Maintenance of X chromosome inactivation after T cell activation requires NF-κB signaling.

X chromosome inactivation (XCI) balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of X-inactive specific transcript (Xist) RNA and heterochromatic modifications on the inactive X chromosome (Xi), which are involved in maintenance of XCI, and these modifications return to the Xi after stimulation. Here, we examined allele-specific gene expression and epigenomic profiles of the Xi in T cells. We found that the Xi in unstimulated T cells is largely dosage compensated and enriched with the repressive H3K27me3 modification but not the H2AK119-ubiquitin (Ub) mark. Upon T cell stimulation mediated by both CD3 and CD28, the Xi accumulated H2AK119-Ub at gene regions of previous H3K27me3 enrichment. T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of the TCR, was required for Xist RNA localization to the Xi. Disruption of NF-κB signaling in mouse and human T cells using genetic deletion, chemical inhibitors, and patients with immunodeficiencies prevented Xist/XIST RNA accumulation at the Xi and altered X-linked gene expression. Our findings reveal a previously undescribed connection between NF-κB signaling pathways, which affects XCI maintenance in T cells in females.

Characterization of cxorf21 Provides Molecular Insight Into Female-Bias Immune Response in SLE Pathogenesis.

Background: Ninety percent of systemic lupus erythematosus (SLE) patients are women. X chromosome-dosage increases susceptibility to SLE and primary Sjögren's syndrome (pSS). Chromosome X open reading frame 21 (CXorf21) escapes X-inactivation and is an SLE risk gene of previously unknown function. We undertook the present study to delineate the function of CXorf21 in the immune system as well as investigate a potential role in the sex bias of SLE and pSS. Methods: Western blot protein analysis, qPCR, BioPlex cytokine immunoassay, pHrodo™ assays, as well as in vitro CRISPR-Cas9 knockdown experiments were employed to delineate the role of CXorf21 in relevant immunocytes. Results: Expressed in monocytes and B cells, CXorf21 basal Mrna, and protein expression levels are elevated in female primary monocytes, B cells, and EBV-transformed B cells compared to male cells. We also found CXorf21 mRNA and protein expression is higher in both male and female cells from SLE patients compared to control subjects. TLR7 ligation increased CXorf21 protein expression and CXorf21 knockdown abrogated TLR7-driven increased IFNA1 mRNA expression, and reduced secretion of both TNF-alpha and IL-6 in healthy female monocytes. Similarly, we found increased pH in the lysosomes of CXorf21-deficient female monocytes. Conclusion: CXorf21 is more highly expressed in female compared to male cells and is involved in a sexually dimorphic response to TLR7 activation. In addition, CXorf21 expression regulates lysosomal pH in a sexually dimorphic manner. Thus, sexually dimorphic expression of CXorf21 skews cellular immune responses in manner consistent with expected properties of a mediator of the X chromosome dose risk in SLE and pSS.

Aberrant X chromosome skewing and acquired clonal hematopoiesis in adult-onset common variable immunodeficiency.

Advances in genomic medicine have elucidated an increasing number of genetic etiologies for patients with common variable immunodeficiency (CVID). However, there is heterogeneity in clinical and immunophenotypic presentations and a limited understanding of the underlying pathophysiology of many cases. The primary defects in CVID may extend beyond the adaptive immune system, and the combined defect in both the myeloid and lymphoid compartments suggests the mechanism may involve bone marrow output and earlier progenitors. Using the methylation profile of the human androgen receptor (AR) gene as a surrogate epigenetic marker for bone marrow clonality, we examined the hematopoietic compartments of patients with CVID. Our data show that clonal hematopoiesis is common among patients with adult-onset CVID who do not have associated noninfectious complications. Nonblood tissues did not show a skewed AR methylation status, supporting a model of an acquired clonal hematopoietic event. Attenuation of memory B cell differentiation into long-lived plasma cells (CD20-CD27+CD38+CD138+) was associated with marked changes in the postdifferentiation methylation profile, demonstrating the functional consequence of clonal hematopoiesis on humoral immunity in these patients. This study sheds light on a potential etiology of a subset of patients with CVID, and the findings suggest that it is a stage of an acquired lymphocyte maturation disorder.

Cutting Edge: CXCR3 Escapes X Chromosome Inactivation in T Cells during Infection: Potential Implications for Sex Differences in Immune Responses.

CXCR3, an X-linked gene, is subject to X chromosome inactivation (XCI), but it is unclear whether CXCR3 escapes XCI in immune cells. We determined whether CXCR3 escapes XCI in vivo, evaluated the contribution of allelic CXCR3 expression to the phenotypic properties of T cells during experimental infection with Leishmania, and examined the potential implications to sex differences in immune responses. We used a bicistronic CXCR3 dual-reporter mouse, with each CXCR3 allele linked to a green or red fluorescent reporter without affecting endogenous CXCR3 expression. Our results show that CXCR3 escapes XCI, biallelic CXCR3-expressing T cells produce more CXCR3 protein than monoallelic CXCR3-expressing cells, and biallelic CXCR3-expressing T cells produce more IFN-γ, IL-2, and CD69 compared with T cells that express CXCR3 from one allele during Leishmania mexicana infection. These results demonstrate that XCI escape by CXCR3 potentially contributes to the sex-associated bias observed during infection.

Altered X-chromosome inactivation in T cells may promote sex-biased autoimmune diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that predominantly affects women and is driven by autoreactive T cell-mediated inflammation. It is known that individuals with multiple X-chromosomes are at increased risk for developing SLE; however, the mechanisms underlying this genetic basis are unclear. Here, we use single cell imaging to determine the epigenetic features of the inactive X (Xi) in developing thymocytes, mature T cell subsets, and T cells from SLE patients and mice. We show that Xist RNA and heterochromatin modifications transiently reappear at the Xi and are missing in mature single positive T cells. Activation of mature T cells restores Xist RNA and heterochromatin marks simultaneously back to the Xi. Notably, X-chromosome inactivation (XCI) maintenance is altered in T cells of SLE patients and late-stage-disease NZB/W F1 female mice, and we show that X-linked genes are abnormally upregulated in SLE patient T cells. SLE T cells also have altered expression of XIST RNA interactome genes, accounting for perturbations of Xi epigenetic features. Thus, abnormal XCI maintenance is a feature of SLE disease, and we propose that Xist RNA localization at the Xi could be an important factor for maintaining dosage compensation of X-linked genes in T cells.

Female predisposition to TLR7-driven autoimmunity: gene dosage and the escape from X chromosome inactivation.

Women develop stronger immune responses than men, with positive effects on the resistance to viral or bacterial infections but magnifying also the susceptibility to autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, the dosage of the endosomal Toll-like receptor 7 (TLR7) is crucial. Murine models have shown that TLR7 overexpression suffices to induce spontaneous lupus-like disease. Conversely, suppressing TLR7 in lupus-prone mice abolishes SLE development. TLR7 is encoded by a gene on the X chromosome gene, denoted TLR7 in humans and Tlr7 in the mouse, and expressed in plasmacytoid dendritic cells (pDC), monocytes/macrophages, and B cells. The receptor recognizes single-stranded RNA, and its engagement promotes B cell maturation and the production of pro-inflammatory cytokines and antibodies. In female mammals, each cell randomly inactivates one of its two X chromosomes to equalize gene dosage with XY males. However, 15 to 23% of X-linked human genes escape X chromosome inactivation so that both alleles can be expressed simultaneously. It has been hypothesized that biallelic expression of X-linked genes could occur in female immune cells, hence fostering harmful autoreactive and inflammatory responses. We review here the current knowledge of the role of TLR7 in SLE, and recent evidence demonstrating that TLR7 escapes from X chromosome inactivation in pDCs, monocytes, and B lymphocytes from women and Klinefelter syndrome men. Female B cells where TLR7 is thus biallelically expressed display higher TLR7-driven functional responses, connecting the presence of two X chromosomes with the enhanced immunity of women and their increased susceptibility to TLR7-dependent autoimmune syndromes.

TLR7 escapes X chromosome inactivation in immune cells.

Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key pathogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prevalence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary X chromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is encoded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chromosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7 allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasmacytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chromosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than the monoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations. Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27+ plasma cells. In addition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immunoglobulin G class switch during the TLR7-driven, T cell-dependent differentiation of naive B lymphocytes into immunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with added responsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing to biallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and in men with Klinefelter syndrome.

Intragenomic Conflict and Immune Tolerance: Do Selfish X-Linked Alleles Drive Skewed X Chromosome Inactivation?

In mammalian females, diploid somatic cells contain two X chromosomes, one of which is transcriptionally silenced, in a process termed X chromosome inactivation (XCI). Whereas XCI is largely random in placental females, many women exhibit skewed XCI (SXCI), in which the vast majority cells have the same X chromosome inactivated. SXCI has serious health consequences, associated with conditions ranging from Alzheimer's to various autoimmune disorders. SXCI is also associated with outcomes of pregnancies, with higher rates of recurrent spontaneous abortion in women with SXCI. Here, I suggest that SXCI could be driven by selfish X-linked alleles. Consistent with the association of SXCI with autoimmunity, I first note the possibility that recurrent spontaneous abortion could reflect immune rejection of fetuses inheriting alleles from the largely silenced maternal X chromosome. Preferential abortion of fetuses carrying silenced X-linked alleles implies a transmission advantage for X-linked alleles on the largely expressed chromosome, which could drive the emergence of X-linked alleles that make the chromosome resistant to XCI. I discuss the evolutionary dynamics, fitness tradeoffs and implications of this hypothesis, and suggest future directions.

Serial cytokine expressions in infants with incontinentia pigmenti.

NF-κB dysfunction resulting from NEMO (NF-kappaB essential modulator) mutation can lead to significant alterations in cytokine production. However, little is known about changes in the expression of downstream molecules in patients with incontinentia pigmenti (IP). We aim to investigate serial cytokine expressions during the first 2 years of life in young infants with IP, the period in which skin inflammation and morphological changes are most significant. Gene analysis and X-inactivation test were performed for the two neonates with IP. Peripheral mononuclear cells were obtained after birth and successively at 6-month interval up to the age of two years. Levels of TNF-α and IL-6 were analyzed with ELISA before and after stimulating with Toll-like receptor ligands. The result showed the male IP patient had normal NEMO allele. His cytokine level, although initially lower, had returned to a level comparable with those of controls at 12 months of age. The female infant had mutated NEMO gene. Her baseline TNF-α level was significantly higher than those of the control subjects at birth and remained high by 6 months of age. All cytokine responses had decreased significantly by 2 years of age, the time in which all vesicular skin lesions had resolved. Both infants had normal serum immunoglobulin level and remained infection free during the follow up period. To our knowledge, this is the first report that demonstrates serial changes of cytokine profiles in humans with IP. This study showed that in the presence of NEMO mutation, alteration of cytokine production was remarkable during the first year of life, which may account for the prominent inflammatory changes in skin morphology.

Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies.

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.

Skewed X chromosomal inactivation impacts T regulatory cell function in systemic sclerosis.

OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

The role of epigenetics in aging and autoimmunity.

The decline in immunocompetence with age is accompanied by the increase in the incidence of autoimmune diseases. Aging of the immune system, or immunosenescence, is characterized by a decline of both T and B cell function, and paradoxically the presence of low-grade chronic inflammation. There is growing evidence that epigenetics, the study of inherited changes in gene expression that are not encoded by the DNA sequence itself, changes with aging. Interestingly, emerging evidence suggests a key role for epigenetics in human pathologies, including inflammatory and neoplastic disorders. Here, we will review the potential mechanisms that contribute to the increase in autoimmune responses in aging. In particular, we will discuss how epigenetic alterations, especially DNA methylation and histone acetylation, are accumulated during aging and how these events contribute to autoimmunity risk.

Skewing of X chromosome inactivation in autoimmunity.

Approximately 5% of the population in Western countries is affected by autoimmune diseases (AID), with a significantly higher prevalence in women. Genetic factors are known to be crucial determinants of susceptibility as shown by family and twin studies, although no specific genes predisposing women to autoimmunity have been identified thus far. Several studies indicate that X chromosome abnormalities, such as inactivation patterns, characterize some female-predominant AID. We herein review the most recent evidence on the role of the X chromosome in the breakdown of immune tolerance and discuss its potential implications. Future efforts will help to identify specific X chromosome regions containing candidate genes for disease susceptibility.

The X chromosome and systemic sclerosis.

PURPOSE OF REVIEW: Similar to the majority of autoimmune rheumatic diseases, systemic sclerosis is characterized by a striking female predominance superimposed on a predisposing genetic background. At least two genetic mechanisms have been proposed that play a role in susceptibility to systemic sclerosis; firstly the maintenance of immune tolerance via genes on the X chromosomes and, secondly, fetal microchimerism. Based on these lines of evidence, experimental efforts have been most recently dedicated to investigating the role of X chromosome abnormalities (i.e. monosomy rates and inactivation patterns) in autoimmunity. We will review herein the most recent data on the role of the X chromosome in systemic sclerosis onset and discuss the potential implications. RECENT FINDINGS: Women with systemic sclerosis manifest an enhanced rate of X monosomic cells in peripheral blood compared with healthy age-matched women. Furthermore, a severely skewed X chromosome inactivation pattern is found in women with systemic sclerosis. SUMMARY: These observations, reproduced in other female-predominant autoimmune diseases, strongly support the role of the X chromosome in conferring susceptibility to tolerance breakdown and open novel scenarios to emphasize the unknown etiopathogenesis of systemic sclerosis. The implications of these findings will be discussed.

TLR7 ligands induce higher IFN-alpha production in females.

IFN-alpha exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-alpha upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-alpha and TNF-alpha production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups (n(1) = 120, n(2) = 101, and n(3) = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-alpha levels after TLR7 stimulation (p(1) < 0.0000001, p(2) < 0.0000001, and p(3) < 0.0001) compared with male PBLs. In contrast, no sex differences were evident after TLR9 stimulation. TNF-alpha production after TLR7 stimulation and also total pDC numbers were not different between females and males. X-inactivation escape of the TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17beta-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-alpha production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-alpha with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.