key: cord-0807393-tdycrxtl authors: Sawalha, A. H.; Zhao, M.; Coit, P.; Lu, Q. title: Epigenetic dysregulation of ACE2 and interferon-regulated genes might suggest increased COVID-19 susceptibility and severity in lupus patients date: 2020-04-04 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.03.30.20047852 sha: 711445f8fdaf17fa507602650677d1a7f03e7177 doc_id: 807393 cord_uid: tdycrxtl Infection caused by SARS-CoV-2 can result in severe respiratory complications and death. Patients with a compromised immune system are expected to be more susceptible to a severe disease course. In this report we suggest that patients with systemic lupus erythematous might be especially prone to severe COVID-19 independent of their immunosuppressed state from lupus treatment. Specially, we provide evidence in lupus to suggest hypomethylation and overexpression of ACE2, which is located on the X chromosome and encodes a functional receptor for the SARS-CoV-2 spike glycoprotein. Oxidative stress induced by viral infections exacerbates the DNA methylation defect in lupus, possibly resulting in further ACE2 hypomethylation and enhanced viremia. In addition, demethylation of interferon-regulated genes, NF{kappa}B, and key cytokine genes in lupus patients might exacerbate the immune response to SARS-CoV-2 and increase the likelihood of cytokine storm. These arguments suggest that inherent epigenetic dysregulation in lupus might facilitate viral entry, viremia, and an excessive immune response to SARS-CoV-2. Further, maintaining disease remission in lupus patients is critical to prevent a vicious cycle of demethylation and increased oxidative stress, which will exacerbate susceptibility to SARS-CoV-2 infection during the current pandemic. Epigenetic control of the ACE2 gene might be a target for prevention and therapy in COVID-19. Infection caused by SARS-CoV-2 can result in severe respiratory complications and death. Patients with a compromised immune system are expected to be more susceptible to a severe disease course. In this report we suggest that patients with systemic lupus erythematous might be especially prone to severe COVID-19 independent of their immunosuppressed state from lupus treatment. Specially, we provide evidence in lupus to suggest hypomethylation and overexpression of ACE2, which is located on the X chromosome and encodes a functional receptor for the SARS-CoV-2 spike glycoprotein. Oxidative stress induced by viral infections exacerbates the DNA methylation defect in lupus, possibly resulting in further ACE2 hypomethylation and enhanced viremia. In addition, demethylation of interferon-regulated genes, NFκB, and key cytokine genes in lupus patients might exacerbate the immune response to SARS-CoV-2 and increase the likelihood of cytokine storm. These arguments suggest that inherent epigenetic dysregulation in lupus might facilitate viral entry, viremia, and an excessive immune response to SARS-CoV-2. Further, maintaining disease remission in lupus patients is critical to prevent a vicious cycle of demethylation and increased oxidative stress, which will exacerbate susceptibility to SARS-CoV-2 infection during the current pandemic. Epigenetic control of the ACE2 gene might be a target for prevention and therapy in COVID-19. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Lupus is a chronic autoimmune disease of incompletely understood etiology. We propose that lupus patients might be at an increased risk for infection by SARS-CoV-2 and for developing a more severe form of COVID-19, independent of the possible effect of immunosuppressive medications. Epigenetic dysregulation plays an important role in the pathogenesis of lupus 3 . While most epigenetic studies in lupus have been performed in T cells, there is evidence to suggest reduced DNA methylation levels in multiple cell types in this disease. For example, a robust demethylation signature in interferon-regulated genes in lupus is wide-spread and characteristic of the disease 4-6 . DNA methylation plays an important role in the inactivation of the X chromosome. X chromosome inactivation, whereby gene expression from one gene copy of the X chromosome is silenced by DNA methylation, is essential to maintain normal levels of gene expression and comparable expression levels between male and female cells. In women with lupus, reduced DNA methylation has been shown to result in defective X chromosome inactivation 7 . Hypomethylation of the normally inactivate X chromosome leads to an abnormal increase in the expression levels of affected genes. This abnormal X chromosome inactivation has been demonstrated to mediate increased expression of CD40LG in CD4+ T cells in women with lupus, contributing to T cell autoreactivity in this disease 7 . CD40LG expression in CD4 + T cells isolated from healthy women was similarly increased after treatment with the DNA methylation inhibitor 5-azacytidine. This gene-dose effect of the X chromosome has been proposed to explain, at least in part, sex bias in lupus. Of interest, men with two X chromosomes, Klinefelter syndrome (47, XXY), have a significantly higher risk to develop lupus compared to normal men (46, XY) and a similar risk to women 8 . ACE2 is a functional receptor for the viral spike glycoprotein that allows the entry of SARS-CoV-2 into cells 9 . ACE2 gene is located on the X chromosome, similar to CD40LG. Recent findings suggest the expansion of a demethylated T cells subset in lupus patients characterized by the expression of several genes known to be demethylated in lupus, including CD40LG, CD70, ITGAL (CD11A), and the KIR gene cluster (defined using flow cytometry as KIR + CD11a hi CD4 + CD28 + T cell subset) 10, 11 . Indeed, we can induce this T cell subset in normal healthy individuals using the DNA methylation inhibitor 5-azacytidine 11 . We generated whole-genome DNA methylation data using an array-based approach and observe significant hypomethylation in the ACE2 gene in this T cell subset compared to KIR -CD11a low All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint T cells isolated from the same lupus patients ( Figure 1A ). This hypomethylation involves several CpG sites located in the ACE2 promoter region proximal to the transcription start site, the 5'-untranslated region, and the 3'-untranslated region, suggesting functional regulatory effect of the methylation changes. Furthermore, we can induce hypomethylation in some of these same CpG sites in CD4 + T cells isolated from normal healthy women treated with the DNA methylation inhibitor 5-azacytidine 11 . Importantly, in a subset of female lupus patients characterized by skin, joint, and kidney involvement ACE2 promoter region was significantly hypomethylated in CD4 + T cells compared to age and sex matched normal healthy controls 12 . Gene expression profiles extracted from a publicly available dataset (GEO accession: GSE4588) showed a significant overexpression of ACE2 mRNA in lupus CD4 + T cells compared to normal healthy controls ( Figure 1B) . We further confirm hypomethylation in ACE2 in an independent set of female lupus patients characterized by kidney involvement ( Figure 1C) . Gene expression profiles in the same set of patients showed ~1.5-2-fold ACE2 overexpression compared to healthy controls, although his did not reach statistical significance ( Figure 1D ). Lupus patients are also characterized by expansion of a pro-inflammatory double negative T cell subset (CD4 -CD8 -) 13 . We have previously demonstrated that double negative T cells heavily demethylate cytokine genes including IL-18 and IFNγ, among others 14 . Interestingly, we also observe significant hypomethylation in the ACE2 gene in double negative T cells compared to autologous CD4+ and CD8+ T cells isolated from normal healthy women 14 . Together, these data support that ACE2 expression is regulated by DNA methylation and that a methylation defect in lupus extends to regulatory sequences in the ACE2 gene, which could result in ACE overexpression in lupus patients. It is important to note that a major limitation to these arguments is the absence of data from alveolar epithelial cells in lupus patients. However, it is worth highlighting that overexpression of ACE2 in other cell types might facilitate viremia and organ damage in COVID-19. Indeed, in SARS, SARS-CoV viral particles (which also utilize ACE2 for cell entry) have been shown to infect immune cells in large numbers, including peripheral blood T cells, which can result in viral dissemination throughout the body 15 . Direct tissue damage from viral infection of target tissues, such as the kidneys, intestine, and brain has been reported 16 . Overall, the DNA methylation defect in lupus patients is more evident in patients with increased disease activity, and is exacerbated by increased oxidative stress, such as in the case of viral infections 17 . Oxidative stress has been shown to inhibit DNA methylation in lupus T cells by blocking PKC-δ activation, resulting in attenuated MEK/ERK signaling and reduced expression of the main DNA methyltransferase DNMT1 17, 18 . In addition, oxidative stress depletes NADPH and glutathione levels leading to mTOR activation which also exacerbates the DNA methylation defect by inhibiting DNMT1 19 . Therefore, it is reasonable to suggest the possibility that the DNA methylation defect in lupus patients, exacerbated by All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint the oxidative stress from SARS-CoV-2 infection, will further enhance viral entry in lupus patients through epigenetic de-repression of ACE2 and increased ACE2 expression. which is persistent and present across multiple cells types and disease subsets 20 . We have previously suggested that this epigenetic activation poises interferon-regulated genes for transcription in lupus patients, and likely explains the exaggerated expression response of interferon-regulated genes in the presence of response to type-I interferons 4 Response to SARS-CoV-2 resulting in tissue damage and oxidative stress might further exacerbate the pro-inflammatory epigenetic changes observed in lupus patients resulting in a vicious circle of cytokine response (Figure 2) . Taken together, these observations suggest that epigenetic dysregulation might increase the risk and severity of SARS-CoV-2 infections in lupus patients. The likelihood of disseminated SARS-CoV-2 infection in lupus patients might be increased due to ACE2 overexpression in peripheral blood mononuclear cells. This is independent of any potential effects of immunosuppressive medications. Based on this model, we also expect that lupus flares will increase the likelihood of infection with SARS-CoV-2 due to increased oxidative stress and DNA demethylation of ACE2. Therefore, maintaining remission in lupus patients is of critical importance during this pandemic. Drugs that target epigenetic mechanisms should be considered for further investigation in COVID-19. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the Ethical approval information: All study participants signed informed consents approved by the institutional review boards/ethics committees of our institutions. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint 1 0 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.30.20047852 doi: medRxiv preprint Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Epigenetic Variability in Systemic Lupus Erythematosus: What We Learned from Genome-Wide DNA Methylation Studies Genome-wide DNA methylation study suggests epigenetic accessibility and transcriptional poising of interferon-regulated genes in naive CD4+ T cells from lupus patients Epigenome profiling reveals significant DNA demethylation of interferon signature genes in lupus neutrophils DNA methylation mapping identifies gene regulatory effects in patients with systemic lupus erythematosus Demethylation of CD40LG on the inactive X in T cells from women with lupus Autoimmunity and Klinefelter's syndrome: when men have two X chromosomes SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Characterisation of an epigenetically altered CD4(+) CD28(+) Kir(+) T cell subset in autoimmune rheumatic diseases by multiparameter flow cytometry CD4+CD28+KIR+CD11a(hi) T cells correlate with disease activity and are characterized by a pro-inflammatory epigenetic and transcriptional profile in lupus patients DNA methylation and mRNA and microRNA expression of SLE CD4+ T cells correlate with disease phenotype Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys The DNA methylation signature of human TCRalphabeta+CD4-CD8-double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response. Clinical 16. Gu J, Korteweg C. Pathology and pathogenesis of severe acute respiratory syndrome Oxidative stress, T cell DNA methylation, and lupus Impaired T cell protein kinase C delta activation decreases ERK pathway signaling in idiopathic and hydralazine-induced lupus Oxidative stress in the pathology and treatment of systemic lupus erythematosus Deoxyribonucleic Acid Methylation in Systemic Lupus Erythematosus: Implications for Future Clinical Practice Epigenetic Reprogramming in Naive CD4+ T Cells Favoring T Cell Activation and Non-Th1 Effector T Cell Immune Response as an Early Event in Lupus Flares All rights reserved. No reuse allowed without permission author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the