Multiple sclerosis: Glial cell activation – Biomarker advances and their translational significance

Abstract

The diagnosis of multiple sclerosis (MS) predominantly hinges on the 2017 McDonald criteria, integrated with adjunctive diagnostic modalities including cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI). Notwithstanding their utility, these established methods exhibit inherent limitations in fully elucidating the complex histopathological transformations characteristic of MS. Specifically, they encounter difficulties in precisely quantifying and standardizing patient-specific pathological features, and often lack the requisite precision to delineate the disease's nuanced pathological hallmarks. Recent advancements in neuroimaging, biomarker discovery, and genomic profiling have yielded substantial evidence implicating glial cell activation as a central pathogenic mechanism in MS. In particular, the activation of astrocytes and microglia has been shown to play a pivotal role in disease progression, precipitating a cascade of events that culminate in neuronal axonal demise. The identification of distinct glial cell subsets represents a critical step towards the development of targeted therapeutic strategies. Biomarker-based approaches offer a powerful paradigm for in-vivo interrogation of disease processes, facilitating a more comprehensive understanding of MS pathophysiology and potentially paving the way for personalized treatment modalities. This paper provides a comprehensive overview of the current research on the pathophysiology of glial cells in MS, emphasizing newly identified biomarkers with potential value related to the damage of microglia and astrocytes in MS in recent years.

Introduction

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease characterized by inflammatory demyelination and activation of astrocytes and microglia^1,2 Recent studies have highlighted the significant role of glial cell activation in the development of MS, particularly in astrocytes and microglia, affecting the inflammatory response and degeneration of axons.^3–5 Resting microglia, the central nervous system (CNS) immune sentinels, transform into an activated state via diverse cues, including genetics, Epstein‒Barr virus (EBV) infections, smoking and obesity, pollutants and excessive exposure (e.g. UV), and microbiome. Once activated, microglia release inflammatory cytokines and chemokines-double-edged mediators that support repair but, if dysregulated, fuel neurodamage (Figure 1). Therefore, there is a need for tools to differentiate between disease subtypes and determine disease activity stages. ⁶ Magnetic resonance imaging (MRI) has important value in the diagnosis, prognosis, and monitoring of diseases. However, conventional MRI cannot specifically detect neuronal and axonal injuries, and it lacks sensitivity and specificity for neurodegeneration. Moreover, determining the image acquisition protocol and scanning time of MRI remains a challenge. ⁷ Therefore, there is an urgent need to find new detection methods. Search for biomarkers for MS has gained significant attention due to advancements in research techniques. While technologies such as single-molecule sequencing, proteomics, and single-cell sequencing have contributed to exploring disease progression and potential therapeutic targets, there is still an urgent need for reliable and user-friendly biomarkers to identify the risk of disease progression. ⁸ This article reviews the existing literature, and explores potential links between the glial cell activation and MS, shedding light on its evolving significance in the context of MS pathogenesis. The authors consider the pathophysiological implications of glial cell activation in MS, and the impact of glial cells as biomarker on neuroinflammation or neurodegeneration. By offering an understanding of the intricate interplay between the glial cell and MS, this review provides valuable insights which glial cells as biomarkers are promising in MS.

Figure 1.

Genetic, environmental, and lifestyle factors influencing the activation of microglia of MS.

Glial cells and MS

Pathophysiological functions of glial cells

Microglia and astrocytes play crucial roles in maintaining homeostasis by supporting neuronal activity, development, and survival. ⁶ And this, they may undergo functional alterations in response to inflammation or brain injury, and become activated into two distinct phenotypes: one exhibiting a neuroprotective response and the other a pro-inflammatory on.^7,8 Proinflammatory microglia exhibit increased phagocytic activity and produce higher levels of proinflammatory cytokines, ⁹ while activated astrocytes contribute to scar formation and release inflammatory mediators and are associated with synaptic degeneration and imbalances in the neurotransmitter glutamate.^8,10,11 Furthermore, the activation of microglia and astrocytes may contribute to the progression of neurodegenerative pathways and lead to disability in MS. ¹² The activation of microglia is also linked to the demyelination of the CNS, ¹³ and late-stage microglial activation is associated with chronic neurodegeneration and damage to axons in MS. ¹⁴

Glial cells in MS pathophysiology

At present, the widely recognized pathological mechanism of MS is that, at the beginning of the disease, peripheral activated T lymphocytes infiltrate the CNS by changing the permeability of the blood‒brain barrier, and the activated lymphocytes attack the myelin sheath, triggering an immune inflammatory response. Peripheral immune infiltration is a hallmark of relapsing-remitting multiple sclerosis (RRMS). ² MS begins with a relapsing-remitting course and then progresses to a hidden deterioration of disability, which is unrelated to clinically obvious relapses and is known as secondary progressive multiple sclerosis (SPMS). ³ However, recent studies have found that glial cells not only participate in the initial disruption of the blood-brain barrier, ¹⁵ but also play an important role in the subsequent immune inflammatory response. Research confirms that transition of neural glial cells from a stable physiological state to a reactive pathological state is responsible for the development of focal inflammatory demyelinating lesions in MS. This transition is accompanied by disruptions in the blood-brain barrier and infiltration of immune cells into the surrounding parenchyma. Histopathological studies suggest that the onset of MS and damage to the CNS are associated with the activation of microgliacytes and proliferation of reactive astrocytes. ¹⁶ On one hand of active lesions, reactive astrocytes release chemokines, activate microglia, and increase the permeability of the blood-brain barrier, allowing macrophages and T lymphocytes to migrate into the CNS parenchyma. ¹⁷ On the other hand of chronic lesions, damaged astrocytes contribute to the formation of glial scars. The activation of astrocytes plays a crucial role in triggering the immune system cascade, leading to neuronal damage, inflammatory demyelination, and axonal degeneration. In addition to axonal injury, activated astrocytes are believed to be significant contributors to neural injury, dysfunction, and the progression of the MS. ¹⁸ The progression of MS is characterized by the transition of microglia and astrocytes from a steady-state anti-inflammatory phenotype to a pro-inflammatory phenotype in the cell compartment of the CNS.^10,19,20

Role of astrocytes in MS

Astrocytes are classified into type A1 (pro-inflammatory, present in gray matter) and type A2 (anti-inflammatory, present in white matter), with their activation states dynamically changing throughout the course of MS. ²¹ Reactive astrocytes expressing complement factors (e.g. C3) ²² and complement system regulators (e.g. SerpinG1) are characterized as type A1 and have been identified in lesions of some inflammatory MS cases. ²³ In contrast, other reactive astrocytes, which are associated with cerebral ischemia, are designated as type A2.^23,24

Antigen presentation

By activating β2-adrenoceptor, norepinephrine can inhibit astrocytes expression inflammatory cytokines, including tumor necrosis factor and interleukin-1β. ²⁵ Astrocytes do not express MHC II and B7 molecules under normal conditions and many pathological conditions related to inflammation. This is because the expression induced by IFN-γ is strongly inhibited by some neuromodulators such as norepinephrine and vasoactive intestinal peptide. However, in patients with MS, due to the lack of expression of β2-adrenoceptor on astrocytes, astrocytes can act as antigen presenting cells to perform the function of microglia under the induction of IFN-γ, thus initiating the inflammatory cascade reaction.^26,27Studies have shown that astrocytes stimulated by IFN-γ can activate CD8⁺ and CD4⁺ T cells.

Participate in the destruction of blood-brain barrier

Blood‒brain barrier is composed of a neuroglial membrane surrounded by the continuous capillary endothelium of the brain and its tight connections between cells, intact basement membrane, pericytes, and astrocytes end foot. In inflammatory CNS conditions such as MS, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. By using myelin oligodendrocyte glycoprotein (MOG35-55), Argaw et al. induced the C57BL/6 experimental autoimmune encephalomyelitis mouse model (EAE, recognized as a commonly studied mouse model for MS), which is considered an animal equivalent of MS and found that astrocytes are involved in the disruption of the blood cerebrospinal fluid (CSF) barrier. ²⁸ In addition, Chapouly et al. used the EAE model to study that reactive astrocytes activate VEGF-A and ECGF-1, which jointly producing 2-deoxyribose and acting on the permeability of the blood-brain barrier, ultimately leading to its destruction. ²⁹ After the blood-brain barrier is disrupted, antigen-specific T cells enter the nervous system, causing a chain reaction of cytokines and leading to myelin sheath loss.

Release of active substances

Activated astrocytes can secrete various pro-inflammatory cytokines and chemokines, which will participate in the pathological changes of MS as direct or indirect immune mediators or inflammatory mediators. In MS, activated astrocytes are considered harmful on the one hand. It can release cytokines such as NO, TNF, IL-6 and other pro-inflammatory factors to aggravate the pathological changes of MS. ³⁰ Constantinescu et al. believe that astrocytes play an important role in activating Th1 and Th17 by secreting IL-12/IL −23, and Th1 or Th17 are involved in the pathogenesis of MS. ³¹ Saikali et al. demonstrated that astrocytes could synthesize IL-15, destroyed the activation of CD8⁺ T cells, and aggravated the tissue damage in the pathogenesis of MS. ³² The researchers have found that the inactivation of astrocyte-specific Clec16a increases the activation of NF-κB, NLRP3, and gasdermin D in vivo, exacerbating experimental autoimmune encephalomyelitis in the mouse model of MS. In addition, we have also verified this in the individual samples of MS patients. ⁹Moreover, in both acute and chronic EAE models, the number of ACLY p300 memory astrocytes increases, and re-challenge intensifies the pro-inflammatory response. ¹⁰ On the other hand, astrocytes also have the ability to promote the survival of neurons by synthesizing growth factors and neurotrophic factors. Watzlawik et al. also demonstrated that in EAE, astrocytes had a powerful role in synthesizing Platelet-derived growth factors (PDGF) and promoting the proliferation of oligodendrocyte precursor cells (OPCs) mediated by IgM, thus contributing to myelin regeneration. ³³

Inhibit myelin sheath and axon regeneration

Oligodendrocytes are important cells for regeneration of myelin sheath and axons. In order to ensure myelin regeneration, OPCs must migrate to the demyelinating region, and then mature into oligodendrocytes to promote myelin regeneration. Hyaluronic acid is good at inhibiting the ability of OPC maturation. Study has shown that HA(an acidic glycosaminoglycan and key extracellular matrix component, can regulate cell differentiation and proliferation) is found in astrocytes in the lesion area of MS, and by acting on the Toll-like receptor 2, it can inhibit OPC maturation and myelin regeneration. ³⁴ Some researchers have also found that in MS, astrocytes proliferate and hypertrophy, secrete nutritional factors and cytokines, form glial scars, block the migration of oligodendrocyte progenitor cells, and thus hinder myelin sheath and axon regeneration. ³⁵ It has also been found that astrocytes play an important role in the phagocytosis of myelin debris by recruiting microglia to the demyelination site and producing regenerative mediators. ¹¹

Role of microglia in MS

It is generally agreed that microglia is the main immune effector cell of CNS, which has the function similar to peripheral macrophages and plays a key role in the inflammatory response of the CNS. Research indicated that the activation of microglia occurred 2‒4 weeks before demyelination. ³⁶ In MS and EAE, microglia not only participates in CNS injury caused by immune response, but also presents an important role in disease recovery and nerve regeneration. ³⁷

Antigen presentation

Microglia are the main antigen-presenting cells (APCs) in the brain, and it can express MHC II and co-stimulatory molecules CD80, CD86, and CD40, enabling it to present antigens to CD4 T cells and participate in reactive T cell infiltration. ³⁸ Through cross-presentation function, microglia will present myelin proteins to CD8 T cells in the case of inflammatio. ³⁹ After capturing antigens, microglia present MHC-II molecules to the cell surface to form antigenic peptides-MHC-II molecular complexes, which then bind specifically to T cell receptors, thus providing the first signal of T cell activation CD80, CD86, and CD40 bind to T cell surface adhesion molecules (CD28, etc.) to provide a second signal of activation, which enables T cells to activate and proliferate into effector cells. Minocycline can reduce the severity of EAE by reducing the expression of MHC-1I molecules in microglia, thereby inhibiting the antigen-presenting function of microglia. ⁴⁰

Microglia secrete active substances involved in MS and EAE pathogenesis

Activated microglia can produce a variety of effector molecules, such as TNF-α, IFN-γ, IL-2,IL-4,IL-6, IL-10, IL-1β, IL-1α, IL-12, IL-8, IL-23, IL-17, NO, and prostaglandins, etc., which play an important neurotoxic or neuroprotective role in the pathogenesis of MS/EAE. Centonze et al. found that in the early lesions of EAE, activated microglia can release a large amount of TNF-α, improve the transmission and release of amino acids in the striatum, and damage CNS nerve cells through the excitotoxic effect of glutamic acid, thus promoting synaptic degeneration and dendritic spine loss. ⁴¹ IL-6, IL-1β,IL-23 and IL-12 secreted by microglia can polarize Th0 into Th1 and Th17, and then participate in the pathogenesis of EAE. ⁴² IFN-γ, mainly secreted by TH1, enables microglia to express IL-23. However, IL-23 plays an important role in Thl7 proliferation and maintenance of inflammation. If IL-23 is lacking, EAE cannot be induced. ⁴³ IL-17 and IL-23 bind to the corresponding receptors on microglia, which can make microglia secrete more inflammations and chemokines to further enhance the MS/EAE inflammatory response.^44,45 IL-23 regulates Th17 differentiation via the STAT3/NF-κB pathway ⁴⁶ ; Th1 cells can induce M0 polarization into M1 macrophages, while Th17 cells secrete IL-17 and mediate BBB damage through ROS production. ⁴⁷ Moreover, the proportions of Th17 cells and IL-17 are increased in the CSF and peripheral blood of patients with RRMS,^14,48 and Th17 cells are involved throughout the course of MS. However, the mechanism underlying the role of Th17 cells in MS remains unclear. ⁴⁹

Phagocytosis of myelin fragments

In MS lesions, there are a large number of myelin fragments produced by denatured nerve cells. Microglia can trigger the release of pre-inflammatory factors and NO by phagocytosis of myelin proteins, and promote the occurrence and development of neuroinflammation. On the other hand, myelin fragments contain growth-inhibiting factors such as NogoA, which inhibit the maturation of oligodendrocytes and the growth of axons, and promote axon regeneration by phagocytosis of myelin fragments. ⁵⁰ Recent studies using cuprizone (CPZ) as an animal model of CNS demyelination have shown that the upregulation of signaling proteins in microglia promotes the effective phagocytosis of myelin debris (Figure 2). ¹¹

Figure 2.

Schematic diagram of the pathogenesis of glial cells in multiple sclerosis.

Recruiting stem cells and promoting neurogenesis

Adult neural stem cells and neural progenitor cells are usually in a relatively static state and have the potential to self-renew and differentiate into neurons or glial cells. Some scholars believe that microglia can secrete nerve growth factors such as IGF-1 in MS lesions, promote the recruitment of oligodendrocyte progenitor cells and nerve regeneration, and participate in maintaining the integrity and stability of the CNS. ⁵¹

Glial cell activation in MS imaging

Throughout the progression of lesions and the course of MS, the brain's chronic inflammatory response includes astrocytes activation and microglial activation, as well as recruitment of peripheral macrophages. However, activation of glial cells in multiple sclerotic lesions can be observed and tracked with special imaging technology. Magnetic resonance spectroscopy (MRS) myoinositol and positron-emission tomography (PET) [11C] PBR28, as independent means of measuring the inflammatory process, can appear together in severe inflammatory diseases such as MS. Microglia activation measured by uptake of [11c] PBR28 combined use of MRS is associated with loss of neuronal integrity and gray matter atrophy. ⁵² the 18 kDa translocator protein (TSPO) has a low expression in the brain under physiological conditions and can be upregulated by activated glial cells. ¹⁸ TSPO-PET imaging can quantify the activation of microglia/macrophages and is also an important indicator for predicting the progression of MS. ¹⁹ The visualization of astrocytes can be samely achieved using 18F-THK5351 PET imaging, which accumulates in plaques of MS. Additionally, astrocytes activation also can be indirectly assessed by measuring astrocytes metabolism using 11C-acetate ¹⁰ Proton MR spectroscopy (1H-MR spectroscopy) is a well-established method for the in vivo investigation of the normal-appearing white matter in patients with MS. A significant increase of the activity of the glial cells can only be observed in patients with an established diagnosis of MS but not in patients with clinically isolated syndromes (CIS). Axonal damage occurs already during the first demyelinating episode in patients with CIS as well as in patients with definite MS. ⁵³ While these technologies have played a role in the detection of MS, the potential and usefulness of new advanced detection and diagnostic technologies in MS patients still need to be fully evaluated and created.

Comparison of biomarkers in serum versus cerebrospinal fluid

The use of serum-based biomarkers offers a unique advantage, as blood can be easily and routinely collected in a minimally invasive manner from large cohorts of patients. These biomarkers are likely to reflect peripheral immune mechanisms and may indirectly provide insights into immune mechanisms in the CNS.^54,55 On the other hand, CSF represents the most direct source of biomarkers and is closely related to the pathology of the disease. ⁵⁶ The CSF biomarkers may better represent pathological processes in the CNS due to their proximity to the brain and spinal cord compared to serum biomarkers. The levels of neurofilament light chain in the cerebrospinal cord (CNfL) and the immunoprotein IgG index increase. Among them, oligoclonal bands and the IgG index are helpful for the diagnosis of MS, but they are not ideal biomarkers for predicting the disease progression. Oligoclonal bands (OCB) lack specificity, ²⁰ CNS infection, autoimmune encephalitis, and even noninflammatory neurologic diseases can also be associated with OCB.^57,58 While the IgG index was included as a subanalysis, its sensitivity for detecting intrathecal IgG synthesis is limited. ⁵⁸ Glial fibrillary acidic protein (GFAP) in the CSF of patients with SPMSis higher than that in patients with RRMS. ²⁵ Astrocytes in acute lesions are negative for aquaporin 4 (AQP4), a water channel molecule, ²⁶ but AQP4 cannot be detected in the cerebrospinal fluid. Changes in fatty acid and lipid metabolism have also been detected in the CSF. ²⁷ Bile acid receptors are present on glial cells. Appropriate supplementation with bile acids can prevent astrocytes and microglia from polarizing into a neurotoxic phenotype. ²⁸ 27-hydroxycholesterol (27OHC) is formed outside the brain and crosses the blood-brain barrier proportionally to the barrier dysfunction. CSF 27OHC is associated with extensive neurodegeneration. ²⁹ However, there is a lack of data regarding astrocyte-associated biomarkers in serum in clinical practice, and the application of CSF biomarkers still requires thorough validation. Additionally, despite the extensive investigation of many CSF biomarkers in MS, inconsistent results have hindered their clinical application (Figure 3). ⁵⁹

Figure 3.

Biomarkers, clinical roles, limitations.

Biomarkers

Early identification of biomarkers for MS can help us refining the clinical stages and the risk stratification of early treatment to reduce disability. ⁶⁰ In recent years, scientists have sought to explore heterogeneity from novel perspectives, such as the gut-brain axis and gut microbiota, Because MS displays a notable heterogeneity in terms of imaging, histopathology, and clinical course, it raises a challenge for identifying biomarkers. Activation of glial cells has always been considered a marker of neuroinflammation or neurodegeneration. With the change of environment, glial cells can show different phenotypes and gene expression profiles, and the more common ones are astroglia and microglia. The biomarkers of astrocytes activation were GFAP, S100B; and biomarkers of microglial activation include sTREM-2, CHI3L1, and CD14.

Biomarkers of glial cell activation play a crucial role in clinical practice

In MS, the activation of microglia and astrocytes can contribute to neurodegenerative pathways. Neuroinflammation is a key factor in all types of MS, and inflammation in the CSF promotes disease activity and increases disability in patients with RR-MS.^61,62 Glial cell involvement is necessary in both PP-MS and SP-MS, and the activation of glial cells is believed to be closely associated with the progression of disability.

The limited effectiveness of immunomodulatory treatments in progressive MS suggest that factors other than innate immune activation may contribute to disability progression in later stages of MS. Current treatments for RRMS is to target the peripheral immune system, but have not been successful in progressive MS. Therefore, a better understanding of inflammation driven by CNS-resident cells, such as astrocytes and microglia, is needed to identify new potential therapeutic opportunities. ⁶³ In conclusion, in the emerging therapeutic era of PPMS, it is more important than ever to assess glial cell activity in vivo. ⁶⁴ Due to the complex relationships between biomarkers, further studies are necessary to identify the origins of these markers, deepen our understanding of their biological roles, explain their uses as biomarkers, and recognize new therapeutic targets.^65–68 Several studies have demonstrated a correlation among GFAP, sTREM-2, and cytokines in the CSF, suggesting a link between central inflammation and the pathological reactivity of microglia and astrocytes in the early stages of MS. These biomarkers have been proposed as prognostic tools for MS.^64,69,70 Furthermore, both CSF and serum levels of these biomarkers correlate with clinical disability and neuroimaging activity in MS.^71,72

Biomarkers related to glial cell activation

Many studies have shown that neuronal and axonal damage play a significant role in the progression of disability in MS. However, it has been observed that glial cells also contribute to the pathophysiological mechanisms of MS, especially in late stages where immunomodulatory treatments are less effective. Pathological studies have identified abnormal activation of astrocytes and microglia, leading to oligodendrocyte and neuronal degeneration. Despite this knowledge, there is a lack of measurement of glia-specific processes in the CNS. Therefore, the clinical management of MS can be greatly facilitated by the identification of biomarkers of neurons and glial cells that can aid in early diagnosis, improve patient care, and facilitate disease management. In addition, biomarkers that contribute to the objective monitoring of disease diagnosis, patient typing, disease progression, and treatment response are essential for the development and implementation of various therapies. ⁷³

GFAP

Is a cytoskeletal protein expressed by astrocytes and represents an established marker associated with astrocytes activation and proliferation, ^{10 74–76}. Studies have shown that increased expression of GFAP in CSF may characterize patients with a higher risk of progression, and its levels are higher in progressive MS than in RRMS, correlating with disability progression.^64,77,78

The level of CSF-GFAP is positively correlated with the EDSS score and can serve as a supplementary indicator for prognostic assessment .Furthermore, serum GFAP levels have been found to correlate with clinical severity and MRI lesion counts, particularly in patients with progressive MS, making it a potential marker of disease progression. ⁷⁶ GFAP is also a useful biomarker of acute inflammation in patients with RRMS and CIS. ⁷⁰ Serum GFAP levels have been also demonstrated to correlate with disease progression, higher extended disability scale, and longer disease duration. ⁷⁸ Consequently, it can be concluded that elevated GFAP levels in the CSF of MS patients are indicative of astrocytes damage and astrocytes proliferation, GFAP expression is associated with astrocyte activation, and its regulatory mechanism involves the complement C3 and IL-6 pathways^79,80 potentially identifying patients at a greater risk of progression.

sTREM-2

sTERM-2 is currently recognized as a biomarker for the activation of macrophages and microglia in the CNS . ⁸¹ sTREM-2 is increased in CSF in MS patients and can be used as a surrogate indicator of microglia activity. ⁶¹ This finding supports the hypothesis that both microglia and astrocytes are involved in initiating and sustaining the inflammatory response in MS. Furthermore, it suggests that the inhibition of anti-inflammatory function by relevant receptors on the cell surface may be associated with the correlation between microglia and astrocytes activation. Several studies have reported a strong positive correlation between GFAP and sTREM-2 levels in the CSF. TREM-2 acts synergistically with TLR2/4; TLR4 activates the NF-κB pathway to promote TREM-2-mediated inflammatory responses.^82,83 Additionally, some studies have found a positive correlation between GFAP and sTREM-2 levels, as well as the levels of various inflammatory cytokines, indicating the association between microglia and astrocytes activation and CSF inflammation. ¹⁰ Moreover, sTREM-2 levels in the CSF have been found to correlate with the Expanded Disability Status Scale and disease severity. Studies have shown that oxidized phospholipids (OXPC) are identified as effective neurotoxins, and the enhancement of OXPC clearance mediated by TREM2 by microglia may contribute to the prevention of neurodegeneration in MS. ³⁴ However, this correlation has not been consistently reported in all studies, possibly due to variations in the disease course among different patients. These findings suggest that measuring sTREM-2 in the CSF could be useful for monitoring disease progression and treatment effectiveness in clinical trials. ⁸⁴ Future studies could be conducted to determine whether sTREM-2 or its indicated activation of macrophages and microglia causes lesions within the CNS, or whether lesions within the CNS lead to the activation of macrophages and microglia and an increase in sTREM-2 production.^82,83

S100B

A small Ca2⁺-binding protein, highly expressed in the CNS following injury, is a crucial factor in the inflammatory process of MS lesions and a promising target for therapy. S100B binds to the RAGE receptor, activates the NF-κB signaling pathway, and promotes Th1 cell differentiation. In the EAE model, elevated S100B levels are associated with Th1 cell infiltration and glial cell proliferation.^85–87 At the time of RRMS diagnosis, S100 B levels were significantly higher in both CSF and serum. Furthermore, S100B expression was notably increased in active lesions, primarily found in reactive astrocytes. ⁸⁸ There was a trend toward elevated S100B levels from PPMS to SPMS to RRMS. S100B can differentiate between different types of MS and assess therapeutic efficacy. ⁸⁹ These findings suggest that S100B has the potential to be a valuable biomarker for distinguishing between different types of MS. However, it is controversial that other researchers also elaborated that there was no significant difference in S100B among patients with different types of MS. ⁹⁰ Additionally, high concentrations of S100B can stimulate microglia to secrete proinflammatory cytokines. ⁹¹ Inhibition of S100B in an ex vivo demyelination model has shown reduced inflammatory responses. ⁹² From the above description, it is not difficult to conclude that the high expression of S100B in MS patient samples indicates its effectiveness as a diagnostic biomarker for MS, and the beneficial results of inhibiting S100B in demyelinating models suggest that S100B is an emerging therapeutic target for MS. However, further studies are necessary to determine whether varying levels of S100B correlate with different stages of MS and can be used as a prognostic tool.

CD14

A receptor that is upregulated in activated microglia, plays a crucial role in inflammation of the innate immune response. Recent evidence suggests that the innate immune system contributes significantly to the development and progression of MS. ⁹³ Studies have shown that local microglia and migrating hematopoietic macrophages are involved in antigen presentation and myelin destruction, indicating a disease-promoting role of macrophages in MS pathogenesis. ⁹⁴ CD14 is highly expressed on macrophages and monocytes, and it can also exist in a soluble form called sCD14. Research has demonstrated the upregulation of CD14 in MS, ⁹⁵ and elevated levels of sCD14 have been detected in experimental autoimmune encephalomyelitis, as well as in the serum of MS patients.^96,97 As a receptor for LPS, CD14 activates signaling pathways by binding to the LPS-LBP complex, thereby promoting the production of pro-inflammatory cytokines (e.g. IL-6 and TNF-α) and exacerbating inflammatory responses in MS. Additionally, studies have demonstrated that serum levels of soluble CD14 (sCD14) are significantly elevated in MS patients and negatively correlated with clinical disease activity. Specifically, sCD14 levels are higher in patients in the stable phase compared to those in the acute or progressive phase, suggesting that it may serve as a predictive indicator of disease stability. ⁹⁸ Measurement of sCD14 serves as a biomarker for microglial activation and is a potential indicator of MS severity, enabling the monitoring of disease activity and responding to therapy.

YKL-40

Also known as CHI3L1, is a glycoprotein belonging to the family of chitinase-like proteins. Obesity is both a contributing factor to CHI3L1 elevation and more prevalent in MS patients.^99,100 It is primarily produced by reactive astrocytes, ¹⁰¹ with some contribution from microglia. YKL-40 levels are increased in CSF of MS patients, ¹⁰² although its physiological role remains unknown. ⁶⁸ Both YKL-40 and GFAP levels in the CSF are independent prognostic markers for MS disability progression, suggesting that YKL-40 could be a valuable biomarker for monitoring disease progression in SPMS. ¹⁰³ Elevated levels of YKL-40 and GFAP have been associated with early disability progression in MS. ⁶⁰ In patients with MS, serum YKL-40 levels are positively correlated with EDSS scores, suggesting that YKL-40 can serve as a quantitative indicator for assessing disease severity.^104,105Moreover, YKL-40 has been found to be overexpressed in the CSF of CIS patients who later develop MS, and it is linked to a faster accumulation of disability.^71–74,75 In addition to CSF, serum levels of CHI3L1 also have diagnostic value in distinguishing MS from CIS, as well as in predicting MS progression. However, its ability to differentiate between different types of MS, such as SPMS, PPMS, and RRMS, has shown inconsistent results among studies.^106–108

Research progress

The heterogeneity in the course and pathological changes of MS necessitates the identification of multiple biomarkers for diagnosis, prognosis, and therapeutic response. Currently, a single biomarker may not be effective, and future studies should focus on combinations of several biomarkers. ⁷³ Statistical analyses and pattern detection tools will play a crucial role in identifying the most appropriate biomarker combinations that reflect clinical status. ¹⁰⁹

In addition, future research should (1) develop new diagnostic prognostic models,^4,97,110 (2) analyze different biomarkers individually or in combination with MRI, (3) further characterize the origins and biological roles of each biomarker, and(4) The Role of Dynamic Changes in Glial Cell Phenotypes at Different Stages of MS. This ongoing research will not only enhance our understanding of biomarker use but also aid in the identification of new therapeutic targets. ¹⁰⁸

Challenges

Clinically useful and relevant markers in MS include MRI markers.^110,111 However, one of the main challenges in studying glial cell biomarkers is their specificity. Changes in blood or CSF levels of these biomarkers must be correlated with glial activity and CNS lesions. It is important to note that the glial cell biomarkers discussed in studies also be expressed by various other cells in the peripheral immune system, such as hepatic stellate cells, testicular mesenchymal stromal cells, chondrocytes, and osteoblasts. Although some preliminary studies have shown promising results, further analyses in large patient cohorts, matched for age and sex, with full clinical validation are necessary to develop biomarkers for clinical use in progressive MS. Additionally, there is a high level of interindividual variability in glial cell biomarker levels. Therefore, it has been suggested that the use of composite metrics, such as combining biomarkers with MRI markers, should be explored to mitigate the effects of confounding and non-disease-related factors. ⁶³ In terms of using biomarkers that have not yet been clinically validated for further research, it is important to note that biomarkers studied in MS subjects cannot currently be used as surrogate endpoints. The challenges of the current study include the need for (1) further studies in clinical applications, (2) further investigation into the role of these biomarkers in differentiating between different subtypes and disease courses, (3) a larger sample size to study and determine the diagnostic thresholds for the different biomarkers, and (4) considering that these biomarkers can also be expressed in different demyelinating diseases.

Conclusion

For MS, there is still no clear and unified standard in the diagnostic criteria. This has led to significant heterogeneity in diagnostic strategies in clinical practice. Second, most studies are based on animal models and in vitro experiments, and their results may not be fully consistent with those in humans with MS. With the increasing urgency of early intervention for the disease, the development of new auxiliary detection technologies has become a key area that urgently needs to be broken through. Due to the natural existence of the blood-brain barrier, the detection and intervention of CNS diseases face unique challenges. Currently, the research focus has gradually been placed on the roles of innate immunity and glial cells in the pathological mechanism of MS. Exploring the biomarkers related to glial cells can not only provide an objective basis for the precise staging diagnosis and prognosis assessment of MS, but also serve as an important breakthrough for discovering potential therapeutic targets and promoting the innovation of disease-modifying treatment (DMT) strategies.