Glial Cells as Key Regulators in Neuroinflammatory Mechanisms Associated with Multiple Sclerosis

Even though several highly effective treatments have been developed for multiple sclerosis (MS), the underlying pathological mechanisms and drivers of the disease have not been fully elucidated. In recent years, there has been a growing interest in studying neuroinflammation in the context of glial cell involvement as there is increasing evidence of their central role in disease progression. Although glial cell communication and proper function underlies brain homeostasis and maintenance, their multiple effects in an MS brain remain complex and controversial. In this review, we aim to provide an overview of the contribution of glial cells, oligodendrocytes, astrocytes, and microglia in the pathology of MS during both the activation and orchestration of inflammatory mechanisms, as well as of their synergistic effects during the repair and restoration of function. Additionally, we discuss how the understanding of glial cell involvement in MS may provide new therapeutic targets either to limit disease progression or to facilitate repair.


Introduction
Multiple sclerosis (MS) is the most common demyelinating neurodegenerative disease that affects young adults, and it is characterized by neuroinflammation of the central nervous system (CNS), during which the infiltrating peripheral immune cells cause damage to the myelin and axons of the neuronal cells [1].MS symptoms and clinical signs are variable depending on which areas of the brain and spinal cord are affected, and the disease course is heterogeneous as it may follow different degrees of severity and progression.MS is broadly categorized into the following three phenotypes: relapsing-remitting MS (RRMS), primary progressing MS (PPMS), and secondary progressive MS (SPMS), depending on the disease course.The pathological hallmark of the disease, plaque formation, is evident in multiple CNS areas, including the cerebral white matter (WM) and gray matter (GM), brainstem, spinal cord, and optic nerve.The evaluation of the lesions with MRI has proven to be a sensitive method to diagnose a patient.Gadolinium enhancement on the MRI is a marker of active and focal inflammation as the dye penetrates the broken blood-brain barrier (BBB) [2].
The loss of BBB permeability is due to the secretion of highly harmful reactive oxygen and nitrogen species (ROS, NOS) [3] from the perivascular astrocytes resulting in edema formation and infiltration of immune cells into the CNS [4].As the extracellular matrix (ECM) relaxes, there is a degradation of the molecules including elastin, generating elastinderived peptides like serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) [5].Elastin-derived peptides have been shown to interact with various receptors, leading to the activation of intracellular signaling pathways like inflammation and cell proliferation, especially with glial cells, and have also been implicated in various neurodegenerative diseases [6].Finally, ECM proteins released were shown to be deposited in lesional and perilesional areas and, since they are known potent pro-inflammatory molecules, they are thought to contribute to disease severity and remyelination failure [7].
Aside from the disruption of the BBB, T cells and, in particular, the CD4+ T cells play a key role in the disease pathology.When naïve CD4+ T cells are exposed to interleukin (IL)-12, they are polarized to the T helper (Th) 1 phenotype and produce cytokines including interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNFα).In the presence of transforming growth factor (TGF)-β and IL6, naïve CD4+ T cells become Th17 cells and secrete IL-17 and granulocyte macrophage colony stimulating factor (GM-CSF) that are pro-inflammatory in many animal models.High levels of circulating IL-17 were measured in the active MS patients compared to the healthy controls, suggesting its involvement in CNS inflammation [8].In addition to the T cells, B cells are also essential players in MS pathogenesis.B cells not only produce cytokines that are proinflammatory (IFN-γ) and regulatory (IL-10), but they also present antigens to T cells [9].Moreover, B cells form ectopic structures, follicles, in MS patients that express CD20 [10].Treatments with anti-CD20 monoclonal antibodies have been proven to be very effective and lead to disease stabilization.
During early MS, glial activation plays a key role in the initial reactive process, and is thought to be key in determining the fate of the disease progression [11].MS lesions have different characteristics depending on their state of activation and position, as well as regional glial subtype diversity [12] (Figure 1).Active lesions are characterized by reduced oligodendrocytes, the accumulation of microglia and macrophages throughout the lesion area, and myeloid cells (granulocytes and monocytes) phagocytosing myelin proteins [13], for which recent evidence has suggested that the vast majority is residentderived microglia [14].Additionally, hypertrophic astrocytes, which express proinflammatory cytokines, chemokines, and remyelination signaling molecules, are present and act as phagocytes in MS lesions [15,16].Astroglial scar formation is limited or absent at this stage [17].Chronic active lesions, where complete demyelination is evident, have a hypocellular lesion core, a sharp edge, with microglia and macrophages to be limited to the lesion border with increased major histocompatibility complex (MHC) presentation [14].Chronic active lesions also contain myeloid cells which secrete proinflammatory molecules [18].An astroglial scar is present at the core, while hypertrophic astrocytes surround the lesion border.Most importantly, remyelination can be seen at the border, indicating the healing process [19].In contrast, in chronic inactive lesions, microglia and macrophages are almost absent [20], as is remyelination.In remyelinated shadow plaques, there is limited axonal degeneration, some astroglial scarring, few microglia and macrophages, and reappearance of oligodendrocytes [21].
At present, MS management strategies mainly include disease-modifying therapies (DMTs) and monoclonal antibodies.Therapies focus on treating acute attacks, improving symptoms and reducing inflammatory activity to prevent further relapses using immunomodulatory and/or immunosuppressive molecules, while little progress has been made into treating relapses themselves.Additionally, in cases of flares-ups, corticosteroids such as methylprednisolone, which has anti-inflammatory and immunosuppressive effects, may be prescribed to limit the inflammatory response, as well as symptomatic medications for other disease manifestations.Nevertheless, depending on the disease course and clinical picture, different personalized therapeutic strategies are followed.While therapeutic strategies show sufficient efficacy in controlling or suppressing disease activity, they mainly concentrate on the initial dominating inflammation driven by adaptive immunity and not on the chronic inflammation driven by CNS innate immunity and the associated neurodegeneration.Efficient remyelinating therapy, especially at early stages of MS, may halt long-term axonal degeneration and possibly prevent secondary progression [22].This underscores the importance of investigating the glial cell involvement in the inflammatory processes and repair.During the course of the disease, lesions change in respect to glial cell population.Active lesions contain macrophages at the center, while astrocytosis starts to be present.The rim of the lesion is populated with microglia which recruit other inflammatory cells to the site, as well as myelin-phagocytosing macrophages.Mixed active/inactive lesions do not show significant astrocyte population, while there is distinct formation of microglia around the rim.Additionally, there is recruitment of OPCs.Remyelination can be seen as a shadow plaque, where oligodendrocytes slowly initiate the Figure 1.Glial cells populate lesions in a heterogeneous manner during the course of MS lesions.During the course of the disease, lesions change in respect to glial cell population.Active lesions contain macrophages at the center, while astrocytosis starts to be present.The rim of the lesion is populated with microglia which recruit other inflammatory cells to the site, as well as myelin-phagocytosing macrophages.Mixed active/inactive lesions do not show significant astrocyte population, while there is distinct formation of microglia around the rim.Additionally, there is recruitment of OPCs.Remyelination can be seen as a shadow plaque, where oligodendrocytes slowly initiate the repair process.Astrocytes and glial scar formation are clearly present at the center of the lesion.Inactive lesions show some degree of scar with few astrocytes and microglia present.Last but not least, recruitment of OPCs, and microglia nodules, can be seen in the NAWM, even in significant distance from the lesion site.OPC, Oligodendrocyte precursor cells; NAWM, normal-appearing white matter (figure was created with BioRender.com).
MS has been extensively studied using experimental animal models to both investigate pathological mechanisms as well as assess treatment efficiency.The most studied animal model is the immune-mediated experimental autoimmune encephalomyelitis (EAE) mouse model as it replicates the inflammation, demyelination, gliosis, and axonal loss that are present in MS [23].While it provides the opportunity to understand the consequences of the inflammatory response in the CNS, it shows significant differences compared to the human disease and does not always cause myelin loss [24].The viral-induced demyelination mouse model caused by the Theiler's murine encephalomyelitis virus (TMEV) is also used, as the virus-induced pathology is very similar to the human chronic progressive MS; however, the persistent viral infection of the CNS is not commonly seen in the human disease.Since the neuroinflammatory models of MS, including EAE and TMEV, have limited application in the study of the remyelination process and myelin repair, over the last decades, toxin-induced models such as cuprizone and lysolecithin (LPC) are increasingly used to study demyelination and remyelination in the CNS.Overall, the different animal models of MS, despite their limitations in not recapitulating all the pathological and clinical manifestations of the disease, they provide insights into the mechanisms of disease initiation and progression and offer useful tools for testing the safety and efficacy of novel therapeutics.
The proper immune system function is an absolute prerequisite in the regeneration process via tissue damage healing and the initiation of the repair process through cellautonomous and non-cell-autonomous machinery.Many factors control this sequence of events, including reactive astrocytes and microglia, the demyelinated axons themselves, as well as various inflammatory molecules.The degree of remyelination varies in MS lesions, while the failure of remyelination is a common finding in MS lesions and it seems to be linked to inadequate activation, recruitment, and/or differentiation of oligodendrocyte precursor cells (OPCs) [25] and the presence of significant inflammation [26,27].
The biological pathways and therapeutic potential of remyelination efficiency have gained increasing interest over the past decades.Enhancement of endogenous remyelination could be one of the most promising interventions to delay, prevent, or reverse disease progression, and several candidate remyelination targets have emerged, including macrophage-induced inflammation antioxidants and nanoparticles [28].As remyelination is a complex multifactorial process, a fine-tuning of the coordination of immune cell responses is needed to acquire a regulated reaction in terms of magnitude and duration of the response [29].Glial cells are key regulators in the orchestration of such activity in the CNS, and dissecting the functional mechanisms behind their interactions is key to both understand and target these responses.
This review aims to address the underlying pathological mechanisms by which glial cells contribute to the inflammatory process during the course of MS and how these cells have increasing potential to be targeted for future therapeutic interventions.More and more evidence has implicated oligodendrocytes, the key cells involved in myelination, in immune reactivity modulation; while their exact role has not yet been thoroughly investigated, they seem to have an active role in orchestrating the inflammatory process.Additionally, the role of astrocytes and microglia in their contribution to MS will be discussed, as they have a known role in immune mechanisms.Additionally, they show distinct patterns in and around lesions, possibly predicting disease progression.Finally, glial cell function targeting will be addressed as potential modulators of the disease course, especially during progressive MS.

Glial Cell Function and Communication
The CNS is comprised of neurons and glia, which include oligodendrocytes, microglia, astrocytes, and ependymal cells.Initially, they were regarded as passive cells, serving the physical support of neurons.However, their crucial role in the support and protection of neurons and their axons was subsequently identified, as well as their regulatory role in the efficient communication between all cells of the CNS, the maintenance of homeostasis, the facilitation of synapse formation and neurotransmission, and the detection of inflammation and tissue damage [30].Glial cells are highly plastic and, by altering their phenotype, they can adjust to their environmental needs.During early MS, it is considered that glial cell activation is mainly triggered by autoimmune responses; however, the persistence of their activation, independent of autoimmune interactions, is key to the progression of the disease.
Intercellular communication is crucial for the normal function of the nervous system.Glia and neurons communicate via the bidirectional exchange of ions, neurotransmitters, metabolites, cell adhesion molecules, and extracellular vesicles [31].Moreover, oligodendrocyte communication with the axon is crucial for the preservation of the axon survival and proper myelination, while oligodendrocytes promote neuronal survival via secretion of neurotrophic factors including insulin-like growth factor 1 (IGF-1) and glial cell linederived neurotrophic factor (GDNF) [32].However, disturbed axo-glial communication has been recognized in MS normal-appearing white matter (NAWM) and correlates to microglial inflammation [33].
The bi-directional exchange between glial cells regulates and controls their function, migration, and activity, and therefore treatments to block or induce their activation may prove effective for various immune-mediated inflammatory diseases, including MS. Glial communication is a complex, multifactorial process.Oligodendrocytes, astrocytes, and microglia have interdependent functions, exerting both beneficial and detrimental effects in MS degeneration.They facilitate various processes by secreting signaling molecules like cytokines and chemokines and therefore play a key role in the regulation of acute and chronic inflammation.
Glia cells communicate with other glia cells via the intercellular diffusion of chemical messengers [34] to maintain homeostasis and regulate synaptic transmission during development and various pathologies.Glial-glial interactions are recognized to be vital regulators in myelin health, especially astrocyte-oligodendrocyte crosstalk during myelination.The survival of mature oligodendrocytes is also dependent on astrocyte function by a downregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and an upregulation of cholesterol synthesis/export [35].Moreover, astrocytes induce OPC proliferation, which in turn upregulates the factors involved in the tightening of the BBB as well as the regulatory function of astrocytes.Oligodendrocyte-astrocyte crosstalk exploration may provide novel insights in controlling their modulatory effects and potential therapeutic targets.
Crosstalk between astrocytes and microglia is an emerging topic of interest.It is maintained via the secretion of mediators such as growth factors, cytokines and chemokines, NOS, ROS and metabolic mediators, and others [36], and has a potent role as disease modulators [37].However, they are modulated by inflammatory insults as microglia regulate the innate functions of astrocytes, their pro-or anti-inflammatory phenotype, and vice versa [38].Additionally, astrocytes modulate microglial migration and phagocytic activity.Moreover, astrocyte-microglia crosstalk may be induced through the gut-brain axis after the metabolites of dietary tryptophan act directly on microglia, and the production of vascular endothelial growth factor (VEGF)-β and tumor growth factor (TGF)-α regulate astrocyte activation [39].Astrocyte-microglia communication is also essential during injury to support neuronal survival.
A correlation between the microglial activity and oligodendrocyte damage has been established, as a wide range of inflammatory molecules have been detected in MS lesions [40].NO production is induced by the presence of IFN-γ, TNF-α, and IL-1β secreted by microglia under stress.LPC-induced microglial activation leads to the secretion of molecules that mediate the arrest of OPC proliferation and the induction of OPC death, as well as the secretion of extracellular vesicles in MS lesions [41].
Glial cell function depends on intra-and intercellular communication mediated by gap junction (GJ) channels that connect the cytoplasm of adjacent cells and are permeable to ions and various small molecules including second messengers and metabolites [42].
Myelin integrity and function depends on the proper function and communication of the glial cells and connecting neurons [48].As previously mentioned, glial cells communicate by receiving signals from neighboring cells via the Cx-formed GJ channels, which modulate a coordinated response.Previous studies have shown alterations in glial GJ expression in the brain of postmortem MS patients, both in the NAWM [49] and GM [50], while similar results were reproduced in the EAE mouse model of MS [51].Oligodendrocyte GJ connectivity was shown to have a regulatory role in inflammatory demyelination, since the ablation of Cx47 or Cx32 led to increased EAE severity compared to the wildtype (WT) [52], while EAE induction in Cx47 −/− mice led to the dysregulation of the bloodspinal cord barrier (BSCB) [53].Additionally, the ablation of Cx43 promoted remyelination by containing local inflammation [54], while the inducible conditional knockout of Cx47 increased inflammatory activation upon the induction of EAE [55].Finally, molecules that serve as GJ blockers have been investigated for the treatment of neurodegenerative diseases [56][57][58].

Oligodendrocytes and Neuroinflammation
Oligodendrocytes are the cells responsible for forming myelin and providing insulation to the axons by ensheathing them with multiple concentric layers of membrane.The assembly of a tightly packaged membrane to form a compact myelin layering allows for the electrical insulation of the axons, increased velocity of propagation, as well as reduced axonal energy consumption [59].Myelin compartments, called internodes, are formed with periodic interruptions, creating the nodes of Ranvier, which, by being highly enriched in ion channels, mainly clusters of voltage-gated sodium (Na + ) channels, facilitate the propagation of the action potential via saltatory conduction.In unmyelinated axons, the action potential travels continuously, making the propagation slower, further indicating the crucial role of myelin.Myelin is composed of water, lipids, and protein molecules, including myelin basic protein (MBP), which is expressed on the cytoplasmic surface of the plasma membrane, the surface marker myelin oligodendrocyte protein (MOG), myelinassociated glycoprotein (MAG), and the transmembrane proteolipid protein (PLP) [60].These are the main targets of autoimmune attack in MS [61].Myelin not only facilitates saltatory conduction, but it is also important to support the axon by regulating the axonal cytoskeleton and transport of molecules and to protect it from damage [62].Damage to myelin leads to demyelination and eventually axonal degeneration, which is the main feature of MS clinical manifestations [63].
Oligodendrocytes derive from OPCs, which, during development, differentiate and mature into myelinating oligodendrocytes via a tightly controlled process of activation and repression of transcription and growth factors, and then spread evenly into the GM and WM of the CNS.However, the adult CNS maintains a number of undifferentiated OPCs responsible for the production of myelinating oligodendrocytes during adulthood [61], with the white matter having a slightly higher abundance of OPCs than the grey matter [64].When axonal myelination is needed, OPCs migrate from their site of origin to the developing WM tracts in a crawling mode along the blood vessels, a process highly dependent on the Wnt signaling [65,66], leading to an excessive pool of progenitors.Oligodendrocyte generation dynamics remains a crucial step into understanding the myelination process [67].
Recent technological advances have allowed for a deeper and more delicate classification of oligodendrocytes, in relation to their differentiation state, developmental origin, and expression site.Single-nucleus and single-cell RNA sequencing (snRNA-seq, scRNA-seq) from the WM of a postmortem human brain has shown that specific sub-clusters of oligodendroglia are under-represented in MS tissue compared to the healthy controls, while others are over-represented [68,69].As the maturation state determines the mechanism of cell death that oligodendrocytes undergo in response to insult [70], these differences indicate that different functional states of oligodendrocytes are present upon tissue injury.A better understanding of their role can lead to new ways of treatment regimens [71].
Oligodendrocytes are thought to be the most vulnerable cells of the CNS due to their highly complex architecture, unique differentiation program of proliferation, migration, and myelination, as well as high metabolic demands [72,73].They are affected by various stimuli from either neighboring astrocytes and microglia or the immune cells, which may trigger oligodendrocyte injury or apoptosis.Oligodendrocytes express different cytokines, chemokines, antigen-presenting molecules (APCs), and complement receptors and have an active role in the immune modulation in the CNS.Additionally, they assess their microenvironment by extending filopodia, especially in response to inflammatory cues [74], and migrate short distances [75].
Oligodendrocytes were reported to express at least four chemokine receptors, CXCR1, CXCR2, CXCR3, and CXCR4 [77,78], which, upon binding with their respective ligands, are involved in the triggering of multiple signaling pathways.CXCL1 is upregulated in the peripheral areas of demyelination and, when recognized by CXCR2, prevents OPC migration in vivo [79].Additionally, CXCR2 signaling protects oligodendrocytes from demyelination in a viral-induced mouse model of demyelination [80], while CXCR2-positive neutrophils are necessary for cuprizone-induced demyelination [81].An important molecule in the migration, proliferation, and differentiation of neural precursor cells, CXCL12, is upregulated in the corpus callosum of mice after cuprizone-induced demyelination, along with increased astrocyte and microglia activation [82].CXCR4 is a receptor for CXCL12 signaling, and the loss of CXCR4 signaling leads to decreased OPC maturation and remyelination blockage.The CXCL12-mediated migration of OPCs is facilitated by the CXCR4-activated mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositol 3-kinase/Akt (PI3K/AKT) pathways [83].The inhibition of the mitogenactivated protein kinase (MAPK/ERK) pathway promotes oligodendrocyte differentiation and recovery from demyelination [84].Additionally, cuprizone-induced demyelination leads to high levels of CCL2, which is critical for monocyte recruitment, and IL-1β, which is involved in many immune-mediated responses [85].
In order to interact with the neighboring immune cells, oligodendrocytes secrete a broad range of cytokines.They express IL-17A, a pro-inflammatory cytokine involved in the pathogenesis of autoimmune diseases [86], with a subpopulation expressing IL-17 in active lesions [87].IL-17 −/− mice showed milder EAE pathogenesis compared to the WT, while the IL-17 neutralizing antibody administration partially relieves symptoms [88].IL-4 and IL-10 presence in primary oligodendrocyte cultures led to reduced OPC differentiation and immune responses, while exposure to TNFα led to increased phagocytic activity, cytokine production, and MHC-II expression [89].Additionally, oligodendrocytes and OPCs express MHC-II molecules in response to IFNγ, which regulated T cell survival and proliferation and activated CD4+ T cell memory and effect [67].Additionally, OPCs exposed to IFNγ can act as antigen-presenting cells to cytotoxic CD8+ T cells in vivo and in vitro, leading to cytotoxic death [67,90].Contrary to this, intrathecal and peripheral administration of IFNγ ameliorated EAE pathology, while IFNγ ablation in mice increased EAE susceptibility [91].
recombinant antibodies activate the classical complement pathway, leading to oligodendrocyte death and rapid demyelination [100].In corroboration with this, the C5b-9 complex was shown to prevent oligodendrocyte cell death via regulating the PI3K/AKT pathway [101] and inhibiting caspase-8 activity [102].
Helper T cells that drive adaptive immunity also play a key role in MS and the EAE animal model.Th17 polarization induces cytotoxic inflammation and inhibits remyelination in vitro [103].Oligodendrocytes have close contact with Th17 cells upon EAE induction, which may worsen the pathology and increase cell death, possibly by the release of cell stress-inducing glutamate due to the increased expression of integrin CD29 [104].

Oligodendrocyte and Remyelination
The process of remyelination depends on a time-tight activation and recruitment of OPCs to the lesion site, followed by proliferation, differentiation, and maturation into myelinating cells, which contribute to the regeneration process.OPCs attempt to re-establish the myelin sheath of previously demyelinated axons [105] by coming into close contact with the denuded axon, expressing myelin-specific genes, and finally forming the new myelin sheath [106].Remyelination is mostly found in acute and RRMS active lesions and borders [107], whereas mixed active/inactive lesions show a decrease in or absence of Additionally, the disruption of the BBB in MS is present also upon interaction of the vasculature with oligodendroglia, which triggers CNS inflammation by pronounced microglia/macrophage activation [65].Adhesion molecules like intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein (VCAM-1) are upregulated upon EAE induction, while blocking ICAM-1 could reduce the number of Th1 binding with oligodendrocytes in vitro, suggesting the interaction of the adhesion molecules with the CD4+ T cells [92].Kv1.4 was previously identified to be involved in controlling OPC proliferation in vitro [93] and in vivo [94] and may interact with cells of the immune system, as blocking Kv1.4 expression exerts beneficial results in the acute EAE model but not in the cuprizone.
Oligodendrocyte and OPC survival, proliferation, and differentiation depends on the inflammatory microenvironment.Presence of effector T cells, IFNγ, and ROS affects the fate of OPCs by inhibiting differentiation and therefore remyelination [61].The presence of inhibitory molecules in the inflammatory microenvironment of OPCs, like Lingo-1 that inhibits axonal regeneration after spinal cord injury [95], may also affect their fate.Similarly, the absence of proliferating factors, such as IGF1, TGFβ, and integrins can limit remyelination.Fibroblast growth factor (FGF) signaling is likely to play a role in oligodendrocyte regeneration and myelin formation, as FGF2 and/or FGFR were upregulated in MS patients and mouse models of demyelination.Moreover, FGF1/FGFR2 −/− mice showed less efficient myelin recovery in chronic but not in acute cuprizone demyelination [96].
The proinflammatory phenotype of OPCs that promotes damage and blocks remyelination suggests that the suppression of these pathways may ameliorate tissue destruction and promote OPC proliferation and differentiation into myelin-producing cells [90].Oligodendrocytes were shown to express tumor necrosis factor receptor 2 (TNFR2) in the early phase of EAE pathogenesis, while TNFR2 ablation leads to an earlier onset and increased motor dysfunction after EAE induction, with a higher infiltration of immune cells [97].
Helper T cells that drive adaptive immunity also play a key role in MS and the EAE animal model.Th17 polarization induces cytotoxic inflammation and inhibits remyelination in vitro [103].Oligodendrocytes have close contact with Th17 cells upon EAE induction, which may worsen the pathology and increase cell death, possibly by the release of cell stress-inducing glutamate due to the increased expression of integrin CD29 [104].

Oligodendrocyte and Remyelination
The process of remyelination depends on a time-tight activation and recruitment of OPCs to the lesion site, followed by proliferation, differentiation, and maturation into myelinating cells, which contribute to the regeneration process.OPCs attempt to re-establish the myelin sheath of previously demyelinated axons [105] by coming into close contact with the denuded axon, expressing myelin-specific genes, and finally forming the new myelin sheath [106].Remyelination is mostly found in acute and RRMS active lesions and borders [107], whereas mixed active/inactive lesions show a decrease in or absence of remyelination.Early remyelinated lesions are characterized by increased numbers of OPCs distributed in increasing gradient from the core of the lesion towards the periphery [95,108,109].Pre-existing mature oligodendrocytes are able to generate further myelin sheaths; however, their remyelination capacity is limited [110], with adult OPCs being the main cell population responsible for the re-establishment of myelinated healthy axons.Mature oligodendrocytes at the lesion borders limit demyelination and favor myelin repair [111].Interestingly, Schwann cells were shown to mediate remyelination within astrocyte-deficient areas where some of these also derive from OPCs in a transdifferentiation process [112].However, OPC differentiation and efficient remyelination ultimately depends on the resolution of local inflammation [113].
Demyelination induces the secretion of various chemoattractants from astrocytes and microglia to activate OPC recruitment, survival, migration, proliferation, and differentiation to lesion sites, including IGF1, FGF2, IL-1β, TNFα, CXCL12, and platelet-derived growth factor (PDGF)-AA [114].OPCs express functional receptors for cytokines like IL-10, IL-6, IFNγ, and others, which have a predominantly negative impact on their maturation and proliferation [115].Migration and differentiation of OPCs is promoted by CCL2 and IL1β in vitro and in the cuprizone-induced demyelination model [85].Additional mechanisms including the PDGF-A-induced ERK pathway promotes OPC migration in vitro [116], as well as CXCL12 stimulation of the MEK/ERK and PI3K/AKT pathways [83].On the other hand, glia cells release a wide range of chemorepellents, such as ECM-remodeling enzymes, that inhibit the remyelination process by impairing OPC recruitment and enhancing the in-flammatory process [117].Furthermore, the Wnt signaling [118] and Notch pathways [119] inhibit OPC differentiation.
A potent contributor to OPC recruitment failure is the proinflammatory cytokine, IFNγ.IFNγ promotes the senescence of OPCs by upregulating the transcription factor paired related homeobox protein 1 (PRRX1), which prevents the proliferation and tissue colonization of transplanted human OPCs, and upon LPC-induced demyelination [120].Moreover, IFNγ was shown to induce MHC-I expression on OPCs [121], which inhibits FGF receptor signaling and the proliferation of neural progenitor cells [122].Aging WM OPCs responds to IFNγ, induced by CD8+ T cells, leading to oligodendrocyte loss [123].Consequently, IFNγ signaling contributes to the failure of OPC proliferation at lesion areas and could be a potent target for remyelination-promoting therapies [124].
While myelin-reactive T cells are the key cells releasing chemicals and causing inflammation in the MS lesions, T cells were also shown to target OPCs and inhibit remyelination as well as CD8+ T cell infiltration into the CNS via the IFNγ-mediated promotion of antigen cross-presentation in the cuprizone-induced demyelination model [90].Additionally, a hybrid model of the demyelination model, which combines cuprizone-induced demyelination with the adoptive transfer of myelin-specific CD4+ T cells, showed that infiltration of Th17 cells into the CNS correlates with impaired remyelination [125].In contrast, T cell activation was shown to induce the proliferation of OPCs via releasing vascular endothelial cell growth factor-A (VEGF-A) [126] as well as the Treg-mediated release of cellular communication network 3 (CCN3), a growth regulatory protein [127].
The unmet need for the development of approaches aimed at regeneration and repair has led several groups to attempt to enhance OPC proliferation via the administration of various compounds.One such approach was adopted by Mei et al. [128], which screened for the efficiency of 1000 bioactive molecules and identified eight U.S. Food and Drug Administration-approved antimuscarinic compounds that enhanced oligodendrocyte differentiation and membrane wrapping.Additionally, the direct antagonism of the M1 and/or M3 muscarinic receptors by the drug benztropine enhanced remyelination in the cuprizone-induced demyelination model as well as EAE adoptive transfer by stimulating the differentiation of progenitor cells [129].Moreover, the single-cell transcriptomic analysis of differentiating OPCs treated with various compounds identified two promising bioactive molecules, namely Ro1138452 and SR2211, that promote myelination in human pluripotent stem cells in vitro [130], providing evidence for the possible development of oligodendrocyte differentiation-targeting-based therapies.

Astrocytes and Neuroinflammation
Astrocytes, accounting for approximately 40-50% of all glial cells in the CNS, form starlike processes.They play a key role in efficient interactions with other glial cells to maintain homeostasis and provide support to surrounding cells.Astrocytes promote myelination by facilitating OPC proliferation, differentiation, and mediating the oligodendrocyte-axon initial contact.Additionally, astrocyte end-feet surround blood vessels where they contribute to the formation of the BBB and create the glia limitans (Figure 3).There, they assist in the blood flow regulation and nutrient absorption through the water channel aquaporin 4 (AQP4) [131,132], potassium (K + ) channels [133], and Cx43, which are expressed on the astrocyte end-feet.Furthermore, by taking up glucose from the bloodstream, they supply neurons with crucial energy substrates.They also play a key role in the synapses as they ensheathe them with their fine processes and participate in ion regulation, neurotransmitter release and osmotic gradient, as well as promote synaptogenesis and modulate synaptic pruning by labeling the synapses for microglia-mediated elimination [134].Finally, they are the cells responsible for scar formation upon injury, known as astrogliosis, which prevents inflammation from spreading to the surrounding tissue; furthermore, while less than microglia, astrocytes have a phagocytic capacity [135,136].
rons with crucial energy substrates.They also play a key role in the synapses as they ensheathe them with their fine processes and participate in ion regulation, neurotransmitter release and osmotic gradient, as well as promote synaptogenesis and modulate synaptic pruning by labeling the synapses for microglia-mediated elimination [134].Finally, they are the cells responsible for scar formation upon injury, known as astrogliosis, which prevents inflammation from spreading to the surrounding tissue; furthermore, while less than microglia, astrocytes have a phagocytic capacity [135,136].there is a significant compromise of the BBB, through which activated immune cells extravasate from the periphery into the CNS.Both astrocytes and microglia are activated into A1 and M1 states, respectively, and by secreting various pro-inflammatory molecules induce further in myelin destruction.Additionally, peripheral macrophages and microglia assist in myelin debris clearance.The activation of this proinflammatory state is devastating and determines the lesion formation.Later, both astrocytes and microglia shift their activation state into an anti-inflammatory, namely A2 and M2, respectively, whereby inducing scar formation and OPC recruitment, facilitate in myelin repair.Occasionally, there is activation back to the pro-inflammatory states, depending on Astrocytes arise from neural progenitor cells (NPCs) in the subventricular zone (SVZ) during development and migrate along radial glial processes to populate the brain [137].Ramón y Cajal initially classified astrocytes into protoplasmic and fibrous, depending on their morphology, location and function as well as antigen phenotype [138].Type 1 astrocytes (A1), or protoplasmic, are found in the GM and ensheathe synapses as well as blood vessels, promoting BBB functions, while type 2 astrocytes (A2), or fibrous, are found in the white matter, in close contact with the nodes of Ranvier and blood vessels.In addition to their classification by morphology, they are also divided into inactive (quiescent), active, and reactive.Inactive astrocytes are present in the normal resting tissue and become activated by various mechanisms, leading to various pro-inflammatory molecules release [139].

Astrocyte Dysregulation in MS
The activation of proinflammatory astrocytes leads to loss of their physiological functions and the secretion of cytokines and chemokines that regulate different events in MS development and progression, driving rapid neuronal and oligodendrocytic death [140].Additionally, near the lesions, they present with morphological changes in respect to length and complexity, indicating metabolic changes [141].Reactive astrocytes extend beyond the lesions, suggesting their contribution to lesion development.Moreover, the formation of glial scars, while detrimental in limiting further inflammation, may interfere with the ability of oligodendrocytes to facilitate remyelination.Therefore, understanding the contribution of astrocytes in different stages of MS lesion formation may prove crucial in developing possible strategies for intervention.
Astrocytes express MHC molecules, which gives them a non-traditional APC phenotype [149].Additionally, they express a wide range of immune receptors including pattern-recognition receptors (PRRs), which detect pathogen-associated molecular patterns (PAMPs), and tissue damage-associated molecular patterns (DAMPs).One major PAMP is the Toll-like receptor (TLR)-3, which is activated by double-stranded RNA, usually found in viral genome replication, and promotes neuroinflammation by the upregulation of CCL2, IL-1β, and CXCL10 [150].TLR3 and TLR4 activation, shown to be upregulated in EAE as well as the blood cells of MS patients [151], suggests an inflammatory activation through NFκB and TNFα [149].Conversely, TLR-3-mediated astrocyte activation induces an antiinflammatory cytokine release, including IL-9 and IL-10, suggesting a modulatory role in MS patients [152].
Depending on the environment, anti-or pro-inflammatory stimulating molecules are secreted, which affect the de-and remyelination processes.The stimulation of the astrocytes upregulates or induces the secretion of cytokines like TNFα, IL-1β, brain-derived neurotrophic factor (BDNF), VEGF, as well as chemokines including CCL2 and CXCL10 [37].Cell-specific and region-specific transcriptomics showed increased expression of immune pathways related to astrocyte function in the EAE mouse model [153].Moreover, persistent activation of astrocytes alters astrocytic cell signaling, protecting oligodendrocyte degeneration from cuprizone-induced demyelination via reduced CXCL10-mediated NFκB signaling [154].These studies corroborate with the current theory that astrocytes can have a detrimental effect in the demyelination process by inducing further inflammation as well as promote its resolution when given the appropriate signals.
When astrocytes forming the glia limitans become activated, they may alter the environment of the BBB and increase permeability [155].In MS patients, the perivascular astrocytic end-feet formation was shown to be damaged in early lesions, indicating the possibility that astrocyte lesions may drive demyelination [156,157].Additionally, astrocytes facilitate the T cell entry into the CNS via expression of VCAM-1, as shown in the EAE mouse model [158].Interestingly, AQP4 was upregulated in both the postmortem MS tissue [159] and EAE mouse model [160], while the ablation of AQP4 ameliorates EAE progression [161].Moreover, astrocyte activation leads to increased CCL2 production, which plays a key role in the recruitment of immune cells during chronic EAE [156,157,162].
While astrocytes play a key role in the compromising of the BBB, they also release chemokines that favor the recruitment of circulating leukocytes [163] and the induction of differentiation of CD4+ T cells into a pro-inflammatory state, as well as CD8+ T cell cytotoxic activity [164].Th1-and Th17-derived factors induce a proinflammatory phenotype in astrocytes [165], with an increased expression of IL-1β, IL-6 and NOS2, as well as chemokines CCL2, CCL20, CXCL10 [166].Additionally, during EAE, Th1, and Th17 induced the production of GM-CSF [167], which mediates neuroinflammation by activating microglia, BBB disruption by enhancing expression of adhesion molecules like ICAM-1 and VCAM-1, as well as tight junction disassembly via zonula occludens 1 (ZO-1) transcription downregulation [168].Th1-mediated release of IFNγ triggered the microglia to release IL-1β, which in turn downregulated Cx43 in astrocytes, disrupting the astrocytic intercellular communication [169].
The role of the complement has emerged as a crucial component in the controlling of immunological responses, as the CNS actively produces components of innate immunity, like complement proteins [174].C1q and C3 components determine the cellular and humoral immune responses in the progression of MS, participating in astrocytosis, microgliosis, and synaptic engulfment.Reactive astrocytes express the C5a complement receptor [175,176] as well as C1q and C3 [177].Synaptopathy occurs in MS, independent of demyelination, and further expands the disease progression.The induction of EAE leads to the increased bioavailability of C1q and C3, which modulates glutamate release from astrocytic compartments, while affecting the activity at nerve terminals and impairing astrocytic processes [178].

The Role of Astrocytes in Remyelination
The transition from demyelination to remyelination leads to a shift in activation status of astrocytes.Transcriptomic analysis of the astrocytes after cuprizone-induced demyelination and upon short and longer withdrawal, where remyelination takes place, showed that they underwent significant transcriptional changes in each phase [179].This large spectrum of astrocyte functions contributes to both disease progression and repair.
After CNS injury, astrocytes secrete inhibitory molecules and extracellular matrix components, which are detrimental to the remyelination process [180].The expression of endothelin-1 (ET-1) was shown to inhibit remyelination via the Notch pathway [181], while ET-1 signaling caused an upregulation of the Notch1 receptor ligand, jagged-1, inhibiting the interaction between astrocytes and oligodendrocytes [182].Remyelination is also impaired due to the glial scar formation and astrocyte production of inhibitory molecules, PDGF [183] and FGF2 [184].Additionally, while the depletion of Cx43 did not affect LPCinduced demyelination, it accelerated remyelination, indicating that Cx43 hemichannels may regulate negatively the remyelination process by favoring local inflammation [54].
As remyelination is more efficient in patients of a young age, it is presumed that astrocyte activation is beneficial through the secretion of both permissive and inhibitory molecules.Later, the balance between pro-or anti-inflammatory state is lost, and astrocytes become more toxic rather than regenerative [180].Nevertheless, whether the effects of astrocytes are beneficial or detrimental is much more complex, as it depends on a temporal dynamic interplay between all the cells involved in the process.
Astrocytes are thought to be promising therapeutic targets for MS management, due to their ability to communicate with oligodendrocytes and support myelin formation.Although many studies are focusing on promoting remyelination via OPC differentiationrelated pathways, one should consider the microenvironment in which these cells need to survive and function.Understanding astrocyte diversity as well as crosstalk with other glia cells is crucial to develop astrocyte-mediated therapeutic strategies [189].

Microglia and Neuroinflammation
The microglial cell population, comprising around 5-20% of the total glial population, depending on health state, are the resident macrophages of the CNS.By being members of the innate immune response, they constantly survey their microenvironment for tissue damage [190] and are actively involved in antigen presentation.They also play important roles in myelin development, preservation, and growth, while the absence of microglia leads to altered lipid metabolism in oligodendrocytes through disrupted TGFβ function [191].Microglia mediate the clearance of myelin debris and misfolded proteins in response to injury by phagocytosis, which facilitates the efficient remyelination and recruitment of OPCs [192].They are also involved in promoting synaptic plasticity [193][194][195] as well as neuronal function by maintaining continuous interactions with neighboring cells [56].Chronic activation of microglia during neurodegeneration leads to the prolonged release of proinflammatory molecules, which can be both detrimental and protective depending on the microenvironment [11].
Microglia are highly dynamic cells and, at resting state, they are ramified cells with small somata and multiple processes, which protrude and retract in order to survey large brain areas [196].When activated, they gain an amoeboid morphology which allows for a proinflammatory function, as they exert shorter ramifications and have larger cell somata [197].Phagocytic microglia have no ramifications but have large somata, while dystrophic microglia are considered to be present in the aging brain [198].
Microglia are usually categorized as classically stimulated proinflammatory (M1) or alternatively activated anti-inflammatory cells (M2), depending on their polarization state.M1 microglia are stimulated by IFNγ and secrete proinflammatory cytokines IL-1β, IL-6 and TNFα [139] as well as C1q [140], that contribute to the inflammatory response.Conversely, polarization to M2 by cytokines IL-4 or IL-3 leads to the secretion of the anti-inflammatory and neurotrophic factors IL-10, IGF1, and TGFβ [203].Advances in technologies such as mass cytometry and single-cell RNA sequencing (sc-RNA seq) have allowed a deeper understanding of the microglia phenotype, both in healthy as well as in MS brains, offering new insights into the potential immune mechanisms [196,204,205].

The Role of Microglia in MS
There is emerging evidence that implicates microglia in MS pathology, directly or indirectly.The continuous activation of microglia drives neuroinflammation and neurodegeneration, while microglia nodules are associated with more severe MS pathology [206].Moreover, microglial depletion prevents demyelination, oligodendroglial loss, and reactive astrocytosis upon cuprizone-induced demyelination [207].Additionally, blocking mito-chondrial complex-I in proinflammatory microglia limits neurotoxic damage and improves functionality in the EAE model [208].Hence, the modulation of microglial activation comprises a promising target for MS management.
In early lesion formation, there is increased pro-inflammatory microglia accumulation [14] that later changes into mixed pro-and anti-inflammatory microglia [209,210] (Figure 3).Microglia express TMEM119 at the initial stage of new lesion formation, indicating that resident microglia dominate the lesion site, while their number drops when the lesion matures, and the recruitment of monocytes and other peripheral cells takes place [211].Additionally, microglia expressing MHC-II are initially present, which serve as APCs.Increased microglia numbers are also present in the NAWM of MS patients compared to controls, especially in progressive MS [212].In inactive lesions, microglia density is significantly reduced, while in chronic active lesions microglia, they form a rim surrounding the lesion [213], which is rich in iron and myelin debris.Chronic inactive lesions, however, are devoid of microglia/macrophages.While patterns of microglia activation are similar in different pathological conditions including stroke lesions, MS is dominated by a chronic microglia activation, which is thought to drive lesion transformation.
Microglial activation plays a fundamental role in MS lesions and disease progression where they exert both beneficial and detrimental effects.During demyelination, microglia clear the myelin debris by phagocytosis and secrete proinflammatory cytokines IL-1β, IL-6, IL-12, IL-13, IL-18, IFNγ, and TNFα, and chemokines including CCL2, CCL3, CCL4, CCL5, and CCL12 [214].CCL5 promotes the activation of MMP-9, which is involved in leukocyte extravasation as well as their link with myelin degradation products [201,215].
CX3CL1, also known as fractalkine, is a transmembrane protein produced by neurons that interacts through its receptor CX3CR1, which is located on microglia [216].CX3CR1 −/− show decreased phagocytosis by microglia, and the persistence of myelin debris that inhibits proper remyelination due to impaired OPC recruitment [217].CX3CR1+ microglia exert protective effects by engulfing and destroying Th17 cells upon EAE induction [218], which are predominantly present in the lesions and serum of MS patients [219] and are considered critical regulators of the disease course [220].
Growing evidence indicates that microglia colocalize with T cells in MS lesions [221], and their presence correlates with axonal damage.Interactions between T cells and microglia may be present not only via soluble factors but also via direct cell-to-cell contact [222].Microglia secrete IL-12 and IL-13, which promote Th1 accumulation [223].In turn, Th1 cells activate further resident microglia into the M1 phenotype [224] and upregulate expression of MHC and other costimulatory molecules, further favoring Th1 reactivation and infiltration [225].Moreover, Th1-and Th17-induced astrocyte stimulation releases factors that enhance microglia migration in vitro [166].
As previously mentioned, microglia express various TLRs, including TLR2, TLR4, and TLR5.TLR2 was shown to be upregulated in demyelinating MS lesions [226], while TLR4 was increased in cerebrospinal fluid (CSF) mononuclear cells [227].Increased expression of the latter suppresses the polarization of the M1 to M2 phenotype and therefore prolongs proinflammatory response [228].TLR3, 5, 7, 8, and 9 are expressed by dendritic cells and have been shown to be present in a subset of MS patient lesions [229].
The transcription factor NFκB regulates various aspects of the immune functions of both the innate and adaptive immunity, including encoding cytokines and chemokines, as well as inflammasome activation like the leucine-rich repeat-(LRR-) and NOD-like receptor protein 3 (NLRP3) [230] by inducing pro-IL-1β [231].It is activated by factors like IL-33, IL-1b, IL-12, GM-CSF, TNF-α, and IL-17.The conditional knockout of inhibitory kappa B kinase beta (Ikkβ), a key regulatory kinase in the activation of NF-κB, in mice conditioned with EAE, exhibited ameliorated progression as reduced levels of microglia infiltrated lesions decreased M1 polarization and CD4+ T cell response [232].
Colony-stimulating factor 1 receptor (CSF1R) is required for the proper development of microglia and macrophages and is a critical regulator of homeostasis.CSF1R was shown to be upregulated in microglia-associated diseases [233] as well as in MS [234].Activated microglia drive demyelination through the CSF1R signaling [207], especially in progressive MS, as neuroinflammation persists with constant survival and proliferation of microglia [235], while CSF1R inhibition ameliorates neuroinflammation and microglial activation in the EAE mouse model of MS [234].
TNFα is a major regulator of inflammatory response, mainly secreted by activated microglia via extracellular vesicles as well as macrophages, T cells, and natural killer (NK) cells that mostly exert cell death signals.It drives microglia into M1 polarization, leading to further TNFα secretion [236].TNFα is upregulated in EAE and cuprizoneinduced demyelination models as well as MS lesions [237], upregulating the NFκB signaling pathway and enhancing the secretion of various pro-inflammatory molecules [238].IL-9 was shown to modulate microglia inflammatory activity by reducing the expression of triggering receptors expressed on myeloid cells-2 (TREM-2) and TNF release in the EAE mouse model, improving clinical disability and mitigating synaptic damage [239].However, even though anti-TNFα treatment resulted in the reduced incidence and delayed onset of EAE, no effect on disease severity was observed [240].
IFNγ release is considered a hallmark of Th1-driven inflammation in MS and EAE [241].It can also trigger microglia to act as effector cells, causing damage via the release of cytotoxic factors [242] and, along with TLR4 coactivation, can result in massive dysfunction and cell death [243].However, IFNγ was also shown to induce microglia apoptosis, possibly having a pivotal role in a self-limiting negative feedback mechanism, exerting a beneficial effect in the disease progression.Additionally, IFNγ administration ameliorated EAE progression via decreased CD11b+ myeloid cells and inflammatory cell infiltration [244].
Microglial contribution to MS pathology is a decisive factor in the disease progression.They have attracted increasing attention due to their diverse functions, from maintaining homeostasis and supporting neurons to actively favoring myelination.Extracellular vesicles secreted by microglia can influence their microenvironment, signaling astrocytes into proinflammatory states as well as promoting a pro-regenerative milieu [245].Their versatile roles in the pathogenesis of neuroinflammation, neurodegeneration, and repair, are crucial in biomarker discovery, as well as in more targeted therapeutic interventions.

Microglia in Remyelination
Microglia are highly dynamic cells as changes in activation, transcription, and proteomics allow them to regulate their functions in a temporal-and context-dependent manner in response to de-and remyelination [246,247].Although increasing evidence implicates microglia in the pathology that underpins neurological disruption in MS, microglia function can orchestrate the remyelination process.Microglia and macrophages are typically located at the lesion border, where the first signs of remyelination are present, and their profile can decide the fate of remyelination.Microglia of the M2 phenotype are present in high densities at areas of active remyelination and not where remyelination is impaired, highlighting the importance of transition of microglia into an anti-inflammatory type [248].Corroborating this, recent evidence has shown that microglia polarization to the M2 phenotype regulates spontaneous remyelination after intermittent cuprizone-induced demyelination [249].Much like astrocytes, microglia can create both a beneficial and a hostile environment in respect to tissue regeneration.
Microglia promote remyelination by the efficient phagocytosis of myelin debris, secretion of regenerative factors and regulation of the ECM [250], while persistent activation is associated with impaired remyelination.Even though microglia activation may drive demyelination, myelin debris clearance by phagocytic microglia is thought to be a key step in promoting a favorable environment for remyelination [41,[250][251][252][253][254].However, efficiency of myelin debris clearance declines with aging [255].This complex process involves internalization of the myelin debris, lysosome maturation, and cholesterol recycling.Interestingly, microglia also produce the immediate cholesterol precursor, desmosterol, which aids the resolution of inflammation and facilitates efflux of lipid/cholesterol to oligodendrocytes, a critical requisite for myelin synthesis [256].
One key mediator of myelin debris clearance and breakdown is CX3CR1, which is highly expressed in microglia and macrophages.More specifically, CX3CR1 ablation leads to the phagocytes lacking myelin debris after demyelination, suggesting an impairment in the internalization process [217].However, compensatory mechanisms are present, as even if less OPCs are present, the CX3CR1 −/− mice showed some degree of remyelination.Moreover, myelin debris uptake was shown to be regulated by TREM2, which is regulated by CSF1R signaling.The conditional knockdown of CSF1R led to impaired myelin clearance after cuprizone-induced demyelination and debris accumulation [257].TLR4 signaling has also been associated with myelin debris clearance, as well as regulation of cytokine and growth factor expression, which directly affect OPC replacement and remyelination [258].Moreover, TLR2 ablation enhanced remyelination in the LPC mouse model [226], while reduced TLR2 signaling was associated with improved myelin recovery [259].
Beyond creating a remyelination-friendly environment via phagocytosis, microglia can directly regulate OPC responses [260].Initially, a proinflammatory phenotype is abundant, aiding in the recruitment of OPCs to lesions, which changes into an anti-inflammatory phenotype at the oligodendrocyte differentiation and remyelination phase [250].Microglia express Semaphorin-3F, which is a chemoattractant of OPCs to the lesion site [261].Moreover, the secretion of inflammation-induced factors regulates oligodendrocyte development, especially microglia-secreted TNFα [262], IGF1 [263], and IL-1β [264].Microglia deletion reduces OPC differentiation, even in the presence of phagocytic monocyte-derived macrophages [265], indicating further mechanisms in microglia activation to support remyelination.
Overall, microglia display a heterogeneous profile in activation state depending on their microenvironment, which is strongly correlated with underlying pathological processes, making them potential therapeutic targets in MS.Remyelination-specific MHC-IIrelated molecules, including HLA-DRB1 and CD74, found in human postmortem lesions, indicate the immunoregulatory role of subclusters of microglia [266].However, due to the various characteristics denoted in microglia, and the absence of specific markers and molecular signatures, a more detailed assessment of microglia activity is needed to understand disease progression and plan therapeutic interventions.

Glial Cell Targeting in MS Treatment
While multiple approved DMTs are currently used as safe and effective options in the treatment of MS, they mainly rely on the modulation of the inflammatory response but not on the disease progression or functional recovery.Additionally, depending on the drug response and adverse effects, one needs to consider the escalation, de-escalation, or switching of DMTs to balance the risks and benefits of each treatment [267].Therefore, an unmet need for new therapeutic strategies has emerged, especially for the progressive phase of the disease.More specifically, it is imperative to create new therapies for patients with highly active disease, as they have an increased risk for future disability as well as therapies to slow down or prevent the disease and/or disability with more targeted delivery [268].Consequently, exploring innovative treatments to target the autoimmune reaction more broadly may offer new prospects in addressing the complexity of the disease.
Given the lack of approved therapies targeting myelin maintenance or regeneration, the current therapeutic strategies focus on targeting oligodendrocyte differentiation and maturation to become myelinating cells.Induced pluripotent stem cells (iPSCs) might be used as precursors to mature myelinating oligodendrocytes and may not only address remyelination issues in MS but other dysmyelinating disorders as well [268].Moreover, the stimulation of endogenous remyelination via the promotion of OPC proliferation and maturation has been addressed.Compounds showing promising results include the antibody rHIgM22 [269], the DNA aptamer LJM-3064 [270] and Nogo-related blocking antibodies.Though, thus far, studies have mainly utilized iPSCs-derived neurons, drug development may shift towards iPSC-derived glia, given their emerging potential.
Various studies have shown the involvement of glial cells in orchestrating the complex immune response during the course of MS.Both microglia and astrocytes contribute to myelin injury and axonal damage, making them good candidates for therapeutic targeting.As discussed in the previous sections, glial cells have pivotal roles in the inflammatory cascade, by regulating leukocyte trafficking, releasing neurotoxic and neuroprotective factors, as well as limiting inflammatory damage.These pathways provide additional molecular targets for pharmacological intervention, which need to target specifically the detrimental activity of glia cells while preserving their reparative functions.
Current MS therapies were shown not only to mediate the adaptive immune activity but also influence glial cell function via the modulation of astrocyte or microglia activity.While initially fingolimod effectiveness was attributed to the modulation of T cell populations, non-lymphocyte-related mechanisms were introduced, especially via the regulation of astrocytic activity [271].More specifically, among others, the promotion of astrocyte migration via the sphingosine-1 protein-1 (S1P1) receptor [272] and mediation of ERK phosphorylation [273] were shown to contribute to the drug's effectiveness.Moreover, dimethyl fumarate induces the expression of Nrf2 in astrocytes and therefore upregulation of oxidative stress-induced growth inhibitor (OSGIN1), which is thought to limit astrocyte-mediated damage and leakage of the BBB by preventing the retraction of the perivascular astrocytic feet [274].As the modulation of astrocytic activity is a major target in developing therapeutic strategies, one should take into account the diverse astrocyte functions, including scar formation and neuroinflammation [18].
As microglia can contribute directly or indirectly to the inflammatory and remyelination processes, modulation of their activity may strongly influence MS progression.DMTs were shown to modulate microglia activity [213], with fingolimod showing the most direct effect on S1P receptors on microglia, leading to the downregulation of IL-6, IL-1β, and TNFα [275].Dimethyl fumarate modulates microglia differentiation into an anti-inflammatory type, leading to the reduced secretion of pro-inflammatory cytokines via the NFκB pathway [276].Additionally, glatiramer acetate exerts neuroprotection via the activation of proinflammatory M2 microglia [277].Various other agents are being tested in a pre-clinical setting and, even though targeting microglia seems more plausible than astrocytes, at different stages of the disease, microglia exert different properties, therefore a deeper understanding and characterization of the distinctive pathways influenced by microglia activation may allow for the development of microglia-directed treatments.
A potential therapeutic effect may arise from the nanomodulation of glial cells as well as macrophages migrating to the CNS [278].It has been suggested that macrophages may be used as carriers of therapeutic agents as, due to the limiting crossing of pharmacological agents though the BBB, they make great candidates for transferring cargo via a more natural chemokine-induced migration to the brain.Inflammation in the brain leads to upregulated secretion of ICAM-1 and therefore BBB breakdown.Macrophage-secreted exosomes were shown to interact with the BBB endothelial cells, leading to increased BBB leakage, which can be used as a means for more effective drug delivery [279].Overall, utilizing macrophages as delivery systems of small drugs or RNAs may provide a new perspective in the management of inflammation in MS.
Nanoparticle delivery loaded with myelin antigens and other tolerogenic adjuvants induced antigen-specific tolerance, ameliorating chronic progressive EAE [280,281].Additionally, mRNA vaccines coding for disease-related autoantigens showed disease suppression in the EAE mouse model [282].Overall, these approaches target bystander immune system activation; however, it still remains unclear whether this is sufficient to limit the ongoing inflammation in MS.
A key reason in further understanding the inflammatory response in MS is the potential identification of disease biomarkers, facilitating the prediction of disease future course.Recent studies have proposed the use of profiling the cytokine and chemokine levels in the CSF and serum of MS patients, especially in RRMS [283], as dynamic biomarkers can indicate the disease severity and level of inflammatory activity [284].Moreover, the use of microglia and/or astrocyte-secreted molecules and cytokines may have potential usage as markers for MS [214].Recent studies have identified chitinase-3-like protein 1 (CHI3L1) to be a biomarker for MS progression, especially predicting disability progression in the serum [285], plasma [286], as well as in the CSF [287] of progressive MS patients.More specifically, CHI3L1 release was associated with both microglia/macrophage [288] and astrocytes [289,290], holding promise for targeted therapy of inflammatory demyelination.Finally, immunophenotyping MS patients before the initiation of treatment may serve as a powerful tool to offer more targeted, personalized treatment for each patient and disease course [291].
Given the significant role of glial cells in the pathogenesis and progression of MS, modulation of the detrimental neuroinflammatory process could potentially be a crucial milestone in disease management.However, glial cells function in a synergistic manner, and therefore glial-glial interactions have emerged as a potent target for promoting remyelination.As remyelination tightly depends on the involvement of multiple cell types around the lesion, understanding glial cell interaction may prove to be key in the pathway towards promoting efficient tissue repair.

Concluding Remarks and Future Perspectives
Over the years, increasing evidence has shown the involvement of glial cells in the underlying pathological mechanisms in MS.A milieu of stimuli can determine whether they exert pro-or anti-inflammatory effects and to what extent; however, a deeper understanding of these finely tuned effects of damage and repair shall prove detrimental in developing effective therapies for immune-driven diseases like MS.The proper function of the CNS relies on the communication of glial cells, and understanding these complex dynamic interactions will allow for more specialized targeting when considering potential therapeutic targets that can be combined with the immune intervention of myelin repair.

Figure 1 .
Figure 1.Glial cells populate lesions in a heterogeneous manner during the course of MS lesions.During the course of the disease, lesions change in respect to glial cell population.Active lesions contain macrophages at the center, while astrocytosis starts to be present.The rim of the lesion is populated with microglia which recruit other inflammatory cells to the site, as well as myelin-phagocytosing macrophages.Mixed active/inactive lesions do not show significant astrocyte population, while there is distinct formation of microglia around the rim.Additionally, there is recruitment of OPCs.Remyelination can be seen as a shadow plaque, where oligodendrocytes slowly initiate the

Figure 2 .
Figure 2. The role of oligodendrocytes in immunomodulation.In recent years, there has been increasing evidence of the role of oligodendrocyte in inflammatory activation.OPC proliferation is induced by IGF-2, VEGF-A, and TGF-β, while the presence of IL-1β, IL-17, and IFNγ has an inhibitory function.Nevertheless, the presence of OPCs further induced the recruitment of peripheral immune cells, modulation of other glial cells, as well as some degree of phagocytic activity.Mature myelinating oligodendrocytes may act as APCs, by expressing specific receptors like MHC-I and -II, as well as TLR3 and TNFR2.Most importantly, Cx47 expressed at the soma and proximal processes of the oligodendrocyte support intercellular communication.IGF-2, insulin-like growth factor; VEGF-A, Vascular endothelial growth factor; TGFβ, Transforming growth factor beta; IL-1β, Interleukin 1-beta; IL-17, Interleukin-17; IFNγ, Interferon-gamma; CCL2, C-C motif chemokine ligand 2; FGF, Fibroblast growth factor; TNFR2, Tumor necrosis factor receptor 2; TLR3, Toll-like receptor 3; Cx47, Connexin 47; MHC, Major histocompatibility complex; T reg, B reg, T and B regulatory cells; TCR, T cell receptor (figure was created with BioRender.com).

Figure 2 .
Figure 2. The role of oligodendrocytes in immunomodulation.In recent years, there has been increasing evidence of the role of oligodendrocyte in inflammatory activation.OPC proliferation is induced by IGF-2, VEGF-A, and TGF-β, while the presence of IL-1β, IL-17, and IFNγ has an inhibitory function.Nevertheless, the presence of OPCs further induced the recruitment of peripheral immune cells, modulation of other glial cells, as well as some degree of phagocytic activity.Mature myelinating oligodendrocytes may act as APCs, by expressing specific receptors like MHC-I and -II, as well as TLR3 and TNFR2.Most importantly, Cx47 expressed at the soma and proximal processes of the oligodendrocyte support intercellular communication.IGF-2, insulin-like growth factor; VEGF-A, Vascular endothelial growth factor; TGFβ, Transforming growth factor beta; IL-1β, Interleukin 1-beta; IL-17, Interleukin-17; IFNγ, Interferon-gamma; CCL2, C-C motif chemokine ligand 2; FGF, Fibroblast growth factor; TNFR2, Tumor necrosis factor receptor 2; TLR3, Toll-like receptor 3; Cx47, Connexin 47; MHC, Major histocompatibility complex; T reg, B reg, T and B regulatory cells; TCR, T cell receptor (figure was created with BioRender.com).

Figure 3 .
Figure 3. Glial cells in the center of inflammation and neurodegeneration in MS.During early MS, there is a significant compromise of the BBB, through which activated immune cells extravasate from the periphery into the CNS.Both astrocytes and microglia are activated into A1 and M1 states, respectively, and by secreting various pro-inflammatory molecules induce further in myelin destruction.Additionally, peripheral macrophages and microglia assist in myelin debris clearance.The activation of this proinflammatory state is devastating and determines the lesion formation.Later, both astrocytes and microglia shift their activation state into an anti-inflammatory, namely A2 and M2, respectively, whereby inducing scar formation and OPC recruitment, facilitate in myelin repair.Occasionally, there is activation back to the pro-inflammatory states, depending on