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The X chromosome in immune functions: when a chromosome makes the difference

Claude Libert*, Lien Dejager and Iris Pinheiro*

Abstract | In response to various immune challenges, females show better survival than males; the X chromosome has an important role in this immunological advantage. X chromosome-linked diseases are usually restricted to males, who have only one copy of the X chromosome; however, females are more prone to autoimmune diseases, and the X chromosome may be involved in the breakdown of self tolerance. Several hypotheses have been proposed in recent years that support a role for the X chromosome in shaping autoimmune responses. Here, we review the main mechanisms responsible for increased immune activity in females. This provides a survival advantage in the face of pathogenic insult but can also enhance the susceptibility of females to autoimmunity.
X chromosome inactivation
(Also known as lyonization or silencing). A process that occurs in female mammals in which gene expression from one of the pair of X chromosomes is downregulated. This ensures that the levels of X chromosome gene expression in females matches that of males.

Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), 9052 Ghent, Belgium, and the Department of Biomedical Molecular Biology, Ghent University, Ghent 9000, Belgium. Correspondence to C.L. e-mail: Claude.Libert@dmbr. vib-ugent.be *These authors contributed equally to this work. doi:10.1038/nri2815

In general, females are healthier and live longer than males, and this is true for several mammalian species, including humans1. Additionally, the outcome and survival rates from illnesses such as those caused by infectious diseases, sepsis, trauma or injury are better in women than in men2,3. This immunological advantage of women has been long known, and several studies have shown the superior ability of women to produce more antibodies and serum IgM 4,5. The greater susceptibility of males to infections is evident from birth: male newborns are more prone to septicaemia and meningitis6, and the incidence of tuberculosis is higher in males from infancy to adulthood7. Males experience more frequent and severe infections caused by bacteria or viruses, such as respiratory infections caused by parainfluenza virus and respiratory syncytial virus, as well as those caused by bacteria such as Staphylococcus spp., Escherichia coli, Legionella pneumophila and Campylobacter spp.5,812. Moreover, these clinical observations have been corroborated by theoretical and mathematical models, which confirm that throughout evolution, males have acquired lower immune responsiveness than females13,14. The X chromosome is partly responsible for the hyperresponsiveness of the female immune system. Females carry two X chromosomes, one from each parent, whereas males carry one X chromosome inherited from the mother and one Y chromosome from the father. To avoid double dosage of proteins in females, one of the X chromosomes is randomly silenced during

X chromosome inactivation ,

which occurs in the early stages of female embryogenesis; however, the pseudoautosomal regions of the X chromosome escape inactivation (BOX 1). The process of X chromosome inactivation results in female cellular mosaicism: in a female, approximately half of the cells express genes derived from the maternal X chromosome and the other half express genes derived from the paternal X chromosome. Thus, deleterious or disadvantageous mutations that occur in an X chromosome-linked gene will result in the functional loss of the protein in all cells in a male but in only half of cells in a female. The X chromosome has numerous genes which, directly or indirectly, are involved in immunity, and naturally occurring variations in one gene copy might result in two distinct alleles with different regulatory and response capacities. For females, this means additional physiological diversity: not only do heterozygous females avoid the effects of deleterious gene-mutations, they also benefit from added diversity when facing new immune challenges, such as microbial infections1517. Indeed, it has been proposed that males suffer the equivalent of inbreeding depression involving 5% of their protein-coding genome (the X chromosome transcriptome), leading to a higher rate of mortality, from infancy to adulthood16. Phenotypical differences between females and males result from direct genetic differences18. These genetic differences can be attributed to a number of factors: the expression of genes located on the non-recombining regions of the Y chromosome, sex hormone-mediated
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Box 1 | At a glance: the sex chromosomes
Although the mammalian sex chromosomes were originally a homologous pair of autosomes47, nowadays they barely resemble each other. The human Y chromosome contains only ~100 genes, whereas the X chromosome has ~1100 annotated genes, which is approximately 5% of the human genome51,93. The number of functional genes homologous to both chromosomes is presently limited to 54 (ReF. 51). DNA homology is fundamental to guarantee recombination, and the sex chromosomes can still exchange DNA at the pseudoautosomal regions in order to ensure proper segregation during male meiosis. Human sex chromosomes have two pseudoautosomal regions (PARs): PAR1 at the terminus of the short arms and PAR2 at the terminus of the long arms of both sex chromosomes. Twenty-nine of the homologous genes of the sex chromosomes are located in the PARs, whereas the remaining 25 homologous genes are located in the non-recombining region51. The genes located in the PARs are present in two copies in both sexes and therefore do not require inactivation. However, genes which are X-specific have to be silenced in one of the female X chromosomes in order to ensure that only a single copy functions in both sexes. Inactivation is initiated by the X-inactive specific transcript (XIST) gene located in the X inactivation centre (XIC). The XIC is located in the long arm, which is, evolutionarily, the original and the most conserved portion of the X chromosome, whereas the short arm of the X chromosome was recently added by translocations from autosomes7. Together with the fact that the centromere possibly creates a barrier to efficient spreading of inactivation from the XIST, this may explain why the short arm is more prone to escape inactivation94,95.

Pseudoautosomal regions
Small regions of sequence homology located at the tips of mammalian X and Y chromosomes where recombination still occurs during male meiosis.

Cellular mosaicism
The chimeric state of female tissues that results from random X chromosome inactivation, meaning that normal mammalian females have two genetically distinct types of cells.

Inbreeding depression
(Also known as loss of heterozygosity) is the reduced fitness of a certain population that results from breeding between close genetic relatives.

Sex-determining region of the Y chromosome

(SRY). The gene located on the Y chromosome that is responsible for male sex determination in almost all placental mammals.

effects based on the presence or absence of the sex-determining region of the Y chromosome (sRY), differences in the expression of X-linked genes of maternal or paternal origin (due to imprinting), the gene-dosage effects of sex chromosome-linked genes (namely those genes that escape X chromosome inactivation or are reactivated), nonrandom X chromosome inactivation and, finally, cellular mosaicism of females. X chromosome-located protein-coding genes are susceptible to X chromosome inactivation, cellular mosaicism, silencing escape and unusual patterns of evolution. Here, we describe the most important properties of the X chromosome that may provide females with an immunological advantage, and highlight the differences between the genders in X-linked primary immunodeficiencies (PIDs). It is widely recognized that possessing two X chromosomes can be beneficial to females; however, it is becoming clear that this can also be disadvantageous. Recent studies show that perturbations in X chromosome inactivation are markers of several autoimmune disorders, which are far more common in females1921. The evidence in support of X chromosome involvement in the breakdown of self tolerance and the currently accepted hypotheses by which this occurs are discussed here.

hormones cannot be regarded as totally independent (FIG. 1). on the Y chromosome, SRY is the triggering gene that leads to maleness. The signalling cascade initiated by this gene results in the development of testicles in the male embryo and the consequent production of testosterone. In the absence of SRY activation, the default pathway leads to the development of female embryos and to the secretion of oestrogens by the ovaries. signalling pathways induced by sex hormones, which can be regulated by or regulate sex chromosome-linked genes and/or autosomes, influence the immunological responses of an individual. The suggestion of a partnership between sex hormones and sex chromosomes in immunity is not new. An opposing and compensatory mechanism between the two has been proposed as an evolutionary mechanism that emerged to decrease the differences between males and females26. This study showed that ovariectomized XY female mice lacking Y chromosome-specific Sry expression (XYSry mice) have an increased immune response to an autoantigen than ovariectomized XX female mice. This shows that, independent of the hormonal background, the lack of one X chromosome correlates with an enhanced immune reaction. When testosterone was administered to ovariectomized XYSry mice, the immune response was diminished, suggesting a compensatory mechanism of testosterone in immunity. The authors concluded that the antagonizing actions of male hormones and the XY genome may have evolved to decrease the differences between female and male immune responses, as extreme dissimilarity between genders could be maladaptive26. However, a total balance is not achieved, as males cannot overcome the fact that they only have one X chromosome and are, therefore, more exposed to X-linked deleterious mutations. Moreover, it has also been proposed that evolution has endowed females with a superior immune system in order to face the immunological challenges of pregnancy 5. sex hormones are generated as a result of sex chromosome-induced signalling-cascades, and although the two may influence each other during immunological challenges, they can also independently contribute to the regulation of immune responses. The influence of sex chromosomes in early life. sex differences in susceptibility to infectious diseases is shown by case records from several hospitals27,28. For example, in the John Hopkins Hospital, Baltimore, Maryland, usA, studies carried out over a 30 year period showed that there is a higher incidence of bacterial meningitis and bacterial septicaemia in males than females, from infancy to adolescence27. Plasma testosterone levels in male newborns are, on average, 68 ng per 100 ml in the first 15 days of life. Testosterone concentrations rapidly increase to reach a pubertal level of 202 ng per 100 ml in the first 3 months after birth and drop to almost undetectable levels by the age of 712 months. By contrast, plasma testosterone levels in females are 12 ng per100 ml in the first 15 days of life and drop rapidly to pre-pubertal levels by the third week after birth29.
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Imprinting
An epigenetic process that, through methylation and histone modification, tags the chromosomes inherited from the mother or the father and results in differential gene expression in a parent-oforigin-specific manner.

Sex chromosomes and sex hormones Are they two sides of the same coin? sex hormones such as testosterone and oestrogens are responsible for the development of secondary sexual characteristics and have also a role in modulating inflammatory immune responses2224. As it is easier to study and manipulate the effects of sex hormones than genes on the sex chromosomes, sex hormones have been more extensively studied. sex chromosomes induce gender differences in several organs, even before the gonads begin to differentiate, and so these actions are independent of sex hormones18,25. However, sex chromosomes and sex

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Sex chromosomes XX XY SRY X-linked genes Autosomes

Gonads Ovaries Sex hormones Oestrogens

Testes

Testosterone

Hormone-dependent signalling Sexual characteristics Sex-specific responses Innate and adaptive immunity Inflammation Autoimmunity

Figure 1 | The different mechanisms regulating immunity and triggered by the sex chromosomes. Both X-linked genes and sex hormones produced by the gonads Nature Reviews Immunology influence sex-specific immune responses. X-linked genes, autosomal genes |and sex hormones can operate together or act individually on the different immunoregulatory processes. SRY, sex-determining region of the Y chromosome.

Gene-dosage effects
The nearly linear relationship between the phenotype and the number of copies of the relevant gene present in the genome.

Self tolerance
Tolerance to an individuals own antigens that is achieved through both central and peripheral tolerance mechanisms, including T cell deletion, anergy and immune regulation. Without central and peripheral tolerance, the immune system could not distinguish self from foreign antigens, resulting in autoimmunity.

Chronic granulomatous disease

An inherited disorder caused by defective oxidase activity in the respiratory burst of phagocytes. It results from mutations in any of four genes that are necessary to generate the superoxide radicals required for normal neutrophil function. Affected patients suffer from increased susceptibility to recurrent infections.

Hypomorphic mutations
A type of mutation that results in a reduced level of activity of the product encoded by the mutated gene.

Although there are no studies indicating that early testosterone secretion influences the immune response of male newborns, we cannot exclude this hypothesis. nevertheless, by the age of 12 months, and until adolescence, the sex hormonal background is identical in both sexes29. Therefore, it can be assumed that the differences in mortality rates between male and female infants owing to infectious diseases are independent of sex hormones and that sex chromosomes have an important role during this period. More recently, retrospective studies of explained and unexplained cases of sudden unexpected death in infancy (suDI) showed that the male to female death ratio is 3:2 (ReF. 30). The most common cause of explained suDI is bacterial infections31, and recent studies claim that unexplained suDI has possibly the same aetiology 32. Considering the low number of functional genes on the Y chromosome, it was initially proposed that the difference in X chromosome number accounted for the immunological differences between males and females5,27. Although this was the most straightforward explanation, it was difficult to confirm. However, a recent global dataset, comprising approximately 70,000 autopsies carried out in infants of both sexes with postneonatal suDI, suggested that an X-linked recessive allele could be responsible for the higher susceptibility of males to suDI30. The X chromosome has many genes involved in immune functions, such as Toll-like receptor 7 (TLR7), CD40 ligand (CD40L) and forkhead box P3 (FOXP3) (see supplementary information s1 (table)), and we suspect that further genes and mutations relevant to immunity are still to be discovered. In the context of the gender-specific expression mode of X-linked genes, we speculate that mutations affecting such genes and their associated regulatory pathways can partly account for the differences in immunological responses of males and females.

The disadvantage of being male X-linked primary immunodeficiencies. Mammalian males have only one X chromosome; from an evolutionary point of view this is a tremendous disadvantage, as every newly arisen recessive mutation on the X chromosome will be phenotypically manifested. This survival disadvantage is shown by the existence of numerous X-linked PIDs. These are diseases that typically do not affect females or cause less severe effects in females than in males. Thirteen different X-linked PIDs are presently recognized33 (TABLe 1), although it should be mentioned that diverse mutations occurring in the same gene result in different diseases. usually, these diseases are diagnosed during early infancy, and affected newborn males tend to suffer from recurrent bacterial, fungal and viral infections, which can result in severe septicaemia and, eventually, lead to premature death (reviewed in ReF. 34). The immune system can be impaired at different levels: the mutated genes may be fundamental for killing microorganisms by innate immune cells (for example, mutations in cytochrome b-245 -subunit (CYBB; also known as NOX2) are responsible for X-linked chronic granulomatous disease (X-CgD)35) or crucial for lymphocyte differentiation for example, mutations of FOXP3 and interleukin-2 receptor- (IL2RG; also known as the common cytokine receptor -chain) are responsible for immunodysregulation, polyendocrinopathy enteropathy, X-linked syndrome (IPeX) and X-linked severe combined immunodeficiency (X-sCID), respectively 36,37. Most of these X-linked PIDs are characterized by an absence or decreased number of immune cells or by the presence of non-functional immune cells, and impaired cytokine-mediated signalling may aggravate the clinical outcome of affected patients3840. In some cases, the X chromosome-associated mutation is so severe that it is incompatible with life. lossof-function mutations affecting the inhibitor of nuclear factor-B (nF-B) kinase- (IKBKG) are responsible for incontinentia pigmenti, a disease which is usually diagnosed only in females, as affected males die in utero41. By contrast, hypomorphic mutations of IKBKG are responsible for a rare congenital disease, X-linked anhidrotic ectodermal dysplasia with immunodeficiency (X-eDA-ID). Male patients suffer from abnormal development of ectoderm-derived skin appendages (hair, teeth and nails) and are susceptible to infections, particularly to pyogenic bacteria, non-tuberculous mycobacteria and histoplasma42. IKBKg is an essential molecule in numerous pathways that are triggered by several receptors, such as the TlR family of receptors, CD40, the tumour necrosis factor receptor (TnFR) superfamily and ectodysplasin A receptor (eDAR). The activation of IKBKg leads to nF-B-dependent gene transcription and to subsequent activation of numerous pathways involved in inflammatory responses, cell differentiation and proliferation. Typically, mutations affecting the IKBKG gene result in impaired immune responses, and different point mutations have been associated with an increased risk of viral and bacterial infections in males that can result in sepsis, pneumonia, otitis or sinusitis4345.
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Table 1 | Xlinked primary immunodeficiencies and their genderspecific clinical manifestations*
Disease
X-linked agammaglobulinaemia (XLA) WiskottAldrich syndrome (WAS) X-linked neutropenia and myelodysplasia X-linked chronic granulomatous disease (X-CGD) Properdin deficiency

gene
BTK

Females
Usually not affected

males
Absence of mature B cells and immunoglobulins of all classes; severe immunodeficiency and susceptibility to microbial infections Absence of T cells and platelets; severe immunodeficiency and susceptibility to microbial infections; eczema Severe congenital neutropenia and monocytopenia; recurrent bacterial infections

WAS WAS CYBB

Usually not affected Usually not affected

Reduction in superoxide Deficient superoxide production in phagocytes; severe production; increased incidence of bacterial and fungal infections; colitis SLE; carrier genodermatosis of CGD Low to normal levels of properdin Absent haemolytic activity by the alternative pathway of complement; increased susceptibility to meningococcal (Neisseria spp.) infections Enteropathy; insulin-dependent diabetes mellitus; dermatitis; thyroiditis; haemolytic anaemia; thrombocytopenia; eczema; recurrent infections Absence or diminished numbers of T cells and natural killer cells; non-functional B cells; decreased levels of immunoglobulins; severe susceptibility to infections Severe EBV infection; aplastic anaemia; reduced number of B cells; infectious mononucleosis syndrome Splenomegaly; bone marrow failure; increased apoptosis of peripheral blood lymphocytes Low levels of IgG, IgE and IgA and normal to high levels of IgM; neutropenia; thrombocytopenia; haemolytic anaemia; recurrent infections Dysgammaglobulinaemia; severe susceptibility to infections; defective NF-B signalling pathway Death in utero Deficient superoxide production in phagocytes; severe haemolytic anaemia

PFC

immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX) X-linked severe combined immunodeficiency (X-SCID) X-linked lymphoproliferative disease type 1 (XLP1) X-linked lymphoproliferative disease type 2 (XLP2) CD40L deficiency (also known as X-linked hyper-IgM syndrome) Anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) Incontinentia pigmenti Glucose-6-phosphate dehydrogenase (G6PD) deficiency

FOXP3

Usually not affected

IL2RG

Usually not affected

SH2D1A XIAP CD40L

Usually not affected Usually not affected Usually not affected

IKBKG IKBKG G6PD

Usually not affected Skin, hair and teeth abnormalities Usually not affected

BTK, Brutons tyrosine kinase; CD40L, CD40 ligand; CYBB, cytochrome b-245 -subunit; EBV, EpsteinBarr virus; FOXP3, forkhead box P3; IKBKG, inhibitor of nuclear factor-B kinase-; ILR2G, interleukin-2 receptor-; NF-B: nuclear factor-B; PFC, properdin factor complement; SH2D1A, SH2 domain containing 1A; SLE, systemic lupus erythematosus; XIAP, X-linked inhibitor of apoptosis. *Table based on the reports published by the committee convoked by the World Health Organization, which regularly classifies and updates primary immunodeficiency diseases33. Although incontinentia pigmenti is not considered a primary immunodeficiency per se, it is a classical example of how an X-linked mutation can adversely affect males; although females usually do not manifest immunodeficiencies, the rare male survivors are affected by severe immunodeficiencies and by an increased susceptibility to infections7.

Purifying selection
(Also know as negative selection). The selective elimination of deleterious alleles from the population within the mechanism of natural selection.

Sexual antagonism
When an allele is favoured in one sex and selected against in the other.

Hemizygous
A diploid genotype that has only one copy of a particular gene, as in X chromosome genes in a male, or when the homologous chromosome carries a deletion.

Why are there still X-linked diseases? Deleterious recessive mutations on the X chromosome would be expected to be rapidly eliminated by purifying selection, as males carrying such mutations tend to die before the age of reproduction. But, as exemplified above, this does not seem to be happening. one explanation for this is that de novo mutations can occur in the parental germ cells, particularly during male meiosis, and be transmitted to the offspring. second, the X chromosome has special modes of inheritance and evolution, and it is under more efficient selection than the autosomes and accumulates genes with a sex-biased expression pattern (see below)46. In support of such efficient selection, the X chromosome content in mammals is highly conserved; this indicates that there is a barrier to the exchange of genes between the X chromosome and the autosomes 47. This happens for two reasons: first, tight regulation by means of X-linked inactivation has to ensure equal expression of X-linked genes in females and males and, second, a coordinated upregulation of X chromosome-specific

genes of nearly twofold in both sexes is necessary to balance autosomal gene expression, in which there are two copies48. This explains why so many important genes, such as those involved in immunity, have not been swapped to other chromosomes. The sex-biased content of the X chromosome can be explained by two antagonizing evolutionary forces: sexual antagonism, which results in the feminization of the X chromosome (and masculinization of the Y), and hemizygous exposure, which results in some masculinization of the X chromosome49. If mutations are neutral or advantageous to females, they are likely to persist in the male lineage. X chromosome genes spend twice as much of their evolutionary life in females as they do in males. Therefore alleles that are beneficial to females have a higher net selective advantage and are likely to become fixed in the population if they are not too disadvantageous for males18. so, sexual antagonism predicts that the X chromosome will accumulate genes that are expressed specifically in females if there is a strong sex-biased fitness effect46.
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By contrast, the hemizygous exposure theory predicts that the frequency of a new recessive mutation arising on the X chromosome will increase in the population if it is beneficial to males even if deleterious for females. In males, the advantageous effects are immediately evident, whereas the detrimental effects in females are masked by recessivity 46. However, if the sex-biased fitness effects are strong, expression of male-beneficial genes may become restricted to male-specific tissues50. The melanoma antigen (MAge) family of genes, which are members of the cancertestis antigen group, are, under normal conditions, exclusively expressed in the testes51, which may indicate a role for these genes in spermatogenesis. Indeed, some studies show that the X chromosome is abundant in genes expressed in spermatogonia50. Together, these data show that the X chromosome has a biased gene content that, depending on the circumstances, can benefit either males or females18. These particular evolutionary features of the X chromosome might have resulted in the retention of genes involved in immunity on this chromosome, which immediately exposes males to the lethal effects of deleterious mutations, and ultimately contributes to their genetic disadvantage when facing immunological challenges. to pathogens such as Listeria monocytogenes56,57. several other human and mouse XY homologous genes vary in expression levels and tissue distribution between sexes, and this might have contributed to their retention on the Y chromosome54, leading to a different number of functional copies in females and males. We speculate that females, being XX, would have elevated amounts of DDX3X in all tissues when compared with males (as this gene escapes inactivation), possibly resulting in an enhanced IFn response following bacterial infections in the cells in which only DDX3X protein is expressed. In addition, particularly in humans, several X chromosome genes that lack a Y counterpart also escape inactivation. one study carried out in fibroblast cell lines indicated that up to 15% of genes from the inactive X chromosome can escape permanent silencing 52. This means that these genes are expressed by both X chromosomes in females, although the expression level is frequently lower from the inactive X chromosome than from the active X chromosome. However, a second study carried out in lymphoblastoid cell lines from four human populations showed elevated female expression in only 5% of X-linked genes53. This suggests that there is possibly variation in the number of genes escaping inactivation, in a cell-type dependent manner, or the existence of inter-individual variability. Indeed, 10% of X-linked genes have variable patterns of inactivation52. This indicates heterogeneity of gene expression among females, meaning that in some females these genes will be inactivated in one of the copies and in other females the same genes will be expressed by the two copies. nevertheless, variability in the number of genes escaping inactivation may be an indication that tight control of their expression is not needed for the survival of the cell. Moreover, the hypothesis that mechanisms of posttranslational control may restore imbalanced expression to avoid gene dosage effects cannot be excluded. An example of polymorphism due to escape from X chromosome inactivation is tissue inhibitor of metalloproteinase 1 (TIMP1) (see supplementary information s1 (table)). Two studies have shown TIMP1 expression from the inactive X chromosome in four out of nine human female cell lines58,59. The mechanism behind polymorphic TIMP1 expression from the inactive X chromosome is not known, but the medical implications of this may be important as TIMP1 is an inhibitor of matrix metalloproteinases, a family of proteins involved in responses to bacterial infections and septic shock60. Indeed, TIMP1 expression levels are elevated in patients with sepsis61, and genome-wide association studies indicate that polymorphisms in the TIMP1 gene are associated with rheumatoid arthritis62. In addition to TIMP1, variable patterns of expression from the inactive X chromosome were found for other genes involved in immunity such as Rho gTPase activating protein 6 (ARHGAP6), dual specificity phosphatase 21 (DUSP21), WiskottAldrich syndrome (WAS) gene, gATA-binding protein 1 (GATA1), EDA2R, Brutons tyrosine kinase (BTK), nF-B activating protein (NKAP), ARHGAP4, Il-1R-associated kinase (IRAK1), IKBKG, and gRB2-associated binding protein (GAB3)52 (see supplementary information s1 (table)).
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X chromosome inactivation and immunity X chromosome inactivation has evolved as a mechanism to equal gene expression dosage in males and females. However, there are exceptions, and some genes on the X chromosome are known to escape silencing 52. In instances in which such genes are involved in immunity, this might result in elevated levels of functional immune proteins in females, which may be directly correlated with gender differences in immune responses. Moreover, inactivation of the X chromosome may be nonrandom, and different females may have different levels of mosaicism according to the extent that one of the parental X chromosomes is preferentially inactivated. Together, these exceptions may result not only in differences in X chromosome gene expression levels between sexes, but also in heterogeneous patterns of expression of immune genes between females.
Escape from X chromosome inactivation. Mosaicism of female cells is not only an advantage in case of deleterious X-linked mutations, it also confers added diversity to biological and immune responses. This diversity may be increased when X-linked genes escape inactivation. In reality, most XY homologous genes do not require X chromosome inactivation (BOX 1). These genes, in particular from the pseudoautosomal regions, behave as autosomal genes and are equally expressed in both sexes53; however, there is mounting evidence that some genes, common to both the X and Y chromosomes, do not have equivalent expression levels and tissue distribution54. For example, the human DDX3X (DeAD-box helicase 3, X-chromosomal) and DDX3Y homologues are widely transcribed, but only DDX3X is widely translated, whereas DDX3Y expression is exclusive to the male germline55. DDX3X has recently been shown to be important for interferon (IFn) production in response

Fitness effect
The expected contribution of an individual to the next generation.

XY homologous genes
Genes common to both sex chromosomes and therefore present in two copies in both females and males.

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Direct consequences of imbalanced X chromosomegene expression are not known, but we speculate that escape from silencing by incomplete X chromosome inactivation or reactivation of portions of the inactivated X chromosome may affect immune signalling pathways in a cascade-like effect and be responsible for different immunological responses between individual females and between the two sexes. Skewing of X chromosome inactivation. In addition to escape from X chromosome inactivation, skewing of X chromosome inactivation can also occur in females. The somatic mosaicism expected from random inactivation does not occur in females with this skewing. This means that these females have only one of the parental X chromosomes expressed in almost all cells. skewing can occur by chance alone or when X-linked mutations negatively affect cell growth or impede a proper response to immune challenges63. Differences in proliferation potential between cell types can lead to competition between them and, eventually, cells carrying a normal X chromosome outgrow the mutant cells, with the rapidity of elimination depending on the growth disadvantage63,64. skewing of X chromosome inactivation is often tissue specific and usually only occurs in the tissues expressing the mutant gene. Furthermore, even if expressed in different cell types, skewing may be only observed in some cell classes, depending on the role of the gene in the survival, proliferation and/or differentiation of the various cell lineages. Thus, immune cells with a functional advantage may become overrepresented in the bone marrow, blood or other organs involved in immune responses. There are several examples in the literature of diseases that are associated with such skewing 65. In female patients with incontinentia pigmenti, peripheral blood mononuclear cells and hepatocytes expressing a mutant IKBKG allele are usually rapidly eliminated, and cells expressing the normal IKBKG allele outgrow the mutant cells66. Although female carriers of incontinentia pigmenti typically have extreme skewing of X chromosome inactivation, which ameliorates the deleterious phenotype associated with IKBKg loss of function, some of them display several symptoms of the disease, the severity of which depends on the proportion of mutant cells in the body and on the tissues affected. For example, skewing that favours the expression of the normal allele seems to be less effective in the skin than in other organs; therefore, affected females can have a variety of skin-associated pathologies42. similarly, in female carriers of WiskottAldrich syndrome (WAs), there is selection against mutated cells during the development of haematopoietic precursor cells, resulting in preferential expression of the normal allele in T cells, platelets, monocytes, granulocytes and erythrocytes, although skewing is more pronounced in T cells than in myeloid cells67. As a result, females are usually unaffected by this disease15,67. These examples show that when female cells carry a mutation in an X-linked gene that can be detrimental for proper cellular function, the mechanism of X inactivation leads to preferential expression of the X chromosome carrying the normal allele, allowing females to respond properly to immune challenges and to avoid disease development. nevertheless, extreme skewing of X chromosome inactivation that favours expression of the mutant allele can also occur. normal cells can be lost for stochastic reasons or by selection for the mutant cells, leading to increased manifestation of symptoms in females. In such cases, females may become hemizygous for the mutant allele and manifest male-specific diseases; these females are therefore called manifesting heterozygotes63,64. For example, in female carriers of properdin (also known as factor P) deficiency, extremely low or high properdin levels correspond to preferential inactivation of the normal or the mutated X chromosome, respectively 68. In conclusion, heterogeneity in female mosaicism and skewing of X chromosome inactivation leads to different degrees of disease manifestation: male-specific symptoms in female carriers of certain X-linked diseases, if present, can either be mild or resemble those in males. Together, the phenomena of X chromosome silencing escape and X chromosome inactivation skewing tell us that balanced gene expression is not always maintained between the sexes.

Skewing of X chromosome inactivation

When the process of X chromosome inactivation selects for or against alleles on the active X chromosome. This nonrandom pattern of inactivation results in overrepresentation of one of the parental X chromosomes in female tissues. Mutations in the X-inactive specific transcript (XIST) gene or chromosomal rearrangements resulting from translocations from autosomes can also lead to inactivation skewing.

WiskottAldrich syndrome
(WAS). A life-threatening X-linked immunodeficiency caused by mutation in the WAS gene. It is characterized by thrombocytopenia with small platelets, eczema, recurrent infections caused by immunodeficiency, and an increased incidence of autoimmune manifestations and malignancies.

Fetal microchimerism
The presence of fetal cells in the mother after pregnancy.

Haploinsufficiency
Occurs when only one copy of a certain gene is present in the genome, and both copies are required for proper function.

The disadvantage of being female Approximately 5% of the population in Western countries is affected by autoimmune diseases, and over 80% of the patients are women69,70. of the more than 70 known autoimmune disorders, the most prominent gender differences in prevalence of disease are observed in autoimmune thyroid diseases (such as Hashimotos thyroiditis and graves disease), sjgrens syndrome, Addisons disease, systemic sclerosis, systemic lupus erythematosus (sle) and primary biliary cirrhosis (FIG. 2). In each of these disorders, more than 80% of the patients are women. For rheumatoid arthritis, multiple sclerosis, and myasthenia gravis, this is ~6075%. Although a few hypotheses have been proposed to explain the preponderance of females for autoimmune diseases, there is still no convincing evidence supporting a single hypothesis. Autoimmunity is the result of both environmental and genetic components and, to date, only a few gene defects have been assigned to a particular autoimmune disease. Breakdown of self tolerance can be caused by hormones, immunological challenge during pregnancy, fetal microchimerism, skewing of X chromosome inactivation or X chromosomeassociated abnormalities, such as gene duplication or microdeletions19. some hypotheses have been suggested to explain why X chromosome inactivation skewing or other X chromosome-associated anomalies can contribute to disturbances in self recognition and ultimately to autoimmunity. There are three main hypotheses (the loss of mosaicism hypothesis, the reactivation hypothesis and the haploinsufficiency hypothesis) that have been proposed based on findings in female patients with specific autoimmune disorders (FIG. 3). However, the same disease can be caused by each of the proposed mechanisms in different patients, and
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Hashimotos disease Sjgrens syndrome Addisons disease Systemic sclerosis Systemic lupus erythematosus Primary biliary cirrhosis Autoimmune chronic hepatitis Graves disease Antiphospholipid syndrome Idiopathic thrombocytopenic purpura Rheumatoid arthritis Myasthenia gravis Giant cell arteritis Pernicious anaemia Myositis Multiple sclerosis Vitiligo 0 Male Female 10 20 30 40 50 60 % Incidence 70 80 90 100

Figure 2 | Sex distribution of the most important autoimmune diseases. The depicted diseases are the ones which usually show female predominance21,102.
Nature Reviews | Immunology

some autoimmune disorders do not show characteristics of any of these mechanisms. This suggests that these mechanisms are not universal and that the same disease can have different aetiologies. The loss of mosaicism hypothesis. The loss of mosaicism hypothesis states that disturbances in random X chromosome inactivation result in autoimmunity 20,71,72. This mechanism, first proposed by Kast 71, and then further developed by stewart 72 and ozcelik20, affirms that deviations from the expected random cellular mosaicism are responsible for breakdown of tolerance mechanisms (FIG. 3a). If females are random mosaics, they possess two populations of dendritic cells (DCs) that express either maternal or paternal X-linked self antigens for thymic negative selection, and when potentially autoreactive thymocytes pass through the thymus, they are negatively selected by both DC populations. But, if a female has a high degree of X chromosome inactivation skewing with, for example, preferential inactivation of the paternal X chromosome, T cells will be only tolerized by DCs expressing maternal X-linked self antigens. so, autoreactive T cells specific for paternal X chromosome self antigens will escape negative selection and enter the periphery 72. This hypothesis has been corroborated by two types of evidence. First, nonspecific, polyclonal T cell activation has been described in several female patients with sle73. self antigens encoded by the X chromosome may prime the non-tolerized X-reactive T cells, and then these T cells will activate B cells presenting the same endogenous X-chromosome self antigen72,73. sle disease activity is associated with polyclonal B cell activation and,
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according to this model, the polyreactive T cells are each specifically reactive to a particular B cell-derived X-chromosome-associated self antigen72. secondly, mosaic XXY men (that is, men who have Klinefelters syndrome) have a 14-fold higher risk of developing sle than XY men74. one of the X chromosomes of XXY men is subject to inactivation, and evidence shows that X chromosome skewing is a common characteristic of Klinefelters syndrome, that could contribute to the abnormalities associated with the disorder and be correlated with sle prevalence in these patients72,75. studies carried out in patients with other autoimmune disorders showed a similar pattern. For example, autoimmune thyroid diseases and systemic sclerosis are also associated with a high degree of X chromosome inactivation skewing 7679. However, patients with sjgrens syndrome and primary biliary cirrhosis do not have X chromosome inactivation skewing in blood cells, indicating that this is not a common characteristic of all autoimmune diseases20,80. Moreover, even if skewed inactivation pattern is a common feature of women affected by autoimmune diseases, it does not necessarily lead to autoimmunity in all women, as X chromosome inactivation patterns comparable to those in peripheral blood cells from female patients with sle, multiple sclerosis or rheumatoid arthritis were observed in age-matched healthy controls81. The reactivation hypothesis. This hypothesis states that reactivation of genes from the inactive X chromosome may contribute to autoimmunity 82 (FIG. 3b). The mechanism behind inactive X chromosome reactivation is not known, but this could lead to overexpression of
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a Loss of mosaicism hypothesis
Thymocyte specific for paternal X-linked antigens Thymocyte specific for maternal X-linked antigens 1:1 inactivation ratio

TReg cells specific for X-linked antigens from both parents

Naive T cells specific for parental X-linked antigens are deleted

Random Female embryo Xm Xp X chromosome inactivation

APC presenting paternal X-linked antigens

APC presenting maternal X-linked antigens TReg cells specific for only maternal X-linked antigens Naive T cells specific for maternal X-linked antigens are deleted

APCs in the thymus

Skewed Thymocyte specific for maternal X-linked antigens Thymocyte specific for paternal X-linked antigens Autoimmunity Preferential inactivation of Xp Naive T cells specific for paternal X-linked antigens are not deleted

b Reactivation hypothesis
Female embryo Xm Xp X chromosome random inactivation

One of the parental X chromosomes is randomly inactivated in each cell Reactivation of genes from inactivated X chromosome by unknown mechanism

Overexpression of X-linked genes genes in T cells (such as CD40L) promotes hyper-responsiveness

Autoimmunity T cell with reactivated X chromosome

T cell with inactive maternal X chromosome

T cell with inactive paternal X chromosome

c Haploinsufficiency hypothesis
Female embryo Xm Xp X chromosome random inactivation

One of the parental X chromosomes is randomly inactivated in each cell Loss of one X chromosome by unknown mechanism

Haploinsufficiency for X-linked genes in pseudoautosomal regions

Autoimmunity

Pseudoautosomal regions

T cell (or B cell) with inactive maternal X chromosome

T cell (or B cell) with inactive paternal X chromosome

T cell (or B cell) T cell (or B cell) with no maternal with no paternal X chromosome X chromosome X0 cells

Figure 3 | Current hypotheses attesting the involvement of X chromosome anomalies in autoimmunity. a | The loss of mosaicism hypothesis states that extreme skewing of X chromosome inactivation causes breakdown of self tolerance in the thymus. b | The reactivation hypothesis claims that reactivation of genes, such as CD40 ligand (CD40L), on the inactivated X chromosome in T cells leads to overexpression (cells depicted in purple and bigger size) of X-linked genes and to Nature Reviews | Immunology autoimmunity. c | The haploinsufficiency hypothesis states that loss of one X chromosome in B cells or T cells (X0 cells) leads to haploinsufficiency of X-genes from the pseudoautosomal regions, resulting in autoimmunity. APC, antigen-presenting cell; TReg, regulatory T cell; Xm, maternal X chromosome; Xp, paternal X chromosome.

X-linked genes involved in immune functions, which in turn would be responsible for overproduction of autoantibodies82. Alternatively, overexpression of X-linked genes could disrupt the equilibrium in the mechanism of fine-tuning protein expression and generate protein aggregates that would trigger responses against self antigens83.
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Reactivation of CD40L on the inactive X chromosome has been found in T cells of female patients with sle82,84. It was proposed that regulatory sequences on the inactive X chromosome would be demethylated in T cells from these patients, resulting in CD40L overexpression82. Indeed, CD4+ T cells from sle female patients express twofold higher CD40L than males, and
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Box 2 | MicroRNAs located on the X chromosome and immunity
MicroRNAs (miRNAs) are a family of small (1923 nucleotides) double-stranded non-coding RNAs that negatively regulate gene expression by translational repression and/or messenger RNA degradation. Bioinformatics studies predict that mammalian miRNAs may regulate up to 30% of all protein-coding genes and participate in the regulation of almost all cellular processes96. The same miRNA can target several genes and be expressed in an organ- or cell-specific manner. In general, miRNAs are strongly conserved among species, an indication that they are subject to strong selective pressures, possibly owing to their important role in development and disease. A recently published study claims that no miRNAs can be found on the Y chromosome of any of the species studied to date and, intriguingly, the X chromosome seems to have more miRNAs than autosomes in several mammals, including humans, mice and rats97. Moreover, this higher miRNA density on the X chromosome seems to be mammal-specific, which means that X-linked microRNAs (X-miRNAs) may have X-related functions in mammals97. X-miRNAs may be of great importance in both cancer onset and progression (for example, miR-221 and miR-222)98, as well as in immune regulation (for example, miR-223, miR-106a and miR-424)99101. Most X-miRNAs have no described functions, and we think that X-miRNAs escaping inactivation or being subject to silencing skewing affect female immunity.

these CD40L overexpressing CD4+ T cells were shown to stimulate increased Igg production by autologous B cells. This enhanced Igg production was found to be significantly correlated with sle disease84. gene dosage effects may be the consequence of anomalies other than reactivation of methylated X-linked genes. For example, duplication of Tlr7 has also been associated with an sle-like disease in mice85. Furthermore, a recent study showed that XX female mice are more susceptible to pristane-induced sle and experimental autoimmune encephalomyelitis (a model of multiple sclerosis) than XYSry female mice86. The two groups consist of female mice with the same gonadal and hormonal background, which suggests that double dosage of the X chromosome is the sole factor conferring susceptibility to the two autoimmune disease models. The haploinsufficiency hypothesis. A third hypothesis states that haploinsufficiency for X-linked genes results in some autoimmune disorders80,87 (FIG. 3c). one of the first pieces of evidence corroborating this hypothesis came from the observation that patients with X chromosome monosomy (loss of one X chromosome; X0), such as those with Turners syndrome, have several autoimmune manifestations88. This syndrome is characterized by the absence of all or part of one of the X chromosomes, and women with Turners syndrome have, for example, an increased risk of developing autoimmune thyroid and inflammatory bowel diseases89,90. Haploinsufficiency for genes located in the pseudoautosomal region 1 of sex chromosomes has been proposed to contribute to the defects observed in Turners syndrome91. These genes, present in two copies in both XX females and XY males, are only present in one copy in X0 females, and this is apparently insufficient for proper function. Pseudoautosomal region 1 contains around 26 genes and, although some have been associated with the mental retardation observed in Turners syndrome, only a few have been shown to be involved in immune functions (see supplementary information s1 (table)). However, women with isochromosome-Xq

are also more prone to developing autoimmune thyroid and inflammatory bowel diseases, again suggesting that an imbalanced expression of genes from the short arm of the X chromosome may be a predisposing factor for developing these disorders89. More recently, autoimmune disorders, such as primary biliary cirrhosis, systemic sclerosis and autoimmune thyroid disease, were found to be characterized by a higher X chromosome monosomy rate in peripheral B and T cells, suggesting again that haploinsufficiency for X-linked genes may be a crucial factor 80,87,92. The mechanism behind loss of one X chromosome from these cells is not known but seems to increase with age. It has been proposed that a gradually acquired haploinsufficiency for specific X chromosome-linked genes (from pseudoautosomal regions, or genes that permanently escape inactivation in normal females) in peripheral B and T cells could be responsible for the generation of autoantibodies87. Despite these theories, few genes contained in the X chromosome have been specifically associated with one or more autoimmune disorders. However, it is unlikely that mutations in a single gene would be responsible for the higher susceptibility of females to autoimmunity. If this was the case, we would expect a higher frequency of autoimmune diseases in males because males have only one X chromosome (similar to what is observed for X-linked PIDs). Although the role of sex hormones cannot be excluded, there is sufficient evidence indicating that X chromosome-associated anomalies, such as inactivation skewing, overexpression or haploinsufficiency for portions of the X chromosome, may be relevant for the aetiology of some, but not all, autoimmune diseases. nonetheless, even if these abnormalities may account for the higher prevalence of autoimmune diseases in females, the incidence of autoimmunity in males, even if small, indicates that other mechanisms must be involved.

Isochromosome-Xq
A genetic defect that results from the total deletion of the short arm of the X chromosome owing to the fusion of two long arms.

Conclusions The sex chromosomes have intrigued researchers for a long time. Their special mode of inheritance and atypical patterns of evolution have shaped their present structure and are at the root of differences between genders. X chromosome inactivation, a high percentage of genes escaping X chromosome inactivation, female mosaicism and heterogeneity in X chromosome inactivation patterns are likely to contribute to differences in immune responses between women and men at both the cellular and molecular levels. A better understanding of these phenomena would enhance the accuracy of diagnosis and improve the success of treatment65. Familial cases where males are affected by a certain recessive disease, but the same or a few symptoms are also present in the mother or sisters, may not rule out the presence of an X-linked disorder, as these women may have skewed X chromosome inactivation that favours the expression of the mutant allele. The same is true in identical twins, as discordant phenotypes for an X-linked disease may be the result of different X chromosome inactivation patterns.
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Recent evidence shows that X chromosome-specific abnormalities, such as extreme inactivation skewing, inactive X chromosome reactivation, translocations and microdeletions may contribute to the breakdown of self tolerance and to autoimmunity. However, complete mechanistic explanations are still lacking, and the aetiology of several autoimmune diseases has not been studied. These questions open the door to a field that is still to be explored. The effects of such X chromosome-specific properties in regulating X-linked microRnA (miRnA) expression and function are also not known. The X chromosome encodes several
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miRnAs involved in immunity (BOX 2), and we predict that in the coming years much will be discovered about their modes of action and possible targets. The contribution of X-linked miRnAs to gender-specific immune responses is not known, and this demands further research. It is clear that the X chromosome can have a great influence in immunity and that these effects can be detrimental or beneficial to either sex depending on the immune context. Therefore we think that this chromosome has been essential in shaping immunity in humans.
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75. 76.

92. 93. 94. 95.

77. 78.

79. 80. 81.

96.

97. 98.

82.

99. 100.

83. 84.

101.

85. 86.

102.

Acknowledgements

The authors acknowledge A. Bredan for critical review of the manuscript. Research in the authors laboratory is sponsored by the Fund for Scientific Research-Flanders, the Interuniversity Attraction Poles Program of the Belgian Science Policy (IAP VI/18), the Belgische Vereniging tegen Kanker and the Flanders Institute for Biotechnology (VIB).

87. 88. 89.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION
C. Liberts homepage: http://www.vib.be/Research/EN/ Research+Departments/Department+for+Molecular+Biom edical+research/Claude+Libert/

90. 91.

SUPPLEMENTARY INFORMATION
See online article: S1 (table)
All lInkS Are ACTIve In The onlIne pDF

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