Reproductive Dysfunction in Experimental Autoimmune Encephalomyelitis, an Animal Model of Multiple Sclerosis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). The latter is a human organ-specific autoimmune disease of the central nervous system (CNS). EAE is characterized by systemic inflammation associated with increased blood levels of pro-inflammatory mediators that potentially trigger inflammation of both reproductive organs and the CNS. Pathological changes in the hypothalamus-pituitary gland-gonadal axis have occasionally been reported in both the MS and EAE contexts. Such changes may affect the reproductive organs. We used the phrase “EAE and hypothalamus-pituitary-gonads (testis and ovary)” to retrieve relevant papers from PubMed. We postulated that EAE might be associated with inflammation of the hypothalamus, pituitary gland and gonads, in turn indicating reproductive dysfunction. This paper overviews evidence supporting the roles of both hormonal and inflammatory alterations in animals with EAE. This aids our understanding of how certain autoimmune diseases are associated with infertility.

INTRODUCTION

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) characterized principally by infiltration of the nervous system by inflammatory cells [1]. Multiple sclerosis, one of neurodegenerative disease, is an autoimmune disease in which demyelination occurs in CNS. EAE is the representative animal model of human MS by artificial immunization, such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). While B cells play an important role in the pathogenesis of MS, autoreactive CD4+ T cells play an important role in the pathogenesis of EAE [2]. Multiple sclerosis is classified into relapsing-remitting MS, which is most common type, secondary progressive MS, and primary progressive MS. Clinical symptoms vary, including sensory abnormalities, blurred vision, fatigue and impairment in memory [3]. It occurs more in women, nearly three times more than man, due to genetic factors. Recently, other risk factors of MS include Epstein-Barr virus, smoking and low vitamin D level [3]. EAE is traditionally mediated by Th1 cells, and recovered by promotion of a Th2 bias [1]. After immunization such as MBP and MOG, autoreactive T cells migrate to target organ, in conjunction with the upregulation of adhesion molecules [4].

As neuroinflammatory diseases, including EAE and MS progress, the blood-brain barrier (BBB) is disrupted, associated with perivascular cuffing. Cerebrospinal fluid (CSF) diffuses out of the meninges [4]. In EAE, inflammatory cells that accumulate in meninges infiltrate either the associated perivascular space or/and the glial limitans that covers the central nervous system (CNS) [4, 5]. Thus, the hypothalamus may be exposed to inflammatory cells that evade the BBB or/and pro-inflammatory mediators in the CSF of various ventricles, including the third ventricle. An earlier review found that both stress and hypothalamic-pituitary-adrenal (HPA) axis activity greatly influenced EAE and MS development [5]. Decreased serum levels of adropin are observed along with hypothalamic atrophy in relapsing-remitting MS (RRMS) patients [6, 7]. Because sex hormones such as prolactin, estrogen, progesterone, and testosterone are involved in regulating the progression of MS, hypothalamus atrophy is thought to be closely related to sex hormone imbalance [8, 9]. Therefore, we postulated that EAE might be associated with inflammation of the hypothalamus, pituitary gland, and gonads, in turn indicating reproductive dysfunction.

Brain EAE is a tissue-specific autoimmune disease associated with marked changes in serum cytokine levels and, thus, systemic inflammation [10]. It is possible that the increased levels of pro-inflammatory mediators produced during the course of EAE affect non-neuronal tissues. Such tissues may be infiltrated by inflammatory cells, including macrophages and neutrophils [11]. Recent RNA sequencing analysis clearly confirmed that organs associated with reproduction, including the hypothalamus, ovary, and adrenal glands, exhibited both hormonal and inflammatory changes as EAE progressed [12, 13]. Thus, it is important to explore further the reproductive dysfunction associated with EAE and MS. This article examines whether hormones and inflammation might explain the reproductive dysfunction associated with autoimmune CNS neuroinflammation.

Various online databases, including Pubmed, were searched using the following MeSH terms: “EAE” and “hypothalamus” or “pituitary gland” or “gonads (testis and/or ovary),” or “hypothalamus-pituitary-gonadal (HPG) axis.” Only manuscripts published in English after 2000 were retrieved. Drug trials and reviews were excluded. The remaining abstracts were screened, and papers exploring how EAE affected reproduction extracted. A manuscript was eligible for consideration if all authors so agreed.

Inflammatory response of the hypothalamus in EAE

Inflammatory cells infiltrate the subarachnoid space in the early stage of EAE [1, 4]. These cells may include T cells, macrophages, B cells, natural killer cells, and mast cells [1]. Then, encephalitogenic CD4+ T cells preferentially enter the spinal cord parenchyma, followed by CD8+ T cells [14]. Macrophages have been implicated in CNS damage in EAE patients, and the removal of macrophages alleviates symptoms [15]. In the early stages of EAE, macrophages and activated microglia are the main sources of pro-inflammatory cytokines, such as interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-1-beta, and IL-6 [1]. Furthermore, glial cells, including microglia and astrocytes, are strongly associated with the progressive neurodegeneration observed in MS [16] and EAE [17].

The subarachnoid space can thus be viewed as a CNS buffer. Perivascular cuffing is occasionally detected around ventricles, where extravasation easily occurs [1]. The space lies in close proximity to the third ventricle of the hypothalamus. Various pro-inflammatory mediators both stimulate ependymal cells and facilitate cell migration into the hypothalamus. Kisspeptin of hypothalamus controlled the releasing of reproductive hormones in HPG axis, and influenced by stressful conditions, such as heat stress in domestic sows. Furthermore, down-regulation of kisspeptin expression and activity in hypothalamus is deeply related to seasonal infertility [18] GnRH neurons of hypothalamus is associated with sexual development, puberty, stressful condition, and ovulation [18]. Therefore, the pro-inflammatory mediators stimulate neuroglial cells, including microglia and astrocytes which lead to downregulation of kisspentin1 and GnRH in neurons (Fig. 1). The BBB may not be breached during EAE. All brain regions, including the hippocampus, become inflamed to various extents [19]. This is also the case during the course of MS. A dysfunctional hypothalamus might induce muscle wasting, sleep disorders, insulin-resistance, fatigue, and reproductive disorders.

Fig. 1.

The effect of third-ventricle cerebrospinal fluid (CSF) on the adjacent hypothalamus. Proinflammatory mediators in the CSF enter the hypothalamus after passing through ependymal cells, and then stimulate both microglia and astrocytes. This, in turn, downregulates the neuronal expression levels of Kisspentin 1 and GnRH. The EAE-induced hypothalamic changes may alternatively be attributable to migration of inflammatory cells, thus both T cells and macrophages, across the BBB.

Dysregulation of the HPG axis in EAE

The hypothalamus is a component of the limbic system. The hypothalamus lies under the thalamus and can be divided into three different regions. From anterior to posterior, these are the preoptic, supraoptic, tuberal, and mammillary regions. Each region contains a number of nuclei with different functions [20]. The hypothalamus regulates body temperature, food intake, sleep, aggression, and sexual behavior [5, 12]. HPG axis activation during EAE is strongly associated with changes in cognitive-emotional behavior [21], stress responses [22, 23], and food intake [8, 24]. HPG axis activation induces hyperleptinemia [25] and sex-specific changes in hormone levels [26].

The hypothalamus maintains bodily homeostasis by regulating metabolic processes, the autonomic nervous system, and neurohormonal secretion [12]. To evaluate changes in the HPG axis, hormone and gene expression levels have been measured in animal models of EAE [8]. Dark Agouti (DA) rats with EAE exhibited elevated levels of Npy and Argp in the hypothalamus. The concentrations of growth hormone (Gh), growth hormone-releasing hormone receptor (Ghrhr), and growth hormone receptor (Ghr) rose in the pituitary gland [8]. Corticotropin-releasing hormone (Crh) expression was down-regulated in both sexes. On the other hands, EAE-induced animals showed the decreasing insulin like growth factor-1 (IGF-1) in serum. These results are closely associated with chronic inflammation-induced GH resistance, with body weight loss during EAE progression (Zivkobic et al., 2024). Male DA rats exhibited decreased levels of plasma adrenocorticotropic hormone on onset stage, but there were no significant changes [27]. Mice in which EAE was induced via injection of MOG exhibited changes in the levels of hypothalamic neuropeptides associated with stress and feeding behavior, revealed via longitudinal profiling of the transcriptome [12]. The hypothalamus-pituitary-adrenal (HPA) axis became less sensitive to inflammation. In chronic-relapsing remitting EAE model of female DA rats, the corticosterone level decreased, associated with disease progression, hind-limb paralysis, and infiltration of inflammatory cells [28].

The pituitary gland or hypophysis features two main lobes, the anterior adenohypophysis and posterior neurohypophysis, which together control energy production, metabolism, and reproductive function [29]. The pituitary gland responds to GnRH secreted from the hypothalamus by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the bloodstream [29]. Growth hormone-releasing hormone (GHRH) produced by the the arcuate nucleus of the hypothalamus is a representative neuropeptide that stimulates GH secretion by the pituitary gland. GHRH-deficient mice do not develop MOG-induced EAE [30]. In DA rats with EAE, the levels of Gh, Ghrhr, and Ghr increased in the pituitary gland [8].

In summary, inflammatory mediators of CSF in EAE patients diffuse into the brain parenchyma, including the hypothalamus and pituitary gland. This in turn reduces the secretion of GnRH, LH, and FSH. These hormonal imbalances may affect the reproductive organs and cause reproductive dysfunction.

Testicular changes in EAE

To assess the role played by the hypothalamic-pituitary-testis axis in MS, hormone levels and sperm quality have been evaluated [31]. The serum levels of LH, FSH, and testosterone were decreased. Sperm numbers were low and sperm motility poor; fertility was greatly reduced [20, 31] The serum level of testosterone was low. Testosterone injections improved the sexual dysfunction [32].

In SJL/J mice with PLP-induced EAE, it has been suggested that the observed decrease in testosterone might be attributable to reduced activity of Leydig cells induced by both pro-inflammatory cytokines and an increased level of serum LH. The HPG axis was thus adversely affected [32]. A high LH level indicates that testosterone production is too low to support reproduction. This is often observed in patients with autoimmune conditions.

In Lewis rats with EAE, the mRNA expression levels of IL-17 and IFN-γ diminished but increased at the late phase of the disease [33], suggesting that the testes also mounted an inflammatory response. Testicular steroidogenesis was suppressed in DA rats with EAE but there was no evidence of testicular inflammation. Expression of the insulin-like 3 gene was reduced at the peak of disease, implying that Leydig cells were then functionally suppressed [34]. Although testicular inflammation appeared to be absent, PCR tests revealed increased expression of pro-inflammatory cytokines that functionally suppressed Leydig cells. The testosterone levels were reduced in animal models of EAE, and humans with MS, reflecting the action of LH produced by the hypothalamus [34].

An inverse correlation between the circulating total testosterone and corticosterone levels was apparent in Wistar rats in the acute phase of EAE induction [35]. This suggests that hormonal changes greatly contribute to EAE progression.

Ovarian changes in EAE

DA rats with EAE exhibit various ovarian changes such as prolonged diestrus, maintenance of the corpus luteum, increased intraovarian progesterone levels, and a pseudopregnancy state evidenced by an increased level of steroidogenic acute regulatory (StAR) protein [36]. The corpus luteum is the temporal endocrine gland of ovary and is deeply associated with the maintain of estrous cycle and pregnancy [37]. The residual corpus luteum and increased progesterone levels may affect reproductive function by preventing the formation of a normal menstrual cycle. The ovarian mass tends to be reduced in mice with MOG-induced EAE, associated with follicular atresia and fewer viable follicles [12]. Such findings suggest that EAE induces the follicular atresia which is the loss of ovarian follicles via apoptosis. We postulate that ovarian developmental disorders, such as ovarian atrophy, may be caused by abnormalities in the secretion of hormones, including GnRH, LH, and FSH.

Hyperplasia of the adrenal glands in EAE

It is generally agreed that the adrenal glands are enlarged in both mice with MOG-induced EAE [13, 38] and DA rats with EAE [26, 27]. Notably, MOG-induced EAE accompanied by hyperplasia of the adrenal gland and ovarian atrophy has been associated with dysregulated metabolic pathways, including peroxisome proliferator activated receptor γ-dependent fatty acid metabolism and cholesterol biosynthesis [13]. Elevated levels of glucocorticoids in male DA rats with hyperplasia of the adrenal gland are strongly linked to the suppression of HPA activity, such as decreased hypothalamic Gnrh 1 expression [26]. This is especially marked in males, suggesting increased production of adrenal corticosterone that may suppress both ovarian and testicular function [27]. The between-gender differences require further study.

CONCLUSION AND PERSPECTIVES

This review describes occasional dysfunction of the reproductive system during the course of human MS and in animal models of EAE. This is associated with a varying extent of CNS inflammation, including the hypothalamus. This affects hormonal production by both the hypothalamus and pituitary gland. Ultimately, the HPG axis becomes dysfunctional (Fig. 2). Alternatively, reproductive dysfunction may be attributable to inflammation of the reproductive organs, the ovaries and testes, and is mediated by both direct infiltration of inflammatory cells and the actions of serum pro-inflammatory peptides. It is possible that both HPG axis dysfunction and local inflammation synergistically compromise reproduction in the presence of EAE. The mean age of diagnosis in MS is 32 years old, and the prevalence of MS is twice as high in women as in men [39]. As we know it, no specific relationship has been identified between the MS/EAE and infertility. However, recent research suggests that there is a correlation between MS progression and infertility. It is thought that there is a high correlation between MS and reproductive dysfunction. Therefore, there is a need to further investigate the relationship between disease and reproduction using the EAE model.

Fig. 2.

A schematic of changes in the hypothalamus-pituitary-gonadal (HPG) axis in animal models of experimental autoimmune encephalitis (EAE). Normal: GnRH from the hypothalamus stimulates the pituitary gland. The anterior pituitary gland secretes LH and FSH that target either the ovaries or the testis. EAE: In EAE, inflammatory mediators in cerebrospinal fluid (CSF) diffuse into the brain parenchyma, including the hypothalamus, and downregulate the expression of Kisspeptin 1 and GnRH. This in turn reduces secretion of LH and FSH by the pituitary gland. Spermatogenesis and testosterone production are reduced in male animals with EAE. In females, the ovaries become atrophied and the uterus is then dysfunctional. Red arrows: upregulated genes. Blue arrows: Downregulated genes. The genes of the HPG axis are those defined by Carver et al. (2024a, b). Argp, Agouti-related peptide; Crh, corticotropin-releasing hormone; Gh, growth hormone; Ghrhr, growth hormone-releasing hormone receptor; Npy, neuropeptide Y; FSH, follicle-stimulating hormone; LH, luteinizing hormone; StAR, Steroidogenic acute regulatory protein.