The X-linked histone demethylases KDM5C and KDM6A as regulators of T cell-driven autoimmunity in the central nervous system

T cell-driven autoimmune responses are subject to striking sex-dependent effects. While the contributions of sex hormones are well-understood, those of sex chromosomes are meeting with increased appreciation. Here

Sex differences are crucial in infection and immunity.While women are thought to have more robust immune responses, and thus greater susceptibility to autoimmune diseases, men are often prone to worse outcomes and higher severity in different immune mediated diseases such as multiple sclerosis (MS) and COVID-19 (Dehingia and Raj, 2021;Peckham et al., 2020).The impact of hormones in these sex differences is well established in the literature; however, the role of sex chromosomes is yet to be elucidated.Of note, recent advances have shown the role of sex chromosome located histone lysine demethylases (KDMs) in sex-biased responses to immune diseases.In this review, we discuss the role of KDM proteinsmost notably X chromosome-encoded KDM5C and KDM6A -in immune cell function, with a particular focus on T cell responses.

MS
MS is a chronic inflammatory disease of the central nervous system (CNS) in which peripheral autoreactive T and B cells cross the bloodbrain barrier (BBB) to launch an inflammatory attack that targets CNS myelin.This immune response causes chronic inflammation resulting in demyelination.MS is characterized by episodes of neurologic dysfunction characterized by symptoms that include optic neuritis, ocular motility disorders, fatigue, depression, and eventually motor dysfunctions, amongst others (McFarland and Martin, 2007).While its etiology remains unclear, multiple lines of evidence suggest that MS occurs as a result of a combination of genetic and environmental factors such as genetic polymorphisms in the HLA gene locus (Correale et al., 2017) and exposure to infectious agents such as Epstein-Barr virus (Bjornevik et al., 2022;Lanz et al., 2022).Around 80% of patients will initially present relapsing/remitting disease (RRMS), and 40-60% of these will transition to a secondary progressive form that is currently refractory to treatment (SPMS).
While traditionally depicted as a T cell mediated disease, it is now widely accepted that B cells play a central role in MS pathophysiology; indeed targeting the B cell surface molecule CD20 are now front line therapies.However, CD20 is not expressed on antibody-secreting plasmablasts, which has led to the view that the role of B cells in MS might lie in facilitating and reinforcing T cell activity.Indeed, poor outcomes in SPMS have been recently linked to the presence of tertiary lymphoid organs (TLOs) in the CNS meninges.These structures, composed principally of B cells, T follicular helper (Tfh) cells and dendritic cells (DCs), are sites at which CNS antigen-specific effector T cells can be restimulated.They can also be the source of potentially neurotoxic inflammatory mediators (Pikor et al., 2015).

Experimental autoimmune encephalomyelitis as an animal model of MS
Experimental autoimmune encephalomyelitis (EAE) is the most used animal model of MS and CNS inflammation (Rangachari and Kuchroo, 2013).EAE recapitulates many of the inflammatory processes seen in MS pathophysiology, such as peripheral immune cell invasion, demyelination, axonal loss, and gliosis (Yeola et al., 2019).The relevant immunogens are derived from CNS self-proteins like myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG).The classic form of EAE is marked by an ascending paralysis that starts at the tail and eventually affects the hind and forelimbs.The severity of the model is most frequently assessed on a semi-quantitative 5-point scale (Stromnes andGoverman, 2006a, 2006b).Immunization of SJL/J background mice with the immunodominant epitope of PLP (PLP 139-151 ), triggers a relapsing-remitting disease course, while C57BL6/J mice immunized with the immunodominant MOG 35-55 peptide tend to develop a chronic form of the disease.By contrast, when non-obese diabetic (NOD) mice are actively immunized with MOG 35-55 , the resulting disease can often replicate the relapsing-remitting phase followed by a progressive form seen in secondary progressive multiple sclerosis (Ignatius Arokia Doss et al., 2015).
EAE can be induced in mice either by active immunization with encephalitogenic protein or peptide, or by passive transfer of encephalitogenic T cells.Active immunization protocols elicit a T cell-driven response and can also be used to examine the role of antigenpresenting cells (APCs) or B cells in the disease process.Another commonly used approach involves transferring activated autologous encephalitogenic T cell clones or activated transgenic T cells that have antigen specificity to encephalitogenic epitopes.This latter approach is particularly useful in isolating the contributions of effector T cells to the immune pathogenesis of EAE, as it bypasses the initial steps in T cell activation (Rangachari and Kuchroo, 2013).

Contributions of sex hormones and chromosomes: an overview
As is observed in other autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Crohn's disease, the incidence of relapse-onset MS is skewed towards females: in the case of MS, at a ratio of 3:1.However, males tend to experience more aggressive progression of the disease and worsened outcomes: they display a shorter median time between disease onset and reaching a given disability level relative to females, as well as a shorter mean time to convert from relapsing-remitting to secondary progressive MS (Confavreux et al., 2003;Koch et al., 2010).Moreover, men exhibit greater gray matter atrophy than women with MS (Antulov et al., 2009).These observations indicate that exploring sex-related variables could provide valuable insights into understanding the underlying pathological mechanisms involved in MS.
Biological sex differences arise from the separate and collaborative impacts of sex hormones and chromosomes.The role of sex hormones in MS has been an area of substantial interest.Natural history studies reveal that both the overall incidence of MS, as well as the sex bias favoring females, increases after puberty (Ucciferri and Dunn, 2022).These findings indicate that physiological levels of estrogens may favor MS incidence.Notably, though, the incidence of MS relapse is strikingly reduced during pregnancy (Confavreux et al., 1998), pointing to an ameliorative role for heightened circulating concentrations of estrogens and/or progesterone.Given their proven safety profile in vivo, it is unsurprising that several trials of exogenous sex hormones in people with MS have been attempted.Exogenous estrogens can reduce MS relapse rate (De Giglio et al., 2017) and cognitive impairment (Voskuhl et al., 2016), while testosterone can reduce gray matter atrophy (Kurth et al., 2014).Overall, the evidence suggests that sex hormones play complex roles in MS incidence and severity that are linked in part to their dosage.Males and females differ by virtue of the former having XY chromosomes and the latter having XX.Using EAE models, there is accumulating evidence that sex chromosome-encoded factors can impact CNS autoimmunity (Doss et al., 2021;Golden et al., 2019;Itoh et al., 2019;Smith-Bouvier et al., 2008).While the Y chromosome is relatively gene-poor, the Teuscher group showed that increased copy number of the Y-linked multicopy genes Rbmy and Sly can correlate with worsened EAE (Case et al., 2013).Notably, while female mammals randomly inactivate one of their two X chromosomes in order to normalize X gene dosage with males, silencing of genes on the second X chromosome is incomplete with up to 20% of X-genes escaping this process (Berletch et al., 2015).In the following sections, we will present animal models that allow the disaggregation of hormonal and chromosomal sex.We will also discuss the potential roles of the X-linked histone demethylases KDM5C and KDM6A in regulating T cell function in the inflamed CNS.

The FCG model to study the contributions of chromosomal vs hormonal sex
Early studies using gonadectomized rodents demonstrated that sex hormones are not exclusively responsible for sex differences in EAE.This was particularly the case for male animals, as gonadectomy of male Lewis rats did not ameliorate their severe chronic EAE (Trooster et al., 1996), while a large study of intercrossed B10.S:SJL/J mice revealed no histopathological differences upon male gonadectomy (Fillmore et al., 2004).These findings led researchers to address the distinct contributions of sex hormones and chromosomes to disease processes.
The double-transgenic four-core genotypes (FCG) mouse model is a powerful tool that enables such analyses (reviewed extensively in (Arnold and Chen, 2009)).It exploits the fact that the Y-linked gene Sry is the sole driver of sex determination; XX individuals develop as females due to a lack of Sry.FCG mice thus consist of a Sry knockout (Y SryKO ) crossed to a knock-in of Sry on chromosome 3 (Tg-Sry).They are thus chromosomally male (XY) as well as hormonally male, as the Sry knockin allele complements the missing allele on Y.When Tg-Sry:XY S- ryKO FCG males are bred to WT females, "four core" sex genotypes are revealed in their progeny: two that are concordant with respect to hormones and chromosomes (hormonally and chromosomally male Tg-Sry:XY SryKO ; hormonally and chromosomally female XX) and two that are discordant (hormonally male yet chromosomally female Tg-Sry: XX; hormonally female yet chromosomally male XY SryKO ).By examining these phenotypes, one can better understand how sex hormones and chromosomes interact to shape biological processes.

SJL/J background: interplay of female and male chromosomal elements in a tissue compartment-specific manner
Female SJL/J-background mice are prone to EAE of greater severity than males (Bebo et al., 1996;Papenfuss et al., 2004); this phenotype is dependent on the increased encephalitogenicity of female T cells (Bebo M.R. Fazazi et al. et al., 1998b).While gonadectomy studies in male SJL/J revealed a protective role for androgens (Bebo et al., 1998a;Palaszynski et al., 2004), Voskuhl and colleagues used the FCG model on this background to discover unexpected roles for chromosomal factors in EAE outcomes.Upon active immunization, XX mice exhibited a more severe form of the disease when directly compared to XY SryKO counterparts.Likewise, Tg-Sry:XX mice showed more severe EAE symptoms than Tg-Sry:XY SryKO mice.Thus, worse outcomes were linked to female sex chromosomes, regardless of the hormonal context (Smith-Bouvier et al., 2008).Further experiments involving adoptive transfer of primed XX and XY SryKO T cells demonstrated that the effects were localized in the T cell compartment, with XX T cells leading to a more severe form of the disease than XY SryKO T cells (Smith-Bouvier et al., 2008).Hypothesizing that increased expression of X-linked genes might explain the increased pathogenicity of XX T cells, they then uncovered the X-linked H3K27 demethylase KDM6A as being expressed at higher levels in XX vs XY SryKO SJL/J T cells (Itoh et al., 2019).KDM6A escapes second-allele silencing (Greenfield et al., 1998), and indeed the group found that it was also increased in XX vs XY SryKO B6 T cells as well as in T cells from healthy women vs healthy men (Itoh et al., 2019).Functionally, conditional deletion of KDM6A in T cells reduced the severity of EAE, showing that expression of this X-linked gene is linked to exacerbated CNS autoimmunity (Itoh et al., 2019).
Curiously, in parallel investigations, Voskuhl and colleagues uncovered an opposing pathogenic role for XY chromosomes within the CNS itself.They generated bone marrow (BM) chimeras on the FCG strain in which hormonally and chromosomally female (XX), or hormonally female yet chromosomally male (XY SryKO ) immune cells were implanted in donor mice of either genotype (Du et al., 2014).They found that XY SryKO host mice developed EAE of greater severity compared to XX, regardless of whether XX or XY SryKO BM was implanted.As both XY SryKO and XX mice are hormonally female, it suggests that male sex chromosomes in non-immune tissues were the cause of increased disease susceptibility.Indeed, XY SryKO hosts showed greater CNS pathology and synaptic loss than XX hosts, suggesting that XY chromosomes render the male CNS uniquely sensitive to insult.Worsened disease in XY SryKO hosts was linked to increased CNS-specific expression of the immune pattern recognition receptor TLR7.While TLR7 is X-linked, its increased expression in XY rather than XX suggests that it is not an escapee of second-allele silencing; the molecular mechanisms underlying increased expression of TLR7 in XY CNS tissues remain to be elucidated.

NOD background: male sex chromosomes exacerbate Th17-mediated disease
While active immunization of NOD mice reveals no overt sex differences (Papenfuss et al., 2004), we somewhat unexpectedly uncovered a sex bias in an adoptive transfer model on this background (Doss et al., 2021).Using NOD-background 1C6 T cell receptor transgenic (TcR-Tg) CD4 + T cells, which are specific for MOG  , we generated T helper 1 (Th1) and Th17 effector subsets using well-described in vivo culture conditions; these being the CD4 + T cell effector types most closely linked to CNS autoimmune pathology (Rangachari and Kuchroo, 2013).We next adoptively transferred these cells to sex-matched NOD.Scid animals.Under both Th1 and Th17 conditions, the transfer of male 1C6 CD4 + T cells resulted in disease of increased severity relative to female cells.This was particularly the case for male Th17 transfers, with > 50% of recipients developing a severe progressive form of EAE resulting in ethical endpoints being attained (Doss et al., 2021).Interestingly, in the in vivo context, male Th17 cells preferentially adopted expression of the Th1-associated cytokine interferon (IFN)γ, a phenotype associated with Th17 cell pathogenicity (Kebir et al., 2009;Zielinski et al., 2012).
After crossing the (B6 background) FCG strain to 1C6, we generated Th17 cells from the resulting F1 Tg-Sry:XY SryKO :1C6 and Tg-Sry:XX:1C6 mice and adoptively transferred them to NOD.Scid hosts.Tg-Sry:XY Sry- KO :1C6 Th17 cells induced progressive disease of greater severity than Tg-Sry:XX:1C6 counterparts, indicating that male XY chromosomes are required for severe disease in a male hormonal context; further, CNSinfiltrating Tg-Sry:XY SryKO :1C6 Th17 cells were more highly positive for IFNγ (but not IL-17) as compared to Tg-Sry:XX:1C6 Th17 cells.
In investigating potential X-linked genes that can inhibit Th17 responses in female Th17 cells, we identified Jarid1c/KDM5C as one such regulator (Doss et al., 2021).Curiously, KDM5C, like KDM6A, is a histone lysine demethylase.Together, our data were in line with previous findings in myocarditis and arthritis (Andersson et al., 2015;Li et al., 2013) that Th17 cell responses may be exacerbated in males.Further, they suggested that the functionally similar X-linked molecules KDM6A and KDM5C might exert opposing effects on T cell function.In the following sections, we will discuss the roles played by these genes in immune pathology.

Nucleosomes and histone modifications
The basic building block of chromatin is the nucleosome (Fig. 1A), which consists of 146 bp of DNA wrapped around a histone octamer, which itself is comprised of a dimer apiece of each of the core histone proteins H2A, H2B, H3 and H4.(Mariño-Ramírez et al., 2005).Histones have N-terminal tails that are prone to multiple post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, ubiquitination, citrullination and SUMOylation.These modifications affect the chromatin structure by making it more condensed (heterochromatin) or less condensed (euchromatin).The level of compaction of the chromatin has a direct impact on the transcriptional state of the genes.Each PTM alone can affect chromatin architecture, but it is the combined effect of these modifications that is the ultimate determinant of the chromatin state in a specific DNA region, which governs gene transcription.Acetylation (on lysine, K) and methylation (on K and also on arginine, R) are the PTMs best described as impacting the epigenetic status of gene loci via histone modification.Arginine residues can be mono and di-methylated, while lysines can be mono, di or tri-methylated (Bannister and Kouzarides, 2004).As each nucleosome contains a histone octameric core, and as each histone has a tail of > 20 amino acids that can modified post-translationally, one can readily imagine that chromatin modifications can have highly nuanced impacts on gene expression/repression (Fig. 1B).It is traditionally considered that a globally hyper-acetylated/hypo-methylated histone state corresponds to euchromatin and more active gene transcription (Fig. 1C).Nevertheless, methylation can also render chromatin more accessible in a number of contexts (Shi, 2007).

Lysine methylation and demethylation
The impact of methylation of key histone residues on transcriptional regulation is well documented.Tri-methylation of H3K27 at transcription start sites (TSS) is generally associated with the repression of active transcription.By contrast, mono-methylation of the same lysine (H3K27) is associated with an enhanced transcription activity (Shi, 2007;Shi and Whetstine, 2007), as is trimethylation of H3K4 (Lauberth et al., 2013).In the early 2000's, a major milestone was attained in understanding of the mechanisms underpinning DNA methylation with the first description of a histone methyltransferase (Rea et al., 2000).It was followed shortly by the first description of a histone demethylase (LSD1, now known as KDM1A), which in contrast to methyltransferases, acts to remove methyl groups from histones (Shi et al., 2004).Evidence rapidly accumulated that histone methylation was an important modification associated with both activation and repression of transcriptional activity (Martin and Zhang, 2005).Known histone lysine methyltransferases (KMTs) and demethylases (KDMs) are presented in Fig. 1D.

Sex chromosome-encoded KDMs
KDMs demethylate their target residues by oxidizing methyl (CH3) groups.KDM5 and KDM6 family members contain a zinc finger domain (PHD) responsible for histone recognition and binding.They use α-ketoglutaric acid (αKG) as an essential factor, with an active catalytic site in the Jumonji-C domain that incorporates a single oxygen atom from O 2 into their substrates (Hua et al., 2021).Out of the 20 KDMs proteins currently identified, 3 -KDM5C, KDM6A, and KDM5D -are situated on sex chromosomes.KDM5C and KDM6A are located on the X chromosome, whereas KDM5D is located on the Y chromosome.It has been reported by multiple groups that KDM6A and KDM5C escape X-inactivation in immune cells, resulting in heightened expression in females (Greenfield et al., 1998;Xu et al., 2002;Xu et al., 2008;Yang et al., 2010).
KDM5C is a H3K4 demethylase that targets trimethylated and demethylated, but not monomethylated, residues.Demethylation of permissive H3K4 is generally considered to repress gene transcription.Curiously, however, it has been reported that at enhancer regions, KDM5C can have the opposite effect by augmenting gene expression (Outchkourov et al., 2013).Y chromosome-linked KDM5D, also a H3K4 demethylase, regulates transcription of the androgen receptor (AR) transcriptional activity and can also suppress the metastatic capacity of prostate cancer cells by repressing the expression of multiple matrix metalloproteinases (Komura et al., 2016;Li et al., 2016).
KDM6A, on the other hand, targets trimethylated and dimethylated H3K27.As removal of me3/me2 marks on H3K27 generally activates transcription, KDM6A is considered a broad transcriptional activator (Van der Meulen et al., 2014).However, it should be noted that KDM6A has been described as having demethylase-independent functions by which it can also repress transcription (Cheng et al., 2023;Gozdecka et al., 2018;Wang et al., 2017).

KDM5D, 5C and 6A in pathologic states
4.3.1.1.Cancer.KDM dysregulation is an important determinant in different cancers (Greer and Shi, 2012;Schwartzentruber et al., 2012;Varier and Timmers, 2011).Dysregulation of KDM5C, KDM5D and KDM6A are associated with sex-dependent malignancies such as prostate, breast and pancreatic cancer (Chang et al., 2019;Duan et al., 2022;Varghese et al., 2021).For instance, loss of KDM5D expression leads to androgen receptor activation and resistance to treatment in prostate cancer in male patients (Komura et al., 2016).KDM5D deficiency is also used as a negative prognostic indicator in male lung cancer (Willis-Owen et al., 2021).A number of somatic mutations of KDM6A have been linked to sex-biased incidence in different types of cancers (Tricarico et al., 2020).For example, mutations leading to loss of function of KDM6A in T-cell acute lymphoblastic leukemia (T-ALL) are indicators of a poor outcome and are more likely to occur in males ( Van der Meulen et al., 2015).Similarly, KDM5C promotes breast cancer tumorigenesis by activating the target genes of the estrogen receptor alpha (ERα) and also by preventing immune cell surveillance by repressing type 1 interferons (Shen et al., 2021).It was also shown to facilitate castration-resistant prostate cancer cell growth by downregulating PTEN (Hong et al., 2019).However, KDM5C can also act as a tumor suppressor in the case of HPV-associated cancers by regulating the oncogenic genes E6 and E7.KDM5C knock-down was increasing E6/E7 expression levels suggesting a role as tumor suppressor for KDM5c (Smith et al., 2010).

Neurological disorders. KDM dysregulation is pivotal in multi-
ple neurological diseases such as autism, intellectual disability and epilepsies (Pinto et al., 2010) (Greer and Shi, 2012).Here again, sex-related KDMs are associated with sex-specific mental disorders such as X-linked intellectual disability, or XLID (Abidi et al., 2008 et al., 2016;Iwase et al., 2007).Mutations in KDM5C resulting in loss of function are a common cause of XLID and are associated with autism, hyperreflexia, and short stature among other phenotypes.XLID is much more prevalent in males compared to females; as KDM5C can escape second copy X silencing, heterozygous females can transcribe wild-type KDM5C from the non-mutated allele.Mechanistically, it was shown that KDM5C knockdown in neurons results in impaired dendrite development and neuronal survival (Iwase et al., 2007).Similarly, different groups have linked congenital mutations in KDM6A to seizures and epilepsy (Lindgren et al., 2013;Van Laarhoven et al., 2015).

Sex-linked KDMs and immune regulation
Both hematopoietic cell differentiation and immune cell function depend on the tightly coordinated regulation of master regulators and effector molecules in a time-and context-dependent manner.It is thus unsurprising that KDMs are linked to immune regulation (Kang et al., 2017).Here, we discuss what is known about the function of KDM6A and KDM5C in immune cells.

KDM6A
Accumulating evidence indicates that KDM6A has several important roles in T cell development and function.Together with KDM6B, it drives the late stages of CD4 + thymocyte differentiation by derepressing the expression of Klf2, and it promotes thymic egress by then derepressing S1pr1 (Manna et al., 2015).In the immune periphery, KDM6A is crucial for Th17 cell differentiation.Pharmacological inhibition of KDM6A results in increased H3K27me3 marks at the locus encoding Rorgt, the master regulator of Th17 cell differentiation.In mature Th17 cells, KDM6A inhibition causes metabolic reprogramming, reduced proliferation and decreased production of IL-17 (Cribbs et al., 2020).KDM6A was also shown to support T follicular helper (Tfh) cells responses, which are necessary for plasmablast help (Cook et al., 2015).Specific T cell deletion of KDM6A in mice results in their inability to resolve chronic viral infections, which was linked to reduced Tfh cell frequency, germinal center responses and immunoglobulin (Ig) production.
Having shown that female (XX) chromosomes are linked to exacerbated T cell pathogenicity in the SJL/J strain, Voskuhl and colleagues assessed the expression of X-genes that were more highly expressed in female vs male T cells.Kdm6a transcript was found to be substantially upregulated in CD4 + T cells from the SJL/J and C57BL6/J backgrounds, as well as from CD4 + T cells from healthy human females versus males (Itoh et al., 2019).Kdm6a conditional knock-out (cKO) in CD4 + T cells resulted in a profound reduction in EAE severity upon active immunization, accompanied by reduced immune cell infiltration in the CNS as well as reduced axonal damage.Transcriptional profile revealed that genes associated with neuroinflammatory signaling were reduced in Kdm6a cKO T cells; interestingly, those associated with Th1 and Th2 CD4 + T cell responses were increased.Given the evidence suggesting that KDM6A is essential for Th17 cell differentiation, these findings raise the possibility that alternate, Th17-antagonistic cell fates are induced in its absence.

KDM5C
Several reports have shown that KDM5C can regulate innate immune cell activity.Krawczyk and colleagues found that in association with the transcriptional repressor PCGF6, it promotes dendritic cell (DC) quiescence by repressing expression of MHC class II, the costimulatory molecule CD86 and the T cell differentiation cytokine subunit IL-12p40 (Boukhaled et al., 2016).The same group recently observed that KDM5C-deficient bone marrow macrophages display defects in oxidative phosphorylation, resulting in defective osteoclastogenesis and increased trabecular bone mass in females (Liu et al., 2023).In our recent work examining sex differences in NOD-background 1C6 adoptive transfer EAE, we found that female XX chromosomes confer an inhibitory phenotype on Th17 cells.Hypothesizing that, in contrast to the SJL/J strain, X-linked genes expressed at higher levels from XX T cells might repress 1C6 Th17 pathogenicity, we reanalyzed a previous dataset that compared the transcriptome of "non-pathogenic" vs "pathogenic" Th17 cells in EAE (Gaublomme et al., 2015).We identified a number of X-linked genes that were enriched in the non-pathogenic population.Three of these genes -Kdm5c, Pbdc1 and Utp14a -had previously been described as escaping 2nd-allele silencing in mouse spleen cells (Berletch et al., 2015).When we assessed transcript expression of these 3 targets in 1C6 CNS-infiltrating Th17 cells, only Kdm5c was more highly expressed in female cells as compared to male cells (Doss et al., 2021).We found that overexpression of KDM5C in male 1C6 Th17 cells suppressed their capacity to induce severe EAE.Intriguingly, we found that KDM5C expression is sharply reduced in circulating T cells from MS-affected individuals versus healthy controls, and that it was further downregulated in T cells from MS-affected men versus MS-affected women.Together, these data demonstrated that KDM5C repress Th17-mediated CNS autoimmunity in a sex-specific manner.
4.4.3.Investigating the impact of KDM5 inhibition on effector T cell signature cytokines 4.4.3.1.Rationale.Epigenetic inhibitors can potently regulate effector T cell function (Hammitzsch et al., 2015) and histone lysine-modifying enzymes have been of intense interest therapeutically (Diao et al., 2022).However, the bulk of these studies have targeted KMTs (Bhat et al., 2021;Pan et al., 2022;Wang and Patel, 2013), with the consequences of KDM inhibition on inflammation comparatively understudied (McAllister et al., 2016).Nonetheless, inhibition of the H3K27 demethylase KDM6B with the pharmacological reagent GSK-J4 decreased the expression of LPS-inducible genes in macrophages (Kruidenier et al., 2012), suggesting that this class of molecule is targetable.Given our finding that KDM5C can regulate CD4 + T cell pathogenicity (Doss et al., 2021), we wanted to examine the impact of pharmacological inhibition of this enzyme class on cytokine secretion of male and female Th1/Th17 cells.

Pharmacological inhibition of KDM5 molecules reduces IFNγ production from effector T cells.
In recent years, a number of KDM5 inhibitors have been described that target cysteine residues unique to KDM5 family members (Vazquez-Rodriguez et al., 2019) or that interfere with the ability of KDM5 molecules to bind histones (Liang et al., 2017).We used KDM5A-IN-1/KD-2119a, an inhibitor with the latter mode of action (Liang et al., 2017), to assess the capacity of KDM5 family molecules to impact effector CD4 + Th1 and Th17 cells, as these are the effector subsets most relevant in EAE (Rangachari and Kuchroo, 2013).Th1 cells are characterized by their production of IFNγ.Th17 cells produce IL-17 in vitro, but not IFNγ (Bettelli et al., 2006), despite their ability to generate both cytokines in the in vivo context.
Using both male and female NOD mice, we generated Th1 and Th17 cells in vitro in the presence or absence of KDM5A-IN-1.Treatment of male Th1 cells with KDM5A-IN-1 resulted in augmented expression of IFNγ relative to vehicle treated cells (Fig. 2A); by contrast, KDM5 inhibition resulted in no change in IFNγ expression from female Th1 cells.Interestingly, no differences in IL-17 production were observed in the presence of KDM5A-IN-1 from either male or female Th17 cells (Fig. 2B).
Our data suggest that production of IFNγ from male T cells is preferentially repressed by KDM5 family molecules.There are several potential explanations for these findings.Male cells have only one copy of the X-linked KDM5C gene; female cells, by contrast, can express increased levels of KDM5C due to incomplete X-silencing.They therefore could be relatively resistant to its effects, at least under in vitro culture conditions.Alternately, it is possible that Y-linked KDM5D plays a role in repressing IFNγ production specifically in male cells.It should also be noted that pan-KDM5 inhibitors, such as KDM5A-IN-1, can impact the function of autosomally encoded KDM5A and KDM5B that might have as-yet-unexplored sex-specific effects.
Curiously, we find that KDM5 inhibition upregulates IFNγ, but not IL-17, in male effector T cells.This is despite our observation that treating Th17 cells with KDM5A-IN-1 exacerbates their pathogenicity in vivo (Doss et al., 2021).It is possible that KDM molecules directly repress IFNγ; indeed, genetic deletion of KDM5B increases IFNγ production in response to respiratory syncytial virus (RSV) infection (Ptaschinski et al., 2015).Characterizing the ability of KDM5 family members to regulate the expression of IFNγ and other inflammatory cytokines will be an important future step.using publicly available transcriptomics data (GEO accession # GSE221786), we assessed KDM5C and KDM6A expression from IL-17secreting cells taken from the peripheral blood of female and male humans with ankylosing spondylitis (AS), a Th17-mediated autoimmune disease (Jethwa and Bowness, 2016), or healthy donors (HD).

Decreased KDM5C:KDM6A ratio may characterize female Th17 cells in AS.
While neither target, on its own, was differentially expressed between AS versus HC females, or between AS versus HC males (Fig. 3A), we found that the ratio of KDM5C:KDM6A (Fig. 3B) was significantly lower in AS females versus HC (p < 0.0321).By contrast, no difference was observed between male conditions (p < 0.5026).These data provide preliminary support for a model by which the balance of KDM5C and KDM6A can regulate Th17 pathogenicity in autoimmunity in female individuals, though it will be important to validate these findings in larger cohorts and in other autoimmune conditions such as MS.

Conclusions
While biological sex plays a critical role in T cell-driven autoimmune diseases, attempts to target this variable have been limited principally to the manipulation of sex hormones.Here, we have outlined the evidence supporting a parallel role for sex chromosome-encoded molecules in autoimmunity.In particular, the X-linked histone lysine modifying enzymes KDM5C and KDM6A play opposing functions in regulating inflammatory T cell responses.They therefore represent appealing, therapeutically tractable, targets in autoimmune diseases such as MS.

Mice
All experimental animal protocols were approved by the Animal Protection Committee of Université Laval (2021-80 to M.R.).1C6 mice were a kind gift of Vijay K. Kuchroo (Brigham & Women's Hospital, Boston).
Staining for CD4 was carried out for 20 min at 4 • C. Cells were then fixed and permeabilized using Fixation and Perm/Wash buffers (Biolegend) prior to staining for intracellular IFNγ and IL-17.Data were collected by a LSR II flow cytometer (BD Biosciences).Analysis was conducted on live cells (negative for Fixable Viability Dye) using FlowJo (Treestar).Gate were based on fluorescence minus one (FMO) controls.

Declaration of Competing Interest
The authors declare no competing interests.

Fig. 1 .
Fig. 1.Histone and chromatin regulation and its impact on gene expression.(A) The nucleosome consists of 146 bp of DNA wrapped around a histone octamer.Histones have N-terminal tails that are subject to multiple post-translational modifications (PTMs) such as methylation and acetylation.Multiple nucleosomes, compacted together, form the 30 nm fiber which is a condensed structure of the DNA.(B) The impact of tri-di and mono-methylation of different lysines on the H3 histone regarding the activation state of the transcription starting site.(C) Acetylation and methylation are PTMs impacting the epigenetic status of gene loci via histone modification.It is traditionally considered that a globally hyper-acetylated/hypo-methylated histone state corresponds to euchromatin and more active gene transcription, with the opposite (hypo-acetylated/hyper-methylated) correspond to heterochromatin and silenced genes.(D) Known histone lysine methyltransferases and demethylases.Generated using Biorender.
Table 1 outlines the nomenclature and histone substrates of KMTs and KDMs.