X-chromosome linked genes associated with myeloid cell CNS trafficking contributes to female–male differences in the disease outcome for neuroinflammatory diseases

Introduction

Autoimmune diseases, including those related to the central nervous system (CNS) such as multiple sclerosis (MS), are a leading cause of morbidity and mortality in women, who can have up to a four-fold increased risk than men [1]. MS is characterized by many typical and atypical attributes that are found in autoimmune disorders, such as organ-specific or systemic immune reactions [2]. MS particularly afflicts women between the ages of 20–40 years. Remission during pregnancy can be observed, with transient reduction or augmented presence in the puerperium, which shows a diverse response to immunosuppressive therapies. The production of autoantibodies is well-documented in MS; however, this examination lacks significant impact due to the absence of linkage between autoantibodies and MS clinical manifestation [2].

This female–male bias is not unique to MS but applies to other autoimmune conditions too. Of the approximately 8 percent of the human population affected by autoimmune illnesses, 78 percent are women. Connective tissue diseases like Hashimoto’s thyroiditis, Sjogren’s syndrome (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), and systemic sclerosis (SSc) also disproportionately affect women compared to men (reviewed in [3]). This is particularly apparent in SLE where females are at greatest risk factor for the disease, and like other illnesses, can disparagingly affect the well-being of a patient across significant areas of life, including mental health and social functioning. Consequences of a disease can range from depression to illness-linked disability and can be associated with worse health outcomes [4]. Depending on the condition, these can include adverse maternal and fetal consequences for pregnant women [5], hypertensive disorders during pregnancy leading to longer hospitalizations [6], pulmonary arterial hypertension [7], negative self-perceptions due to body mass index (BMI) [8], lower quality of life (QoL) [9], and both resilience, and health literacy scores [10]. Comorbidities like major depressive disorder (MDD) and anxiety further complicate treatment and affect progression and cognition, resulting in earlier mortality [11]. Table 1 enlists common female–male biased diseases in addition to their incidence and ratios, emphasizing the importance of studying female–male differences.

To balance gene products, a process known as X-chromosome inactivation (XCI) occurs. Among its regulatory functions, XCI ensures that X-chromosomal genes are not overly expressed in males [37]. While XCI occurs primarily in females, this process is also been observed in males with female–male dimorphic developmental timing. In females, inactivation occurs during embryonic development [38]; however, in males, it is transient and is confined to the late phases of initial meiotic prophase during spermatogenesis [39]. This phenomenon is called meiotic sex chromosome inactivation (MSCI) [39]. Interestingly, mRNA genes are not known to escape MSCI in males, but about 80 percent of X-linked miRNA genes are shown to escape this process. Thus, it has been suggested that RNA interference may participate in controlling XCI in female cells.

Interestingly, female–male bias is linked at the gene as well as chromosome level since males and females possess different copies of the X-chromosome (1:2). However, to maintain the balance, one X-chromosome copy is inactivated (explained in detail later). Nevertheless, some disease-related genes especially those escaping X-inactivation are present in different ratios between females and males. Table 2 summarizes those genes along with their disease prevalence. For example, LAMP-2 and IRAK-1 that escape X-chromosome inactivation are involved in Danon disease and inflammatory autoimmune disorders like SLE, respectively [40, 41]. Another gene of interest is TLR7, which participates in lupus [42]. This condition has a particularly high female-to-male prevalence ratio (9:1). Additionally, USP27X has been suggested to participate in various neurodegeneration diseases like Parkinson’s as well as infection and cancer, along with other disorders [43]. Interestingly, this gene has also been attributed to X-linked intellectual disability. DDX3X participates in various antiviral signaling pathways and its role has been implicated in DDX3X syndrome [44]. Individuals impacted by this disease experience both developmental and intellectual delays that can range from moderate to severe. Notably, a variation on the DDX3X gene may contribute to X-linked mental disability [45]. Furthermore, CXORF21 is another gene that escapes the inactivation process. Like TLR7, this gene’s abnormalities can promote SLE with a high female-to-male ratio (9:1) as discussed later in further detail.

Particularly in MS, there is an underlying dysregulation of neurological inflammation significantly contributing to disease onset. However, the initiating pathology and why it is more prevalent in women is a growing area of research [55]. One of the most common neurological illnesses among young adults, particularly women, is MS and a primary element of such neurodegenerative diseases includes persistent neuroinflammation [56]. As previously mentioned, disease symptoms are attenuated during pregnancy [2]. This is evidenced by the significantly diminished rate and potency of relapses over time, especially in the final trimester [57]. It is a chronic, prevalent rising inflammatory condition with accumulations of neurological deficits, progressing over time being a key characteristic of its pathogenicity [58]. Particularly, MS results in the demyelination of neuron fibers and reduced muscular mobility associated with underlying inflammation mounted by infiltrating leukocytes following a cascade of events for CNS entry [59]. In both MS and a commonly used animal model (experimental autoimmune encephalomyelitis; EAE), infiltrating macrophages and CNS-resident microglia both demonstrate increased reactivity that is known to contribute to both the onset and maintenance of a proinflammatory environment [60–63]. Sustained inflammation can then both initiate and exacerbate demyelination, axonal damage, and oligodendrocyte cell death [64–66]. Figure 1 demonstrates how immune cells behave in the leukocyte trafficking cascade. This review discusses markers and genes related to each stage of leukocyte trafficking in particular interest to those associated with X-chromosome and MS pathogenesis.

Figure 1:

The process of leukocyte trafficking is illustrated, demonstrating the behavior of immune cells during chemoattraction, initial contact, firm adhesion, rolling, and transmigration. Specifically, markers interleukin 2 receptor subunit alpha, forkhead box protein 3 (FoxP3) gene, and C-X-C motif chemokine receptor 3 are X-chromosome linked and are shown with an “X” in the figure, along with other genes and markers who are not known to be X-linked.

Among several drugs with differing mechanisms of action, it has been shown that blocking immune cell entry into the CNS can have a major impact on MS [47, 67]. FDA-approved drugs, such as Tysabri and Gilenya, that target immune cell invasion at the BBB have shown efficacy in treating autoimmune disorders like MS. In addition, antibodies and disease-modifying therapies such as glatiramer acetate, block the leukocyte integrin VLA-4 [68]. However, current marketed drugs have several limitations that include cost inefficiency, they only have a moderate effect in alleviating disease symptoms, and may even be linked with opportunistic infections like progressive multifocal leukoencephalopathy (PML) [67]. Since disease culminates in severe disability, patients experience significant financial and symptom burdens. Consequently, advancing therapies that would ameliorate these issues may be highly beneficial. Thus, addressing the female–male bias present in MS in the context of therapies is needed.

Recent evidence suggests dendritic cells (DCs) are critically involved in the initiation and progression of autoimmune disorders, including MS. In both EAE and MS, they are essential for the effector phase and immune response cascade. DCs, one of the most potent antigen-presenting cells (APCs), play a crucial role in inducing inflammatory or tolerogenic responses contingent upon local environmental cues and lineage [69]. DC accumulation within the CNS has been observed in ongoing MS and EAE [70, 71], Notably, DCs are detected within inflammatory demyelinating lesions of MS patients and EAE animals, indicative of a role in both disease initiation and progression [70]. We too have demonstrated the most efficient recruitment of DCs into EAE lesions compared to other leukocytes [71]. This is why contemporary therapies for MS, such as disease-modifying drugs, target the function of these APCs [72]. Chemokines and their receptors play a crucial role in regulating the migration of immune cells to inflammatory sites and thus, their contribution to autoimmune diseases like MS, is well-studied [73].

This paper focuses on the female–male bias seen in X-linked genes in the context of myeloid cell trafficking in MS. We also elaborate on the steps involved in leukocyte trafficking following neuroinflammatory induced immune cell entry along with chemokines and their receptors. Other topics of inquiry that we touch upon include X-chromosome inactivation, dosage compensation, and how X-linked differences in both DCs and T cells affect disease progression. Overall, the paper offers unique and novel perspectives on the mechanism of myeloid cell trafficking across the BBB, examining uncontrolled activation and antigen presentation.

Female–male biased autoimmune and neuroinflammatory diseases

Heightened immune response in women may result in a predisposition for loss of self-tolerance, leading to the development of skin disorders as well as autoimmunity [1]. Female skin is “autoimmunity prone,” with close examinations depicting the upregulation of proinflammatory genes. This section will discuss autoimmune disorders, including MS, highlighting the role of skin, autoantibodies, gut microbiota, and sex hormones.

One possibility that can elucidate female–male bias seen in autoimmune disorders, such as lupus, is skin processes that include mediating inflammation, immune responses, wound healing, and angiogenesis [74]. Vestigial-like 3 (VGLL3) is a component of the VGLL family of proteins and despite the absence of a precise and transparent physiological role [75, 76], its augmented expression in females is evident and also encourages autoimmunity [76]. A previous study has identified VGLL3 as a putative transcription cofactor enriched in female skin. Markedly, this study found that the skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash due to autoantibody production and immune complex deposition. It is hypothesized that this sex-linked increase in VGLL3 transcription contributes to increased lupus rates in females. The autoantibodies are associated with autoimmune diseases and female–male differences are also seen with antibody production. It has been shown that generally, women possess higher CD3⁺ and CD4⁺ T-cell counts, increased basal and stimulated antibody reactions to viruses and vaccines, and enhanced cytokine production in response to infection [77]. This difference may be due to an evolutionary development to protect offspring from infection [78]. Meanwhile, another notion regarding increased autoimmunity occurrence in women includes the pregnancy compensation hypothesis (PCH), a concept that assumes female–male specific immune differences arise in response to challenges of maintaining protection against pathogens, simultaneously tolerating an invasive placenta, and the introduction of foreign fetal antigens [77].

While the origins of increased female–male differences in antibody production still need further study, it has been shown that B cells play a major role in autoimmunity, especially for women. Several studies have concluded the integral role of B cells and the CD40 gene in autoimmune disorders. One study found that the administration of an anti-B-cell agent called rituximab, delayed disease onset in pre-Rheumatoid arthritis (RA) patients [78]. For individuals with SLE, there is an overexpression of CD40L on T-cell surfaces as well as in serum. Also, CD40 serves as a pathogenic factor in many other autoimmune illnesses, including MS [78]. Similarly, it has been noted that expression of CD40L on activated CD4⁺ T cells was greater in patients with Sjogren’s syndrome compared to healthy controls [79]. CD40 has been found to play a pathogenic role in other autoimmune diseases including type 1 diabetes [80], inflammatory bowel disease [81], MS [82], and lupus [83] as epigenetic deregulation of genes occurring on the X-chromosome. The CD40L gene is X-linked, its duplication can yield an overexpression of CD40L and has been associated with various autoimmune illnesses, including lupus [84].

Another X-linked dysregulation that is associated with autoimmune diseases is gut microbiota dysfunction. SLE disease progression rates in humans have been associated with certain compositions of gut microbiota [85]. It has been demonstrated that people with MS have altered microbiome and gut dysbiosis, possessing both enrichment and/or depletion of certain types of bacteria [86]. Common trends show that those with MS have decreased amounts of Prevotella, Parabacteroides, and Lactobacillus, among others. Meanwhile, there was heightened concentrations of Akkermansia, Eggerthella, and Blautia. There is some evidence that females are more effected by gut dysbiosis. Utilizing lupus-prone mice, one study found that a pro-inflammatory immune response in the gut mucosa of female lupus-prone mice can be detected at a juvenile age [85]. Correspondingly, a microbiota-dependent pro-inflammatory immune response is seen in female mice gut mucosa at a juvenile age. Alternatively, there seems to be androgen-dependent protection of males, as these SNF1 mice are safeguarded from this disease through this hormone, indirectly and/or gut microbiota dependently [85]. These observations can contribute to female–male differences in the intestinal immune phenotype and systemic autoimmune progression. Along with gut microbiota, another significant factor contributing to MS development is sex hormones. Generally, sex hormones partake in B-cell development, function in physiology, and have a significant role in the abnormalities that occur in autoimmune diseases [87]. The most prominent female sex hormone, estrogen, modulates humoral responses, B-cell differentiation, and immunoglobulin production. Several complex roles are attributed to estrogen and its protective contribution in MS and RA is evident in other published work. However, its pathogenic effects can be noted in other conditions such as SLE. While progesterone and androgen have more immune-suppressive effects, estrogen is more dose and cell-type dependent increasing cell activation, intracellular signaling, and antibody production in the context of autoimmunity [88]. Additionally, estrogen has been demonstrated to produce an anti-inflammatory response and decrease inflammation in the CNS via ERalpha, and its activation of ERbeta results in myelin and axonal restitution [89]. To better understand how female–male differences influence autoimmunity in general as well as in MS, the elucidation of complex and intricate interactions transpiring between sex hormones, X-chromosomes, and immune response genes is indispensable.

Impact of immune cell CNS entry on neuroinflammation

Although there is no solid explanation for the female–male bias in autoimmune diseases, theories with respect to a more comprehensive immune response, the microbiome, sex hormones, and the X-chromosome, have been proposed. Due to increased phagocytic activity, more effective antigen presentation, and higher expression of pathways linked to PRRs such as TLRs, women have been shown to produce a more robust innate response. Changes in the gut microbiota composition called gut dysbiosis can disrupt homeostasis and put individuals at risk for various inflammatory and neurological conditions like MS [104]. Increasing evidence suggests that along with genetic factors, numerous hormonal elements influence autoimmune susceptibility [105]. Sex hormone outcomes can differ contingent upon their concentration and target cell and receptor subtype expressed on any cell form. Due to the existence of hormone receptors on immune cells, sex hormones like progesterone and estrogens can impact various components of the immune system function, with the likelihood of influencing risk, activity, as well as the progression of the condition. Of these factors, X-linked genes, and markers play an important role in determining female–male bias vs. diseases. Specifically, X-linked gene expression such as TLR7 and CD40L in healthy females and Klinefelter syndrome (KS) individuals implies that X-chromosomes may function as major factors in enhancing both innate and adaptive immune responses, possibly explaining female–male based differences in immune biology as well as female–male bias in autoimmune disease predisposition [106]. XCI (also known as lyonization) is an epigenetic process that occurs in females and results in the silencing of one X-chromosome through dosage compensation to balance X-associated gene expression levels among females and males [107].

Recent evidence suggests that innate, as well as adaptive, immune responses are vital contributors to neuroinflammation [108]. The role of T cells was examined in the previous section, but here we will concentrate on innate immune cells, particularly DCs. There is evidence that neuroinflammation attracts both mature and immature DCs. In previous studies, we reported CNS trafficking of DCs across the BBB during EAE via intravital video microscopy (IVM) which has enabled direct visualization of the inflamed white matter (WM) of the spinal cord [71]. DCs can present neuroantigens (i.e. myelin basic protein, MBP) to prime naïve autoreactive CD4⁺ T cells in the peripheral immune system, and within the CNS. Depending upon the pathological conditions, these cells can mature into T helper type 1 (Th1), Th2, and Th17 effector cells or regulatory T cells (Tregs) [109]. In MS/EAE, circulating neuroantigen-specific T cells gain access to the CNS and promote neurodegeneration contributing to the disease progression. The source of the inflammation, however, appears to be the DCs, which not only prime T cells, but also maintain a chronic inflammation within MS lesions. Furthermore, the influx of leukocytes contributes to inflammation and their accumulation can lead to abnormalities observed in MS, such as the dysfunction of the BBB [110].

During MS and EAE, accumulation of DCs is observed within inflammatory infiltrates in the CNS parenchyma and as well as the CSF [69]. Its presence in the CSF of affected patients suggests that DCs might play a key role in determining the outcome of CNS inflammation. This notion of EAE pathogenesis is underlined by observations that CD11c−/− mice are resistant to EAE and that CD11c⁺ DCs are sufficient to present Ag to primed myelin-reactive T cells in vivo to mediate CNS inflammation and clinical EAE [71]. In addition, DCs have been implicated in epitope spreading during chronic EAE and are particularly well-suited to polarize myelin-specific T cells toward Th1 and Th17 effector T cells in vitro. Our observations in prior studies confirm the conviction of the migratory ability of DCs being intrinsically linked to their maturation state and differences in traffic signals displayed on the surface of immature vs. mature DCs might influence their efficiency to enter certain inflammatory sites [71].

In addition to the role of DCs and neutrophils in leukocyte trafficking, T cell involvement is also apparent, especially in MS. CD4⁺ T cells are essential in the pathogenesis of MS, and they depart from the lymphoid organs and move through the BBB following activation and differentiation [59]. This step is vital for CD4⁺ T-cell entry into the CNS. Contemporary data depict that the paths that regulate the departure and migration of these cells are perhaps contingent on their type. Additionally, their hindrance could have ramifications outside of migration. In MS, abnormally activated autoimmune T cells recognize components of the CNS myelin and initiate the disease [111]. EAE models have shown that the representation of the disease may be passively transferred by activated myelin-specific T cells. Thus, it may be suggested that T-cell autoimmune reactions to myelin can be involved in the inflammatory response inception observed in the CNS. Likewise, the pathogenic mechanisms of MS development include Ag-specific T-cell activation and Th1 differentiation, followed by T-cell and macrophage migration into the CNS.

Chemoattraction, initial contact, firm adhesion, rolling, and diapedesis are the complex steps involved in the leukocyte trafficking cascade (Figure 1 and [112]). The final step of the leukocyte extravasation cascade, migration, is attributed to multiple cell trafficking molecules, located on surfaces of moving leukocytes as well as BBB endothelial cells. Participation of molecules such as very late antigen 4 (VLA4) and vascular cell adhesion molecule 1 (VCAM1) is evident, as they engage in advancing the movement of various immune cell subsets. Other contributors such as IL-17 cytotoxic CD8⁺ T (TC17) cells and activated leukocyte cell adhesion molecule (ALCAM) observed in both monocytes and lymphocytes, partake in expression and cell trafficking. Leukocyte trafficking also involves other genes that are not associated with the X-chromosome. Details on each are provided below in accordance with the step of the cascade.

Chemokine and chemokine receptors

Chemokines are proteins expressed by various cells. They are a critical contributor to leukocyte extravasation, guiding cells to their eventual migration across the endothelium and into the inflamed tissue. They, along with their receptors, will be discussed in detail in the following section. Clinical manifestations of autoimmune illnesses arise from a strong immune response to a self-antigen, leading to leukocyte activation and accumulation. Through the recruitment of chemokine-dependent cells, structural cells potentially exhibit a role in chronic inflammation [132]. Chemokines play a crucial role in the regulation of leukocyte trafficking. By binding to their G protein-coupled receptors (GPCR), they guide defined leukocyte subsets to inflammatory sites or coordinate homing and recirculation of lymphocytes [133]. These proteins are critical in sustaining the typical function of immune responses, including instances of cellular destruction and inflammatory reactions. As a result, they impact leukocyte cell survival, and effector functions, and act on tumor cells to contribute to angiogenesis, tumor growth, and metastasis [132, 133]. Upon tissue injury or infection, there is an inflammatory response involving the recruitment of inflammatory leukocytes by chemokines, guiding appropriate cell migrations to the site of injury. An upregulation of chemokine receptor expression and function is essential for a significant leukocyte response. However, influxes of leukocytes require monitoring to prevent excessive accumulations, as unrestrained buildup may lead to a continuous inflammatory response that can originate both tissue damage as well as chronic inflammation. Figure 2 illustrates the mechanism and signaling pathways associated with CXCR3 activation.

Figure 2:

Cellular migration and proliferation via CXCL10 and CCR3 signaling. CXCL10 binding to CXCR3 activates the G-couple protein receptor via the Gαi subunit and induces the activation of SRC/Ras pathways and downstream PI3K/ERK/AkT signaling. These pathways are responsible for promoting cell proliferation. At the same time, via the Gαq subunit, activation triggers downstream phospholipase C-beta (PlCβ) signaling. This results in the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-triphosphate (IP3). This promotes the release of calcium from the endoplasmic reticulum, which activates the calpain protease, which is responsible for cell mobility and increased migration.

Chemokines can increase immune cell infiltration across the BBB and cause uncontrolled activation and antigen presentation [134]. They can also modulate neuroinflammation and neurodegenerative processes and are crucial to the activation and migration of immune cells to lesion sites. C–C chemokine ligand 2 (CCL2), a potent chemoattractant released by astrocytes and microvascular endothelial cells, permits chemoattraction of patrolling immune cells of the BBB vasculature. CCR2 is a receptor for CCL2 which is commonly known as monocyte chemoattraction protein 1 (MCP-1). MCP-1 participates in monocyte infiltration seen in inflammatory conditions like RA [135]. Increased CCL2 expression has been noted in several EAE models and a reduced accumulation of CD11c⁺ DCs was seen within the spinal cord homogenates of CCL2−/− chimeric mice induced with EAE. Interestingly, these mice also display less demyelination than wild-type EAE controls [134].

In a prior study, we imaged and evaluated the ability of endogenous DCs to transmigrate across the BBB during EAE and assessed both mechanism and extent of chemotaxis and diapedesis in the presence of CCL2 [92]. Our transmigration and immunofluorescence studies determined that DCs were more potent responders to CCL2 than T cells. Furthermore, we noted that the DC transmigration pattern was primarily paracellular and dependent on signaling molecule ERK1/2 phosphorylation compared to that of T cells. This observation was transcellular and dependent on phosphorylation or signaling molecule p38-mitogen-activated protein kinase (MAPK). DCs also transmigrated with greater efficiency than T cells, potentially due to specific adhesion molecules used to tether to endothelia. We previously determined that DC transmigration correlates with the severity of inflammation during EAE, and confirmed via ex vivo histology that CCL2 was present in EAE lesions. Additionally, CCL2facilitates DC transmigration in an ERK1/2-dependent manner. In one study, we used an EAE model known to exhibit peak leukocyte activation between days 12 and 30 post-inoculation. The sclerotic lesions of this model are known to induce CCL2 generation and release [92].

A 2013 genome-wide association study conducted by the International Multiple Sclerosis Genetics Consortium (IMSGC), highlighted the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway as a key player in MS pathology [136]. Accordingly, its activation results in the rise of inflammation [136]. Comparably, CCL2 expression is dependent on NF-kB signaling, and its mRNA has been noted to increase within the demyelinated MS hippocampus. However, there are discrepancies regarding the spatial and quantitative distribution concerning gray matter (GM) and WM lesions. This chemokine-induced monocyte migration also results in the breakdown of the BBB via the downregulation of endothelial tight junction proteins. CCL2 is a pro-inflammatory chemokine that attracts DCs, monocytes, T cells, and natural killer (NK) cells to inflammatory sites via its CCR2 receptor. CCL2 can increase the permeability of the BBB allowing for infiltration of circulating systemic immune cells and potentially recruits monocytes and microglia to CNS inflammatory sites [137]. This chemokine induces migration of monocytes, memory T cells, and DCs [138]. Picomolar levels of CCL2 have been shown to limit inflammation in inflammatory bowel disease models. CCR2 is expressed on immune cells and mediates monocyte recruitment. Inadequate quantities of CCR2 within T cells promote the buildup of FoxP3⁺ regulatory T cells (Tregs) and simultaneously reduce levels of Th17 cells in vivo. Low doses of CCL2 are influential in repressing MOG-affected EAE and can downregulate severe EAE. EAE is modulated by CCL2 and was linked to the downregulation of Th1/Th17 cells as well as upregulation of TGF-β and induction of regulatory CD4⁺ FoxP3 T cells. Accordingly, high levels of CCL2 and receptor CCR2 in the CNS have been associated with EAE relapse.

Chemokine receptor CXCR3 is a GPCR expressed by T cells that participates in mediating chemotactic migration, cell proliferation, and survival; studies have identified its expression in CNS diseases [139]. Intriguingly, increased presence of CXCR3 is observed in neurological disorders like MS, Alzheimer’s disease, and bipolar disorder. Meanwhile, its antagonistic agents have demonstrated therapeutic effects. CXCL10, a ligand of CXCR3, was expressed by astrocytes in MS brain lesions, however, an equivalent observation was not made in control patients’ WM. Another study displayed IFN-β’s therapeutic effects where following treatment, CXCR3 expression on CD4⁺ and CD8⁺ T cells is significantly decreased [140].

CCR7 is critical in eliciting an immune response and initiation of immunological tolerance and its chemokine ligands include CCL19 and CCL21 [141]. Under physiological conditions in the human brain, CCL19 can be transcribed and measured as a protein in both tissue lysates and CSF. Notably, in both active and inactive relapsing-remitting and secondary progressive MS patients, this chemokine ligand is elevated in the CSF and it has been implicated to participate in both normal and abnormal immunosurveillance [141].

CCR1 and CCR2 are chemokine receptors regulating monocyte trafficking to the location of inflammation [142]. Particularly, CCR1 is essential for the control of the acute phase of inflammation that transpires in both EAE and MS. It responds to inflammation through CCL3 and CCL5 and targeting the actions of the former in vivo via antibody or genomic deletion significantly reduces MS. Current therapies target leukocyte trafficking and inflammation in the CNS. However, despite showing adequate safety profiles in the treatment of MS, CCR2-targeted drugs have failed in efficacy and continued research into their development has ceased [142].

X-linked genes in the context of leukocyte trafficking

Several genes on the X-chromosome have been implicated in increasing susceptibility to MS and other autoimmune disorders. Some of these genes include members of the interleukin family of cytokines and receptors such as Interleukin 2 receptor A (IL-2RA) and Interleukin 3 receptor A (IL-3A), which are discussed in detail as follows.

X-chromosome inactivation/dosage compensation and escape

Remarkably, the human X-chromosome has a peculiar inheritance pattern and unique biology [182]. Genes on the X-chromosomes and the X-inactivation process are the root cause of a considerable portion of the health disparities between females and males. Autoimmunity has been linked to X-chromosome structural and number abnormalities, as deletions and translocations of the X autosome are associated with early ovarian failure. Disturbances in the XCI process are one possibility for these deviations. Interestingly, the Kast-Stewart hypothesis is based on two observations: first, women are disproportionately affected by autoimmune disorders, and second, X-inactivation is a crucial biological regulator that primarily affects women [182]. Notably, XCI occurs early, particularly during embryonic development [38]. Following fertilization, the X-chromosome inherited from either parent is silenced. This process is perpetual, and every daughter cell can recognize which X-chromosome is to be inactivated. Additionally, the level of expression from Xi is not as high compared to the Xa and a large portion of Xi genes are silenced [183, 184]. However, studies suggest that silencing is not invariably successful; genes below 20 percent can avoid silencing and variability exists in activation between females [38]. Interestingly, as mentioned before, in MSCI, the XCI process in males, no mRNA genes are found to be escapees [39]. Thus, this process is incomplete in humans as up to one-third of X-chromosomal genes are expressed from the active as well as the inactive chromosome, separately [185]. As a result of vast number of genes present on the X-chromosome, it has been suggested that female–male differences in MS susceptibility are plausibly due to X [12]. Figure 3 depicts the process of X-chromosome inactivation.

Figure 3:

Process of X inactivation or lyonization during embryonic development (A). Noncoding RNAs, known as XIST and TSIX begin and regulate this complex process. Of the two X-chromosomes, one is randomly selected for inactivation. (B) Inactive chromosome condenses and forms a Barr body structure. (C) X-chromosome remains inactive throughout subsequent cell divisions.

The X-chromosome, unlike the gene-poor Y-chromosome, contains over 1,000 genes essential to development and cell viability. Additionally, it can be subjected to X-chromosome dosage compensation (XDC). XDC occurs when an X-chromosome associated gene attains equivalent expression between females and males [13]. It has been proposed that XDC is realized by two-fold upregulations of X-associated genes in both males and females. This transpires by the silencing of one X-chromosome, hence XCI in females [13]. Interference in the X inactivation process can produce a loss of dosage compensation, imbalance of gene products, as well as changed endogenous material originating from typical epigenetic context [13]. Consequently, these anomalies can lead to the initiation of autoimmune disorders [48]. Importantly, the gravity of such diseases is reliant upon the status of the XCI and whether it is X-linked, influenced by XCI escape, or due to X-chromosome aneuploidy. The human X-chromosome is unique in comparison to other species. Thus, the alterations that accompany such differences can both instances and intensities of autoimmune illnesses in humans in addition to encountered autoantigens [48]. Particularly, even non-autoimmune disorders characterized by X-chromosome aberrations can contribute to autoimmune disorders. This notion has been supported by the presence of lupus-like symptoms. However, the underlying mechanism behind this process is still not well understood. Reports exist in which dosage alterations could solely influence X-linked dosage-sensitive genes like the protein complex coding genes (PCGs) [13]. In one study, it has been exhibited that human PCGs have a greater probability of escaping XCI and avoiding PCGs (EsP) depicting a two-fold greater expression vs. the inactivated PCGs (InP) or other X-linked genes at the level of both RNA and protein. Therefore, this suggests that EsP could potentially achieve upregulations as well as XDC [13]. The same study also indicated that EsP genes are enhanced in the TLR pathway, NF-kB pathway, and apoptotic pathway and many phenotypes including abnormal, mental, and developmental. Findings from this study provide the conviction that anomalies of EsP can be a decisive contributing factor in autoimmune, reproductive, as well as neurological disorders [13]. XCI is a mechanism of dosage compensation in mammalian females, flies, and worms [14]. Through XCI, female mammals transcriptionally silence one of the two Xs, and the inactivated X-chromosome condenses into a compact structure called a Barr body, stabilized in a silent state. Two noncoding RNAs, XIST and TSIX, initiate and control the inactivation process [14].

One study looked at whether female–male differences were caused by differences in X-chromosomes using EAE [105]. An in-depth look at FoxP3 suggested that maternal compared to paternal imprinting of X-chromosome genes may be responsible for female–male differences seen in autoimmunity. EAE mice with XY chromosomes in the CNS vs. EAE mice with XX exhibited worse clinical symptoms. This was apparent in various brain regions like the cerebellum as demonstrated by Purkinje cell and myelin loss and cerebral cortex via synaptic deprivation. The mapping of the transcriptome, as well as methylome in T cells and neurons, has the capability of better comprehending underlying female–male differences seen in both autoimmunity and neurodegeneration.

Overall, there is a supported notion that demonstrates that women suffer higher immunoreactivity and a more vigorous humoral and cell-mediated immune response to microbial infection and vaccination than men [106]. Genes encoded by the Xi may “escape” inactivation and lead to the expression of X-related genes from the active and inactive chromosome female cells. One study observed the expression of the X-linked gene CD40L, involved in adaptive immunity, and TLR7, a participant in innate immunity. Levels of expression were determined in varying karyotypes including males, females, and those with X-chromosome aneuploidy. An increase in these X-linked genes was observed in healthy females and patients with KS. There seems to be a role for X-chromosome gene dosage expression in innate and adaptive immune responses and a cause for female–male bias in autoimmune disease predisposition. TLR7 plays a major role in SLE development, and it can escape XCI in about 30 percent of immune cells in healthy women. Various genes can escape XCI and ultimately contribute to the development of X-linked diseases. These genes include LAMP-2, IRAK-1, TLR7, USP27X, DDX3X, and CXORF21 and are presented in detail in the next subsections.

Conclusion and perspective

Women are disproportionately affected by autoimmune diseases that cause significant morbidity and mortality such as SLE, SSc, SS, and MS. This is evident through occurrences of greater incidence and prevalence among females. The explanations behind this female–male bias can vary, ranging from the gut microbiota to the expression of certain genes, i.e., FoxP3. It is through a variety of factors that developments of autoimmune conditions transpire and there is no sole source that can be attributed to them. Consequently, a combination of genetic and environmental factors can lead to their pathogenic initiation and eventual progression. Leukocyte trafficking via immune cell entry and migration is critical in disease pathogenesis. It is through this cascade that immune cells, with chemokines serving as their mediators, travel to the site of inflammation and contribute to their accumulation. There is a integral role for immune cells, particularly T cells and DCs, in the development and progression of MS and EAE. Proinflammatory Th1, as well as Th17, have been associated with MS pathology [133]. CD4 Tregs are also involved by restricting pathogenic T cell responses via mechanisms such as inhibitory cytokine production, metabolic disruption, and cell-contact-dependent cytolysis. Dysregulation of autoimmune T cell response linked with MS pathology is thought to be due to a variety of factors such as reduced Treg suppressive activity and a decline in Treg numbers. CD8⁺ T cells have one of the most paramount roles in MS pathology. Large quantities of CD8⁺ T cells are observed in the injured WM and GM of MS patients. CD8⁺ T-cell participation in CNS damage is evident through the production of granzymes and perforins as well as secretion of proinflammatory cytokines. The innate immune response encompasses the activation of DCs [25]. These cells take up and process antigens while traveling through lymphatic vessels to tissue-draining lymph nodes, advancing T-cell activation. Therefore, they prepare the immune response and immune memory against future attacks of the same antigen. Numerous amounts of DCs are detected in brain lesions of MS patients and modified phenotype or function is shown vs. healthy controls [26]. Studies indicate the robust presence of DCs in the CSF, where plasmacytoid DCs (pDCs) are reliant on disease terms. Once relapses occur, the amount of pDCs in the CSF increases vs. patients in remission. Thus, based on these T cell and DCs functions, it can be suggested that these cells have a critical role to play in the disease progression of MS. The X-chromosome is another element to consider, as anomalies in chromosome structure and number are linked to autoimmune symptoms. Whether they’re random or imprinted, XCI disturbances may contribute to autoimmune disorders. Genes on the X-chromosome have essential roles in development and cell viability, and XCI helps balance their expression. X-linked genes may escape XCI and be expressed on both the active and inactive chromosomes in female cells. This phenomenon could affect the immune response and contribute to the female–male bias observed in autoimmune diseases. For instance, TLR7, an X-linked gene, plays a significant role in the development of SLE, and its escape from XCI has been observed in the immune cells of healthy women. Thus, a greater understanding of the mechanism of XCI is warranted.

MS is a demyelinating CNS disease with various explanations as to its origins/causation. In this review, we highlighted these topics and describe the risk factors, gene involvement, how X-linked and non-X-linked genes contribute to autoimmune diseases, and how they can escape from dosage compensation. We have also discussed in detail various immune cells such as DCs and T cells, the X-chromosome, chemokines and their receptors, and the role of leukocyte trafficking, among other areas of interest. We have identified the importance of these topics and their cumulative contribution of knowledge to autoimmunity and female–male biased disorders. Taken together, they provide a more comprehensive outlook on autoimmune disorders and their overall pathogenicity, yielding more robust explanations for autoimmunity.

Autoimmune disorders inordinately impacting women can be very concerning; it has been observed that several areas of life are negatively affected, including overall quality. Additionally, it can be very important to investigate the mechanisms underlying female–male bias in autoimmune illnesses since they can highlight and showcase features of diseases that would otherwise go neglected. As more research is conducted into the emergence as well as the mechanism of autoimmune disorders, we expect that over time, the female–male bias observed in these conditions could be further illuminated and ultimately addressed so that fewer women will be affected compared to the present incidence and prevalence. Greater comprehension of autoimmune diseases could pave the way for more advanced remedial options for patients, potentially improving daily life and standard of care.

Even if there is no one specific cause for autoimmune disease, there needs to be a greater understanding of pathogenesis and the reasons for the higher frequency of these illnesses in some regions of the world than others need to be better understood. For instance, certain environmental factors like exposure to EBV, vitamin D deficiency, and lack of sunlight have been demonstrated to increase risk of developing MS. As previously mentioned, females display a more robust immune response and combined with genetic factors are more likely to develop female–male biased diseases emphasizing the need to identify causative factors. X-chromosome inactivation provides another avenue of research worth further exploration in the context of disease etiology and pathogenesis. Additionally, targeting the recruitment and accumulation of immune cells could be beneficial as mechanisms of leukocyte trafficking remains poorly understood. Based on the growing acknowledgment amongst researchers and current literature, there is a recognized need for further research on understanding the observed female–male bias among autoimmune disorders. Future studies should address the aforementioned research gaps discussed in this review so as to better develop targeted pharmacological treatments that ease disease burden and improve quality of life among those affected.