Epigenetic features, methods, and implementations associated with COVID-19

The infection and life cycle of severe acute respiratory syndrome coronavirus 2 are widely studied, yet multiple gaps exist in the knowledge that affects therapeutic developments against coronavirus disease 2019 (COVID-19). Predominantly caused by a respiratory virus, COVID-19 is not restricted to the respiratory tract but affects multiple organs of the body including the cardiovascular, neurological, immunological, and renal systems. COVID-19 affects all age groups, although the elderly population inherently presenting with multiple comorbidities are disproportionately affected. The majority of the patients experience mild symptoms, although moderate, severe, and critical symptoms occur in a smaller group of patients. Interestingly, the effects of the disease can be acute or chronic and present an ongoing health care challenge. Epigenetic mechanisms of COVID-19 (DNA methylation, histone posttranslational modifications, histone citrullination, etc.) are an emerging field and present enormous potential toward the medical management of COVID-19. Angiotensin converting enzyme 2, an important protein in the cardiovascular system, is a receptor for viral entry into cells, and the epigenetic processes that regulate this protein have been widely studied. Identification of the epitranscriptomic profile has led to the identification of putative biomarkers for disease diagnosis and trials of novel epidrugs for targeted therapy. © 2023 Elsevier Inc. All rights reserved.

Sex chromosome complement and sex steroid signaling underlie sex differences in immunity to respiratory virus infection.

Epidemiological studies have revealed sex differences in the incidence and morbidity of respiratory virus infection in the human population, and often these observations are correlated with sex differences in the quality or magnitude of the immune response. Sex differences in immunity and morbidity also are observed in animal models of respiratory virus infection, suggesting differential dominance of specific immune mechanisms. Emerging research shows intrinsic sex differences in immune cell transcriptomes, epigenomes, and proteomes that may regulate human immunity when challenged by viral infection. Here, we highlight recent research into the role(s) of sex steroids and X chromosome complement in immune cells and describe how these findings provide insight into immunity during respiratory virus infection. We focus on the regulation of innate and adaptive immune cells by receptors for androgen and estrogens, as well as genes with a propensity to escape X chromosome inactivation. A deeper mechanistic knowledge of these pathways will help us to understand the often significant sex differences in immunity to endemic or pandemic respiratory pathogens such as influenza viruses, respiratory syncytial viruses and pathogenic coronaviruses.

Unusual X chromosome inactivation maintenance in female alveolar type 2 cells is correlated with increased numbers of X-linked escape genes and sex-biased gene expression.

Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA "clouds" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.

Genetic and hormonal mechanisms underlying sex-specific immune responses in tuberculosis.

Tuberculosis (TB), the world's deadliest bacterial infection, afflicts more human males than females, with a male/female (M/F) ratio of 1.7. Sex disparities in TB prevalence, pathophysiology, and clinical manifestations are widely reported, but the underlying biological mechanisms remain largely undefined. This review assesses epidemiological data on sex disparity in TB, as well as possible underlying hormonal and genetic mechanisms that might differentially modulate innate and adaptive immune responses in males and females, leading to sex differences in disease susceptibility. We consider whether this sex disparity can be extended to the efficacy of vaccines and discuss novel animal models which may offer mechanistic insights. A better understanding of the biological factors underpinning sex-related immune responses in TB may enable sex-specific personalized therapies for TB.

Stem cell-derived trophoblast organoids model human placental development, X chromosome inactivation, and susceptibility to emerging pathogens

Problem: Trophoblast organoids derived from human placental villi provide a powerful 3D model system of placental development, but access to first-trimester tissues is limited due to ethical and legal restrictions. Here we sought to establish a methodology for establishing 3D trophoblast organoids from naïve human pluripotent stem cells (hPSCs), which have an expanded potential for extraembryonic differentiation. Method of Study: We previously demonstrated that naïve hPSCs readily give rise to self-renewing human trophoblast cells (hTSCs) that resemble post-implantation cytotrophoblast (CTB) progenitors and can further differentiate into specialized trophoblast lineages. Here we examined whether hTSCs derived from three distinct sources (naïve hPSCs, human blastocysts, and first-trimester placental tissues) have the potential to self-organize into 3D trophoblast organoids by transfer to Matrigel droplets in the presence of trophoblast organoid medium. The expression of protein markers in the resulting stem cellderived trophoblast organoids (SC-TOs) was examined by immunofluorescence and light-sheet microscopy, while their single cell transcriptome was analyzed using the 10X Genomics platform. We also investigated the X chromosome inactivation (XCI) status of organoids derived from female naïve hPSCs and their ability to differentiate into invasive extravillous trophoblast (EVT) organoids. Finally, we evaluated whether SC-TOs are susceptible to infection by various emerging pathogens (SARS-CoV-2 and Zika virus), as a basis for establishing a stem cell-based model system of placental infections during the first trimester. Results: Trophoblast organoids generated from naïve and primary hTSCs displayed comparable tissue architecture, placental hormone secretion, microRNA expression, and capacity for long-term selfrenewal. In-depth single cell transcriptome profiling revealed that SCTOs encompass a variety of trophoblast identities that closely correspond to CTB progenitor, syncytiotrophoblast (STB) and EVT cell types found in human post-implantation embryos. Interestingly, the cellular composition in trophoblast organoids derived from naïve and primary hTSCs was highly similar, which suggests that trophoblast organoid culture represents a powerful attractor state in which the influence of subtle epigenetic differences between naïve and primary hTSCs is mitigated. These organoid cultures displayed clonal XCI patterns previously described in the human placenta.Upon differentiation into specialized EVT organoids, extensive trophoblast invasion was observed in co-culture assays with human endometrial cells. We further demonstrated that SC-TOs display selective vulnerability to infection by SARS-CoV-2 and Zika virus, which correlated with the expression levels of their respective entry factors. Conclusions: The generation of trophoblast organoids from naïve hPSCs provides an accessible and patient-specific 3D model system of the developing placenta and its susceptibility to emerging pathogens. The ability to genetically manipulate naïve hPSCs prior to differentiation into SC-TOs enables functional interrogation of regulatory factors implicated in placental organogenesis.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

Why Females Do Better: The X Chromosomal TLR7 Gene-Dose Effect in COVID-19.

A male sex bias has emerged in the COVID-19 pandemic, fitting to the sex-biased pattern in other viral infections. Males are 2.84 times more often admitted to the ICU and mortality is 1.39 times higher as a result of COVID-19. Various factors play a role in this, and novel studies suggest that the gene-dose of Toll-Like Receptor (TLR) 7 could contribute to the sex-skewed severity. TLR7 is one of the crucial pattern recognition receptors for SARS-CoV-2 ssRNA and the gene-dose effect is caused by X chromosome inactivation (XCI) escape. Female immune cells with TLR7 XCI escape have biallelic TLR7 expression and produce more type 1 interferon (IFN) upon TLR7 stimulation. In COVID-19, TLR7 in plasmacytoid dendritic cells is one of the pattern recognition receptors responsible for IFN production and a delayed IFN response has been associated with immunopathogenesis and mortality. Here, we provide a hypothesis that females may be protected to some extend against severe COVID-19, due to the biallelic TLR7 expression, allowing them to mount a stronger and more protective IFN response early after infection. Studies exploring COVID-19 treatment via the TLR7-mediated IFN pathway should consider this sex difference. Various factors such as age, sex hormones and escape modulation remain to be investigated concerning the TLR7 gene-dose effect.

B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells.

The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine.

Sex Differences in Immunity: Implications for the Development of Novel Vaccines Against Emerging Pathogens.

Vaccines are one of the greatest public health achievements and have saved millions of lives. They represent a key countermeasure to limit epidemics caused by emerging infectious diseases. The Ebola virus disease crisis in West Africa dramatically revealed the need for a rapid and strategic development of vaccines to effectively control outbreaks. Seven years later, in light of the SARS-CoV-2 pandemic, this need has never been as urgent as it is today. Vaccine development and implementation of clinical trials have been greatly accelerated, but still lack strategic design and evaluation. Responses to vaccination can vary widely across individuals based on factors like age, microbiome, co-morbidities and sex. The latter aspect has received more and more attention in recent years and a growing body of data provide evidence that sex-specific effects may lead to different outcomes of vaccine safety and efficacy. As these differences might have a significant impact on the resulting optimal vaccine regimen, sex-based differences should already be considered and investigated in pre-clinical and clinical trials. In this Review, we will highlight the clinical observations of sex-specific differences in response to vaccination, delineate sex differences in immune mechanisms, and will discuss the possible resulting implications for development of vaccine candidates against emerging infections. As multiple vaccine candidates against COVID-19 that target the same antigen are tested, vaccine development may undergo a decisive change, since we now have the opportunity to better understand mechanisms that influence vaccine-induced reactogenicity and effectiveness of different vaccines.

A theory on SARS-COV-2 susceptibility: reduced TLR7-activity as a mechanistic link between men, obese and elderly.

I hypothesize that chronic stimulation of tlr7 by intrinsic substrates in old and obese people leads to a desensitization of tlr7-signalling such that upon viral infection the immune response in those patients will be delayed, allowing the virus to more easily establish itself in these at-risk patient groups. Upon severe viral infection, resensitization of tlr7-signalling occurs so as to better fight the viral infection. This, however, leads to a situation where old and obese patients develop an overwhelming tlr7-response due to the presence of not only the viral RNA, but also the intrinsic tlr7 substrates. Thus, old and obese patients with serious COVID-19 develop a state that is similar to SLE-patients and consequently should be treated in a similar manner. The theory predicts that drugs and treatment regimens that help to control SLE-flares might also help in late-stage COVID-19 patients.