University of Groningen
Genomic medicine in inflammatory bowel disease
Voskuil, Michiel
DOI:
10.33612/diss.136307453
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Publication date: 2020
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Voskuil, M. (2020). Genomic medicine in inflammatory bowel disease. University of Groningen. https://doi.org/10.33612/diss.136307453
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TNFα-ANTAGONIST USE AND MUCOSAL
INFLAMMATION ARE ASSOCIATED WITH
INCREASED INTESTINAL EXPRESSION OF
SARS-CoV-2 HOST PROTEASE TMPRSS2 IN
PATIENTS WITH INFLAMMATORY BOWEL
DISEASE
Amber Bangma1,2*, Michiel D. Voskuil1,2*, Shixian Hu1,2, Werna T.C. Uniken Venema1,2, Arno R. Bourgonje1,
Eleonora A.M. Festen1,2, Harry van Goor3, Gerard Dijkstra1, Rinse K. Weersma1
Gastroenterology 2020
* Contributed equally to this work
1Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the
Netherlands; 2Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 3Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
Letter to the Editor
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral transfection is dependent on ACE2 and TMPRSS2, and increased intestinal receptor expression might support viral replication[1,2,3].
Therefore, we read with great interest the work by Krzysztof et al, reporting that anatomical location, intestinal inflammation and age are key determinants of intestinal expression of
angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in patients
with inflammatory bowel disease (IBD)[1]. However, several additional questions remain as the
authors themselves also describe. It is important to understand the effects of immunomodulating drugs on ACE2 and TMPRSS2 expression, and the cell-type specific context in which these genes are expressed[1]. In addition, it is important to understand genetic determinants of intestinal ACE2 and TMPRSS2 expression. To address these remaining questions, we queried host genetic
data and RNA sequencing data of intestinal biopsies from patients with IBD. We independently confirm that anatomical location, intestinal inflammation and age are determinants of intestinal
ACE2 and TMPRSS2 expression. Furthermore, we demonstrate that immunomodulating drugs
are associated with increased intestinal TMPRSS2 expression, and highlight the cell-type specific context of ACE2 and TMPRSS2 expression.
We analyzed bulk RNA sequencing data of 92 ileal and 199 colonic snap frozen mucosal biopsies from 168 patients with IBD, as part of the 1000IBD project[4]. Genotypes were obtained using both
imputed Global Screening Array data and whole exome sequencing. We analyzed clinical data regarding the use of immunomodulating drugs, age, sex, diagnosis (CD, UC or IBD-U) and BMI. Multivariate linear mixed regression analyses were performed to assess the effects of the above-mentioned clinical factors on intestinal gene expression levels of ACE2 and TMPRSS2 (R v.3.6.0). In addition, we assessed the effects of host genetic variation on gene expression (cis-expression quantitative trait loci). All of the above-mentioned clinical factors, as well as sequencing batch, were included as covariates. We performed deconvolution analyses on bulk RNA sequencing data to assess cell-type-specific expression of ACE2 and TMPRSS2. We validated this approach using single-cell RNA sequencing data from an independent set of 18 colonic biopsies from 11 patients with ulcerative colitis (unpublished data).
First, we replicated the effects of anatomical location, intestinal inflammation and age on intestinal expression of ACE2 and TMPRSS2. Expression of ACE2 was higher, and TMPRSS2 was lower in ileum compared to colon (P < 2.2 x 10-16). Moreover, expression of ACE2 was lower, and expression
of TMPRSS2 was higher in inflamed ileum compared to non-inflamed ileum, independent of medication use, age, sex, diagnosis and BMI (P = 4.4 x 10-6 and P = 8.6 x 10-8, respectively) (Figure
1A). Ileal TMPRSS2 expression was associated with increasing age (P = .04) (Figure 2). Furthermore, we found increased TMPRSS2 expression in intestinal (ileal and colonic) biopsies of male patients (P = .02).
Second, we studied the effects of immunomodulating drugs on ACE2 and TMPRSS2 expression in intestinal biopsies. We found increased TMPRSS2 expression in ileal biopsies of patients using Tumor Necrosis Factor alpha (TNFα)-antagonists (P = 8.8 x 10-6) independent of intestinal
inflammation, age, sex, diagnosis and BMI (Figure 1B).
Intestinal expression of SARS-CoV-2 host protease TMPRSS2 in patients with IBD
Figure 1. Intestinal TMPRSS2 gene expression in patients with inflammatory bowel disease. Panel A: Violin plots representing TMPRSS2 gene expression levels in ileal and colonic biopsies with and without active mucosal inflammation. 60 non-inflamed ileal, 32 inflamed ileal, 118 non-inflamed colonic, and 81 inflamed colonic biopsies were included. Differences in gene expression (log2-transformed counts per million reads mapped [cpm]) between inflamed and non-inflamed biopsies were assessed for ileal and colonic biopsies separately, using multivariate linear mixed regression analyses correcting for use of immunomodulating drugs (including TNFα-antagonists, steroids, thiopurines, and aminosalicylates), age, sex, BMI, diagnosis, and RNA-sequencing batch. P < .05 was considered significant. Panel B: Violin plots representing
TMPRSS2 gene expression levels in ileal and colonic biopsies from patients with and without TNFα-antagonist use. 71
ileal biopsies from TNFα-antagonist users, 20 ileal biopsies from non-users, 161 colonic biopsies from TNFα-antagonist users, and 38 colonic biopsies from non-users were included. Differences in gene expression (log2-transformed cpm) between patients using and not using TNFα-antagonist therapy were assessed for ileal and colonic biopsies separately, with multivariate linear mixed regression analyses correcting for mucosal inflammation, use of immunomodulating drugs (including steroids, thiopurines, and aminosalicylates), age, sex, BMI, diagnosis, and RNA-sequencing batch. Data points are each represented by a dot, and refer to log2-transformed cpm. Data is presented as boxplots indicating the median and quartiles with whiskers reaching up to 1.5 times the interquartile range (horizontal dashed lines). The violin plot outlines illustrate the kernel probability density, i.e. the width of the shaded area represents the proportion of the data located there.
Ileum Colon
Non−inflamed Inflamed Non−inflamed Inflamed
6 8 10 Mucosal inflammation TMPRSS2 (log2[cpm])
A
Mucosal inflammation No Yes P = 8.6 x 10-8 n.s. Colon No Yes No Yes 6 8 10 TNFα−antagonist use TMPRSS2 (log2[cpm])B
TNFα−antagonist use No Yes n.s. P = 8.8 x 10-6 Ileum Chapter 9Because aminosalicylates were only used in the context of ulcerative colitis, we assessed the influence of aminosalicylates in colonic tissue only, and observed an increased TMPRSS2 expression (P = .02). The use of thiopurines or steroids was not associated with differential expression of ACE2 or TMPRSS2. Furthermore, host genetic variation was not associated with differential expression of ACE2 and TMPRSS2.
Third, we interrogated the cell-type-specific context of intestinal ACE2 and TMPRSS2 expression. Using bulk RNA sequencing data, we quantified cell-type proportions and observed that an enrichment of epithelial cells is associated with increased expression of ACE2 and TMPRSS2 in both ileum and colon (all P < .004). Using single-cell RNA sequencing data from colonic biopsies, we observed that TMPRSS2 is mainly expressed by absorptive enterocytes, and that intestinal inflammation is associated with increased TMPRSS2 expression within absorptive enterocytes (P = 3.2 x 10-22). ACE2 was also primarily expressed by absorptive enterocytes, but expression was not
affected by intestinal inflammation.
Figure 2. Intestinal TMPRSS2 gene expression by age. The x-axis of the scatterplot represents the age of the patient at time of biopsy collection, while the y-axis represents TMPRSS2 gene expression levels (log2-transformed counts per million reads mapped [cpm]). Here, 91 ileal biopsies and 191 colonic biopsies were included. Data points are each represented by a dot, and the line illustrates the best-fitting straight line through the data points using linear regression. Correlation between gene expression levels and age was assessed for ileal and colonic biopsies separately using multivariate linear mixed regression analyses correcting for mucosal inflammation, use of immunomodulating drugs (including TNFα-antagonists, steroids, thiopurines, and aminosalicylates), sex, BMI, diagnosis, and RNA-sequencing batch. P < .05 was considered significant.
Intestinal expression of SARS-CoV-2 host protease TMPRSS2 in patients with IBD
Ileum Colon 20 40 60 80 20 40 60 80 6 7 8 9 10 11 Age TMPRSS2 (log2[cpm]) P = .04 n.s.
9
In conclusion, ACE2 and TMPRSS2 are key proteins for cellular entry of SARS-CoV-2, and are highly expressed in the intestinal mucosa. Next to intestinal inflammation, age, sex and anatomical location, also the use of TNFα-antagonists and aminosalicylates influence intestinal expression of TMPRSS2. We demonstrate that intestinal inflammation is associated with increased expression of TMPRSS2 in absorptive enterocytes, suggesting that the increased expression is not merely an effect of change in cellular composition during inflammation. Altered intestinal expression could render patients with IBD particularly susceptible to COVID-19 and absorptive enterocytes could provide targets for interventional studies. Indeed, clinical studies are needed to monitor the impact of COVID-19 on patients with IBD.
Acknowledgements
We thank all the participants of the 1000IBD cohort.
Author contributions
AB and MDV contributed equally to this work. Concept and design: AB, MDV, RKW. Acquisition, analysis, or interpretation of data: AB, MDV, SH, WTCUV, ARB, RKW. Drafting of the manuscript: AB, MDV, RKW. Critical revision of the manuscript: SH, WTCUV, ARB, HvG, EAMF, GD. All authors approved the final version of this manuscript.
Conflicts of interest
EAMF received an unrestricted grant from Takeda. GD received an unrestricted research grant from Takeda, and received speaker fees from Pfizer and Janssen Pharmaceuticals. RKW acted as consultant for Takeda, received unrestricted research grants from Takeda, Johnson and Johnson, Tramedico and Ferring and received speaker fees from MSD, Abbvie and Janssen Pharmaceuticals. The remaining authors disclose no (potential) conflicts of interest.
Funding
RKW is supported by a Diagnostics Grant from the Dutch Digestive Foundation (D 16-14). EAMF is supported by a MLDS Career Development grant (CDG 14-04).
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Lamers MM, Beumer J, van der Vaart J, et al. SARS-CoV-2 productively infects human gut enterocytes. Science. Published Online First: 01 May 2020. doi: 10.1126/science.abc1669
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Intestinal expression of SARS-CoV-2 host protease TMPRSS2 in patients with IBD
