Epigenetic remodeling by sex hormone receptors and implications for gender affirming hormone therapy.

Sex hormone receptor signaling in immune cells influences various functions such as cell proliferation, reactivity, and overall function ( 81 ). Table 2 summarizes studies on how sex hormone signaling affects immune cells. Studies on sex hormone receptors and immunology. CD4-ERα knockout (KO) female mouse model showed a mild autoimmune phenotype with increased autoantibody and follicular helper T cells (TFH) production ( 82 ), while polymorphisms in ER binding site affect rheumatoid arthritis by introducing a sex bias Cd2 expression to regulate T cell activation ( 86 ). Skin inflammation also depends on endogenous estradiol in mice where a psoriasis ER mouse KO increased IL-17A and IL-1ß production ( 85 ). In the context of infection, response to Chlamydia muridarum and hepatitis B virus (HBV) in mouse models were both influenced by polymorphisms in ESR1 ( 84 , 101 ). The role of ERs in inflammation in the colon, liver, and airway is also evident in mouse studies where TNFα activates intestinal Er-β, while a reduction in ERα increases NF-kB activity through the liver receptor homolog (LRH-1) ( 87 , 102 ). Additionally, ERα plays a role in allergy, elevating IL-33 release and ILC2-mediated airway inflammation upon allergen challenge ( 83 ). In human studies, sex-based differences in immune cell composition are also observed where males have higher proportion of nature killer (NK) cells subsets, while females exhibit greater abundance of B cell subset ( 88 ). Male neutrophils also show higher TNF expression after lipopolysaccharide (LPS) stimulation, which is attributed to increased TLR4 expression in males which may influence sepsis response ( 89 ). In females with severe asthma, dual activation of ERα and glucocorticoid receptor synergistically enhances the production of circulating T helper (Th2) cells and type 2 cytokines ( 90 ). Finally, ESR1 has been identified to be an integral regulator in HIV-1 infection with females displaying lower inducible HIV-1 RNA reservoirs compared to males ( 91 ). AR signaling has been reported to exert inhibitory actions in a number of immune responses ( 103 ). For example, in mouse models of influenza, testosterone inhibits influenza A virus (IAV) pathogenesis by systematically modulating CD8+ T cell reactivity ( 92 ). Similarly, in a mouse model of asthma, treatment with an allergen, Alterneria extract, decreases helper T cell reactivity, while the suppressive function of Tregs is promoted ( 93 , 94 ). AR signaling also plays an important role in the inflammatory response in the kidney and liver. Treatment of male rats with flutamide, an AR antagonist, has been reported to systematically downregulate cytokines in renal fibrosis, while an increased AR expression was observed in mice during severe infection with HBV ( 95 , 104 ). Sex-specific differences in anti-tumor immune responses in mice have been attributed, at least in part, to AR-mediated epigenetic remodeling of CD8+ T cells leading to lower reactivity and stemness in the tumor environment ( 97 ). In human studies, testosterone reduces oxidative stress in neutrophils, increasing their phagocytic capacity while decreasing their microbicidal activity ( 100 ). AR signaling also increased Foxp3 expression in regulatory T cells of females during the ovulatory phase of the menstrual cycle, leading to changes in the acetylation profile of histone H4 ( 99 ). The importance of AR signaling is further highlighted by a study that showed that inhibition of AR activity in CD8+ T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy ( 96 ). Finally, AR expression inversely correlated with the production of M2 tumor-associated macrophage, CD3+, and CD8+ T cell infiltration in trastuzumab-treated HER 2-positive breast cancer patients, suggesting a stronger role of AR in immune cells within cancer metastasis and proliferation ( 98 ). This section mainly reviewed previous animal studies due to the scarcity of information on how sex hormone signaling affects functional human immune response in various contexts such as autoimmunity and other diseases. Several previous reviews have elucidated the role of sex hormone signaling in immune cells ( 7 , 103 , 105 ) but it is still unclear on how this signaling pathway affects immunity and mechanistically affects diseases associated with it. Overall, these studies highlight that both the ER and AR regulate a wide range of immune cell functions, explaining the sexual dimorphism in inflammation, cancer response, and disease susceptibility. Epigenetic remodeling at promoters and distal regulatory elements is a key mechanism by which transcription factors establish immune lineages from hematopoietic stem cells ( 106 ). Further, exogenous stimuli such as microbial compounds and certain vaccines can induce new regulatory elements in differentiated immune cells, such as monocytes, through changes in histone modifications ( 107 ), or DNA methylation ( 108 ). Likewise, the tissue microenvironment shapes the epigenetic landscape in tissue resident macrophages ( 109 ) and T cells ( 110 ). As noted previously, several regulators of immune cell identity and function are located on the X chromosome. For example, in vitro -activated B cell subsets from adult and pediatric systemic lupus erythematosus (SLE) patients were found to exhibit disrupted X-chromosome inactivation (XCI), which is speculated to result from an increase in inflammatory cytokines and type I interferons ( 111 ). Further, UTX, a master epigenetic regulator that escapes XCI in both human and mouse models was found to control chromatin accessibility and gene expression patterns in NK cells ( 112 ). Therefore, sex-differences in immune cell phenotype and function can arise through epigenetic errors in X chromosome inactivation or epigenetic remodeling by sex hormone receptors.

Sexual dimorphism describes differences between males and females across various factors, including but not limited to behavior and immunity ( 1 ). Sex hormones are one contributor to this dimorphism, where males have higher testosterone and lower estrogen levels, while females have higher estrogen and lower testosterone levels, with age being a major factor in this ratio ( 2 ). Sex hormone signaling via sex hormone receptors is a major transcription pathway that influences cellular function, namely cytokine production, cell proliferation, and reactivity ( 3 ). Sex differences in immune function between males and females are also influenced by genetics, with several key genes involved in immunity expressed on the X chromosome. This includes receptors such as TLR7, TLR8, and ACE2 ( 4 , 5 ), as well as FOXP3, which controls regulatory T cell production ( 6 ). Further, the X-chromosome encodes 10% of all miRNAs, including miRNA-18 and 19 which are associated with sex-biased immune response ( 7 , 8 ). Functional programming of innate and adaptive immune cells depends on epigenetic remodeling, which alters the regulatory landscape of the genome and controls gene expression ( 9 , 10 ). These changes influence immune cell identity and can predict how the cell will respond to exogenous stimuli ( 11 ). For example, T-cell exhaustion has been linked to epigenetic reprogramming that leads to changes in differentiation trajectory ( 12 , 13 ). Autoimmune disease is associated with altered cytokine production and immune cell reactivity due to epigenetic changes that are caused by environmental influences, genetic variants, and medication ( 14 ). Additionally, certain infections, including SARS-CoV-2 and malaria, as well as immunizations, such as influenza and BCG vaccines, induce changes in the epigenomes of hematopoietic stem cells and monocytes ( 15 – 18 ). This mini review will summarize recent data on sex hormone receptor-mediated epigenetic remodeling and immune function modulation, with implications for understanding immune function changes in transgender individuals following gender affirming hormone therapy (GAHT). Although not covered in this mini-review, the sex hormone progesterone also plays a vital role in immunity, particularly the promotion of maternal-fetal tolerance during pregnancy, through expanding regulatory T cells and regulation of reactivity of other immune cells ( 19 ).

Epigenetic remodeling through sex hormone signaling may have wide-ranging impacts on the immune profile of transgender men and women. We highlight that most of our knowledge about how sex hormone receptors remodel chromatin come from cancer studies, and that human studies in the context of hormone change are warranted. Considering that AR and ERs are expressed in a range of tissues and immune cell types, changes in circulating sex hormone concentrations can influence cell responses in the circulation, as well as progenitor populations in the bone marrow. Based on the literature we presented, estrogen and testosterone do not simply promote or inhibit immune responses, but changes in their concentrations would lead to a unique immune cell phenotype in the context of GAHT. Profiling the effects of GAHT on epigenetic remodeling will not only provide insight into the role of sex hormones in immune function and the development of complex immune diseases, but will help inform the healthcare of transgender people on GAHT.

Epigenetics literally means ‘above DNA’ and refers to the study of molecular interactions that influence DNA structure, compaction, and function ( 35 ). Epigenetic marks can be ‘written’, ‘erased’, and ‘read’ by specific nuclear proteins to regulate gene expression in a range of physiological processes ( 36 , 37 ). Three major epigenetic modifications are (i) DNA methylation, where a methyl (-CH 3 ) group is added to the cytosine nucleotide within a cytosine-guanine sequence (CpG dinucleotide) ( 38 – 40 ); (ii) histone post-translational modifications, such as acetylation and methylation ( 41 ); and (iii) non-coding RNAs, which are transcribed RNAs that are not translated into proteins, but can regulate gene expression through mediation of chromatin structure ( 42 ). Most of the genome (over 95%) does not code for proteins. Much of this noncoding landscape plays an important role in regulating the transcription of coding genes ( 43 ). Indeed, hormone receptors such as ESR1 bind large swaths of the noncoding genome, influencing the activity of coding genes ( 44 ). Changes in gene expression due to epigenetic remodeling via sex hormone receptors is observed in various diseases and physiological processes ( Table 1 ). Both ER -α and -ß are expressed in more than 70% of all breast cancers, with estrogen signaling being a driver of carcinogenesis ( 63 ). Therefore, much of what we know about how the ER remodels chromatin is based on cancer studies ( Table 1 ). These studies illustrate that sex hormone receptors can alter the 3D chromatin landscape, DNA methylation and histone post-translational modifications, by interacting with different chromatin modifiers ( Table 1 ). Studies on sex hormone receptors and epigenetic remodeling. P, progesterone. Epigenetic remodeling by ERs also plays a role in non-cancer settings, such as breast development, where the DNA methylation signature of adolescent girls is dependent on estrogen response and breast composition, which may influence breast cancer risk in adulthood ( 50 ). Hormonal changes in females and males during puberty were found to influence DNA methylation near predicted estrogen-responsive genes ( 56 ). In addition, disruption of DNA methylation at the ESR1 gene locus by endocrine-disrupting chemicals has been associated with decreased male fertility due to the decline of sperm quality ( 53 , 57 ). AR can also remodel chromatin, but these effects are much less studied than for ER. Most studies looked at the role of AR in prostate cancer, where the AR drives epigenetic heterogeneity at enhancers through AR binding sites, affecting response to therapy ( 62 ). There is a significant knowledge gap in our understanding of how these AR-mediated changes within the epigenome shape cellular function in other tissues and outside the prostate.

At least 1.5 million people in the United States are transgender (~0.5%), of whom 90% have considered or are undergoing GAHT ( 113 ), with transgender women having a disproportionately higher rate of HIV infection ( 114 ). A cross-sectional study comparing transgender women and men receiving GAHT to cis-men and women, revealed that GAHT influences both the proportions in the circulation and the transcriptome of regulatory T (Treg) cells ( 115 ). Interestingly, sex hormones and chromosomes may collectively influence some cell proportions. Peckham et al. found that CD19+ CD27+ IgD- classical-switched memory B cells were sensitive to estrogen only in the XX karyotype, decreasing in transgender women following GAHT, but in post-menopausal cis-women following HRT, but not influenced by estrogen GAHT in transgender women ( 116 ). Collectively, these findings show that GAHT leads to a unique impact on different immune cell subtypes. The long-term effect of GAHT on the immune system has not been extensively studied which has clinical implications given the known sex-specific prevalence and risks in many inflammatory, infective and autoimmune diseases. To address this significant knowledge gap, we have longitudinally profiled the immune system of transgender women and transgender men newly commencing GAHT. A significant advantage to this approach is it allows us to dissect the contribution of sex hormone action relative to sex chromosomes in immune function, which is usually difficult to separate from the underlying genetic differences between males and females. Using longitudinal GAHT cohorts, we have shown that this therapy can influence epigenetic marks in blood cells affecting the immune response. A notable example of this is differentially methylated CpG site in the promoter of IL-21 , which gained DNA methylation after 12 months of masculinizing GAHT, but lost DNA methylation after 12 months of feminizing GAHT ( 117 ). We further elucidated the effects of feminizing GAHT in the metabolome wherein a cyproterone-acetate specific decrease in glutamine levels, an important amino acid related to immune cell metabolism, has been observed ( 118 ). Altogether, these findings suggest that GAHT influences the changes within the immune cell in a transcriptional level. Recently, Lakshmikanth et al., showed that masculinizing GAHT increased chromatin accessibility at canonical NFkB binding sites in T and NK cells after 12 months of therapy together with the promotion of monocyte responsiveness, together with downstream upregulation of NF-kB and interferon-γ production in NK cells ( 119 ). Modulation of interferon signaling by masculinizing GAHT was previously reported, with a decline in IFN-I production by plasmacytoid dendritic cells through the regulation of TLR7/8 ( 120 ). This testosterone-associated reduction in IFN-I responses was in line with lower TLR7 responses in males compared to females ( 121 ). Therefore, approaches that study immune cell phenotypes, including membrane and nuclear receptors, and downstream chromatin remodeling will provide insights into how sex hormones signal in primary human cells, and potentially explain the contribution of sex hormones to sexual dimorphism in inflammation and development of complex immune diseases.