the key example of a complex immune-mediated entity where environmental and microbial factors 387 modulate the immune response in a genetically susceptible host, 57 the differences in ACE2 expression 388 upon inflammatory stimuli between colon and ileum in patients with IBD may also be attributed to 389 differences in the intestinal microbiome. Lipopolysaccharides, comprising the wall of gram-negative 390 bacteria, was indeed identified as one of the key drivers of the ACE2 gene cluster in colon, but not in ileum. 391
However, blind use of antibiotics or probiotics for COVID-19 is not recommended until a better 392 understanding of the effect of SARS-CoV-2 on gut microbiota is obtained. 58 393 394
In line with our findings, national and international registries suggest active IBD as a risk factor for 395 (complicated) 15, 16, 17, 18 Adequate disease management, by appropriate dampening of 396 intestinal inflammation, therefore seems key in preventing IBD patients from COVID-19. Because of the 397 significant ACE2 upregulation in colon, one might consider that active UC patients and/or CD patients with 398 colonic involvement could be at higher risk for (complicated) COVID-19, compared to ileal CD. Although 399 international registries did not yet report any outcome data split by disease location, our data would suggest 400 an increased risk for complicated COVID-19 depending on disease location and disease activity. 