Immune response and auto-immune diseases: gender does matter and makes the difference

Immune response and auto-immune diseases: gender does matter and makes the difference

Sandra Brunelleschi

Department of Health Sciences, School of Medicine, University of Eastern Piedmont, Novara;
IRCAD (Interdisciplinary Research Center of Autoimmune Diseases), School of Medicine, Novara, Italy.

Received 7 April 2016; accepted 13 April 2016.

Summary. Women produce a more robust immune response to infection and this fact has been suggested as a tool to explain why women usually live longer than men; however, this increased immune reactivity may be responsible for the higher risk of developing autoimmune diseases (AID). Indeed, a female to male predominance occurs in AID and about 65% of all patients are women. Different factors have been implicated in underlie this striking gender difference, sex hormones being the mostly investigated. Gonadal hormones affect both the phenotype and the function of immune cells through interaction with specific receptors that are expressed in these cells. As a general rule, estrogens enhance the immune reactions, while progesterone and testosterone may exert an immunosuppressive role; however, the mechanisms involved in this complex scenario are not yet completely identified. In addition to sex hormones, genetic and environmental factors, innate and adaptive immune cells, fetal microchimerism, X chromosome inactivation and abnormalities have been proposed as key players in the development of AID and female gender bias, but their relative value is not yet fully appreciated. This review will try to critically describe the most important elements involved in the women’s predominance in AID.

Key words: gender, immune response, autoimmune diseases, sex hormones, genetic factors, environmental factors.

Risposta immunitaria e malattie auto-immuni: il genere conta e fa la differenza

Riassunto. Le donne producono una più intensa risposta immunitaria e ciò contribuisce a spiegare perché le donne vivano più a lungo degli uomini; tuttavia, questa aumentata reattività immunitaria predispone le donne a un aumentato rischio di sviluppare malattie autoimmuni (AID). Infatti, nelle AID, c’è una chiara predominanza del genere femminile e circa il 65% di tutti i pazienti sono donne. Tra i fattori implicati quali responsabili di questo bias di genere, gli ormoni sessuali sono sicuramente i più studiati. Gli steroidi sessuali modulano sia il fenotipo che la funzionalità delle cellule immunitarie interagendo con specifici recettori, che sono espressi nelle varie popolazioni cellulari. In linea generale, gli estrogeni potenziano le risposte immuni, mentre il progesterone e il testosterone possono esercitare un effetto di immunosoppressione; tuttavia, i meccanismi coinvolti in questo complesso scenario non sono ancora completamente identificati. Oltre agli ormoni sessuali, altri fattori (quali, ad es., alcuni determinanti genetici e ambientali, l’attività delle cellule della risposta immunitaria, il microchimerismo fetale, l’inattivazione del cromosoma X e le anomalie cromosomiali) possono giocare un ruolo chiave nello sviluppo delle malattie autoimmuni e nel determinare il bias di genere, anche se il loro peso non è ancora completamente apprezzato. Questa review cercherà di descrivere in maniera critica i più importanti elementi che sottendono la predominanza femminile nelle malattie autoimmuni.

Parole chiave: genere, risposta immunitaria, malattie auto-immuni, ormoni sessuali, fattori genetici, fattori ambientali.

Introduction

Autoimmune diseases (AID) represent an important cause of morbidity and mortality, affecting 8.5 million people in USA1,2 and about 6% of the population in industrialized countries3,4.

In spite of ethnic and geographic differences in the incidence of selected AID, some groups being at higher risk for some diseases and lower risk for others4, and, despite a large variability in the age of onset, clinical setting and drug responses, most AID share a common characteristic: the prevalence of female sex.

Indeed, about 65% of all AID patients are women and this percentage is even higher in Sjogren’s syndrome, systemic lupus erythematosus (SLE) and primary biliary cirrhosis4,5.

In spite of this well-known sex bias and multiple efforts to elucidate this situation, the reasons for the female predominance are still unknown. Genetic and environmental factors, innate and adaptive immune cells, sexual hormones, fetal microchimerism, X chromosome inactivation and abnormalities, have been proposed as key elements6,7, but the precise cause is still lacking.

This review will try to critically describe the most important elements involved in the women’s predominance in AID.

The immune response

As known, the immune response, a complex and tightly regulated one, is orchestrated by the immune system in order to protect the body from pathogens or other foreign damaging elements. When functioning properly, the immune system identifies a variety of threats, including viruses, bacteria and parasites, and distinguishes them from the body’s own healthy tissues. While the innate immune system represents the evolutionarily oldest defense mechanism and provides an early first line of defense against invading pathogens, the adaptive immune system allows for a stronger immune response as well as immunological memory, each pathogen being “remembered” by a signature antigen.

The components of nonspecific immune responses are monocytes, macrophages, natural killer (NK) cells, dendritic cells and granulocytes (neutrophils, eosinophils and basophils). These cells phagocyte bacteria and produce oxy-radicals (neutrophils, monocytes and macrophages), lyse infected cells (NK cells), produce cytokines to enhance nonspecific and specific immune responses. Dendritic cells, as well as monocytes and macrophages, act as antigen presenting cells (APC): they take up foreign antigens, process them and present on their surface antigen peptides for the specific immune system, mainly helper T lymphocytes.

The specific immune response comprises the humoral immune response (that is, B lymphocytes producing antibodies) and the cell-mediated immune response (that includes phagocytes, specific T lymphocytes and various cytokines).

T lymphocytes are divided into 3 distinct populations: a) cytotoxic T lymphocytes (Tc cells) that kill foreign or infected cells, b) helper T lymphocytes (Th cells) that produce cytokines and are further sub-divided into Th1 cells (producing IFN-γ that promotes cellular immune responses), Th2 cells (producing IL-4, IL-13 and IL-5 to help humoral immune responses) and Th17 cells (producing IL-17 that plays a key role in autoimmunity and allergen-specific responses), and c) regulatory T lymphocytes (Treg cells) that exert immunoregulation and can suppress both Th1- and Th2-mediated responses8.

The most relevant functions (as well as the major cell types involved; see also below) of innate and adaptive immunity are reported in Table 1.

Disorders of the immune system can result in immunodeficiency (when the immune system is less active than normal; chronic granulomatous disease represents a congenital, inherited immunodeficiency, whereas AIDS/HIV or immunosuppressive medications are examples of acquired immunodeficiency) or autoimmune diseases. AID represent a condition of hyperactive immune system and occur when the immune system, failing to properly distinguish between self and non-self, attacks and destroys tissues and organs of its own host.

Cells involved in immune responses and differences between males and females

Gender differences in autoimmunity can be attributed, at least partly, to differences between male and female immune systems, women presenting stronger cellular and humoral immune reactions than men7.

Sex differences in infectious diseases are common, but often neglected, at any age9,10, males being more susceptible than females11. This male bias has been documented for bacterial, parasitic and viral infections such as tubercolosis, leishmaniasis, leprosy, leptospirosis, HIV12,13.

Response to vaccination, too, differs, either in childhood than in adult life, between sexes: healthy females present a more robust protective antibody response to the influenza and the measles-mumps-rubella vaccines13-16 and it has been also demonstrated that women could be given half dosage of the vaccine17. Thus, the enhanced female immune response to vaccination can ensure a more effective and long lasting protection but can also cause a greater prevalence of adverse effects in women14-16,18. Indeed, following monovalent 2009 pandemic influenza A (H1N1) vaccines, the female:male ratio of adverse effects was > 4:1 for healthy people aged 20-59 years18.

Sex hormones (see below) exert potent effects on immune cell subsets, estrogen and androgen receptors being present in the majority of immune cells; the “reproductive function” (including pre-puberty, puberty, pregnancy and menopause) deeply affects immune responses and AID.

As known, T lymphocytes secrete cytokines that underlie cell-mediated adaptive immunity, while B lymphocytes produce IgG and IgM antibodies.

Men and women have the same total number of lymphocytes, but males present a lower number of T cells19 and post-menopausal women present less Th cells20. The overall number of B lymphocytes does not change between men and women; nevertheless, females aged > 6 years have increased IgM levels, secrete higher amounts of IL-4, IFN-γ and IL-1, present higher CD4+ T lymphocytes and higher plasma IgM levels than men7 and this has been associated to the female susceptibility to AID1.

Upon antigen challenge, men’s T-helper lymphocytes produce a milder “anti-inflammatory” mix of cytokines – the Th2 response, in which antibody production predominates. On the contrary, female lymphocytes tend to generate a more “pro-inflammatory” mix of cytokine, the Th1 response, in which production of cytotoxic T cells predominate, except during pregnancy. In fact, in pregnancy, women’s immune system shifts towards the milder Th2 response: this may explain why some women with multiple sclerosis or rheumatoid arthritis ameliorate, especially during the third semester, while a few weeks after delivery, the disease rebounds2. Indeed, IFN-γ production, that is the paradigm of Th1 response, is regulated by estrogens21 and is secreted at higher levels after menopause and decreased over the years after reaching a plateau22. On the contrary, no difference in IL-10 production has been documented between males and females19 and at different moments of the menstrual cycle23.

Higher numbers of NK cells are more often observed in women than in men24,25, their activity being modulated by estrogens in a biphasic manner: high dosage suppresses NK activity, whereas low dosage has no effect26.

Monocyte and macrophage activity is also regulated by sex steroids: estrogens stimulate TNF-α secretion from monocytes27 whereas testosterone has no effect28. Male monocytes have been reported to produce more IL-1β than female ones19; this cytokine is also regulated by estrogens in a biphasic manner26. Indeed, 17 beta-estradiol modulates cytokine release through modulation of CD16 expression in human monocytes and macrophages and inhibits the release of pro-inflammatory cytokines29.

Gender differences have been observed also in polymorphonuclear leukocytes, females showing a decreased neutrophil apoptosis, as compared to males30. Moreover, chemotaxis is enhanced by progesterone, inhibited by estrogens and unaffected by testosterone31. As far as the respiratory burst is concerned, contrasting and unconclusive results have been reported26.

Gender differences have been reported also in autophagy32.

Auto-immune diseases (AID)

Autoimmune diseases (AID) include more than 70 chronic disorders, affecting about 5% of population in the United States (with a cost of about 100 billion US dollars per year33) and presenting a large variability in terms of age of onset, targeted organs and response to therapy, but sharing a common feature: the female predominance6,11,34-36.

Indeed, as shown in Table 2, the most striking sex differences are detected in Sjogren disease, systemic lupus erythematosus (SLE), systemic sclerosis, primary biliary cirrhosis and autoimmune thyroid diseases (Graves’ disease and Hashimoto’s thyroiditis)4,5,34,37-39. A female to male predominance also occurs for multiple sclerosis (MS), rheumatoid arthritis (RA), dermatomyositis and myasthenia gravis37,40. On the contrary, type 1 diabetes, idiopathic pulmonary fibrosis and myocarditis are more frequent in men than in women37,41 (Table2).

Also the severity of AID may vary between males and females, even if this is not so clearly defined as in the case of gender prevalence. As an example, psoriasis, SLE and disability progression in MS are more severe in males42-44 and men present autoimmune hepatitis at a younger age and have high relapse rates than women45; on the contrary, Crohn’s disease is more severe in girls46. Moreover, MS women have poorer survival outcomes47 and relative mortality for type 1 diabetes is higher in females than in males, at least in Finland48. Increased mortality in SLE patients has been also associated with female sex49,50.

Despite the female susceptibility to AID has long been recognized, the precise cause of this bias is still unknown and both genetic and environmental factors have been suggested as major determinants. A susceptible genetic background is necessary but does not explain by itself both AID onset and female predominance, while environmental factors act as additional players in tolerance breakdown6,51.

The most reputed mechanisms include sex hormones, fetal microchimerism, sex chromosomes and their major defects; however, none of these determinants has gathered till now enough convincing data and conflicting results are often present.

Environmental factors

Increasing evidence supports a role for the environment in the development of AID52 and at least two well-defined environmentally-associated diseases - i.e., the toxic oil syndrome that occurred after oleic anilide and 1,2-di-oleyl ester (DEPAP) addition to rapeseed oil53 and the eosinophilia myalgia syndrome, occurring after ingestion of tryptophan that had been produced by an alternative manufacturing procedure54 - have been described.

Infections, tobacco smoke, sun exposure, stress situations, diet and drugs have been all implicated in the development of AID3,35.

Various AID have been linked to microorganisms, e.g., Streptococcus pyogenes for rheumatic heart disease, Enterovirus for type 1 diabetes55,56; tobacco smoke has been found to play a relevant role in some AID, as it may trigger the development of autoantibodies and act on pathogenic mechanism possibly related with an imbalance of the immune system57.

Sun exposure (i.e., ultraviolet radiation) is reported to play a role in systemic sclerosis, RA, SLE and phospholipid syndrome35 and a varied sunlight exposure may occur between males and females, depending on lifestyle and/or occupation.

Cosmetics (especially hair dyes and nail polish) may also trigger primary biliary cirrhosis, an AID with a striking female predominance that affects middle-aged women, mainly58. Food intake and food composition affect immunity and auto-immunity, as vitamins and micronutrients are necessary for immune cells’ development and functioning26,35. As an example, low levels of vitamin D are associated with an increased risk for MS, SLE, autoimmune thyroid diseases and others59-61.

Differences in the exposure to chemicals in the workplace between males and females are well documented and may contribute to the gender bias. As a general rule, exposure to pesticides results in anti-nuclear antibody formation62, while exposure to organic solvents is a risk factor for systemic sclerosis, primary systemic vasculitis and MS63.

Genetic factors

Genetic polymorphisms largely contribute to AID susceptibility and may form the basis of ethnic differences in disease presentation and/or severity; as an example, in the United States, the black population presents a higher risk for SLE than whites64. Genome wide association studies (GWAS) are available for the commonest AID3,65; however, multiple genes are involved in disease susceptibility and the genetic patterns vary largely, so that most of the associations disclosed by GWAS are relatively modest3. Genetic factors may contribute to the sexual dimorphism of AID; several studies have focused on the interactions between gender and genes that affect antigen processing and presentation, lymphocyte proliferation and differentiation or encode immunoglobulins3,35.

Human leukocyte antigen (HLA) genes are located in a region that includes many genes regulating the immune response, and there is a close association between HLA genes and AID such as Graves’ thyroiditis66, MS67, RA68 and SLE35,69.

The majority of these associations are with HLA-DR and HLA-DQ genes, which encode for proteins that are mandatory for antigen presentation to CD4+ T cells35. The association between HLA genes and AID usually presents a gender bias towards female35, with the exception of SLE, where a higher HLA associated genetic risk is present in men70.

Also non-HLA genes have been associated with AID susceptibility. For instance, polymorphisms in IL-10 are associated with disease severity in RA (an AA-1087 IL-10 genotype being more frequent in females71,72) and Sjogren’s syndrome73, while polymorphisms in acid phosphatase locus 1 (ACP1) and discs large homolog 5 (DLG5) have been linked to Crohn’s disease74,75.

Polymorphisms in apolipoprotein E (APOE) have been related to Sjogren’s syndrome (women carrying APOE epsilon 4 allele presenting an earlier onset of disease than non-carriers76), and MS77. Indeed, in this latter disease, females who have the APOE epsilon2 allele present a less severe disease78, while men carrying the APOE epsilon 4 allele experience the highest cognitive impairment79.

Due to their pivotal role in innate immunity, toll-like receptors 7 (TLR7) and 8 (TLR8), too, have been intensively investigated. As an example, following TLR7 ligation, women responded with a significantly enhanced interferon (IFN)-alpha (but not TNF-alpha) production as compared to men80.

Moreover, gender-specific association between TLR7 and TLR8 polymorphisms and TNF-alpha response after ligand stimulation were observed in measles virus and vaccine81 (please, see also below).

Sex hormones and their role in the incidence of AID

Sex hormones, as well as genes encoded on the sex chromosomes and gender-specific behavior, largely contribute to AID and influence the different immune cells by modulating their responses. The role of sex hormones and gender disparity in immunity and autoimmunity has been reviewed in a previous issue of this journal82; therefore, in the present work, I’ll provide just a few examples relative to some AID.

As known, estrogens stimulate B cell production of specific antibodies in response to infection, vaccination or autoantigens41,82 and may further increase the risk of AID. However, estrogen therapy in MS83 and RA84 may be beneficial, as well as the use of contraceptives, at least in the case of RA women < 35 years of age85. On the contrary, estrogen worsens disease severity in SLE and, in this case, blockade of ER may be beneficial86. As with the contraceptive pill, diverging results are present in the literature concerning AID35.

At high gestational levels, by inhibiting Th1 and Th17 pathways82, progesterone significantly ameliorates RA and MS87,88. Consistently, RA, which was remitted during pregnancy, usually worsens post-partum87,88. Moreover, combined estrogen and progesterone hormone replacement therapy may induce lupus flares in post-menopausal women89.

As far as androgens are concerned, Klinefelter’s patients (that is, males with XXY karyotype) have an increased risk to develop SLE and androgen therapy reduces immunogloblulin levels90.

One-year transdermal testosterone treatment was beneficial in MS male patients, even if it did not affect the number of lesions91, while skin patches with testosterone did not mitigate disease severity in SLE females92. In this condition, conflicting results were also reported with oral dehydroepiandrosterone, a precursor of both androgens and estrogens35. Moreover, men with RA present low testosterone levels93, men with low cortisol and androgen levels have an increased risk to develop RA94 and androgen therapy in RA patients has provided some benefits90.

While mostly secreted in the anterior pituitary gland, prolactin is also produced by human lymphocytes and binds the prolactin receptor (a member of the cytokine receptor superfamily) that is located on monocytes, T and B lymphocytes35,95.

Activation of the prolactin receptor results in gene transcription, T cell proliferation and antibody secretion96. Thus, prolactin may potentiate AID, while hyperprolactinemia is often documented during different AID97. Moreover, antipsychotics-induced hyperprolactinemia is often associated with increased levels of thyroid autoantibodies98. It has been repetitively reported that bromocriptine reduces disease flares in SLE patients99-102; therefore, the issue of bromocriptine and prolactin antagonists for AID therapy warrants further investigations.

Fetal microchimerism in autoimmunity

Microchimerism (i.e, cells’ trafficking from mother to fetus and vice-versa) occurs during pregnancy and usually persists for years after delivery, fetal microchimerism being the presence of fetal cells in the maternal circulation, whereas maternal microchimerism is the persistence of maternal cells into adult life35,51,103.

A possible protective role has been proposed for fetal microchimerism, as fetal stem cells represent a potential source of cells for tissue repair, regeneration and immune suppression, but other evidences suggest that fetal microchimerism may favour neoplastic progression35,104-106. Fetal microchimerism was first evidenced in peripheral blood mononuclear cells from women with scleroderma who presented an increased level of male DNA, as compared to controls107, but this finding was not confirmed by others108.

Maternal microchimerism might result in detrimental effects, given that maternal cells are a possible source of graft vs host responses; however, it was recently shown to protect against asthma35,109.

Despite microchimerism has been observed in autoimmune thyroid diseases, type 1 diabetes, RA and other AID51,105-107,110, its role in autoimmunity and AID seems to be modest.

Sex chromosomes, especially X chromosome

Sex determination in mammals is mediated by the Sry gene on the Y chromosome, which induces the male developmental program11. Mice with the Sry gene deleted from the Y chromosome or trans-located to an autosomal region have been used to assess the role of sex chromosomes apart from the gonadal sex11.

The X chromosome encodes about 1100 genes (that are distinct from the fewer than 100 genes on the Y chromosome)90 and carries a large number of immune-related genes, including CD40L, CXCR3, OGT, FOXP3, TLR7, TLR8, IL12RG51,111-113. This is partly responsible for the female immune advantage117 as, in general, women produce a more vigorous immune response to infection and this fact has been suggested as a tool to explain why women usually live longer than men2.

In females, one copy of the X chromosome is inactivated to allow equal gene expression dosage between XX females and XY males. At early development, one of the X chromosomes is silenced, resulting in a mosaic expression of either the maternal or paternal X chromosome; therefore, each X-linked gene mutation is potentially expressed in 50% cells in females but in 100% cells in males. The loss of mosaicism hypothesis states that alterations in the random X chromosome inactivation may result in autoimmunity and has been proposed to explain the female predominance in AID51,114-116.

The first support to this hypothesis came from the non-specific, polyclonal T cell activation that activated B cells presenting the same endogenous X-chromosome self antigen in females with SLE51,116-117. Indeed, the frequency of Klinefelter’s syndrome (males with XXY karyotype) is 14-fold higher in men with SLE than normal men118 and is comparable to the risk in females, while women with a particular X chromosome deletion (as found in the Turner’s syndrome) are at lower risk for SLE119. Moreover, enhanced frequency of X monosomy has been found in women with primary biliary cirrhosis and autoimmune thyroid diseases, but not SLE120,121.

It has also been estimated that about 10% of the X chromosome escapes inactivation122: this may determine the over-expression of some gene products in females, potentially positive or negative effects depending on the gene. Over-expression and/or hypomethylation of CD40L, CXCR3 and OGT have been reported in female, but not male, SLE patients123,124. FOXP3, a gene that localizes in the short arm of the X chromosome, is essential for Treg cells and its deficiency or mutation leads to aggressive and often fatal multi-organ AID125.

Small non-coding microRNAs (miRNAs) regulate post-transcriptional gene expression by targeting mRNAs and are emerging as new players in AID. The X chromosome (but not the Y chromosome) is highly enriched for miRNAs, whose expression can be regulated by estrogens: an altered miRNA expression has been documented in some AID, including MS, RA, SLE126,127.

In relation to autoimmunity, poor attention has been dedicated to the Y chromosome. It has been suggested to play a role in the inheritance of coronary artery disease128 and has been demonstrated to undergo an age-dependent loss is some AID, including thyroid autoimmune diseases and primary biliary cirrhosis129,130.

Conclusions

Gender differences in immunity, affecting both the innate and the adaptive immune responses, contribute to differences, between males and females, in the pathogenesis of infectious diseases, the response to vaccination and the prevalence of AID. Women have a lower burden of infections, most evident during their fertile years, but experience a higher incidence of AID. The gonadal hormones contribute to this clear gender bias, but alone are not enough. Other main players, e.g., genetic and environmental factors, sex chromosomes and their flaws, participate in such complex scenario, even if none of them has so far obtained a series of incontrovertible data, discrepant results being often reported.

Further investigation is needed to broaden our knowledge on sex and gender differences in immunity and AID; anyway, the differences so far highlighted are sufficient to suggest the need for gender-oriented therapeutic strategies in AID.

References

1. Whitacre CC, Reingold SC, O’Looney PA. A gender gap in autoimmunity. Science 1999; 283: 1277-8.

2. McCarthy M. The “gender gap” in autoimmune disease. Lancet 2000; 356: 1088.

3. Moroni L, Bianchi I, Lleo A. Geoepidemiology, gender and autoimmune diseases. Autoimmunity Rev 2012; 11: A386-A392.

4. Cooper GS, Stroehla BC. The epidemiology of autoimmune diseases. Autoimmun Rev 2003; 2: 119-25.

5. Cooper GS, Bynum MLK, Somers EC. Recent insights in the epidemiology of autoimmune diseases: improved prevalence estimates and understanding of clustering of diseases. J Autoimmun 2009; 33: 197-207.

6. Lleo A, Battezzati PM, Selmi C, et al. Is autoimmunity a matter of sex? Autoimmun Rev 2008; 7: 626-30.

7. Nussinovitch U, Shoenfeld Y. The role of gender and organ specific autoimmunity. Autoimmun Rev 2012; 11: A377-85.

8. Murphy KM, Stockinger B. Effector T cell plasticity: flexibility in the face of changing circumstances. Nat Immun 2010; 11: 674-80.

9. van Lunzen J, Altfeld M. Sex differences in infectious diseases: common but neglected. J Infect Dis 2014; 209 (Suppl 3): S79-S80.

10. Muenchhoff M, Goulder PJR. Sex differences in pediatric infectious diseases. J Infect Dis 2014; 209 (Suppl 3): S120-S126.

11. Markle JG, Fish EN. SeXX matters in immunity. Trends Immunol 2014; 35: 97-104.

12. Guerra-Silveira F, Abad-France, F. Sex bias in infectious disease epidemiology: pattern and processes. PLoS One 2013; 8: e62390.

13. Klein SL, Marriott I, Fish EN. Sex-based differences in immune function and responses to vaccination. Trans R Soc Trop Med Hyg 2015; 109: 9-15.

14. Klein SL, Jedlicka A, Pekosz A. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis 2010; 10: 338-49.

15. Klein SL, Hodgson A, Robinson DP. Mechanisms of sex disparities in influenza pathogenesis. J Leukoc Biol 2012; 92: 67-73.

16. Shohat T, Green MS, Nakar O, et al. Gender differences in the reactogenicity of measles-mumps-rubella vaccine. Isr Med Assoc J 2000; 2: 192-5.

17. Engler RJ, Nelson MR, Klote MM, et al. Half- vs full-dose trivalent inactivated influenza vaccine (2004-2005): age, dose and sex effects on immune responses. Arch Intern Med 2008; 168: 2405-14.

18. Halsey NA, Griffioen M, Dreskin SC, et al. Immediate hypersensitivity reactions following monovalent pandemic influenza A (H1N1) vaccines: Report to VAERS. Vaccine 2013; 31: 6107-112.

19. Bouman A, Schipper M, Heineman MJ, et al. Gender difference in the non-specific and specific immune response in humans. Am J Reprod Immunol 2004; 52: 19-26.

20. Yang JH, Liang CD, Wu MY, et al. Hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverse the decrease in the T-cell subpopulation of interferon-gamma production in postmenopausal women. Fertil Steril 2000; 74: 261-67.

21. Giron-Gonzales JA, Moral FJ, Elvira J, et al. Consistent production of a higher Th1:Th2 cytokine ratio by stimulated T cells in men compared with women. Eur J Endocrinol 2000; 143: 31-6.

22. Kamada M, Irahara M, Maegawa M, et al. Transient increase in the levels of T-helper 1 cytokines in postmenopausal women and the effects of hormone replacement therapy. Gynecol Obstet Invest 2001; 52: 82-8.

23. Oertelt-Prigione S. Immunology and the menstrual cycle. Autoimmun Rev 2012; 11: A486-A492.

24. Sandberg JK, Bhardwaj N, Nixon DF. Dominant effector memory characteristics, capacity for dynamic adaptive expansion, and sex bias in the innate Valpha24 NKT cell compartment. Eur J Immunol 2003; 33: 588-96.

25. Montoya CJ, Pollard D, Martinson J, et al. Characterization of human invariant natural killer T subsets in health and disease using a novel invariant natural killer T cell-clonotypic monoclonal antibody, 6B11. Immunology 2007; 122: 1-14.

26. Oertelt-Prigione S. The influence of sex and gender on the immune response. Autoimmun Rev 2012; 11: A479-A485.

27. Rogers A, Eastell R. The effect of 17beta-estradiol on production on cytokine production in cultures of peripheral blood. Bone 2001; 29: 30-34.

28. Pesma E, Moes H, Heinemann MJ, et al. The effects of testosterone on cytokine production in the specific and non-specific immune response. Am J Reprod Immunol 2004; 52: 237-43.

29. Kramer PR, Kramer SF, Guan G. 17beta-estradiol regulates cytokine release through modulation of CD16 expression in monocytes and monocyte-derived macrophages. Arthritis Rheum 2004; 50: 1967-175.

30. Molloy EJ, O’Neill AJ, Grantham JJ, et al. Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone. Blood 2003; 102: 2653-9.

31. Miyagi M, Aoyama H, Morishita M, et al. Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes. J Periodontol 1992; 63: 28-32.

32. Lista P, Straface E, Brunelleschi S, et al. On the role of autophagy in human diseases: a gender perspective. J Cell Mol Med 2011; 15: 1443-57.

33. Jacobson DL, Gange SJ, Rose NR, et al. Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clin Immunol Immunopathol 1997; 84: 223-43.

34. Whitacre CC. Sex differences in autoimmune diseases. Nat Immun 2001; 2: 777-80.

35. Ngo ST, Steyn FJ, McCombe PA. Gender differences in autoimmune diseases. Frontiers in Neuroendocrinology 2014; 35: 347-69.

36. Gleicher N, Barad DH. Gender as risk factor for autoimmune diseases. J Autoimmun 2007; 28: 1-6.

37. Hayter SM, Cook MC. Updated assessment of the prevalence, spectrum and case definition of autoimmune disease. Autoimmun Rev 2012; 11: 754-65.

38. Brandt JE, Priori R, Valesini G, et al. Sex differences in Sjogren’s syndrome: a comprehensive review of immune mechanisms. Biol Sex Diff 2015; 6: 19.

39. Gershwin ME, Selmi C, Worman HJ, et al. Risk factors and comorbidities in primary biliary cirrhosis: a controlled interview-based study of 1032 patients. Hepatology 2005; 42: 1194-202.

40. Zandman-Goddard G, Peeva E, Shoenfeld Y. Gender and autoimmunity. Autoimmun Rev 2007; 6: 366-72.

41. Fairweather D, Frisanco-Kiss S, Rose NR. Sex difference in autoimmune disease from a pathologic perspective. Am J Pathol 2008; 173: 600-9.

42. Sakai R, Matsui S, Fuhushima M, et al. Prognostic factor analysis for plaque psoriasis. Dermatology 2005; 211: 103-6.

43. Weinshenker BG, Rice GP, Noseworthy JH, et al. The natural history of multiple sclerosis: a geographically based study. 3. Multivariate analysis of predictive factors and models of outcome. Brain 1991; 114: 1045-56.

44. Crosslin KL, Wiginton KL. Sex differences in disease severity among patients with systemic lupus erythematosus. Gend Med 2011; 8: 365-71.

45. Al-Chalabi T, Underhill JA, Portmann BC, et al. Impact of gender on the long-term outcome and survival of patients with autoimmune hepatitis. J Hepatol 2008; 48: 140-7.

46. Gupta N, Bostrom AG, Kirschner BS, et al. Gender differences in presentation and course of disease in pediatric patients with Crohn disease. Pediatrics 2007; 120: e1418-e1425.

47. Grytten Torkidsen N, Lie SA, Aarseth JH, et al. Survival and cause of death in multiple sclerosis: results from a 50-year follow-up in Western Norway. Multip Scl 2008; 14: 1191-8.

48. Asao K, Sarti C, Forsen T, et al. Long term mortality in nationwide cohorts of childhood-onset type 1 diabetes in Japan and Finland. Diabetes Care 2003; 26: 2037-42.

49. Bernatsky S, Boivin JF, Joseph L, et al. Mortality in systemic lupus erythematosus. Arthritris Rheum 2006; 54: 2550-7.

50. Ruiz E, Ramalle-Gomara E, Elena A, et al. Trends in systemic lupus erythematosus mortality in Spain from 1981 to 2010. Lupus 2014; 23: 431-5.

51. Selmi C, Brunetta E, Raimondo MG, et al. The X chromosome and the sex ratio of auto-immunity. Autoimmun Rev 2012; 11: A531-A537.

52. Miller FW, Pollard KM, Parks CG, et al. Criteria for environmentally associated autoimmune diseases. J Autoimmun 2012; 39: 253-8.

53. Gelpi E, de la Paz M, Terracini B, et al. The Spanish toxic oil syndrome 20 years after its onset: a multidisciplinary review of scientific knowledge. Environ Health Perspect 2002; 110: 457-64.

54. Hertzman PA, Clauw DJ, Kaufman LD, et al. The eosinophilia-myalgia syndrome: status of 205 patients and results of treatment 2 years after onset. Ann Intern Med 1995; 122: 851-5.

55. Fae KC, da Silva DD, Oshiro SE, et al. Mimicry in recognition of cardiac myosin peptides by heart-intralesional T cell clones from rheumatic heart disease. J Immunol 2006; 176: 5662-70.

56. Richardson SJ, Willcox A, Bone AJ, et al. The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 2009; 52: 1143-51.

57. Perricone C, Versini M, Ben-Ami D, et al. Smoke and autoimmunity: the fire behind the disease. Autoimmun Rev 2016; 15: 354-74.

58. Bianchi I, Lleo A, Bernuzzi F, et al. The X-factor in primary biliary cirrhosis: monosomy X and xenobiotics. Autoimmun Highlights 2012; 3: 127-32.

59. Alharbi FM. Update in vitamin D and multiple sclerosis. Neurosciences 2015; 20: 329-35.

60. Yap KS, Morand EF. Vitamin D and systemic lupus erythematosus: continued evolution. Int J Rheum Dis 2015; 18: 242-9.

61. Wang X, Zynat J, Guo Y, et al. Low serum vitamin D is associated with anti-thyroid-globulin antibody in female individuals. Int J Endocrinol 2015; 2015: 285290.

62. Rosenberg AM, Semchuk KM, McDuffie HH, et al. Prevalence of antinuclear antibodies in a rural population. J Toxicol Environ Health 1999; 57: 225-36.

63. Barragan-Martinez C, Speck-Hernandez CA, Montova-Ortiz G, et al. Organic solvents as risk factor for autoimmune diseases: a systematic review and meta-analysis. PLoS One 2012; 7: e51506.

64. Youinou P, Pers JO, Gershwin ME, et al. Geo-epidemiology and autoimmunity. J Autoimmun 2010; 34: J163-167.

65. Invernizzi P, Gershwin ME. The genetics of human autoimmune diseases. J Autoimmun 2009; 33: 290-9.

66. Li H, Chen Q. Genetic susceptibility to Grave’s diseases. Front Biosci 2013; 18: 1080-7.

67. Cree BA. Multiple sclerosis genetics. Handbook Clin Neurol 2014; 122: 193-209.

68. Jin H, Arase N, Hurayasu K, et al. Autoantibodies to IgG/HLA class II complexes are associated with rheumatoid arthritis susceptibility. Proc Natl Acad Sci USA 2014; 111: 3787-92.

69. Deng FY, Lei SF, Zhang YH, et al. Functional relevance for associations between genetic variants and systemic lupus erythematosus. PLoS One 2013; 8: e53037.

70. Hughes T, Adler A, Merrill JT, et al. Analysis of autosomal genes reveals gene-sex interactions and higher total genetic risk in men with systemic lupus erythematosus. Ann Rheum Dis 2012; 71: 694-9.

71. Hee CS, Gun SC, Naidu R, et al. Comparison of single nucleotide polymorphisms in the human interleukin-10 promoter between rheumatoid arthritis patients and normal subjects in Malaysia. Mod Rheumatol 2007; 17: 429-35.

72. Padyukov L, Hytonen AM, Smolnikova M, et al. Polymorphism in promoter region of IL-10 gene is associated with rheumatoid arthritis in women. J Rheumatol 2004; 31: 422-5.

73. Qin B, Wang J, Liang Y, et al. The association between TNF-alpha, Il-10 gene polymorphisms and primary Sijogren’s syndrome: a meta-analysis and systemic review. PLoS One 2013; 8: e63401.

74. Browning BL, Annese V, Barclay ML, et al. Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts. J Med Genet 2008; 45: 36-42.

75. Gloria-Bottini F, Bottini N, Renzetti G, et al. ACP1 and Th class of immunological diseases: evidence and interaction with gender. Int Arch Allergy Immunol 2007; 143: 170-6.

76. Pertovaara M, Lehtimaki T, Rontu R, et al. Presence of apolipoprotein E epsilon4 allele predisposes to early onset of primary Sjogren’s syndrome. Rheumatology 2004; 43: 1484-7.

77. Rafiei M, Zarif Yeganeh M, Sheikholestami S, et al. Apolipoprotein E polymorphisms status in Iranian patients with multiple sclerosis. J Neurol Sci 2012; 320: 22-25.

78. Kantarci OH, Hebrink DD, Achenbach SJ, et al. Association of APOE polymorphisms with disease severity in MS is limited to women. Neurology 2004; 62: 811-4.

79. Savatteri G, Messina D, Andreoli V, et al. Gender-related effect of clinical and genetic variables on the cognitive impairment in multiple sclerosis. J Neurol 2004; 251: 1208-14.

80. Berghofer B, Frommer T, Haley G, et al. TLR7 ligands induce TNF-alpha production in females. J Immunol 2006; 177: 2088-96.

81. Clifford HD, Yerkovich ST, Khoo SK, et al. Toll-like receptor 7 and 8 polymorphisms: associations with functional effects and cellular and antibody responses to measles virus and vaccine. Immunogenetics 2012; 64: 219-28.

82. Ortona E, Delunardo F, Maselli A, et al. Sex hormones and gender disparity in immunity and autoimmunity. Ital J Gender-Specific Med 2015; 1: 45-50.

83. Sicotte NI, Liva SM, Klutch R, et al. Treatment of multiple sclerosis with the pregnancy hormone estriol. Ann Neurol 2002; 52: 421-8.

84. Bijlsma JW, Huber-Bruning O, Thijssen JH. Effect of estrogen treatment on clinical and laboratory manifestations of rheumatoid arthritis. Ann Rheum Dis 1987; 46: 777-9.

85. Spector TD, Roman E, Silman AJ. The pill, parity, and rheumatoid arthritis. Arthritis Rheum 1990; 33: 782-9.

86. Abdou NI, Rider V, Greenwell C, et al. Fulvestrant (Faslodex), an estrogen selective receptor downregulator, in therapy of women with systemic lupus erythematosus. Clinical, serologic, bone density, and T cell activation marker study: a double-bind placebo-controlled trial. J Rheumatol 2008; 35: 797.

87. Hughes GC. Progesterone and autoimmune diseases. Autoimmun Rev 2012; 11: A502-A514.

88. Ostensen M, Villiger PM. The remission of rheumatoid arthritis during pregnancy. Semin Immunopathol 2007; 29: 185-91.

89. Buyon JP, Petri MA, Kim MY, et al. The effect of estrogen and progesterone hormone replacement therapy on disease activity in systemic lupus erythematosus: a randomized trial. Ann Intern Med 2005; 142: 953-62.

90. Pennell LM, Galligan CL, Fish EN. Sex affects immunity. J Autoimmun 2012; 38: J282-J29.

91. Sicotte NI, Giesser BS, Tandon V, et al. Testosterone treatment in multiple sclerosis: a pilot study. Arch Neurol 2007; 64: 683-8.

92. Gordon C, Wallace DJ, Shinada S, et al. Testosterone patches in the management of patients with mild/moderate systemic lupus erythematosus. Rheumatology 2008; 47: 334-8.

93. Tengstrand B, Carlstrom K, Hafstrom I. Bioavailable testosterone in men with rheumatoid arthritis: high frequency of hypogonadism. Rheumatology 2002; 41: 285-9.

94. Masi AT, Cutolo M. Perspectives on sex hormones and the systemic rheumatic diseases. Clin Exp Rheumatol 1995; 13: 201-2.

95. Chavez-Rueda K, Hernandez J, Zenteno E, et al. Identification of prolactin as a novel immunomodulator on the expression of co-stimulatory molecules and cytokine secretions on T and B human lymphocytes. Clin Immunol 2005; 116: 182-91.

96. McMurray RW. Estrogen, prolactin and autoimmunity: actions and interactions. Int Immunopharmacol 2001; 1: 995-1008.

97. Orbach H, Zandman-Goddard G, Boaz M, et al. Prolactin and autoimmunity: hyperprolactinemia correlates with serositis and anemia in SLE patients. Clin Rev Allergy Immunol 2012; 42: 189-98.

98. Poyraz BC, Aksoy C, Balcioglu L. Increased incidence of autoimmune thyroiditis in patients with antipsychotic-induced hyperprolactinemia. Eur Neuropsychopharmacol 2008; 18: 667-72.

99. McMurray RW, Weidensaul D, Allen SH, et al. Efficacy of bromocriptine in an open label therapeutic trial for systemic lupus erythematosus. J Rheumatol 1995; 22: 2084-91.

100. Alvarez-Nemegyei J, Cobarrubias-Cobos A, Escalante-Triay F, et al. Bromocriptine in systemic lupus erythematosus: a double-blind, randomized, placebo-controlled study. Lupus 1998; 7: 414-9.

101. Yang XY, Liang LQ, Xu HS, et al. Efficacy of oral bromocriptine in protecting the post-partum systemic lupus erythematosus patients from disease relapse. Zhonghua Nei Ke Za Zhi 2003; 42: 621-4.

102. Qian Q, Liuqin L, Hao L, et al. The effects of bromocriptine on preventing postpartum flare in systemic lupus erythematosus patients from South China. J Immunol Res 2015; 2015: 316965.

103. Maloney S, Smith A, Furst DE, et al. Microchimerism of maternal origin persists into adult life. J Clin Invest 1999; 104: 41-7.

104. Nelson JL. The otherness of self: microchimerism in health and disease. Trends Immunol 2012; 33: 421-7.

105. Fugazzola L, Cirello V, Beck-Peccoz P. Fetal microchimerism as an explanation of disease. Nat Rev Endocrinol 2011; 7: 89-97.

106. Cirello V, Rizzo R, Crippa M, et al. Fetal cell microchimerism: a protective role in auto-immune thyroid diseases. Eur J Endocrinol 2015; 173: 111-8.

107. Nelson JL, Furst DE, Maloney S, et al. Microchimerism and Hla-compatible relationships of pregnancy in scleroderma. Lancet 1998; 351: 559-62.

108. Murata H, Nakauchi H, Sumida T. Microchimerism in Japanese women patients with systemic sclerosis. Lancet 1999; 354: 220.

109. Thompson EE, Myers RA, Du G, et al. Maternal microchimerism protects against the development of asthma. J Allergy Clin Immunol 2013; 132: 39-44.

110. Ye J, Vives-Pi M, Gillespie KM. Maternal microchimerism: friend or foe in type 1 diabetes? Chimerism 2014; 5: 21-3.

111. Libert C, Dejager L, Pinheiro I. The X chromosome in immune functions: when a chromosome makes the difference. Nat Rev 2010; 10: 594-604.

112. Rubtsova K, Marrack P, Rubtsov AV. Sexual dimorphism in autoimmunity. J Clin Invest 2015; 6: 2187-93.

113. Bianchi I, Lleo A, Gershwin ME, et al. The X chromosome and immune associated genes. J Autoimmun 2012; 38: J187-J192.

114. Ozcelik T. X chromosome inactivation and female predisposition to autoimmunity. Clin Rev Allergy Immunol 2008; 34: 348-51.

115. Kast RE. Predominance of autoimmune and rheumatic diseases in females. J Rheumatol 1977; 4: 288-92.

116. Stewart JJ. The female X-inactivation mosaic in systemic lupus erythematosus. Immunol Today 1998; 19: 352-7.

117. Takeno M, Nagafuchi H, Kaneko S, et al. Auto-reactive T cell clones from patients with systemic lupus erythematosus support polyclonal autoantibody production. J Immunol 1997; 158: 3529-38.

118. Scofield RH, Bruner GR, Namjou B, et al. Klinefelter’s syndrome (47,XXY) in male systemic lupus erythematosus patients: support for the notion of a gene-dose effect for the X chromosome. Arthritis Rheum 2008; 58: 2511-7.

119. Conney CM, Bruner GR, Aberle T, et al. 46,X,del(X)(q13) Turner’s syndrome women with systemic lupus erythematosus in a pedigree multiplex for SLE. Genes Immun 2009; 10: 478-81.

120. Invernizzi P, Miozzo M, Battezzati PM, et al. Frequency of monosomy X in women with primary biliary cirrhosis. Lancet 2004; 363: 533-5.

121. Invernizzi P, Miozzo M, Selmi C, et al. X chromosome monosomy: a common mechanism for autoimmune diseases. J Immunol 2005; 175: 575-8.

122. Lockshin MD. Non-hormonal explanations for sex discrepancy in human illness. Ann N Y Acad Sci 2010; 1193: 22-4.

123. Hewagama A, Gorelik G, Patel D, et al. Overexpression of X-linked genes in T cells from women with lupus. J Autoimmun 2013; 41: 60-71.

124. Lu Q, Wu A, Tesmer L, et al. Demethylation of CD40LG on the inactive X in T cells from women with lupus. J Immunol 2007; 179: 6352-8.

125. Zheng Y, Rudensky AY. FOXP3 in control of the regulatory T cell lineage. Nat Immunol 2007; 8: 457-62.

126. Dai R, Ahmed SA. microRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl Res 2011; 157: 163-79.

127. Munoz-Culla M, Irizar H, Saenz-Cuesta M, et al. SncRNA (microRNA & snoRNA) opposite expression pattern found in multiple sclerosis relapse and remission is sex dependent. Scientific Report 2016; 6: 20126.

128. Charchar FJ, Bloomer LD, Barnes TA, et al. Inheritance of coronary artery disease in men: an analysis of the role of Y chromosome. Lancet 2012; 379: 915-22.

129. Lleo A, Oertel-Prigione S, Bianchi I, et al. Y chromosome loss in male patients with primary biliary cirrhosis. J Autoimmun 2013; 41: 87-91.

130. Persani L, Bonomi M, Lleo A, et al. Increased loss of the Y chromosome in peripheral blood cells in male patients with autoimmune thyroiditis. J Autoimmun 2012; 38: J193-196.