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Sexual dimorphism in immunity: improving our understanding of vaccine immune responses in men Expert Rev. Vaccines Early online, 1–11 (2014)

David Furman Institute for Immunity, Transplantation and Infection, Stanford University, 279 Campus Drive, B240 Beckman Center, Stanford, CA 94305-5124, USA Tel.: +1 650 498 7759 Fax: +1 650 498 7771 [email protected]

Weaker immune responses are often observed in males compared to females. Since female hormones have proinflammatory properties and androgens have potent immunomodulatory effects, this sexual dimorphism in the immune response seems to be hormone dependent. Despite our current knowledge about the effect of sex hormones on immune cells, definition of the factors driving the sex differences in immunoclinical outcomes, such as the diminished response to infection and vaccination observed in men or the higher rates of autoimmunity observed in females, remains elusive. Recently, systems approaches to immune function have started to suggest a way toward establishing this connection. Such studies promise to improve our understanding of the mechanisms underlying the sexual dimorphism observed in the human immune system. KEYWORDS: endocrine • gene expression • immune profiling • immune sexual dimorphism • influenza • systems immunology • testosterone • vaccination

The immune response clearly differs between sexes. Across a variety of species, males mount weaker antibody and cell-mediated responses than females. In addition, females also exhibit lower rates of infection. The evolutionary principles that underlie these observations are not obvious, but it has been proposed that males spend a substantial amount of energy on competition with other males to maximize their reproductive success. As a result, males may exhibit diminished energy expenditure on their immune system [1]. Further, females invest more energy in mounting potent immune responses to survive infection and preserve their offspring [1]. Consequently, the sex differences in infection prevalence should peak at reproductive ages and be lowest during childhood and at advanced ages. Such an effect has been observed in epidemiological data [2]. However, the molecular bases of the sexual dimorphism in the immune system are not fully understood. Sex hormones and sex chromosomes are attractive targets to explain these differences, but, due to the complexity of the immune system, it has been difficult to link

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sex-specific assays with immunoclinical outcomes. This is especially true in human cohorts where, compared to animal models, a huge variation in immunological phenotypes is the rule. It is also important to note that for statistical considerations researchers tend to choose to work with a single sex, thus diminishing phenotypic variance. However, as will become clear from this review, inclusion of both sexes in the study design is a crucial consideration for the analysis of immune functions. Specifically, recent studies using systemswide approaches of immune function and seeking to embrace the variation in human cohorts in an unbiased fashion have demonstrated that establishing a connection between immunophenotypes and standard immunoclinical outcomes is achievable. These have identified selective genes that are likely to be modulated by androgens and seem to dampen the vaccine immune responses of men. These studies uncovered novel pathways associated with the sex bias in the immune response in humans.


2014 Informa UK Ltd

ISSN 1476-0584

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Blunted immunity in males

In general, females are healthier and live longer than males. Part of this difference can be attributed to the fact that males experience higher severity and prevalence of bacterial, viral, fungal and parasitic infections [3]. Early studies have shown that in both human and animal models, males have on average a lower ability to produce antibodies. For example, baseline levels of serum IgG, IgM and IgA are significantly reduced in males compared to females [4–7] and the antibody responses to most commercially available vaccines including yellow fever, influenza, measles, mumps and rubella, hepatitis A and B, herpes simplex, rabies, smallpox and dengue are often half as low in males compared to females [8,9]. However, an apparent contradiction is that baseline antibody levels are negatively correlated with the ability to mount serological responses to vaccination [10,11]. In our studies of aging, we demonstrate that specific antibodies recognizing linear hemagglutinin (HA) epitopes at baseline are more prevalent in aged humans and correlate with the preexisting antibody titers as measured by HA-inhibition assays [11] but no sex differences were observed in the reactivity to linear HA epitopes. Using mouse models though we are starting to see that transferring purified IgG from immunized mice to naı¨ve ones causes a reduction in the ability to respond to influenza vaccination [HUANG HUANG, ET LA. UNPUBLISHED DATA]. This would predict that the higher levels of baseline IgG, IgM and IgA observed in females should have a negative impact on their capacity to mount robust antibody responses. However, this is not the case as demonstrated by the vast majority of studies addressing sex differences in vaccine response, including ours. It is therefore expected that other mechanisms may offer an explanation of such sex differences in vaccine response. For example, in recent trials, a HSV recombinant gD vaccine was more efficacious in woman than in men (see below) and further studies have found striking sex differences in the frequency and function of CD4+ T cells recognizing major HLA-DR restricted T-cell gD epitopes with women displaying more potent responses which could explain, at least in part, the improved efficacy of vaccines observed in females. In addition, the CD8+ T-cell-mediated immunity following vaccination has also been shown to be reduced in males [12,13]. The outcome of viral infections is also typically worse in males than in females. For instance, in HIV, viral RNA levels are consistently higher in males than in females [14] and infected males have significantly lower baseline CD4+ T cell counts than females [15]. There have been a number of vaccine trials for HIV, all of which enrolled both women and men, but strikingly none of these have adequately analyzed sex disparities in the immunogenicity of HIV by vaccination [16,17] and therefore, for HIV vaccines to date there are no known sex differences. Mortality rates following exposure to HSV also differ between the sexes; males exhibit more severe pathology following corneal infection with HSV and are more likely to die from infection than females [18]. Interestingly, treatment of female mice with testosterone prior to infection significantly doi: 10.1586/14760584.2015.966694

worsens the disease outcome [18]. This suggests that androgens may be responsible for the decreased immune response to HSV observed in males. In terms of vaccine responses, the HSV vaccine provides protection against the disease in women, but not in men [19]. In Phase I and II studies of a recombinant gD-based HSV vaccine, there was no significant protection when data were combined and included both sexes. However, when the data was partitioned by sex, a significant sex bias in protection was observed in which the efficacy was 73% in females and only 11% in males [20]. This demonstrates that not partitioning and analyzing data by sex can hide major discoveries, and hence can be seen as a flaw in study design. There are also significant differences in HBV with a prevalence of circulating HBV surface antigens being consistently higher in men than in women [21]. This suggests that the ability of males to eliminate the virus is reduced. Furthermore, being male is an independent factor predictive of elevated HBV DNA titers [22]. Androgens have also been implicated in the replication of HBV and in advanced stages of infection. For example, among HBV-infected men, high levels of testosterone and the expression of certain gene alleles for the androgen receptor (AR) are associated with an increased risk of hepatocellular carcinoma [23]. Consistently, the development of chemically induced hepatocellular carcinoma is delayed in AR male knockout mice compared with the wild-type counterparts [24]. This indicates that androgens have an important role in HBVinduced liver cancer. Moreover, in transgenic mice, castration of males reduces the dose of HBV antigen, and replacement of testosterone increases serum HBV antigen concentrations [25]. One mechanism by which androgens could modulate HBV replication is through binding to androgen response elements that have been identified in the genome of HBV [26]. In addition to direct modulation of virus transcription, male hormones could weaken host immune responses to infection and vaccination [11]. Consistently, protective antibody titers against HBV are substantially lower in males than in females [27–29]. Thus, it is very possible that the blunted efficacy of the HBV vaccine in males also contributes to the increased prevalence of HBV. Similar to HBV, in HCV, male sex is a risk factor for HCV infection [30,31]. Interestingly, sex differences in HCV disease are lost after menopause [32], suggesting an important role of sex hormones in the immune response to HCV. Also, male HCV-infected patients exhibit a reduced virological response following antiviral therapy than females [33]. Again, these differences are not apparent in analyses that include women who have entered menopause [34]. Sex differences in the incidence of influenza have been well documented [11,35]. Infection rates are often higher in males; however, fatality following exposure to highly pathogenic influenza A viruses is typically higher in females [36] and this effect seems to be mediated by an amplified inflammatory response causing endothelial dysfunction, vessel leakage, pulmonary edema and lung failure. For instance, influenza-mediated disease in highly pathogenic influenza infections is known to arise Expert Rev. Vaccines

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Sexual dimorphism in immunity

as a result of a profound and uncontrolled proinflammatory response (referred to as ‘cytokine storm’) initiated by the host [37]. Studies in humans, macaques and mice have demonstrated that infection with highly pathogenic strains of influenza results in a massive release of inflammatory cytokines that correlate with elevated mortality [37–39]. Consequently, males experience a lower morbidity and mortality in response to highly pathogenic influenza infection, which is likely mediated by a weak-to-moderate cytokine storm observed in males compared with females [36] and [BUNN ACM, PERS. COMM.]. Antibody responses to the seasonal trivalent inactivated vaccines (TIV) are also lower in men than in women [11,40,41] and in the mice, poorer neutralizing antibody responses against a primary infection and vaccination has also been found for influenza viruses [42]. Sex chromosomes can affect immunity

The notion that sex chromosomes are involved in the differences observed in the immune function between females and males originates mostly in studies of genetic diseases. A mutation in the g-chain subunit common to the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 leads to X-linked severe combined immunodeficiency, a disorder that occurs almost exclusively in men in which the body produces very few T cells and NK cells [43]. Similarly, a mutation in the gene that encodes for the transcriptional regulator FOXP3 leads to IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), a rare inflammatory disease characterized by the absence of functional Treg development and resulting occurrence of multiple autoimmune disorders in affected individuals [44]. X chromosome inactivation gives females protection from IPEX; and although 100% of the cells in males that express FOXP3 display the mutation, normal Tregs that express the wild-type gene are sufficient to maintain homeostasis in females [45]. Escape from X chromosome inactivation constitutes another mechanism by which males’ immune response may differ from that of females. In nominally healthy populations, X chromosome inactivation is not complete. It has been estimated that, for reasons that go beyond the scope of this review, as much as 15% of the genes in the X chromosome (>150 in total) escape inactivation [46]. This may lead to overexpression of certain gene products in females with direct consequences in immunological responses. For example, in patients with scleroderma, X chromosome inactivation is highly skewed [47] and these patients display reduced Treg activity. Moreover, a recent study demonstrated that the dosage of X-linked Toll-like receptor-8 determines gender differences in the development of systemic lupus erythematosus (SLE) [48]. Furthermore, the X chromosome contains 10% of all miRNAs in the genome and because miRNAs are critical regulators of immune function, it is predicted that X chromosome inactivation also leads to additional immune regulation through differential expression of miRNAs [49]. Many X-linked miRNAs are predicted to target immune suppressive genes such as FOXP3, CTLA4, CBL, informahealthcare.com

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SOCS1-3, and PDCD1, among others, with potential effects on the development of autoimmune diseases (reviewed by Hewagama A) [50]. Libert et al. have provided a comprehensive map of all known miRNAs located on the X chromosome, highlighting those involved in immune functions, thereby linking the unique mode of inheritance of the X chromosome with the disadvantage of males following immunological challenges [49,51]. The effect of gene dosage can also be evidenced from studies in patients with abnormalities in the number of sex chromosomes. XXY males with Klinefelter syndrome are at an increased risk for developing SLE equivalent to that of females, and several candidate SLE genes have been identified on the X chromosome [52–54]. In contrast, females with only one X chromosome, who suffer from Turner syndrome, rarely develop SLE. Elegant studies from the group of Teuscher suggest that the Y chromosome is also important for the sex bias observed in immunity. Specific genes encoded by the Y chromosome were shown to act as master regulators of gene expression in pathogenic CD4+ T cells and other immune cells in models of autoimmune disease [55]. The importance of the Y chromosome on disease outcome has also been studied in the context of infection. For instance, it is known that coxsackievirus B3 contributes to the development of myocarditis, an inflammatory heart disease that predominates in males. Recent studies using B6-ChrY consomic mice demonstrated that polymorphisms on the Y chromosome and androgen levels clearly regulate mortality in infected mice [56]. Notably, in humans an inverse correlation was also shown between the number of gene copies for certain Y chromosome genes and the number of significantly upregulated genes in immune cells, thereby supporting a link between copy number variation of such genes and the Y chromosome-dependent regulatory properties [57]. Sex hormones can affect immunity

Sex hormones are responsible for the most dramatic changes that occur in the body, including the development of secondary sexual traits, and the levels of these change with age (FIGURE 1). But in addition, sex hormones can modulate the function of immune cells in many different ways. Immune cells express receptors for a variety of sex steroids including estrogen, progesterone and testosterone. However, it appears that estrogen and testosterone are more active than progesterone in the modulation of the immune response. Estrogens

Cells from both the innate and the adaptive arms of immunity, including T cells, B cells, macrophages, neutrophils, NK cells and others, can express receptors for estrogens, that is, estrogen receptor a and b (ESR1 and ESR2) [58]. Expression of ESR2, however, seems to be less ubiquitous than that of ESR1. These differences are important since they relate to the development of autoimmune diseases. For instance, a genetic deficiency of ERS1 in murine models of lupus results in significantly doi: 10.1586/14760584.2015.966694

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Blood testosterone (nmol/l)

of NF-kB-mediated inflammatory responses [75–77]. Estradiol can also have subE Males stantial effects on the outcome of infection 20 P as shown by the studies of Klein and colleagues where ovariectomized female mice Menopause 13 were treated with 17b-estradiol and infected with influenza virus. Compared Females to controls, the 17b-estradiol-treated 2 1 group exhibited less morbidity and increased recruitment of neutrophils, 25 35 50 60 75 35–44 55–64 85–100 Age Age which enhanced the responses of influenza virus-specific CD8 T cells to promote Figure 1. Sex steroids in humans during lifespan. The levels of testosterone in virus clearance and improve the outcome humans decrease after the age of 40 both in males and females. At advanced ages of infection [78]. compared to young ages, individuals might exhibit less than half of the circulating tesA different mechanism by which estrotosterone (left plot). Since testosterone suppresses inflammation, this decrease in testosgens have proinflammatory properties terone may account for the rise in inflammatory markers with age. Dramatic changes in estrogen (E) and progesterone (P) are experienced by women entering menopause (45– appears to be mediated by their inhibi60 years old). This drop in sex steroids can explain the changes in inflammatory pathtory effect on the expression of caspaseways in older women. 12, a protein closely related to cytokine maturation caspases [79]. In mice, substandecreased disease and prolonged survival, while ERS2 deficiency tial sex differences have been found in the expression of has minimal effect in models of autoimmune disease [59]. Inter- caspase-12 with females expressing significantly lower levels estingly, both ESR1 and ESR2 are also expressed in hematopoi- than males [80]. Administration of estradiol to male mice has a etic progenitors (CD34+ cells) [60] and recent studies have direct suppressive effect on the active form of this protein. demonstrated that estrogens can indeed regulate immune cell Since caspase-12 inhibits the inflammasome by repressing differentiation [61–63]. Importantly, the differential expression of caspase-1 catalysis [80], the production of inflammatory cytoESR1 on plasmacytoid dendritic cells mediates an enhancing kines increases in cells exposed to estrogen. Furthermore, estraeffect of 17b-estradiol observed on TLR-mediated production diol administration to male mice also confers increased of IFN-a [64]. resistance to Listeria spp. infection compared with control It is generally accepted that estrogens are associated with mice [81]. inflammation and can also stimulate proliferation of lymphocytes and monocytes. This broad effect of estrogens could be Progesterone mediated by an increase in the production of nitric oxide [65], Progesterone function on immune cells seems to be particularly which can be induced by STAT1 and the NF-kB system [66]. restricted to specific cell subsets to favor processes relevant to In addition, the proinflammatory effects of estrogens can be pregnancy. For example, blood lymphocytes of pregnant due to its ability to stimulate expression of the receptor for women have been shown to bind a monoclonal antibody advanced glycation products [67], which is known to induce and against the nuclear progesterone receptor (PR) which does not amplify inflammation in response to advanced glycation prod- react with lymphocytes from nonpregnant women or men [82]. ucts. Estradiol can also increase endothelial permeability [68], This transient expression of PR modulates specific immunity thus promoting immune cell extravasation. The effects of estro- during pregnancy as subsets of innate cells often upregulate gens can, however, be divergent. For instance, low doses typi- these receptors to become sensitive and suppress immune cally enhance proinflammatory cytokine production (e.g., IL-1, responses through inhibition of the NF-kB pathway. In addiIL-6 and TNF-a) and Th1 responses, whereas high concentra- tion, during pregnancy progesterone seems to favor tions can cause a reduction in the production of proinflamma- Th2 responses, thereby suppressing the Th1 response [82–84]. In tory cytokines and augment Th2 responses and humoral males and in nonpregnant women expression levels of PR are immunity [69]. This reduction seems to involve the nonestrogen very low and restricted to cells from the innate immune system response element transcription factor AP-1. Interestingly, ESR1 such as NK cells and macrophages. and not ERS2 plays the key role in this effect [70]. Elevated estradiol levels attenuate oxidative stress and inflam- Androgens mation through its antioxidant properties and decreases produc- The effects of androgens on the immune system have been tion of chemokines, including CXCL8, CXCL10, CCL2 and studied for several decades, especially in the context of ecology CCL20, as well as the recruitment of immune cells into several and evolution. Darwin introduced the notion that female preftissues, including the lungs [71–74]. In mouse models, this anti- erence for selective mates may act as a selection force (sexual inflammatory effect of elevated estradiol levels is mediated by selection) resulting in costly male ornamentation and superior signaling through estrogen receptors, which inhibits activation ability to compete and achieve reproductive success [85], all doi: 10.1586/14760584.2015.966694

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Sexual dimorphism in immunity

testosterone-dependent traits. But, as mentioned previously, the severity and prevalence of infection in many species including humans is higher in males than in females and androgens can cause suppression of immunity at different levels (see below). This apparent double-edged effect of testosterone on sexual selection and immunosuppression led to the immunocompetence handicap hypothesis by Folstad and Karter (1992) [86]. This states that (1) testosterone has a dual function in the development of characteristics used in sexual selection and in modulating immunocompetence, and (2) testosterone production is rather plastic influencing and being influenced by parasite burden. This idea predicts that testosterone may participate in the suppression of the immune response at the basal state. Some evidence in support of this prediction was reported in a meta-analysis of studies in different species measuring testosterone levels after infection or immune activation [87]. Strikingly, in all 13 studies analyzed, circulating testosterone levels dropped after immune activation regardless of the species and type of antigen [87]. These observations are in also agreement with in vivo experiments showing that IL-1 suppresses steroid production by Leydig cells [88,89]. This inhibitory effect of IL-1 in the production of testosterone seems to be selfregulated since, as stated earlier, testosterone inhibits inflammatory cytokine and potentiates anti-inflammatory cytokine production. For instance, an Increase in testosterone levels can cause significant reductions in secretion of inflammatory cytokines such as TNF-a and IL-1b and increases in the antiinflammatory cytokine IL-10 [90]. Consistently, men with androgen deficiencies have higher levels of inflammatory cytokines than healthy controls [90–92]. In addition, testosterone may suppress T cells by acting directly on pro-apoptotic pathways [93]. Lastly, The production of IL-2 by T cells has also been found to be reduced in stimulated male lymphocytes as compared to female lymphocytes [82]. The difference in the expression of inflammatory cytokines has been mostly observed in young populations where the largest differences in hormones occur. For instance, rhinovirusinduced IFN-g and IL-13 is significantly higher in £50-yearold women than in age-matched men and in ‡52-year-old women [94]. In our studies, however, we report many inflammatory cytokines whose levels were decreased in the serum from males compared with females of different ages, including the elderly [11]. We also observe diminished antibody responses to an influenza vaccine in men, but no correlation between such diminished inflammatory profile in males and the lowered antibody responses to vaccination (see below) [11]. The mechanisms by which androgens negatively affect cytokine production are not clear, but it appears feasible that they act on two levels: through the NF-kB pathway and the AP-1 complex, since androgen-receptor signaling antagonizes NF-kB and represses AP-1 (FOS/JUN) expression, which mediate the production of proinflammatory and antiviral cytokines [95,96]. Interestingly, activated NF-kB can also induce AR expression, which may serve to increase sensitivity to testosterone creating a negative feedback loop [97–99]. In our studies, we informahealthcare.com

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identify specific genes that correlate with serum testosterone levels and are likely to be negatively regulated by AP-1 partners. Starting from a total of 109 gene modules derived from wholeblood gene expression data [100], we identified only one gene module that could explain the lower response observed in males (see below) [101]. The JUNB, JUND and FOS genes were present in the regulatory program of such a gene module. Further analyses of these data looking at JUN, JUNB, JUND, FOS and FOSB in all testosterone-associated gene modules shows that the regulatory programs of these modules are enriched for AP-1 genes (TABLE 1). This strongly suggests that testosterone could modulate AP-1 genes and thus genes dependent on AP-1 expression. Interestingly, the effect of testosterone on AP-1 and the possibility that this lowers inflammatory cytokine production is reminiscent of the effect of sphingosine-1-phosphate (S1P) signaling on cytokine production [102]. Moreover, two striking similarities exist between testosterone and S1P, both inhibit NF-kb [96,103] and are ligands for GPCR6A [104,105]. Studies in prostate cancer (Pca) have highlighted additional relationships between testosterone and S1P. For example, low levels of circulating S1P correlate with plasma prostatic-specific antigen and with testosterone levels in patients with Pca, and have been considered an early marker of Pca progression to testosterone unresponsiveness [106]. Therefore, similar to the effects of S1P on the inflammatory response, we hypothesize that due to its anti-inflammatory function possibly via direct inhibition of the AP-1 complex, testosterone administration could be utilized in cases of exacerbated immunity such as autoimmune diseases, chronic inflammation or uncontrolled cytokine production during sepsis or infection with highly pathogenic viruses, such as dengue or deadly influenza. Lastly, interesting associations between hormone levels and the gut microbiota in the modulation of immune-mediated diseases have been recently reported. In type 1 diabetes where lower levels of androgens are characteristic [107] and the incidence female-biased, gut microbiota also differs between sexes [108]. These differences are lost in germ-free mice and in castrated male mice, which indicates that androgens can also influence gut microbiota. Thus hormone-dependent expansion of selected microbial lineages can have a negative effect on disease progression contributing to the sexual dimorphism in the development of diabetes and maybe other immune-mediated diseases [108]. Where basic research meets the bedside: linking immunological phenotypes with clinical outcomes

Despite a wealth of literature showing marked immunological differences between the sexes and robust epidemiological data demonstrating clear sex differences in the prevalence and incidence of infectious diseases and in vaccine efficacy, to date no clear associations between the sex differences in immunophenotypes and those of immunoclinical outcomes have been found. Recent studies utilizing a systems approach have identified baseline gene signatures and other immunophenotypes that predict an antibody response to TIV [100]. This systems approach doi: 10.1586/14760584.2015.966694

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Table 1. Testosterone-associated gene modules are connected to AP-1 genes. Gene modules positively correlated with the levels of serum testosterone (n = 27, R >0.2, FDR Q >0.05) were selected and the presence or absence of AP-1 genes was extracted from each module regulatory program to compute enrichment analysis. ID

Functional annotation

p-value*

JUN

JUNB

JUND

FOS

FOSB

mod_005

GSE29614_CTRL_VS_TIV_FLU_VACCINE_PBMC_2007_DN

6.80E-04

0

1

0

0

0

mod_007

GSE14000_UNSTIM_VS_4H_LPS_DC_UP

5.40E-03

0

1

0

0

1

mod_008

GSE2706_UNSTIM_VS_2H_LPS_AND_R848_DC_UP

1.50E-03

0

1

0

0

0

mod_037

KEGG_RIBOSOME

2.30E-15

0

0

1

1

0

mod_038

GSE26495_NAIVE_VS_PD1LOW_CD8_TCELL_DN

1.90E-13

0

1

0

0

1

mod_039

REACTOME_TRANSLATION

1.10E-16

0

0

0

0

0

mod_040

GSE29618_BCELL_VS_MDC_UP

4.40E-16

0

0

0

0

0

mod_041

GSE22886_NAIVE_TCELL_VS_MONOCYTE_UP

5.80E-07

0

1

0

1

0

mod_043

GSE22886_UNSTIM_VS_IL15_STIM_NKCELL_DN

4.00E-04

0

1

0

0

1

mod_044

KEGG_LONG_TERM_DEPRESSION

4.90E-04

0

1

0

1

1

mod_045

GSE25087_FETAL_VS_ADULT_TCONV_UP

7.10E-05

0

0

0

1

1

mod_046

GSE9006_HEALTHY_VS_TYPE_2_DIABETES_PBMC_AT_DX_UP

8.10E-04

0

0

0

0

1

mod_047

PDGF_ERK_DN.V1_UP

1.80E-04

0

0

0

0

0

mod_048

GOLDRATH_NAIVE_VS_EFF_CD8_TCELL_UP

2.20E-04

0

1

1

0

1

mod_049

ACTIN_FILAMENT_ORGANIZATION

3.60E-04

0

0

0

0

0

mod_050

GSE29618_PRE_VS_DAY7_FLU_VACCINE_BCELL_UP

1.50E-03

0

1

0

0

0

mod_052

FATTY_ACID_BIOSYNTHETIC_PROCESS

6.00E-04

0

1

1

1

0

mod_055

REGULATION_OF_ACTION_POTENTIAL

2.00E-04

1

1

1

1

0

mod_083

KEGG_ALDOSTERONE_REGULATED_SODIUM_REABSORPTION

2.00E-03

0

0

0

0

0

mod_084

REACTOME_INFLUENZA_VIRAL_RNA_TRANSCRIPTION_AND_ REPLICATION

3.90E-13

0

0

1

1

0

mod_086

REACTOME_CHOLESTEROL_BIOSYNTHESIS

8.50E-04

0

0

0

0

0

mod_091

GSE22886_NAIVE_TCELL_VS_MONOCYTE_UP

2.50E-04

0

0

0

0

0

mod_096

KEGG_PRIMARY_BILE_ACID_BIOSYNTHESIS

5.30E-03

0

0

0

0

0

mod_097

GSE17721_POLYIC_VS_CPG_0.5H_BMDM_UP

7.90E-04

0

0

0

0

0

mod_098

MITOCHONDRIAL_MEMBRANE_PART

2.00E-03

0

0

0

0

0

mod_101

CARBOHYDRATE_TRANSPORT

4.60E-04

0

0

1

1

0

mod_108

GSE26495_NAIVE_VS_PD1HIGH_CD8_TCELL_UP

5.20E-04

0

0

0

0

0

TRM

1

11

6

8

7

ARM

2

17

13

12

14

p-value**

0.37

0.0001

0.048

0.0012

0.021

*Enrichment for functional annotation of selected gene modules. **Enrichment for the presence of AP-1 genes in the regulatory program of each gene module. ARM: Number of gene modules likely regulated by each AP-1 gene in all gene modules; TRM: Number of gene modules likely regulated by each AP-1 gene in testosterone-associated gene modules; 1/0: present/absent in module the regulatory program.

to vaccinology was pioneered in the studies of Se´kaly [109] and Pulendran [110], who successfully used gene expression data to identify differences between responders and nonresponders to doi: 10.1586/14760584.2015.966694

yellow fever vaccine (YF-17D). Most prominently, these included genes associated with a number of pathways from the innate immune response such as the molecular sensor for Expert Rev. Vaccines

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Sexual dimorphism in immunity

single-stranded RNA, TLR7 and its downstream adaptor molecule MyD88 as well as other several molecules with direct antiviral activity such as ISG20 and OAS1, 2 and 3 [109,110]. Genes controlling important innate responses such as type I interferons, inflammasome, complement system and cytokine signaling, such as IRF7, STAT1, C1QA and C1QB, were also significantly induced by the YF-17D. To note, a reanalysis study from the initial YF-17D gene expression data [109] by Klein and colleagues shows that over 90% of the genes induced by vaccination and reported to be predictive of the antibody response to the YF-17D were only activated in females and not in males [8]. The authors also established that most of the reported TLR-associated genes that activate the interferon pathway are upregulated to a greater extent in women than in men during the first 10 days after vaccination [8]. This highlights the intrinsic differences in vaccine-induced responses between males and females and the importance of unraveling sex-specific immunophenotypes. However, whether this lack of gene activation in males correlates with weaker antibody outcomes remain to be determined. Using a similar approach, we analyzed the neutralizing antibody response to TIV and a large number of immune system components in males versus females of different ages to identify immunophenotypes that can explain the sex differences in vaccine responsiveness. Weaker antibody responses to the vaccine and lower expression of cytokines including LEPTIN, IL1-RA, CRP, IL-5 and GM-CSF were found in males compared to females. This inflammatory profile correlated with the levels of phosphorylated STAT3 proteins in monocytes but not with the weaker response to the vaccine observed in males [11]. However, genes involved in lipid biosynthesis strongly correlated with the difference in vaccine response between males and females. These genes include LTA4H, which activates leukotrienes; MIF, which plays a role in the anti-inflammatory effects of glucocorticoids [111]; PDSS2, whose product synthesizes the prenyl side chain of coenzyme Q; and PEX5, involved in fatty acid metabolism. To note, LTAH4 has also a suppressive function since it encodes for a hydrolase that converts leukotriene A4 to active leukotriene B4, which participates in the generation of suppressive cells both from the myeloid and lymphoid compartments [112–114]. As stated previously, the gene regulators derived for this gene module included the transcription factors FOS, JUNB and JUND (repressors), among others, which is consistent with the suppressing effect of testosterone signaling on the AP-1 complex (FOS/JUN) [95]. Accordingly, men with the highest levels of testosterone and high expression of such gene signatures exhibited the lowest antibody responses to TIV. These studies generate a number of hypotheses that can be further tested in prospective studies, and strongly indicate that via the inhibition of AP-1 androgens may activate genes involved in immunosuppression and lipid metabolism (FIGURE 2). Therefore, these pathways seem to be important drivers of the differences in immune responses between males and females. The differences in the immune response between males and females are important in public health since infections informahealthcare.com

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AP-1 complex

Mod_52

Testosterone

Immunosuppression

Figure 2. Testosterone may mediate immunosuppression through inhibition of the AP-1 complex. Genes involved in lipid metabolism (module 52) also participate in the development of suppressive cells both from the myeloid and lymphoid compartments [27]. The expression of such gene signatures is likely regulated by the transcription factors FOS and JUN (AP-1 complex). Elevated levels of testosterone can inhibit the expression of the AP-1 complex, thereby suppressing the inhibitory effect of AP-1 on module 52, and thus activating lipiddependent immunosuppressive pathways.

are more severe and common in males and autoimmunity is more common in females. In addition, since most clinical studies do not take into consideration the sex of the individual, there might be dozens of potential cases where drugs or vaccine preparations are successful in one sex only, as was the case in the HSV vaccine trial [20]. It is therefore of major importance to include both males and females in research studies and measuring the effect of sex on the observed clinical outcomes. These studies will enable a better comprehension of the bases of the sex differences observed in immunity in humans. Expert commentary

The variation in the immune response of humans in part explains why therapeutic and preventive strategies targeting immune function, including those designed to improve clinical outcomes in cancer, viral and bacterial infections, autoimmune diseases and transplantation have limited success [115]. An important source of immunological variation is known to be the sex of the individual. Males experience a greater severity and prevalence of bacterial, viral, fungal and parasitic infections and exhibit weaker responses to antigenic challenges, while females have a higher risk of developing autoimmune diseases [116,117]. Despite that these clinical differences were noted a long time ago and the existence of extensive evidence showing the significant effect of sex on general immunophenotypes, the identification of specific immunological features that explain differences in immunoclinical outcomes from disparate data has been difficult. One approach to establishing this link is to measure multiple immune parameters of different immune system components, track immunoclinical outcomes in the same individuals and then computationally integrate the data collected across multiple study cohorts to define generalized models of immune failure versus success. This approach, used by a number of laboratories in the last decade, has enabled us to embrace the variation in the human immune system and better understand the immune components contributing to this variation. Despite being in the early phases of this discovery adventure, clinical trials and interventional studies have already been proposed. doi: 10.1586/14760584.2015.966694

Review

Furman

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Five-year view

• Current and future studies on sex differences in immunological function using animal models will provide mechanistic insights into the immunological phenotypes subjected to sexual dimorphism. • An increasing number of laboratories in both the academia and the industry will be adopting systems approaches to immunological problems, and many of these will identify novel immunophenotypes driving the sex differences in immunoclinical outcomes in human populations. • The use of data from the public domain will gain interest and may be crucial for the validation of biomarkers identified in human studies with relatively small sample sizes. • We will experience a degree of inconsistency in the immunophenotypes predictive of immunoclinical outcomes identified

from human studies compared to those in animal models, which will highlight the fact that many of these are speciesspecific and context-dependent. • Systems biology-based interventional studies will continue to emerge.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • Sex differences in immune responses exist; however, the vast majority of studies of immune function do not include both sexes. • Many therapeutic and preventive strategies targeting immune function have failed in one sex and succeeded in the other. • Males have weaker immune responses than females and are at a higher risk of infections. • Females exhibit robust immune responses leading to an increased risk for autoimmunity and virus-induced immunopathology. • The complexity of the immune system makes linking sex differences in immunophenotypes with those observed in immunoclinical outcomes challenging.

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