key: cord-0821056-pdnsyypz
authors: Verstockt, Bram; Verstockt, Sare; Abdu Rahiman, Saeed; Ke, Bo-jun; Arnauts, Kaline; Cleynen, Isabelle; Sabino, João; Ferrante, Marc; Matteoli, Gianluca; Vermeire, Séverine
title: Intestinal receptor of SARS-CoV-2 in inflamed IBD tissue seems downregulated by HNF4A in ileum and upregulated by interferon regulating factors in colon
date: 2020-09-11
journal: J Crohns Colitis
DOI: 10.1093/ecco-jcc/jjaa185
sha: ea59cefeebbb4399c4cf0d6fd1430522ac56330a
doc_id: 821056
cord_uid: pdnsyypz

BACKGROUND: Patients with IBD are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the role of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD. METHODS: We collected inflamed and uninflamed mucosal biopsies from CD (n=193) and UC (n=158) patients, and 51 matched non-IBD controls for RNA sequencing, differential gene expression and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell (sc) sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-TNF therapy, were analyzed. RESULTS: In inflamed CD ileum, ACE2 was significantly decreased compared to control ileum (p=4.6E-07), whereas colonic ACE2 was higher in inflamed colon of CD/UC compared to control (p=8.3E-03; p=1.9E-03). Sc-RNA sequencing confirmed this ACE2 dysregulation, and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2, while pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids (p=1.7E-02), not in non-IBD controls (p=9.1E-01). Anti-TNF therapy restored colonic ACE2 regulation in responders. CONCLUSION: Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing dysregulations in ileum and colon. HNF4A, an IBD susceptibility gene, seems an important upstream regulator of ACE2 in ileum, whereas interferon signaling might dominate in colon.

Since the novel betacoronavirus SARS-CoV-2 was first reported in the province of Wuhan, China, at the end of 2019, the virus has spread worldwide. As of the 19 th of August 2020, SARS-CoV-2 has caused more than 21.9 million infections, including 776.000 death globally. 1 Despite being primarily a respiratory virus, coronavirus disease 2019 can also present with non-respiratory signs, including digestive symptoms as diarrhea, nausea and ageusia. [2] [3] [4] Although it is thought that SARS-CoV-2 primarily infects the lungs with transmission via the respiratory route, the gastro-intestinal tract might be an alternative viral target organ. 3, 5, 6 Indeed, the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes, with strong induction of generic viral response programs upon viral binding. [5] [6] [7] The cellular entry of coronaviruses depends on the binding of the spike (S) protein to a specific receptor, followed by an S protein priming by proteases, with key players ACE2 (receptor for the S protein) and TMPRSS2

(protease) in case of COVID-19. [7] [8] [9] Furthermore, based on protein crystal structures, data predicted that the Middle East respiratory syndrome (MERS)-CoV receptor dipeptidyl peptidase 4 (DDP4) might act as a candidate binding target or co-receptor of SARS-CoV-2. 10, 11 In line, proteomic studies in COVID-19

patients suggested a prognostic role for DDP4. 12 Upon cellular entry in nasal goblet secretory cells, lung type II pneumocytes and ileal absorptive enterocytes, an interferon-driven mechanism is initiated, including the upregulation of ACE2 which further enhances infection. 8 Why ACE2, the S protein receptor, is abundantly expressed on intestinal epithelium, is not entirely understood. Recent studies have addressed the homeostatic role of ACE2 on intestinal epithelial cells demonstrating defective intestinal amino acid absorption in ACE2 deficient mice. 13 Mechanistically, ACE2

independently of its role on the renin angiotensin system (RAS), is essential for regulating epithelial tryptophan absorption, expression of antimicrobial peptides, and consequently regulating the ecology of the gut microbiome promoting homeostasis and preventing intestinal inflammation. 14 

This study was carried out at the University Hospitals Leuven (Leuven, Belgium). All included patients had given written consent to participate in the Institutional Review Board approved IBD Biobank of University Hospitals Leuven, Belgium (B322201213950/S53684 and B322201110724/S52544).

Endoscopy-derived (un)inflamed mucosal biopsies were obtained cross-sectionally from IBD patients requiring colonoscopy during routine care (Supplementary Table S1 ). Samples from individuals undergoing colonoscopy for polyp detection were included as controls. Transmural ileal biopsies, derived during right hemicolectomy from CD patients and patients with colorectal cancer (CRC), were collected, stored in RPMI-1640 medium on ice until single cell isolation.

Mucosal biopsies from both uninflamed and macroscopically inflamed colon segments (UC only) were processed as reported earlier. [22] [23] [24] In short, crypts isolated as described before 23 

Inflamed biopsies were taken at the most affected site at the edge of an ulcerative surface, whereas uninflamed biopsies were taken randomly in macroscopic unaffected areas. All were stored in RNALater buffer (Ambion, Austin, TX, USA) and preserved at -80°C. As described previously, 25 were downloaded and visualized using the SCP data browser. 36 For colonic epithelial single cell data, tSNE coordinates and publicly available annotation with the data was used for visualization and analysis.

Annotation of the ileal data was performed using SingleR R package, with inbuilt Human Cell Atlas data as reference. Quality control, clustering and dimensionality reduction of sc-RNA seq data was performed using Seurat R package (Version 3.1.5). 37 and cytokeratin (IgG1-kappa, clone AE1/AE3, Dako) were applied in 1% BSA, followed by donkey antirabbit Cy3 (Jackson Immuno Research) and donkey anti-mouse Alexa fluor 488 (Invitrogen). Slides were mounted in SlowFade™ Diamond Antifade Mountant (Invitrogen), and stored at 4 °C before imaging.

Images were acquired using a Zeiss LSM 780 at the Cell and Tissue Imaging Cluster (CIC) at KU Leuven.

All samples were genotyped using the Illumina GSA array. All SNPs and samples with more than 10% missingness rate were removed, as were SNPs with minor allele frequency (MAF)<0.001. Genotypes for rs6017342 (HNF4A) were extracted. All steps were performed using PLINK (v1.90b4.9). 40 

Statistical analysis was performed using R 3.6.2 (The R foundation, Vienna, Austria). Pearson correlation coefficients were computed to assess the correlation between individual genes. Multivariate regression analysis was performed using the R package "lm.beta". Continuous variables on graphs were expressed as median and interquartile range (IQR). ACE2, DPP4 and TMPRSS2 comparisons were done using two sample t-tests or Wilcoxon tests, as appropriate; and multiple testing correction was applied (adjusted p [adj. p], Benjamini-Hochberg method).

First, we studied the expression patterns of ACE2, DPP4 and TMPRSS2 in ileum and colon biopsies from 351 IBD patients (193 CD, 158 UC) and 51 non-IBD controls based on bulk RNA sequencing.

In non-IBD controls, ACE2 and DPP4 expression levels were strongly increased in ileum compared to colon (fold change (FC) =32.0, p=6.3E-13, adj. p=1.9E-12; FC=16.5, p=6.3E-13, adj. p=1.9E-12) ( Figure   1A -B). In contrast, ileal TMPRSS2 was lower compared to colon (FC=-2.9, p=6.3E-13, adj. p=1.9E-12) ( Figure 1C ).

When turning to tissue from IBD patients, ACE2 and DPP4 levels in uninflamed IBD ileum were similar to those observed in matched control ileum (p=1.6E-01, adj. p=2.4E-01; p=8.0E-01, adj. p=8.0E-01) ( Figure   1A -B). TMPRSS2 however, was upregulated compared to control ileum (FC=1.2, p=3.4E-02, adj. p=1.0E-01) ( Figure 1C ). In uninflamed IBD colon, expression levels of ACE2, DDP4 and TMPRSS2 did not differ from control colon (p=2.0E-01, adj. p=6.0E-01; p=3.3E-01, adj. p=3.3E-01; 2.2E-01, adj. p=3.3E-01) ( Figure 1A -C).

In inflamed CD ileum, ACE2 and DPP4 expression was significantly decreased compared to control ileum Despite ACE2 being X-linked, multivariate analysis did not reveal any contribution of sex to mucosal Table S2 ).

To get a better understanding of the biological network of ACE2, DPP4 and TMPRSS2, we performed WGCNA on all mucosal biopsies.

At ileal level, we identified 18 co-expression modules (clusters) ranging in size from 106 to 1465 genes (Supplementary Figure S1A) . One module contained both ACE2 and DPP4 (module "blue"; 1134 genes) (Supplementary Table S3 At colonic level, 24 co-expression modules were present ranging in size from 128 to 2267 genes (Supplementary Figure S1B) . In contrast to the ileum, colonic ACE2 and DPP4 were not co-expressed Figure S2) .

As the expression of ACE2-modules was found to be driven by the IBD susceptibility locus, HNF4A, we next studied the genetic variability in rs6017342 (i.e. the causal IBD variant in this locus 41 ), and its relationship with ACE2 and HNF4A expression, both in inflamed ileum and colon. Ileal ACE2 levels were lower in patients carrying the HNF4A-AA genotype, compared to patients carrying the C-allele, i.e.

HNF4A-AC or HNF4A-CC genotypes (p=2.8E-02) (Figure 2) . Colonic ACE2 expression was independent of the HNF4A genotype (p=6.7E-01). Figure S4B) . Sixty-one cell clusters belonging to epithelial, immune and stromal cells were obtained using unsupervised clustering ( Figure 3A, Supplementary Figure S4A ). Cell clusters were annotated by correlating the cluster gene expression profiles with Human Cell Atlas using SingleR, as previously described. 43 ACE2 expression was found exclusively in epithelial cell clusters ( Figure 3B-C) , which could also be confirmed using immunofluorescence staining (Figure 4) . To define the epithelial cell subtypes expressing ACE2 at deeper resolution, clusters annotated as epithelial cells by

SingleR were extracted and re-clustered ( Figure 3D) . The re-clustered epithelial cell subtypes were annotated using a marker panel designed based on previous reports (Supplementary Figure S4C) . 44 Three enterocyte clusters were identified, out of which two clusters co-expressed ACE2, TMPRSS2 and DPP4. Most prominent ACE2 expression was observed in the ACE2/TMPRSS2 Enterocytes 1 cluster ( Figure 3G-I, Supplementary Figure S4D ).

Next, we asked whether ACE2 expression varied across ileal tissue with inflammatory state, as observed in our bulk transcriptomic data ( Figure 1A) . ACE2 expression and frequency of ACE2 positive cells were clearly reduced in ileum of patients with active CD, compared to uninflamed or healthy tissue (Figure 3E-F, Supplementary Figure S4E) . A similar reduction of DPP4 expression was observed in the inflamed samples in the ACE2/TMPRSS2 Enterocytes 1 and ACE2/TMPRSS2 Enterocytes 2 clusters (Figure 4E ).

In line, reduction of ACE2 expression in inflamed ileum compared to healthy tissue was also confirmed with confocal imaging (Figure 4) .

To define ACE2 expression in healthy and inflamed colon, we visualized publicly available colonic sc-RNA seq data containing 366,650 cells from colonic mucosa obtained in 18 (in)active UC patients and 12

healthy individuals (Single Cell Portal, SCP 259) (Supplementary Figure S5A-C) . 36 As for the ileum, ACE2 was solely expressed in colonic epithelium, mainly in a subset of enterocyte ( Figure 5A -B, Supplementary Figure S5D) . As in the ileum, the ACE2 positive colonic enterocyte cluster co-expressed TMPRSS2 and DPP4 (Figure 5B, 5E-G) . However, in contrast to ileum, colonic ACE2 expression was mainly restricted to enterocytes isolated form patient with active UC, while undetectable in colonic enterocytes isolated from the mucosa of healthy subjects. (Figure 5C-D, Supplementary Figure S5E) To compare expression and regulation of ACE2 between colon and ileum, we performed an integrated analysis of epithelial cells from colon and ileum (Supplementary Figure S6A-B) . In colonic ACE2 positive epithelial cells, ACE2 expression was lower compared levels in ileal ACE2 positive epithelial cells ( Figure 5H) . Furthermore, using SCENIC we performed genomic regulatory networks analysis of the epithelial cells to identify specific transcription programs in ACE2 expressing enterocytes, both in ileum and colon. As demonstrated using bulk RNA analysis, we found a relatively higher HNF4A regulon activation in ileal ACE2 positive cells, compared to colonic ACE2 enterocytes ( Figure 5I) . Differently, colonic ACE2 expressing enterocytes were found to have increased regulon activity of interferon responsive factors, such as IRF6 and IRF7, when compared to ileum (Figure 5I ).

We then asked whether particular expression patterns within ACE2 positive cells depend on the tissue and/or inflammatory state, and studied which upstream regulators were linked to these changes. When comparing expression profiles of ACE2 positive cells from inflamed CD ileum with control ileum, we found 56 differentially expressed genes (adj. p<0.05, FC>2.0). Predicted upstream regulators of these genes were HNF4A (inhibited, p=2.3E-04) and IFNγ (activated, p=5.2E-05). At the colonic level, we identified 54 differentially expressed genes in ACE2 positive cells from inflamed colon, as compared to control tissue.

TNF, lipopolysaccharides, IFNγ and IL-1β were predicted as top ranked upstream regulators (activated,

Because of the clear upregulation of ACE2 in inflamed colonic mucosa ( Figure 1A ) and the prediction of TNF as key regulator in ACE2 positive cells, we investigated the effect of an inflammatory stimulus on ACE2 expression in an ex vivo organoid model. In organoids derived from controls, inflammatory stimuli did not affect ACE2 expression (p=9.1E-01, adj. p=9.1E-01) ( Figure 6A) (Figure 6B-C) .

Given the ex vivo model clearly confirmed the effect of a pro-inflammatory mix, including TNF, on epithelial ACE2 expression, we subsequently studied the effect of neutralizing TNF (through administration of infliximab) on intestinal ACE2 expression in IBD patients with active endoscopic disease. Paired transcriptomic data, generated prior to first infliximab administration and 4-6 weeks after treatment initiation, confirmed a significant downregulation of colonic ACE2 in endoscopic remitters, but not in non-remitters (p=1.8E-04, p=6.5E-01 respectively) (Supplementary Figure S7) . In contrast, infliximab therapy did not significantly affect ileal ACE2 expression in remitters and non-remitters (p=7.8E-02, p=2.25E-01 respectively).

Many patients with IBD have long-term exposure to corticosteroids, thiopurines, methotrexate, small molecules and/or biological agents, classifying them as high-risk patients because of their immunosuppression. In addition, intestinal inflammation has shown to be an important risk factor for SARS-CoV-2 infection and prognosis in IBD. [17] [18] [19] [20] [21] However, emerging evidence now suggests that IBD patients do not seem more vulnerable for COVID-19. To reconcile these observations, we investigated the role of intestinal inflammation on the potential viral intestinal entry mechanisms through bulk and single cell transcriptomics, immunofluorescence and ex vivo organoid cultures in patients with IBD.

In contrast to previous bulk data 15 , we observed significant alterations in intestinal ACE2 expression depending on the location and inflammatory state, both at tissue and single cell mRNA level, as at protein level. ACE2 expression was limited exclusively to epithelial cells, both in ileum and colon. Hence, ACE2 dysregulation in bulk transcriptomics as a result of massive influx of immunocytes at the site of inflammation could be excluded.

It is conceived that SARS-CoV-2 infects epithelial cells, causing cytokine and chemokine release, resulting in acute intestinal inflammation characterized by infiltration of neutrophils, macrophages and T cells, 45 with associated shedding of faecal calprotectin and increased systemic IL-6 response, 46 and IFN signaling. 8 Similar to recent data, 16, 47, 48 we found a significant downregulation of ACE2 in inflamed ileum and a significant ACE2 upregulation in inflamed colon. This opposing effect of inflammation on intestinal ACE2 expression in small and large intestine was striking, which could be attributed -based on sc-RNA data -to different key transcription factors active between ileal and colonic ACE2 positive cells.

Being an IBD susceptibility locus, 49 epithelial HNF4A plays a protective role in IBD by consolidating the epithelial barrier, 50 especially in small intestine. 51 HNF4A has also been found as a transcriptional sensor of inflammation 52 , plays a key role as transcription factor in the regulation of angiotensinogen metabolism 53 , and has recently been predicted to regulate intestinal ACE2 expression. 42 The decrease in ACE2 in inflamed ileum does therefore not come as a surprise. In individuals carrying the minor AA genotype at the IBD HNF4A susceptibility locus, ileal ACE2 expression was even further downregulated, without any effect on colonic ACE2. Of note, our sc-RNAseq data showing ACE2 downregulation in enterocytes from inflamed CD ileum further suggest an intrinsic regulation of ACE2. In addition, as we observed significant correlations between enterocyte markers and ileal ACE2 as well as an overall decrease in number of cells expressing ACE2 in inflamed CD ileum, a loss of enterocytes might also explain lower ACE2 levels.

Remarkably, a very recent GWAS study identified 3p21.31 as a genetic locus being associated with COVID-19-induced respiratory failure. 54 This locus covers a cluster of 6 genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, and XCR1), with the identified risk allele (i.e. worse COVID-19 outcome) being associated with increased SCL6A20 expression. Strikingly, SCL6A20 is known to be regulated by HNF4A. 55 Although the colonic ACE2 co-expression cluster in bulk tissue was also enriched for HNF4A as upstream transcriptional regulator, single cell data revealed that colonic ACE2 expression seems primarily driven by interferon regulator factors. Upstream regulating analysis further supported that pro-inflammatory cytokines, including TNF, IFNγ and IL-1β contribute to colonic ACE2 upregulation. Hypothetically, elevated colonic ACE2 levels in patients with active inflammation might promote viral entry and, in theory, could promote COVID-19 disease severity. However, functional data are currently lacking to prove this hypothesis. Furthermore, one could question this hypothesis as downregulated ACE2 in inflamed ileum remains much higher than in normal and IBD colon. However, ACE2 expression is the most abundant in the small intestine, followed by the large intestine, whereas its expression is limited in the respiratory system. [56] [57] [58] Moreover, a recent study in human small intestinal organoids observed similar SARS-CoV-2 infection rates between enterocyte precursors and enterocytes, whereas ACE2 expression was ~1000fold higher differentiating organoids as compared to proliferating organoids. This suggests that lower levels of ACE2 -as observed in the colon -may be sufficient for viral entry. 5 While there is yet no direct evidence that altered expression of intestinal ACE2 directly impacts SARS-CoV-2 intestinal entry and tropisms to different intestinal sites, 59 using ex vivo organoid models we confirmed that pro-inflammatory cytokines can upregulate colonic epithelial ACE2 expression in IBD patients, but not in healthy individuals. Different genetic susceptibility and/or microbial composition may be responsible for difference in response to inflammatory stimuli observed in controls and IBD. Indeed, it has already been demonstrated that organoids from UC patients maintain some inherent differences as compared to non-IBD tissue, 22, 60 presumably reflecting inherent genetic factors which could result in a more sensitive epithelium.

Being the key example of a complex immune-mediated entity where environmental and microbial factors modulate the immune response in a genetically susceptible host, 61 the differences in ACE2 expression upon inflammatory stimuli between colon and ileum in patients with IBD may also be attributed to differences in the intestinal microbiome. Lipopolysaccharides, comprising the wall of gram-negative bacteria, was indeed identified as one of the key drivers of the ACE2 gene cluster in colon, but not in ileum. However, blind use of antibiotics or probiotics for COVID-19 is not recommended until a better understanding of the effect of SARS-CoV-2 on gut microbiota is obtained. 62 National and international registries suggest active IBD as a risk factor for (complicated) COVID-19. [17] [18] [19] [20] [21] Adequate disease management, by appropriate dampening of intestinal inflammation, therefore seems key in preventing IBD patients from COVID-19. The ACE2 upregulation in inflamed colon could potentially impact the viral cell entry in active UC patients and/or CD patients with colonic involvement, although functional data are currently lacking. So far, international registries did not yet report any COVID-19 outcome data in IBD patients split by disease location.

Of note, several key cytokines implicated in IBD pathogenesis, 61, 63 and also key drivers of ACE2 colonic expression in this study, are currently under investigation as potential therapeutic targets for COVID-19, including TNF, IFNγ, IL-1β and IL-6. 64 Although further evidence is warranted if these anti-cytokine therapies can dampen the observed cytokine storm in COVID-19, we demonstrated that anti-TNF therapy does restore intestinal ACE2 dysregulation in a subset of IBD patients.

In this study, we acknowledge the lack of data on SARS-CoV-2 infected patients, a sequencing depth not enabling to look for HNF4A alternative splicing and isoforms with pro-and anti-inflammatory effects, 65 Expression of ACE2, DPP4 and TMPRSS2 in colonic epithelial clusters split between sample types of control, uninflamed and inflamed sample type.

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The manuscript was written by the authors themselves, as specified in author contributions section. There were no additional individuals who provided writing assistance.