Testosterone deficiency promotes arterial stiffening independent of sex chromosome complement.

BACKGROUND: Sex hormones and sex chromosomes play a vital role in cardiovascular disease. Testosterone plays a crucial role in men's health. Lower testosterone level is associated with cardiovascular and cardiometabolic diseases, including inflammation, atherosclerosis, and type 2 diabetes. Testosterone replacement is beneficial or neutral to men's cardiovascular health. Testosterone deficiency is associated with cardiovascular events. Testosterone supplementation to hypogonadal men improves libido, increases muscle strength, and enhances mood. We hypothesized that sex chromosomes (XX and XY) interaction with testosterone plays a role in arterial stiffening. METHODS: We used four core genotype male mice to understand the inherent contribution of sex hormones and sex chromosome complement in arterial stiffening. Age-matched mice were either gonadal intact or castrated at eight weeks plus an additional eight weeks to clear endogenous sex hormones. This was followed by assessing blood pressure, pulse wave velocity, echocardiography, and ex vivo passive vascular mechanics. RESULTS: Arterial stiffening but not blood pressure was more significant in castrated than testes-intact mice independent of sex chromosome complement. Castrated mice showed a leftward shift in stress-strain curves and carotid wall thinning. Sex chromosome complement (XX) in the absence of testosterone increased collagen deposition in the aorta and Kdm6a gene expression. CONCLUSION: Testosterone deprivation increases arterial stiffening and vascular wall remodeling. Castration increases Col1α1 in male mice with XX sex chromosome complement. Our study shows decreased aortic contractile genes in castrated mice with XX than XY sex chromosomes.

Cardiovascular disease is the leading cause of death worldwide. Cardiovascular disease presents differently in men and women. While men develop plaque buildup in large arteries, women develop buildup in the microvessels in the heart. Arterial stiffening, which is the hardening of arteries, increases with age in both men and women. Aging, coupled with the decline in sex hormones, exacerbates cardiovascular disease in women compared to men. Men with XY sex chromosomes have higher circulating testosterone, while women with XX sex chromosomes have increased circulating estradiol. The potential benefits of sex hormone replacement therapy are shown in men and women. Indeed, testosterone replacement deficiency is associated with adverse cardiovascular outcomes in men. Whether adverse events are dependent or independent of sex hormones' interaction with sex chromosomes is unknown. This study used the four core genotype mice comprising males with either XX or XY sex chromosome complement. We show castration increases arterial stiffening and collagen deposition on the arterial wall. We also identified the escapee and smooth muscle contractile genes that may play a role in arterial stiffening. Our data suggests that testosterone deprivation mediates arterial stiffening and remodeling.

Xist Deletion in B Cells Results in Systemic Lupus Erythematosus Phenotypes.

Systemic lupus erythematosus (SLE) is an autoimmune disease preferentially observed in females. X-linked gene expression in XX females is normalized to that of XY males by X-Chromosome Inactivation (XCI). However, B cells from female SLE patients and mouse models of SLE exhibit mislocalization of Xist RNA, a critical regulator of XCI, and aberrant expression of X-linked genes, suggesting that impairment of XCI may contribute to disease. Here, we find that a subset of female mice harboring a conditional deletion of Xis t in B cells ("Xist cKO") spontaneously develop SLE phenotypes, including expanded activated B cell subsets, disease-specific autoantibodies, and glomerulonephritis. Moreover, pristane-induced SLE-like disease is more severe in Xist cKO mice. Activated B cells from Xist cKO mice with SLE phenotypes have increased expression of proinflammatory X-linked genes implicated in SLE. Together, this work indicates that impaired XCI maintenance in B cells directly contributes to the female-bias of SLE.

NF-κB Signaling is Required for X-Chromosome Inactivation Maintenance Following T cell Activation.

X Chromosome Inactivation (XCI) is a female-specific process which balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of Xist RNA and heterochromatic modifications on the inactive X chromosome (Xi), and these modifications become enriched at the Xi after cell stimulation. Here, we examined allele-specific gene expression and the epigenomic profiles of the Xi following T cell stimulation. We found that the Xi in unstimulated T cells is largely dosage compensated and is enriched with the repressive H3K27me3 modification, but not the H2AK119-ubiquitin (Ub) mark, even at promoters of XCI escape genes. Upon CD3/CD28-mediated T cell stimulation, the Xi accumulates H2AK119-Ub and H3K27me3 across the Xi. Next, we examined the T cell signaling pathways responsible for Xist RNA localization to the Xi and found that T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of TCR, is required. Disruption of NF-κB signaling, using inhibitors or genetic deletions, in mice and patients with immunodeficiencies prevents Xist/XIST RNA accumulation at the Xi and alters expression of some X-linked genes. Our findings reveal a novel connection between NF-κB signaling pathways which impact XCI maintenance in female T cells.

The conneXion between sex and immune responses.

There are notable sex-based differences in immune responses to pathogens and self-antigens, with female individuals exhibiting increased susceptibility to various autoimmune diseases, and male individuals displaying preferential susceptibility to some viral, bacterial, parasitic and fungal infections. Although sex hormones clearly contribute to sex differences in immune cell composition and function, the presence of two X chromosomes in female individuals suggests that differential gene expression of numerous X chromosome-linked immune-related genes may also influence sex-biased innate and adaptive immune cell function in health and disease. Here, we review the sex differences in immune system composition and function, examining how hormones and genetics influence the immune system. We focus on the genetic and epigenetic contributions responsible for altered X chromosome-linked gene expression, and how this impacts sex-biased immune responses in the context of pathogen infection and systemic autoimmunity.

Testosterone Deficiency Promotes Arterial Stiffening Independent of Sex Chromosome Complement.

Background: Testosterone plays a vital role in men's health. Lower testosterone level is associated with cardiovascular and cardiometabolic diseases, including inflammation, atherosclerosis, and type 2 diabetes. Testosterone replacement is beneficial or neutral to men's cardiovascular health. Testosterone deficiency is associated with cardiovascular events. Testosterone supplementation to hypogonadal men improves libido, increases muscle strength, and enhances mood. We hypothesized that sex chromosomes (XX and XY) interaction with testosterone plays a role in arterial stiffening. Methods: We used four core genotype male mice to understand the inherent contribution of sex hormones and sex chromosome complement in arterial stiffening. Age-matched mice were either gonadal intact or castrated for eight weeks, followed by an assessment of blood pressure, pulse wave velocity, echocardiography, and ex vivo passive vascular mechanics. Results: Arterial stiffening but not blood pressure was more significant in castrated than testes-intact mice independent of sex chromosome complement. Castrated mice showed a leftward shift in stress-strain curves and carotid wall thinning. Sex chromosome complement (XX) in the absence of testosterone increased collagen deposition in the aorta and Kdm6a gene expression. Conclusion: Testosterone deprivation increases arterial stiffening and vascular wall remodeling. Castration increases Col1α1 in male mice with XX sex chromosome complement. Our study shows decreased aortic contractile genes in castrated mice with XX than XY sex chromosomes.

Modulation of Recovery from Neonatal Hyperoxic Lung Injury by Sex as a Biological Variable.

Recovery from lung injury during the neonatal period requires the orchestration of many biological pathways. The modulation of such pathways can drive the developing lung towards proper repair or persistent maldevelopment that can lead to a disease phenotype. Sex as a biological variable can regulate these pathways differently in the male and female lung exposed to neonatal hyperoxia. In this study, we assessed the contribution of cellular diversity in the male and female neonatal lung following injury. Our objective was to investigate sex and cell-type specific transcriptional changes that drive repair or persistent injury in the neonatal lung and delineate the alterations in the immune-endothelial cell communication networks using single cell RNA sequencing (sc-RNAseq) in a murine model of hyperoxic injury. We generated transcriptional profiles of >55,000 cells isolated from the lungs of postnatal day 1 (PND 1) and postnatal day 21 (PND 21) neonatal male and female C57BL/6 mice exposed to 95% FiO 2 between PND 1-5 (saccular stage of lung development). We show the presence of sex-based differences in the transcriptional states of lung endothelial and immune cells at PND 1 and PND 21. Furthermore, we demonstrate that biological sex significantly influences the response to injury, with a greater number of differentially expressed genes showing sex-specific patterns than those shared between male and female lungs. Pseudotime trajectory analysis highlighted genes needed for lung development that were altered by hyperoxia. Finally, we show intercellular communication between endothelial and immune cells at saccular and alveolar stages of lung development with sex-based biases in the crosstalk and identify novel ligand-receptor pairs. Our findings provide valuable insights into the cell diversity, transcriptional state, developmental trajectory, and cell-cell communication underlying neonatal lung injury, with implications for understanding lung development and possible therapeutic interventions while highlighting the crucial role of sex as a biological variable.

Impaired dynamic X-chromosome inactivation maintenance in T cells is a feature of spontaneous murine SLE that is exacerbated in female-biased models.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a highly female-biased systemic autoimmune disease, but the molecular basis for this female bias remains incompletely elucidated. B and T lymphocytes from patients with SLE and female-biased mouse models of SLE exhibit features of epigenetic dysregulation on the X chromosome which may contribute to this strong female bias. We therefore examined the fidelity of dynamic X-chromosome inactivation maintenance (dXCIm) in the pathogenesis of two murine models of spontaneous lupus-NZM2328 and MRL/lpr-with disparate levels of female-bias to determine whether impaired dXCIm contributes to the female bias of disease. METHODS: CD23+ B cells and CD3+ T cells were purified from age-matched C57BL/6 (B6), MRL/lpr, and NZM2328 male and female mice, activated in vitro, and processed for Xist RNA fluorescence in situ hybridization, H3K27me3 immunofluorescence imaging, qPCR, and RNA sequencing analyses. RESULTS: The dynamic relocalization of Xist RNA and the canonical heterochromatin mark, H3K27me3, to the inactive X chromosome was preserved in CD23+ B cells, but impaired in activated CD3+ T cells from the MRL/lpr model (p < 0.01 vs. B6), and even more impaired in the heavily female-biased NZM2328 model (p < 0.001 vs. B6; p < 0.05 vs. MRL/lpr). RNAseq of activated T cells from NZM2328 mice revealed the female-biased upregulation of 32 X-linked genes distributed broadly across the X chromosome, many of which have roles in immune function. Many genes encoding Xist RNA-interacting proteins were also differentially expressed and predominantly downregulated, which may account for the observed mislocalization of Xist RNA to the inactive X chromosome. CONCLUSIONS: Although evident in T cells from both the MRL/lpr and NZM2328 models of spontaneous SLE, impaired dXCIm is more severe in the heavily female-biased NZM2328 model. The aberrant X-linked gene dosage in female NZM2328 mice may contribute towards the development of female-biased immune responses in SLE-prone hosts. These findings provide important insights into the epigenetic mechanisms contributing to female-biased autoimmunity.

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.

Modulation of recovery from neonatal hyperoxic lung injury by sex as a biological variable.

Recovery from lung injury during the neonatal period requires the orchestration of many biological pathways. The modulation of such pathways can drive the developing lung towards proper repair or persistent maldevelopment that can lead to a disease phenotype. Sex as a biological variable can regulate these pathways differently in the male and female lung exposed to neonatal hyperoxia. In this study, we assessed the contribution of cellular diversity in the male and female neonatal lung following injury. Our objective was to investigate sex and cell-type specific transcriptional changes that drive repair or persistent injury in the neonatal lung and delineate the alterations in the immune-endothelial cell communication networks using single cell RNA sequencing (sc-RNAseq) in a murine model of hyperoxic injury. We generated transcriptional profiles of >55,000 cells isolated from the lungs of postnatal day 1 (PND 1; pre-exposure), PND 7, and PND 21neonatal male and female C57BL/6 mice exposed to 95 % FiO2 between PND 1-5 (saccular stage of lung development). We show the presence of sex-based differences in the transcriptional states of lung endothelial and immune cells at PND 1 and PND 21. Furthermore, we demonstrate that biological sex significantly influences the response to injury, with a greater number of differentially expressed genes showing sex-specific patterns than those shared between male and female lungs. Pseudotime trajectory analysis highlighted genes needed for lung development that were altered by hyperoxia. Finally, we show intercellular communication between endothelial and immune cells at saccular and alveolar stages of lung development with sex-based biases in the crosstalk and identify novel ligand-receptor pairs. Our findings provide valuable insights into the cell diversity, transcriptional state, developmental trajectory, and cell-cell communication underlying neonatal lung injury, with implications for understanding lung development and possible therapeutic interventions while highlighting the crucial role of sex as a biological variable.

The X in seX-biased immunity and autoimmune rheumatic disease.

Sexual dimorphism in the composition and function of the human immune system has important clinical implications, as males and females differ in their susceptibility to infectious diseases, cancers, and especially systemic autoimmune rheumatic diseases. Both sex hormones and the X chromosome, which bears a number of immune-related genes, play critical roles in establishing the molecular basis for the observed sex differences in immune function and dysfunction. Here, we review our current understanding of sex differences in immune composition and function in health and disease, with a specific focus on the contribution of the X chromosome to the striking female bias of three autoimmune rheumatic diseases.

Long Noncoding RNAs That Function in Nutrition: Lnc-ing Nutritional Cues to Metabolic Pathways.

Long noncoding RNAs (lncRNAs) are sensitive to changing environments and play key roles in health and disease. Emerging evidence indicates that lncRNAs regulate gene expression to shape metabolic processes in response to changing nutritional cues. Here we review various lncRNAs sensitive to fasting, feeding, and high-fat diet in key metabolic tissues (liver, adipose, and muscle), highlighting regulatory mechanisms that trigger expression changes of lncRNAs themselves, and how these lncRNAs regulate gene expression of key metabolic genes in specific cell types or across tissues. Determining how lncRNAs respond to changes in nutrition is critical for our understanding of the complex downstream cascades following dietary changes and can shape how we treat metabolic disease. Furthermore, investigating sex biases that might influence lncRNA-regulated responses will likely reveal contributions toward the observed disparities between the sexes in metabolic diseases.

The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients.

Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.

Derivation and investigation of the first human cell-based model of Beckwith-Wiedemann syndrome.

Genomic imprinting is a rare form of gene expression in mammals in which a small number of genes are expressed in a parent-of-origin-specific manner. The aetiology of human imprinting disorders is diverse and includes chromosomal abnormalities, mutations, and epigenetic dysregulation of imprinted genes. The most common human imprinting disorder is Beckwith-Wiedemann syndrome (BWS), frequently caused by uniparental isodisomy and DNA methylation alterations. Because these lesions cannot be easily engineered, induced pluripotent stem cells (iPSC) are a compelling alternative. Here, we describe the first iPSC model derived from patients with BWS. Due to the mosaic nature of BWS patients, both BWS and non-BWS iPSC lines were derived from the same patient's fibroblasts. Importantly, we determine that DNA methylation and gene expression patterns of the imprinted region in the iPSC lines reflect the parental cells and are stable over time. Additionally, we demonstrate that differential expression in insulin signalling, cell proliferation, and cell cycle pathways was seen in hepatocyte lineages derived from BWS lines compared to controls. Thus, this cell based-model can be used to investigate the role of imprinting in the pathogenesis of BWS in disease-relevant cell types.

Time to get ill: the intersection of viral infections, sex, and the X chromosome.

Females have more robust immune responses than males, and viral infections are more severe for males. Hormones and genetic sex, namely the X chromosome, influence sex differences with immune responses. Here, we review recent findings underlying sexual dimorphism of disease susceptibility for two prevalent viral infections, influenza and SARS-CoV-2, which exhibit male-biased disease severity. Viral infections are proposed to be an initiating event for autoimmunity, which exhibits a female bias. We also review recent work elucidating the epigenetic and genetic contribution of X-Chromosome Inactivation maintenance, and X-linked gene expression, for the autoimmune disorder Systemic Lupus Erythematosus, and highlight the complex considerations required for identifying underlying hormonal and genetic contributions responsible for sex differences in immune responses.

Loss of epigenetic modifications on the inactive X chromosome and sex-biased gene expression profiles in B cells from NZB/W F1 mice with lupus-like disease.

The mechanisms underlying the female-bias in autoimmunity are poorly understood. The contribution of genetic and epigenetic factors from the inactive X chromosome (Xi) are beginning to emerge as critical mediators of autoimmunity in females. Here, we ask how epigenetic features of the Xi change during disease development in B cells from the NZB/W F1 spontaneous mouse model of lupus, which is female-biased. We find that Xist RNA becomes increasingly mislocalized from the Xi with disease onset. While NZB/W F1 naïve B cells have H3K27me3 foci on the Xi, which are missing from healthy C57BL/6 and BALB/c mice, these foci are progressively lost in stimulated B cells during disease. Using single-molecule RNA FISH, we show that the X-linked gene Tlr7 is biallelically expressed in ~20% of NZB/W F1 B cells, and that the amount of biallelic expression does not change with disease. We also present sex-specific gene expression profiles for diseased NZB/W F1 B cells, and find female-specific upregulation of 20 genes, including the autoimmunity-related genes Cxcl13, Msr1, Igj, and Prdm1. Together, these studies provide important insight into the loss of epigenetic modifications from the Xi and changes with gene expression in a mouse model of female-biased SLE.

Enjoy the silence: X-chromosome inactivation diversity in somatic cells.

The imbalance of sex chromosomes between females (XX) and males (XY) necessitates strict regulation of X-linked gene expression. X-Chromosome Inactivation (XCI) selects one X for transcriptional silencing in the early embryo, generating an epigenetically distinct and transcriptionally silent X that is maintained into adulthood. Some genes on the inactive X escape XCI, and human somatic cells have a greater number of escape genes compared to mice. Advances with single-cell technologies have revealed human-specific escape genes in fibroblasts and immune cells, some of which exhibit cell and tissue specificity. Here, we review recent discoveries of dynamic XCI in female immune cells, which have changed our understanding of XCI maintenance, and discuss how some X-linked genes might become overexpressed in female-biased autoimmunity.

The long noncoding RNA Morrbid regulates CD8 T cells in response to viral infection.

The transcriptional programs that regulate CD8 T-cell differentiation and function in the context of viral infections or tumor immune surveillance have been extensively studied; yet how long noncoding RNAs (lncRNAs) and the loci that transcribe them contribute to the regulation of CD8 T cells during viral infections remains largely unexplored. Here, we report that transcription of the lncRNA Morrbid is specifically induced by T-cell receptor (TCR) and type I IFN stimulation during the early stages of acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. In response to type I IFN, the Morrbid RNA and its locus control CD8 T cell expansion, survival, and effector function by regulating the expression of the proapoptotic factor, Bcl2l11, and by modulating the strength of the PI3K-AKT signaling pathway. Thus, our results demonstrate that inflammatory cue-responsive lncRNA loci represent fundamental mechanisms by which CD8 T cells are regulated in response to pathogens and potentially cancer.

When the balance is broken: X-linked gene dosage from two X chromosomes and female-biased autoimmunity.

Women and men exhibit differences in innate and adaptive immunity, and women are more susceptible to numerous autoimmune disorders. Two or more X chromosomes increases the risk for some autoimmune diseases, and increased expression of some X-linked immune genes is frequently observed in female lymphocytes from autoimmune patients. Evidence from mouse models of autoimmunity also supports the idea that increased expression of X-linked genes is a feature of female-biased autoimmunity. Recent studies have begun to elucidate the correlation between abnormal X-chromosome inactivation (XCI), an essential mechanism female somatic cells use to equalize X-linked gene dosage between the sexes, and autoimmunity in lymphocytes. In this review, we highlight research describing overexpression of X-linked immunity-related genes and female-biased autoimmunity in both humans and mouse models, and make connections with our recent work elucidating lymphocyte-specific mechanisms of XCI maintenance that become altered in lupus patients.