Sex drives dimorphic immune responses to viral infections

Abstract

Introduction

Age, sex and immune state of the host are considered salient biological factors that determine the extent and strength of pathogen clearance during infectious diseases (1–3). With regard to viral infections, epidemiological studies have revealed that males have a higher mortality rate compared to females, who reportedly display stronger antiviral cellular and humoral immune responses (4). Although stronger immune responses may provide better protection against certain pathogens, in some chronic viral infections, it can lead to aberrant antigenic responses with immunopathology (5, 6). Net sex differences or the degree of sexual dimorphism in biological responses derive from genetic differences in chromosome complement and induce their effects via acute activational or prenatal organizational/epigenetic effects of gonadal sex hormones (estrogen, progesterone and androgens), extra-gonadal effects of sex chromosome encoded genes and compensatory mechanisms, such as reduction in gene dosage differences through X-chromosome inactivation. These processes underlie sex differences in most tissue responses during normal and diseases states including immunological responses to infectious diseases (Figure 1).

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Figure 1

Mechanisms of tissue sexual dimorphism that underlie sex differences in immune responses. Sex hormones, such as estrogens and androgens, contribute to both organizational and activational effects via gene effects that include both promoter activation and chromatin remodeling (epigenetic modifications). Sex chromosome complement exerts its effect in promoting sexual dimorphism independent of sex hormones. X-dosage compensation and escape from X-inactivation influence differential gene expression of innate immune molecules. Y chromosome contributions include Y gene-associated polymorphisms. Studies evaluating sexual dimorphism in immune responses focus on the interdependence of these factors as well as their independent contributions.

Differences in susceptibility and response to viral pathogens observed in male and females have been mostly attributed to activational effects of sex hormones and dosages of genes on X and Y chromosomes (4). The onset of puberty is associated with the numbers and functions of circulating granulocytes and monocytes, which are decreased but activated in females due to rising levels of progesterone in the setting of ovulation or pregnancy (7, 8). In addition, myeloid cells and lymphocytes express receptors for estrogen, progesterone and androgens, which orchestrate transcriptional pathways, ligand dependent or ligand independent signaling cascades that influence innate and adaptive immune responses to viruses (9–12). With regard to gene dosage on sex chromosomes, X-linked genes such as IL-13, IL-4, IL-10, Xist, Tlr7, FoxP3 on X chromosome and Sry (testis-determining factor), Sox9 (SRY-box 9) on Y chromosome may underlie sexually dimorphic responses that contribute to stronger innate, cellular and humoral immune responses and susceptibility to autoimmune diseases in females compared with males (13–15). Studies in humans and animal models indicate sexually dimorphic mechanisms that contribute to virologic control during infections with human immunodeficiency virus (HIV)-1, vesicular stomatitis virus (VSV), hantavirus (Seoul virus), influenza virus (H1N1), hepatitis C virus, Theiler’s murine encephalomyelitis virus (TMEV), herpes simplex virus (HSV-1) and coxsackievirus B3 (CVB3) (5, 16–20). In this review, we will introduce the various mechanisms that impose sexual dimorphism in immune function in males and females. We will then discuss the impact of sexually dimorphic immune responses on pathogenesis during viral infections.

Organizational Effects of Sex Hormones on Immune Function

The initiation of sexual dimorphism occurs through early embryonic development due to effects of genes on sex chromosomes (21, 22). The sex determining region Y (SRY) gene on the Y chromosome induces male supporting cell precursors to differentiate into testosterone-expressing Sertoli cells, leading to masculinization of all tissues within the developing embryo (23–25). During embryonic development, the increased expression of multiple genes maintain the XX sex phenotype and inhibit the expression of genes, such as SOX9, a transcription factor which favor XY male phenotype (15). In males, expression of Sex Determining Region Y-Box 9 (SOX9) protein downstream of Sry is crucial in inducing male sex phenotype. SOX9 can independently induce male to female sex reversal in male embryos through deletion from chromosome 17 (23, 26, 27). Gonadal R-spondin1 (RSPO1) is a secreted protein that is highly expressed in female blastocysts and interacts with Wnt4, which promotes ovarian development and estrogen production via the Wnt4/β-catenin signaling pathway (28, 29). Losses of function mutational studies in RSPO1 and RSPO−/− XX mice have confirmed this observation. (27–30). FOXL2, a forkhead transcriptional regulator, also contributes to ovarian differentiation and maintaininace during embryonic development in females via suppression of testis specific or male sex determining genes such as SRY target gene Sox9 and fibroblast growth factor (Fgf) 9, the latter of which represses Wnt4 (31–34). Although SOX9 deletion is embryonic lethal, homozygous conditional inactivation and mutational studies of SOX9 have confirmed that SOX9 induces suppression of Wnt-4, Foxl2 and β-catenin to prevent feminization of the male embryo (15). In addition, SOX9 also increases expression of genes required for male phenotype development in mammals including steroidogenic factor1 (Sf1), doublesex and mab-3 related transcription factor 1(Dmrt1), and GATA4 binding protein (23, 31, 35, 36).

Studies in rodents show that sexual dimorphism in immune function occurs through embryonic development and is maintained post-natally via the actions of gonadal hormones such as estrogen and testosterone (37–39). Prenatal castration of male mice results in post-pubertal thymic involution and aberrant T cell subset differentiation (40). Females exposed to higher concentration of androgen prenatally due to congenital adrenal hyperplasia, exhibit less modeling of behavior shown to them by other females, suggesting gender-related behavior change is due to prenatal hormonal exposure (41). Surprisingly, immune response to heterologous mixed lymphocyte reaction (MLR-A) is unaffected by perinatal masculinization in both sexes and is strongest in unmanipulated females (42, 43). Consistent with this, loss of feminization at prenatal stages, leads to decreased T-cell/ B-cell ratios compared with normally feminized mice that mirror ratios observed in male animals (44). Several studies also report the role of gonadotropin releasing hormone (GnRH), which maintains early levels of gonadal hormones in both sexes, in the prenatal patterning of the immune system. Postnatal GnRH antagonism, which inhibits expression of gonadal hormones, results in lower number of circulating CD8+ T and B cells in male rodents and primates (45). Similar studies in female rats showed reduced number of CD4+ T cells and reduced immune response of thymocytes and splenocytes to T cell mediated antigen (46, 47). The effects of estrogen and testosterone on B-cell development and differentiation also directly influence the production of IgG (48, 49). Prenatal secretion of testosterone limits the ability of males to produce immunoglobulins compared to females, who maintain much higher default plasma anti DNA immunoglobulin levels compared to males (2, 50). Additional studies in sea gull chicks depicts a reduced T cell and plasma immunoglobulin mediated immunity in prenatal testosterone treated chicks compared to control chicks (51). Activational effects of hormones at puberty further enhance sex differences in antibody production that are present at birth (see below), leading to persistently higher humoral immune response in females throughout their lifetime.

Activational effects of hormones on Immune Function

Post-pubertal expression of gonadal hormones leads to acute and reversible effects in adulthood that maintain physical and behavioral sex differences (52). Estrogen and progesterone in females and testosterone in males are the prime gonadal hormones secreted during the activational period. The effect of hormonal secretion during this period is not limited to reproductive system, but extends to multiple tissues, including those of the immune system (53). The androgen testosterone is synthesized in gonadal and adrenal tissues of both males and females, but is predominantly converted to estrogens via aromatization in the latter (54). Endogenous estrogens produced in female mammals include estrone (E1), 17β-estradiol (E2), and estriol (E3). E2 is the predominant form in females, produced by theca and granulosa cells of the ovaries in premenopausal women. The level of hormones secreted in post-pubertal female mammals varies in a cyclic fashion to facilitate ovulation and subsequent pregnancy.

Estrogen receptors (ER) exist in two forms ERα and ERβ, which bind ligands E1–3 to mediate gene expression. B and T lymphocytes, mast cells, macrophages, dendritic cells and natural killer cells predominantly express ERα (55). Hematopoietic progenitor cells express both ERα and ERβ (56). Based on studies in ERα- and ERβ-deficient mice, ERα appears to be the key regulator in differentiation of hematopoietic progenitor cells (57). Females produce higher levels of estrogen and regulate ER activation on their immune cells, which helps them to exert a stronger humoral and cellular immune response (37, 58).

ERs play vital roles in several signaling pathways, acting as a signal transduction molecule in calcium regulation across cell membrane, and inducing activation of G coupled and other surface receptors, such as receptors that drive expression of insulin growth factor (IGF) 1, and activation of ERK/MAPK, protein kinase C, PI3K and cAMP signaling (59, 60). ERα is also required for proper dendritic cell differentiation and CD40 mediated cytokine production (61). Although ERs can impact signaling pathways in ligand dependent and independent manners (62), signaling in immune cells is ligand dependent whereas signaling through coactivator-associated arginine methyltransferase (CARM)1 is ligand independent. E2 receptor (specifically ERα46 (63)), mediates anti-inflammatory signaling in monocytes and macrophages through suppression of CXC-motif ligand 8 (CXCL8) (63, 64). ERs activate STAT-signaling pathways during T and B cell proliferation, maintenance and activation. Under inflammatory conditions, ER activation also induces nitric oxide synthase and IFN-γ expression in T cells. ER ligands also mediate phosphorylation, nuclear localization and transcriptional activation of STAT1, STAT3 and STAT5 in B cells and circulating monocytes (65, 66). E2 additionally regulates STAT activity by increasing expression of cytokine inhibitor proteins such as Suppressor of Cytokine Signaling 1 and 5 in T cells and macrophages (67–70). In macrophages, ER signaling mediates inhibitory response towards pro-inflammatory genes regulated by NF-κB such as IL-6. On the other hand, E2 directly suppresses the expression of CC-motif ligand 2 (CCL2) in leukocytes, leading to reduction in migration (71, 72).

It is well established that ERs play an important role in promoting sexual dimorphism in the neonatal brain through chromatin remodeling, particularly through promoter methylation and acetylation. This process is mediated by ERα activation and dimerization, followed by nuclear translocation, DNA binding through estrogen response elements (ERE), recruitment of receptor coactivators and corepressors such as nuclear receptor corepressors (NCoR), leading to epigenetic modifications and regulation of downstream transcriptional factors (73–75). Current studies on epigenetics also indicate that methylation of ERα promoter not only contributes to sex differences in the brain, but also maintains sexual dimorphism throughout the life by creating methyl marks on the DNA of the individual (76). However, direct evidence of ER- or AR-mediated epigenetic modifications that contribute to immune cell differentiation and function has not yet been shown.

In addition to endogenous ER ligands, ligands that effect ER signaling are found in environmental sources including food, such as phytoestrogens, and pharmaceuticals, such as tamoxifen, toremifene and raloxifene, which are selective ER modulators (SERM). SERM have been used as therapeutics in multiple sclerosis, ovarian cancer, breast cancer and Ebolavirus infection to suppress the activity of Th1 cells and induce Th2 cytokine expression (77–81). Further studies are required to understand the impact of environmental and exogenous estrogens, either independently or in combination with other factors, on immune function during exposure to pathogens.

Progesterone also mediates both stimulatory and suppressive roles in immune responses. The progesterone receptors are mainly expressed by T and NK cells but recent studies have detected them on dendritic and mesenchymal stem cells, where they suppresses Th1 and increases Th2 cytokine secretion (82, 83). Suppression of T cell cytotoxicity as well as Treg proliferation is also mediated by progesterone (84). Progesterone also inhibits the activity of NK cells via down-regulation of IFN-γ secretion (85). In macrophages, progesterone suppresses nitric oxide levels, inhibits FcγR expression and microparticle release, thereby dampening the initial immune response at initial stages of infection. During pregnancy, progesterone enhances immunomodulatory functions of mesenchymal stem cells through upregulation of PGE-2 and IL-6. This is essential in females to maintain the fetal-maternal interface (83).

Most testosterone (98%) is irreversibly converted to an active metabolite dihydrotestosterone (DHT) (86). DHT binds with higher affinity than testosterone to ARs, which are expressed in varying levels by leukocytes (87). In innate immune cells, such as neutrophils, AR signaling maintains cellular differentiation via induction of granulocyte colony-stimulating factor signaling through activation of ERK1/2 and STAT3 (88). In wound healing studies, AR regulates chemotactic ability of macrophages through upregulation of CCL2, TNF-α and CCR2 (89). AR signaling also regulates T and B cell function and development. CD4⁺ thymocytes express lower levels of inducible AR (iAR), while CD4⁻ CD8⁻ and CD8⁺ thymocytes express the highest levels (90). While AR signaling promotes Th1 mediated T-cell immune response, it also acts as an antagonist to NF-κB and interferon type I signalling pathways (91). Specifically, splenic CD4⁺ and CD8⁺ T cells both express iAR that binds to testosterone in males (92). Castration studies have revealed that in absence of testosterone, AR signaling suppresses the activation of CD4 and CD8+ T cells via by over production of IL-2 and IL-2R (47, 93). Moreover, absence of testosterone leads to dampening of Th1 differentiation from naïve CD4 T cells in autoimmune disease conditions through suppression of IFN-γ and IL-2 expression in males (94, 95).

Sex Chromosome Complement and Immune Function

Sex chromosome complement arises from fundamental genetic differences in XX and XY cells that are mediated by several processes such as X chromosome inactivation, X gene dosage and epigenetic modifications (96). In order to maintain X gene dosage in the female blastocyst, one of the X chromosomes is inactivated by formation of Barr bodies in individual cells (97). Barr bodies are formed as a result of Xist gene expression and histone modification making one of the X chromosomes inactive in every cell (98). This process, which is exclusive to XX somatic cells, is random, leading to cells with active maternal or paternal X, and compensates X gene dosages in all somatic cells (14). The gene products of X or Y gene also facilitate epigenetic modifications on the DNA of an individual. SRY interacts with Kruppel-associated box (KRAB) domain transcriptional factor through the high mobility box (HMG) box on SRY to recruit histone-modifying enzymes such as histone deacetylase (HDAC) and heterochromatin protein 1, which remodels chromatin (99, 100). Few studies have evaluated sexual dimorphism in chromatin remodeling irrespective of hormonal influence and those that have, focused on the central nervous system. Thus, H3 methylation is more prevalent in males compared to females in cortical regions of the brain (101), HDAC 2 and 4 more prevalently bound to promoters of Esr1 and Cyp19a during brain differentiation in males compared to females. This increased interaction leads to stronger deacetylation and higher gene expression (102).

MiRNAs are also sexually dimorphic in nature and are observed to regulate SRY related genes. For example, in prenatal stage miR-124 is highly expressed in female supporting cells and suppresses Sox9 gene expression. MiR-202-5p/3p on the other hand is highly expressed in males through SOX9, which is generated by male supporting cells, and necessary for male sex determination. Data also suggests that miR-202-5p is a direct transcriptional target of SOX9 during testis differentiation (103). Interestingly, miR-124 has also been shown to inhibit STAT3 signaling, which suppresses T cell proliferation and leads to Foxp3+ regulatory T cell induction, including upregulation of interleukin-2, IFN-γ and TNF-α (104, 105). Another study using a murine model of multiple sclerosis found that peripheral administration of miR-124 leads to systemic deactivation of macrophages, reduced activation of myelin-specific T cells and suppression of disease progression (106). Since research regarding the role of sex chromosome complement in many physiologic and disease processes is still in early stages, less is known about their contributions to antiviral immunity. However, studies using four core genotype mice, in which role of XX versus XY genes can be separated from those of gonadal hormonal effects, are providing new insights into the impact of sex chromosome complement on immune responses, particularly during autoimmune diseases.

Female bias in disease expression of autoimmunity is well established with female to male ratios in systemic lupus erythematosis (SLE) and multiple sclerosis approaching 4:1 and 9:1, respectively. A study of sex chromosome aneuploidy in male subjects expressing an excess X chromosome found they were at a higher risk for SLE (107). Thus X chromosome likely plays a crucial role in disease incidence independently of hormonal effects (107, 108). The four-core genotype (FCG) mouse model is a novel tool for examining the effect of sex chromosome complement XX and XY on phenotypic sex differences induced in male and female mice without the confounding effects of hormonal patterning. In the FCG model, the testis-determining gene Sry is deleted from Y chromosome on male B6 or SJL mice (XY⁻) and a transgenic Sry is inserted at multiple sites on an autosome (109). The model generates four genotypes of mice wherein Sry transgene is present in XX and XY⁻ mice, which develop testis in contrast to the XY⁻ mice, which instead develop ovaries similar to XX mice (109). Gonadectomy of FCG allows further examination of genetic contributions of sex chromosomes in adult animals that do not express sex steroids.

Using FCG mice, XXSry mice were found to display increased susceptibility for systemic lupus erythematosus (SLE) and experimental autoimmune encephalomyelitis (EAE) compared to XY⁻Sry mice. Since, both XXSry and XY⁻Sry had testis during development and the only difference between the two groups is the presence of Y chromosome complement, this study confirmed that sex complement alone could promote susceptibility to diseases irrespective of the hormonal secretions (52, 108). Further comparison between XX and XY⁻ mice reported higher levels of IL-13Rα2 and reduced levels of Th2 cytokines in spleen cells isolated from XX mouse. These results suggest that subdued Th2 cytokine levels due to increase in X linked gene IL-13Rα2 expression could be due to X chromosome complement (108).

Sexually Dimorphic Immunity to Viral infections

Differences in susceptibility to viral infections are likely due to inherent differences in the immune system of females and males. Female mount a stronger immune response to viral infections compared to males due to more robust humoral and cellular immune responses (Figure 2). Clinical studies of humans in viral infections are complicated by the impact of non-biological factors, such as exposure rates, social behavior, habitat and diet, on viral pathogenesis in a sex-specific fashion. However, studies in a controlled setting have suggested that levels of estrogen and testosterone differentially alter expression of genes involved in innate immunity, such as those encoding TLRs and interferons (IFN), in females and males thereby contributing to sexual dimorphism in viral infections.

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Figure 2

The interplay of sex chromosomes, sex hormones and immune responses influence sex differences in virologic control. Females display increased innate and adaptive immune responses to most viral infections compared to males, due to difference in effects of sex hormones (estrogen, progesterone and androgen). X and Y chromosome complement also contributes to sexually dimorphic immune responses to viruses in females and males. The relative increase in immune responses in females may contribute to differential levels of virologic control during acute infections and/or immunopathologic effects of anti-viral T cells that may lead to chronic inflammation.

Immune response to viral infections

Innate immune response is the first line of defense against any viral infection. Males and females depict a different pattern of response to viral infections. Innate response is primarily mediated by three classes of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs) (110, 111). These PRRs detect viral components, such as genomic DNA, double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), RNA with 5’-triphosphate ends and viral proteins. The TLRs and RLRs specifically regulate the production of type 1 interferon (IFN) and other cytokines. NLRs on the other hand regulate interleukin (IL)-1β through caspase-1 activation (110, 112, 113). Sexual dimorphism is observed during antiviral responses mediated by TLR and IFN pathway (114, 115). Immune cells in females exhibit a 10-fold higher expression of TLR compared to males (116). In mammals, the number and activity of innate immune cells such as monocytes, macrophages and dendritic cells are higher in females than males. As a result, response to antigens, vaccines and infections is also higher in females compared to males (3, 117). Adaptive immune response also depicts a great share of sexual differences in response to viral infections. Depending on the stage of the infection, females exhibit higher inflammatory Th1, anti-inflammatory Th2 compared to males (3). Additionally, upregulation of anti-inflammatory genes and higher cytotoxic T cell activity is observed in females. Some studies have also shown higher number of regulatory T cells in females compared to males. Clinical investigations in humans have also reported lower CD3+, CD4+, and CD4+: CD8+ ratios, T helper cells in males compared to females (44, 118).

DNA virus members of the Herpesviridae Family

RNA Viruses

Conclusions

This review highlights both early and recent findings regarding the impact of sex on immune cell numbers and function with a focus on sexually dimorphic phenotypes during viral infections. Although still an emerging field, it is clear that mechanisms by which mammals achieve and maintain sex differences can directly and indirectly influence host-pathogen interactions at the cellular and molecular level. These findings support the notion that there are, in fact, “two normals” with regard to manifestations of infectious diseases, with sex-specific responses during acute virologic control and in immunopathologic manifestations of viral infections. Accordingly, the development of therapeutics to treat various phases of viral infections will require continued study and comparisons of viral pathogenesis in female and male animals and humans.

Acknowledgments

References