Иммуномодулирующий и противовоспалительный потенциал S-нитросглутатиона (GSNO) при множественной склерозе: обзор механизмов и терапевтических перспектив.

Introduction

Multiple sclerosis (MS) is a chronic, immune-mediated disorder of the central nervous system (CNS) that affects over 2.8 million people worldwide. It is more prevalent in women and typically manifests between the ages of 20 and 40 ( 1 ). The etiology of MS is multifactorial, involving a complex interplay between genetic predisposition and environmental influences. Among the genetic risk factors, variations within the major histocompatibility complex (MHC) region, especially in human leukocyte antigen (HLA) genes, which play a key role in helping T cells recognize foreign antigens, have been strongly associated with increased susceptibility to MS ( 2 , 3 ). Clinically, MS presents in different forms: most commonly as relapsing-remitting MS (RRMS), which may later evolve into secondary progressive MS (SPMS), or as primary progressive MS (PPMS) in a smaller proportion of patients ( 1 , 4 ). Pathologically, MS is characterized by demyelinating lesions in the CNS driven by autoreactive T cells, B cells, and innate immune mechanisms, with oxidative stress and mitochondrial dysfunction contributing to neurodegeneration and disease progression ( 5 ).

MS remains an incurable disease, and because of its autoimmune-driven inflammatory nature, immune-targeted therapies have become the central focus of treatment. Acute relapses are typically managed with corticosteroids; however, their long-term use is constrained by severe, dose-dependent side effects. To overcome these limitations, more than 20 FDA-approved disease-modifying therapies (DMTs) are currently available, the majority of which act through immunosuppressive or immunomodulatory mechanisms. Among these, injectable (e.g., glatiramer acetate and interferon-β) and infusion-based biologic therapies (e.g., natalizumab and ocrelizumab) are generally considered more mechanism-based immunomodulators ( Table 1 ). For instance, natalizumab prevents the infiltration of activated T cells into the CNS by targeting α4-integrin; glatiramer acetate (Copaxone), a synthetic peptide that mimics myelin basic protein (MBP), promotes anti-inflammatory immune responses by shifting T cell polarization from Th1 to Th2 phenotypes; and interferon-β regulates immune activity by downregulating proinflammatory cytokines such as IL-1 and TNF-α while enhancing anti-inflammatory mediators like IL-10 ( 6 ) ( Table 1 ). Although these therapies represent significant progress in mechanism-based immunomodulation, their reliance on parenteral administration and potential to induce immunogenicity, through the development of neutralizing antibodies, limit their long-term efficacy and clinical utility.

Conversely, small-molecule DMTs, such as dimethyl fumarate, diroximel fumarate, fingolimod, siponimod, and teriflunomide, offer several practical advantages, including oral administration, reduced reliance on clinic-based care, and greater dosing flexibility ( Table 1 ). Nonetheless, their mechanisms of action, which include sphingosine-1-phosphate (S1P) receptor modulation (e.g., fingolimod and siponimod) and inhibition of pyrimidine synthesis (e.g., teriflunomide), result in broad immunosuppression. These effects are associated with significant clinical risks, including treatment-induced lymphopenia in 30-60% of patients and a two- to three-fold increase in the incidence of serious infections ( 7 , 8 ). This highlights a critical therapeutic gap: the pressing need for orally available small-molecule agents that can achieve selective immunomodulation without compromising global immune competence.

To address this unmet therapeutic need in MS, we highlight the therapeutic potential of S-nitrosoglutathione (GSNO), a stable intracellular nitric oxide (NO•) carrier, and reversible inhibitors of GSNO reductase (GSNOR), the key enzyme controlling GSNO metabolism. Unlike conventional small-molecule DMTs, these agents act through redox-sensitive signaling pathways, offering selective modulation of autoimmune inflammation, suppression of neuroinflammation, and attenuation of oxidative stress while preserving immune competence. In this review, we summarize current mechanistic and preclinical evidence supporting the GSNO/GSNOR axis as a promising therapeutic target in MS and discuss the translational potential of reversible GSNOR inhibitors, whose favorable safety profiles in patients with asthma and cystic fibrosis support further investigation of their therapeutic efficacy in MS.

This review was conducted as a narrative review rather than a systematic review and therefore did not follow PRISMA guidelines. Relevant literature was identified through searches of PubMed and Google Scholar, supplemented by manual screening of reference lists from key mechanistic, translational, and clinical investigations. Search terms included combinations of “multiple sclerosis,” “experimental autoimmune encephalomyelitis,” “nitric oxide,” “S-nitrosylation,” “S-nitrosoglutathione,” “GSNOR,” “redox,” “oxidative stress,” “inflammation,” “autoimmune disease,” and “lymphocytes.” Over 400 publications from 2000 through June 2026 were considered, along with seminal earlier studies. Priority was given to original research articles, supplemented by selected reviews for conceptual context. Studies were selected based on their mechanistic, translational, or therapeutic relevance to GSNO/GSNOR signaling in MS, EAE, immune regulation, oxidative stress, and neuroinflammation. Conference abstracts, non-English publications, and studies outside the scope of this review were excluded. As a narrative review, the aim was to provide a representative synthesis of the current evidence rather than an exhaustive systematic assessment.

S-nitrosylation: a stable alternative for NO• signaling regulation under inflammatory and oxidative stress

NO• is a gaseous free radical synthesized endogenously by three distinct nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2) ( 9 ) ( Figure 1A ). Under physiological conditions, NO• is generated at low concentrations (picomolar to nanomolar range) primarily by the constitutively expressed isoforms nNOS and eNOS, whose activity is tightly regulated by cell signaling pathways. In this context, NO• serves as a critical signaling molecule that mediates homeostatic cellular responses. By binding to the heme moiety of soluble guanylyl cyclase (sGC), NO• activates cyclic GMP (cGMP) production, which subsequently stimulates protein kinase G (PKG), cGMP-gated ion channels (CGCs), and phosphodiesterases (PDEs). Through these downstream effectors, physiological NO• signaling governs fundamental processes, including vascular tone, platelet aggregation, neuronal plasticity, and endothelial function ( 10 – 12 ) ( Figure 1B ).

Under inflammatory conditions, however, iNOS becomes the dominant source of NO•, producing markedly higher, sustained concentrations, often reaching the micromolar range ( 13 – 17 ). NO• is not inherently toxic; however, under oxidative stress it reacts with molecular oxygen (O 2 ) and superoxide anion (O 2 • - ) to generate reactive nitrogen species (RNS), including nitrogen dioxide radical (NO 2 •) and peroxynitrite (ONOO - ), which induce cytotoxicity through DNA damage, protein nitration, and lipid peroxidation ( 18 ) ( Figure 1C ). Although RNS play a crucial role in antimicrobial defense, these mechanisms also contribute to host tissue and cell damage, leading to outcomes such as endoplasmic reticulum stress, mitochondrial dysfunction, cell death, and vascular impairment ( 19 ) ( Figure 1C ). RNS also oxidize tetrahydrobiopterin (BH 4 ), a critical NOS cofactor, causing enzyme uncoupling that shifts NO• production toward O 2 • - generation, thereby amplifying oxidative stress and worsening pathology ( 20 , 21 ) ( Figure 1C ). In this context, strategies to elevate NO• levels, whether through enhanced NOS activity or NO•-releasing drugs, often prove ineffective or harmful, as free NO• is rapidly consumed in RNS formation rather than signaling via the canonical cGMP pathway. To address this limitation, recent approaches employ S-nitrosylating agents that bypass free NO• generation, thereby limiting its conversion into ONOO - , and instead directly activate NO•-mediated signaling through targeted protein S-nitrosylation.

S-nitrosylation is a reversible post-translational modification involving the covalent attachment of a NO• group to the thiol group of specific cysteine residues in proteins ( 22 ) ( Figure 1D ). To date, more than 20,000 S-nitrosylation sites have been identified across over 10,000 proteins ( 23 ), influencing protein function, stability, interactions, and subcellular localization across a wide range of species and functional classes ( 24 ). This modification has been implicated in various physiological processes, including neurotransmission, anti-inflammatory responses, immune modulation, and the regulation of vascular tone ( 25 – 29 ).

Currently, the mechanisms by which NO• mediates S-nitrosylation of intracellular proteins are not fully understood. However, growing evidence suggests that NO• does not typically react directly with protein thiols, except under specific conditions. Instead, S-nitrosylation usually occurs indirectly via trans-S-nitrosylation, wherein NO• first reacts with low-molecular-mass thiols such as glutathione (GSH), cysteine, or coenzyme A (CoA) to form low-molecular-mass S-nitrosothiol (LMMSNO) intermediates like GSNO, S-nitrosocysteine, and S-nitroso-CoA ( 30 , 31 ). These intermediates subsequently transfer the nitroso group to protein thiols via trans-S-nitrosylation.

Among LMMSNOs, GSNO is the most abundant and serves as the principal physiological donor for protein S-nitrosylation ( Figure 1D ). Within cells, GSNO exists in equilibrium with protein S-nitrosothiols (PrSNOs) ( 32 ). Therefore, maintaining cellular GSNO homeostasis, tightly regulated by both its synthesis and enzymatic degradation, is essential for the proper control of S-nitrosylation-dependent pathways. GSNO is produced through the reaction of NO• with GSH, the most abundant intracellular reductant; however, this reaction is inefficient in aqueous environments, where direct NO•-GSH interactions typically yield oxidized glutathione (GSSG) and nitroxyl (NO - ) rather than GSNO ( 33 , 34 ). Instead, GSNO formation is favored in hydrophobic compartments such as cellular membranes, where NO• and O 2 accumulate and react to form dinitrogen trioxide (N 2 O 3 ), a potent S-nitrosylating intermediate that subsequently reacts with GSH to generate GSNO ( 35 – 37 ). This process is highly sensitive to the intracellular redox state. Notably, under inflammatory and oxidative stress conditions, large amounts of NO• are produced by iNOS, yet concomitantly elevated O 2 • - reacts with NO• at a near diffusion-limited rate (6.7 × 10 9 M −1 s −1 ) to form ONOO - ( 38 , 39 ). This reaction occurs orders of magnitude faster than the reaction leading to GSNO synthesis, thereby strongly favoring ONOO - generation over S-nitrosothiol formation ( 40 – 43 ).

While GSNO synthesis is tightly influenced by redox conditions, its intracellular levels are also determined by enzymatic degradation, primarily mediated by S-nitrosoglutathione reductase (GSNOR; also known as alcohol dehydrogenase class III/ADH5; EC 1.1.1.1) ( Figure 1D ) ( 44 ). GSNOR is a dimeric, NADH-dependent oxidoreductase that acts on various aliphatic compounds, with GSNO and S-hydroxymethyl glutathione (HMGSH) as preferred substrates. It metabolizes GSNO into an N-hydroxy sulphonamide intermediate (GSNHOH), which reacts with GSH to yield hydroxylamine (NH 2 OH) and oxidized glutathione (GSSG) ( 45 ). GSNOR, a cysteine-rich protein, can itself be S-nitrosylated by GSNO, forming a feedback loop that enhances its activity ( 46 ). Deletion of the GSNOR gene leads to increased levels of both GSNO and protein S-nitrosylation in vivo ( 47 ). However, GSNOR does not directly denitrosylate protein S-nitrosothiols; this function is primarily carried out by the thioredoxin (Trx)–thioredoxin reductase (TrxR) system ( 48 ) ( Figure 1D ).

The pathological relevance of GSNOR dysregulation was first recognized in asthma and cystic fibrosis, where overexpression or hyperactivity of GSNOR reduces GSNO bioavailability, leading to bronchoconstriction and impaired airway relaxation ( 49 , 50 ). Subsequently, increased GSNOR expression and activity have also been observed in animal models of traumatic brain injury ( 51 ), COVID-19 ( 52 ), and MS (EAE) ( 53 ). For the treatment of asthma and cystic fibrosis, reversible small-molecule GSNOR inhibitors, such as the injectable or inhalable N6022 and the orally bioavailable N91115 , have been developed and evaluated in both preclinical models and Phase I/II clinical trials ( 54 , 55 ). These compounds demonstrated favorable safety profiles, high tolerability, and no dose-limiting toxicities. However, neither agent met the primary clinical endpoints in trials for asthma or cystic fibrosis. Despite these setbacks, they offer a unique advantage: the ability to selectively enhance intracellular S-nitrosylation, thereby supporting physiological NO•-mediated signaling while avoiding cytotoxic RNS generation and minimizing off-target effects. More recently, the therapeutic potential of these GSNOR inhibitors has been explored in animal models of traumatic brain injury, COVID-19, and MS, where they exhibited significant disease-modifying effects ( 51 – 53 ). Nonetheless, despite these promising preclinical findings, neither compound has advanced to clinical trials for these indications. Importantly, these findings distinguish two different aspects of translational development. While Phase I/II studies in asthma and cystic fibrosis support the clinical feasibility and tolerability of GSNOR inhibition in humans, the therapeutic evidence for MS remains entirely preclinical and is currently limited to mechanistic in vitro studies and preclinical studies using EAE models. Accordingly, future clinical studies are needed to determine whether the immunomodulatory and neuroprotective effects described in this review translate into clinical benefit for patients with MS.

In summary, GSNOR inhibition enhances GSNO-mediated S-nitrosylation, providing a more sustained means of modulating NO• signaling than direct NO• delivery. Unlike NO•, which has a half-life of only seconds, GSNO is considerably more stable and can persist for hours depending on the cellular environment ( 56 – 59 ). This greater stability enables more prolonged and spatially distributed redox signaling, whereas free NO• primarily acts near its site of production ( 60 , 61 ). Moreover, GSNO does not spontaneously release free NO• and therefore does not directly promote ONOO - formation under oxidative conditions, potentially reducing nitrosative injury while preserving physiological NO• bioactivity ( 62 – 66 ). Nevertheless, because GSNO regulates numerous S-nitrosylation-dependent signaling pathways, the biological consequences of GSNOR inhibition are likely to depend on the magnitude and context of GSNO elevation.

S-nitrosylation-mediated regulation of autoimmune responses in MS and EAE

MS exhibits two distinct inflammatory patterns. In acute and relapsing phases, blood-brain barrier (BBB) disruption permits widespread infiltration of T cells and B cells into white matter, forming active demyelinating plaques with elevated inflammatory cytokines ( 67 , 68 ). In contrast, progressive stages involve gradual lymphocyte accumulation in perivascular and meningeal spaces without major BBB disruption, leading to subpial cortical demyelination and diffuse neurodegeneration in white and gray matter ( 68 ). Progressive MS also features ectopic lymphoid follicles (ELFs) in the meninges that are enriched with T and B cells and other immune cells and provide sites for the local production of pro-inflammatory mediators, antigen presentation, and sustained autoimmune responses within the CNS ( 67 ). Despite differences in inflammatory patterns between relapsing and progressive stages, both are fundamentally driven by T- and B-cell-mediated autoimmune responses, and we explore how the GSNO-GSNOR axis regulates these responses.

S-nitrosylation-mediated down-regulation of effector CD4 + T cells

Studies using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, suggest that CD4 + T helper (Th) cells are crucial in triggering the autoimmune-driven inflammatory demyelination ( 67 ). This finding aligns with genetic evidence linking MS to MHC class II molecules, which present antigens to these CD4 + T cells ( 69 ). Among CD4 + subsets, Th1 and Th17 cells, which produce IFN-γ and IL-17, respectively, are particularly pathogenic during the early and acute phases of neuroinflammation, and elevated levels of these cytokines correlate with disease activity in patients with MS ( 67 , 70 ). Clinical evidence further supports their role: IFNγ administration exacerbates MS, whereas IL-17 blockade reduces lesion formation ( 71 , 72 ).

Th1 and Th17 cells play distinct roles in the pathogenesis of MS and EAE. Th1-derived IFN-γ and TNF-α activate macrophages and microglia and upregulate MHC class II expression on CNS-resident cells, thereby amplifying local inflammation, demyelination, and axonal damage ( 73 ). However, the effects of IFN-γ are stage- and cell-dependent. Although IFN-γ generally promotes inflammation during the acute phase, protective effects have been reported during later or chronic phases of EAE ( 74 , 75 ). IFN-γ also exerts heterogeneous effects on CNS-resident cells, including astrocytes, oligodendrocytes, and microglia, which may partly explain discrepancies among experimental studies. For example, astrocyte-specific IFN-γ signaling was recently shown to limit chronic CNS inflammation through PD-L1 induction ( 76 ). These findings indicate that, although Th1-derived IFN-γ contributes substantially to acute neuroinflammation, its overall effects may vary according to disease stage and the responding CNS cell type. In comparison, Th17 cells are key drivers of sustained neuroinflammation through the production of IL-17A, IL-17F, IL-21, and IL-22, which promote BBB disruption, immune-cell infiltration, and demyelination ( 77 ). Th17 cells also contribute to the formation of ELFs within the CNS in MS and EAE and are critically involved in the pathogenesis of progressive MS ( 78 ).

NO• was initially reported to induce Th1 differentiation ( 79 ). Under in vitro culture conditions, low concentrations (<10 μM) of NO• donors such as DETA-NO enhance Th1 cell differentiation by activating cGMP-dependent signaling pathways, which in turn upregulate the IL-12 receptor β2 subunit (IL12RB2), a critical mediator of Th1 polarization and effector function ( 79 ). In contrast to these effects, GSNO showed no observable impact on Th1 responses in vitro . Specifically, Nath et al. demonstrated that treatment of cultured T cells isolated from EAE mice with GSNO at concentrations up to 100 μM did not affect IL-12-induced Th1 polarization, as evidenced by unchanged levels of STAT4 phosphorylation, T-bet expression, and IFN-γ production ( 80 ). These findings suggest that, unlike NO• donors, GSNO does not directly modulate Th1 responses under in vitro conditions. However, in vivo studies revealed a markedly different outcome. Systemic administration of the GSNOR inhibitor N6022 significantly reduced Th1 cell differentiation, expansion, and IFN-γ production in both the spleen and spinal cord of EAE mice ( 81 ), an effect similarly observed in GSNOR-deficient mice ( 53 ).

The precise mechanisms by which GSNOR inhibition suppresses Th1 immune responses in vivo remain incompletely understood. However, findings that S-nitrosylating agents inhibit LPS- and IFN-γ-induced IL-12 production, an essential factor for Th1 differentiation, by inhibiting NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cell) activation in macrophages and dendritic cells (DCs) ( 82 , 83 ) suggest that GSNOR inhibitors or S-nitrosylating agents may indirectly regulate Th1 differentiation by modulating IL-12 expression in these antigen-presenting cells ( Figures 2A, B ). Consistent with this proposed mechanism, iNOS-deficient mice exhibit exaggerated Th1 responses characterized by elevated IFN-γ production following infections, such as Leishmania major ( 84 ), Staphylococcus aureus ( 85 ), Herpes simplex virus ( 86 ), or in autoimmune models like EAE ( 87 ). Together, these studies indicate that low concentrations of NO• promote Th1 differentiation through cGMP-dependent upregulation of the IL-12 receptor, whereas higher levels, through the formation of S-nitrosothiols such as GSNO, suppress Th1 responses indirectly by inhibiting IL-12 production in antigen-presenting cells.

In addition to their effects on Th1 responses, NO• and S-nitrosylating agents also exert regulatory effects on Th17 responses. The regulatory role of NO• and S-nitrosylating agents in Th17 immune responses is supported by evidence showing that iNOS-deficient CD4 + T cells exhibit enhanced Th17 differentiation under Th17-polarizing conditions compared to wild-type cells ( 88 ). Consistently, iNOS-deficient EAE mice exhibited elevated Th17 cell numbers and IL-17A production relative to wild-type mice, indicating a suppressive role for iNOS-derived NO• in Th17-mediated inflammation ( 89 ). Similarly, treatment of wild-type EAE mice with either GSNO or the GSNOR inhibitors (N6022 and N91115 ) significantly suppressed Th17 responses, improved clinical scores, and reduced spinal cord demyelination ( 53 , 80 , 81 ). Furthermore, similar effects were also observed in GSNOR-deficient EAE mice ( 53 ). Most importantly, the inhibition of Th17 as well as Th1 responses by treatment with GSNOR inhibitors did not cause systemic lymphopenia, unlike fingolimod (FTY720) ( 53 , 81 ), which exerts its therapeutic effect by preventing the egress of lymphocytes from secondary lymphoid organs and thereby reducing their circulation in the periphery ( 90 ). This distinction highlights the potential advantage of GSNOR inhibitors in modulating pathogenic T cell responses without broadly suppressing the immune system.

Although the precise mechanism by which GSNO suppresses Th17 immune responses remains to be fully elucidated, evidence indicates that GSNO can inhibit TGF-β- and IL-6/IL-23-driven expression of RORγt by blocking STAT3 activation (phosphorylation) ( 80 ) ( Figure 2C ). Supporting this, Kim et al. reported that GSNO suppresses IL-6-induced STAT3 activation via S-nitrosylation at Cys259 of STAT3 ( 91 ), indicating a role for S-nitrosylation-dependent signaling in NO•-mediated Th17 suppression. Additionally, NO• has been shown to interfere with TGF-β signaling by inhibiting Smad2/3 phosphorylation and nuclear translocation, via activation of cGMP-dependent PKG-1 ( 92 ). Moreover, NO• is known to inhibit IL-17 promoter activity through tyrosine nitration of RORγt ( 88 ), suggesting that multiple mechanisms, including S-nitrosylation, nitration, and the cGMP/PKG signaling pathway, collectively contribute to the NO•-dependent regulation of Th17 responses.

S-nitrosylation-mediated upregulation of regulatory CD4 + T cells

Regulatory T cells (Tregs) are vital for preserving immune homeostasis and play a pivotal role in suppressing autoreactive immune responses in MS ( 93 – 95 ). In patients with MS, peripheral Treg frequency is generally preserved; however, their immunosuppressive function and migratory capacity to the CNS are markedly impaired. This is reflected by their reduced abundance in brain tissue and enrichment of a highly apoptosis-prone subset in the cerebrospinal fluid, suggesting selective depletion within inflammatory CNS lesions ( 96 , 97 ). Both numerical and functional deficiencies of Tregs contribute to MS pathogenesis, making them a high-priority therapeutic target for restoring immune tolerance and controlling disease progression ( 98 ).

Two main Treg subsets have been described: thymus-derived natural Tregs (nTregs; CD4 + CD25 + Foxp3 + , where Foxp3 is a lineage-defining transcription factor essential for Treg development and suppressive function), which are relatively stable and less influenced by the cytokine milieu, and peripherally induced Tregs (iTregs), which differentiate from naïve CD4 + T cells in response to antigenic stimulation and local inflammatory cues ( 99 ). iTregs are particularly important in MS as they arise at sites of inflammation and can suppress autoreactive T cells that evade central tolerance ( 94 ). Several iTreg subsets exist, including Foxp3 + iTregs with high CD25 expression ( 100 ); Tr1 cells, which produce IL-10 and TGF-β but lack Foxp3 ( 101 , 102 ); and Th3 cells, which predominantly secrete TGF-β ( 103 ).

Emerging evidence highlights a regulatory role for NO• in the differentiation of peripheral Foxp3 + iTreg cells. In vitro cell culture experiments have demonstrated that NO• at low concentrations negatively regulates the generation of Foxp3 + iTregs through activation of cGMP-dependent PKG-1, which suppresses TGF-β-induced Smad3 phosphorylation and subsequent Foxp3 gene expression ( 104 , 105 ). These in vitro findings highlight NO• as a potential inhibitor of peripheral iTreg differentiation and suggest a suppressive effect on key regulatory pathways.

In contrast, findings from in vivo disease models reveal an opposing role for NO•, underscoring the context-dependent complexity of its immunomodulatory functions. In mouse models of colitis and arthritis, administration of the NO• donor DETA-NO alleviated inflammatory pathology by enhancing IL-10 production ( 106 ). Furthermore, genetic deletion of GSNOR or its pharmacological inhibition with N6022 or N91115 led to the expansion of Foxp3 + iTreg cells and increased IL-10 expression in both the spleen and the CNS of MOG 35-55 -immunized C57BL/6 EAE mice ( 53 , 81 ). Notably, NO• has also been shown to induce a distinct subset of iTreg cells, termed NO•-Tregs by Niedbala et al. ( 106 ), which express CD25 but lack Foxp3 expression. These cells are phenotypically and functionally distinct from conventional Treg subsets, including nTregs, Foxp3 + iTregs, Th3 (TGF-β + ), and Tr1 (IL-10-dependent) cells, due to their unique characteristics and differentiation requirements ( 106 ). Consistently, both GSNO treatment and GSNOR knockout have been reported to increase CD4 + CD25 + Foxp3 - iTreg cell populations in EAE mice [65, 88]. These findings suggest that GSNO may modulate CNS inflammation in EAE by promoting both IL-10-producing Foxp3 + and Foxp3 - iTregs, although the underlying molecular mechanisms remain to be elucidated ( Figure 2D ).

Potential involvement of histone deacetylases in S-nitrosylation-mediated regulation of effector vs. regulatory T cells

Histone acetyl transferases (HATs) and histone deacetylases (HDACs) regulate gene expression by catalyzing the acetylation and deacetylation of histone and non-histone proteins ( 107 – 109 ). In MS, HDACs contribute to disease pathogenesis by promoting pro-inflammatory functions of DCs and T cells. Specifically, HDACs enhance DC expression of costimulatory molecules and cytokines such as IL-6, IL-12, and TNF-α, which drive Th1 and Th17 differentiation ( 110 – 113 ) ( Figure 2A ). Simultaneously, multiple studies have reported that HDACs suppress Treg generation and function, compromising immune tolerance ( 114 – 120 ). Specifically, HDACs are known to inhibit the acetylation of Foxp3, thereby reducing its stability and transcriptional activity, ultimately impairing Treg polarization ( 121 ) ( Figure 2D ).

In EAE, HDAC inhibitors such as sodium phenylbutyrate and trichostatin A (TSA) attenuate clinical severity and neuropathology by reducing spinal cord inflammation, demyelination, and neuronal loss, while concurrently upregulating neuroprotective genes and downregulating apoptotic markers ( 122 , 123 ). HDAC inhibitors also impair DC activation and T cell proliferation, in part by inducing the immunoregulatory enzyme IDO (indoleamine 2,3-dioxygenase) ( 112 , 124 , 125 ). Beyond immune modulation, HDAC inhibitors also promote neuronal survival by enhancing antioxidant responses and stabilizing axonal structures ( 126 – 128 ). These dual anti-inflammatory and neuroprotective effects support their therapeutic potential in MS.

S-nitrosylation is increasingly recognized as a key post-translational modification that regulates the activity, localization, and interactions of specific HDACs, including HDAC2 ( 129 , 130 ), HDAC6 ( 131 ), and SIRT1 ( 132 ), thereby influencing gene expression and diverse cellular functions. For instance, neurotrophin-generated NO• S-nitrosylates HDAC2 at Cys262 and Cys274, disrupting its chromatin binding and thereby promoting histone acetylation, chromatin relaxation, and transcription of genes critical for neuronal development, cardiac function, and neuroprotection ( 129 , 130 ). Similarly, HDAC6 is S-nitrosylated by iNOS-derived NO• in cytokine-treated cells, leading to reduced deacetylase activity and increased acetylation of target proteins ( 131 ), while SIRT1, a Sir2 family HDAC, is inactivated by nNOS-generated NO• through S-nitrosylation of its CXXC motif, causing increased acetylation of target proteins, such as PGC-1α ( 132 ). These findings underscore S-nitrosylation as a dynamic and site-specific regulatory mechanism that modulates HDAC function in both physiological and pathological contexts, including those relevant to neuroprotection and immune regulation.

Taken together, both pharmacological inhibition of HDACs and redox-dependent S-nitrosylation modulate HDAC function, ultimately promoting the expression of anti-inflammatory and neuroprotective genes. Notably, HDAC inhibitors have also been reported to suppress Th1 and Th17 immune responses while promoting Treg cell differentiation ( 110 – 120 ), effects that closely resemble those of GSNO and GSNOR inhibitors reported in EAE models ( 52 , 53 , 80 , 81 , 133 , 134 ) ( Figure 2 ). Since HDAC activity can be inhibited through S-nitrosylation, future studies should investigate the potential of GSNO treatment and GSNOR inhibition to therapeutically reprogram autoreactive T cells by targeting HDACs, thereby alleviating disease in MS and EAE through both immune regulation and neuroprotection.

S-nitrosylation-mediated regulation of B cells in MS and EAE

B cells play a central role in autoimmunity by presenting autoantigens to T cells and producing autoantibodies following their differentiation into plasma cells ( 135 ) ( Figures 2A, E ). In addition, B cells also contribute to autoimmune-driven inflammation by secreting pro-inflammatory cytokines, such as IL-6, IFN-γ, and GM-CSF, that exacerbate immune pathology ( 135 , 136 ) ( Figure 2E ). Among these, IL-6- producing B cells are particularly important in the pathogenesis of MS, as their expansion in patients promotes Th17 responses, while targeted deletion of IL-6 in B cells has been shown to reduce disease severity in EAE models ( 137 , 138 ) ( Figure 2E ). These findings support the use of anti-CD20 monoclonal antibodies (mAbs) for B-cell depletion, as demonstrated in EAE models where such treatment selectively reduced IL-6-producing B cells and alleviated disease severity ( 137 ). Building on this rationale, anti-CD20 mAb therapies, including ocrelizumab, rituximab, and ofatumumab, have been tested in MS and are now approved for clinical use in both MS and rheumatoid arthritis ( 139 , 140 ). However, their therapeutic efficacy in MS remains modest, and broad B-cell depletion is associated with notable risks, such as increased susceptibility to opportunistic infections ( 140 , 141 ). Moreover, in other autoimmune diseases, including systemic lupus erythematosus, ulcerative colitis, and psoriasis, anti-CD20 mAb therapies have shown limited benefits or even adverse outcomes ( 142 – 144 ).

Recent evidence suggests that the limited efficacy of anti-CD20 mAbs in autoimmune diseases may stem from their indiscriminate depletion not only of pathogenic effector B (Beff) cells but also of immunosuppressive regulatory B (Breg) cells, whose loss can further exacerbate autoimmunity ( 145 ). Bregs exert their effects primarily through anti-inflammatory cytokines such as IL-10, IL-35, and TGF-β, with IL-10 being the most extensively studied in MS ( 136 , 146 ). Multiple IL-10-producing Breg cell subsets have been identified, including CD24 hi CD27 hi B cells and CD24 hi CD38 hi immature B cells in humans ( 147 , 148 ) and CD1d hi CD5 + B cells in mice ( 149 ). IL-10-producing Breg cells are known to suppress Th1 and Th17 responses, inhibit antigen presentation by macrophages and dendritic cells, and promote Treg induction ( 150 – 153 ) ( Figure 2E ). Consistent with this, IL-10-producing Breg cells have been implicated in the remission and recovery phases of EAE ( 154 , 155 ). Collectively, these findings highlight the potential drawbacks of pan-B cell depletion with anti-CD20 mAbs and underscore the need for targeted therapies that inhibit effector B cell activity while preserving or enhancing regulatory B cell functions.

Mouse and human B cells are known to express iNOS; however, its role in humoral immunity remains uncertain due to conflicting reports ( 156 , 157 ). For instance, iNOS-deficient mice display elevated antiviral IgG2a antibodies and enhanced resistance to influenza A virus infection, pointing to a suppressive role of iNOS in systemic antibody responses ( 158 ). In contrast, other studies indicate that iNOS deficiency impairs IgA class switching and weakens mucosal defense against pathogens ( 159 ). Moreover, iNOS-derived NO• has been shown to promote B cell and plasma cell survival by preventing apoptosis and extending plasma cell longevity through S-nitrosylation-mediated inhibition of caspases ( 160 – 162 ). Collectively, these findings highlight the context-dependent roles of NO•, which can either restrain or support humoral immunity depending on the anatomical site, disease setting, and type of immune challenge.

Although the impact of iNOS-derived NO• and S-nitrosylating agents on humoral immune responses in MS and EAE remains poorly defined, a recent study reported that treatment with the GSNOR inhibitor N6022 after EAE disease onset in a B cell-dependent EAE model (C57BL/6 mice immunized with the recombinant human MOG extracellular domain, rhMOG 1-125 ) led to reduced circulating levels of anti-rhMOG 1–125 autoantibodies ( 163 ). These observations suggest that S-nitrosylating agents may modulate pathogenic autoantibody production in EAE, providing a potential therapeutic avenue for targeting humoral immune responses in MS.

Beyond their role in humoral immunity, NO• also regulates B cell cytokine expression and thereby influences CD4 + T cell-mediated autoimmune responses. Kim et al. showed that in vitro treatment of mouse and human B cells with GSNO (50 μM) reduced IL-6 and increased IL-10 expression following BCR activation with anti-IgM and co-stimulation with CD40 ligand (or anti-CD40 mAb) and recombinant BAFF ( 133 ). Consistent results were observed in EAE mice immunized with either MOG 35-55 (a CD4 + T cell-dependent EAE) or rhMOG 1-125 (a model dependent on both CD4 + T cells and B cells), where treatment with GSNO or the GSNOR inhibitor N6022 upregulated IL-10 and downregulated IL-6 expression in B cells from both the spleen and spinal cord ( 133 , 163 ). Notably, GSNO treatment of cultured B cells and N6022 treatment in EAE mice enhanced IL-10 and suppressed IL-6 expression not only in CD1d hi CD5 + regulatory B cells (Bregs or B10 cells) but also across a broad range of peripheral B cell subsets, including B1a, B1b, transitional (T1 and T2), marginal zone (MZ) and MZ precursor cells, memory B cells, plasmablasts, and plasma cells ( 133 ). Furthermore, transfer of B cells from N6022-treated or GSNOR-deficient EAE mice alleviated disease severity in recipient mice with active EAE ( 133 ). This therapeutic benefit was associated with elevated IL-10, reduced IL-6, expansion of iTreg cells, and a reduction in Th17 cells in the recipients. Together, these findings indicate that B cells from N6022-treated or GSNOR-deficient EAE mice mitigate disease by reprogramming CD4 + T cell-mediated autoimmune responses.

Although the mechanism by which GSNO-mediated S-nitrosylation selectively regulates IL-10 and IL-6 in B cells remains unclear, hypoxia-inducible factor-1 (HIF-1) appears to play a role. HIF-1, composed of a constitutive HIF-1β subunit and an oxygen-sensitive HIF-1α subunit, is a central regulator of metabolic gene expression under hypoxic and inflammatory conditions ( 164 , 165 ). Under normoxic conditions, HIF-1α is rapidly degraded through PHD2 (prolyl hydroxylase domain protein 2)-dependent proline hydroxylation, followed by pVHL (von Hippel–Lindau protein, a component of the E3 ubiquitin ligase complex)-mediated ubiquitination and proteasomal degradation ( 166 – 169 ). By contrast, under hypoxic conditions, PHD2 activity is inhibited, allowing HIF-1α to escape degradation, accumulate, and translocate to the nucleus where it dimerizes with HIF-1β to activate target gene transcription.

In a murine model of colitis, HIF-1 has been shown to suppress IL-6 and IL-23 production in dendritic cells ( 170 ), while promoting IL-10 production by macrophages under hypoxic conditions ( 171 ). Activated B cells also utilize this transcription factor to enhance IL-10 expression. Meng et al. demonstrated that HIF-1α directly binds to the hypoxia-responsive element (HRE) in the IL-10 promoter, thereby increasing its transcription in B cells ( 172 ). In addition, conditional deletion of HIF-1α in B cells led to reduced IL-10 levels and decreased CD1d hi CD5 + Breg cell numbers, while aggravating Th17-mediated inflammation in models of arthritis and EAE ( 172 ).

Interestingly, NO• has been reported to stabilize HIF-1α via S-nitrosylation of HIF-1α itself, as well as of PHD2 and pVHL, thereby inhibiting proline hydroxylation and subsequent ubiquitination and degradation even under normoxic conditions ( 173 – 175 ). These findings suggest that GSNO, by promoting S-nitrosylation, may enhance HIF-1α stabilization in B cells, leading to increased binding to the IL-10 promoter and amplification of hypoxia-responsive transcriptional programs ( Figure 2E ).

Interferon Regulatory Factor 4 (IRF4) is a transcription factor essential for multiple aspects of the humoral immune response, including B cell differentiation and cytokine production ( 176 – 179 ). It has been shown to enhance IL-10 expression in B cells while suppressing IL-6 production and Th17-mediated immune responses ( 180 – 183 ). In a mouse model of EAE, B cell-specific IRF4 conditional knockout resulted in the absence of IL-10-producing plasmablasts in draining lymph nodes and led to exacerbated disease severity ( 180 ). NO• has been reported to induce IRF4 expression in T cells ( 184 ), where it negatively regulates proinflammatory gene expression ( 183 ). Consistently, Kim et al. found that GSNO treatment enhanced IL-10 and suppressed IL-6 expression in activated B cells through upregulation of both HIF-1α and IRF4 ( 133 ). Mechanistically, IRF4 is induced in B cells via BCR-mediated activation of phosphoinositide 3-kinase (PI3K) signaling ( 185 ), and GSNO is known to promote PI3K activation by S-nitrosylating and inhibiting its negative regulator, PTEN (phosphatase and tensin homolog) ( 186 ). Additionally, GSNO activates c-Src kinase ( 187 ), which phosphorylates and activates IRF4 ( 188 ). These findings suggest a potential role for IRF4 in GSNO-mediated induction of Breg cells and IL-10 expression in B cells ( Figure 2E ).

Taken together, these findings demonstrate that S-nitrosylation, mediated by iNOS-derived NO• and GSNO, exerts context-dependent regulatory effects on B cells in MS and EAE by modulating their survival, cytokine profiles, and functional phenotypes. Through HIF-1α and IRF4 signaling, S-nitrosylation may contribute to the enhancement of IL-10 and suppression of IL-6 expression, thereby promoting a regulatory B cell phenotype. These immunomodulatory effects not only provide mechanistic insight into the limited efficacy of current pan-B cell depletion therapies, such as anti-CD20 monoclonal antibodies, but also underscore the therapeutic potential of redox-targeted interventions that selectively suppress pathogenic B cells while preserving or boosting regulatory B cell function in MS and EAE.

Potential roles of GSNO-mediated S-nitrosylation in emerging immune cell populations in MS and EAE

Although CD4 + T cells and B cells are the major immune populations driving MS pathogenesis, increasing evidence indicates that additional immune cell subsets, including CD8 + T cells, natural killer (NK) cells, and Th9 cells, also contribute to disease progression. Compared with conventional CD4 + T cells and B cells, relatively little is known about the role of GSNO in regulating these immune populations. Nevertheless, numerous studies have demonstrated that NO• modulates their differentiation, activation, and effector functions through redox-dependent mechanisms, suggesting that GSNO-mediated S-nitrosylation may similarly influence their biological activities.

CD8 + T cells are now recognized as important contributors to disease pathogenesis. They are abundant in active MS lesions and directly damage oligodendrocytes and neurons through perforin- and granzyme-mediated cytotoxicity, as well as amplify CNS inflammation by producing IFN-γ and TNF-α ( 189 ). Importantly, accumulating evidence indicates that distinct CD8 + T-cell subsets possess immunoregulatory activity capable of suppressing autoreactive immune responses, underscoring the dual pathogenic and regulatory roles of CD8 + T cells in MS ( 189 ). At present, whether GSNO regulates CD8 + T cells in MS or EAE remains poorly understood. However, NO• promotes CD8 + T-cell survival through reversible S-nitrosylation of caspases, thereby inhibiting activation-induced apoptosis ( 190 ). At low concentrations, NO• also enhances CD8 + T-cell differentiation and effector function by upregulating IL-12Rβ2 through the sGC/cGMP pathway, thereby increasing IL-12 responsiveness and promoting Tc1 differentiation and IFN-γ production ( 191 , 192 ). However, at high concentrations, NO• suppresses these CD8 + T-cell responses ( 191 , 192 ). Evidence from a viral lung inflammation model further indicates that GSNO or pharmacological GSNOR inhibitor treatment reduces the infiltration of hyperactivated cytotoxic CD8 + T cells and attenuates tissue inflammation ( 52 ). Although these findings suggest a potential role for GSNO in regulating pathogenic CD8 + T-cell responses, whether similar mechanisms operate during autoimmune neuroinflammation in MS or EAE remains unknown and warrants further investigation.

NK cells are increasingly recognized as important regulators of autoimmune neuroinflammation through their interactions with T cells, B cells, dendritic cells, and microglia ( 193 , 194 ). Human NK cells are broadly divided into CD56 bright and CD56 dim subsets ( 195 ). CD56 bright NK cells primarily exert immunoregulatory functions by eliminating autoreactive CD4 + T cells as well as immature dendritic cells, thereby limiting Th1- and Th17-mediated inflammation ( 196 , 197 ). In contrast, CD56 dim NK cells represent the major circulating NK-cell population and exhibit potent cytotoxic activity through perforin- and granzyme-mediated killing ( 198 ). Consistent with these functional differences, reduced numbers or impaired function of CD56 bright NK cells are associated with increased MS disease activity, whereas excessive activation of CD56 dim NK cells may further amplify CNS inflammation under pathological conditions ( 196 , 199 ). Although direct evidence for GSNO-mediated regulation of NK cells is limited, the effects of NO• on NK-cell biology have been investigated more extensively. High levels of NO• impair NK-cell function by inducing mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential, increased intracellular ROS levels, and apoptosis ( 200 ). Interestingly, CD56 bright NK cells possess greater antioxidant capacity and exhibit increased resistance to oxidative stress than CD56 dim NK cells ( 201 ). These findings suggest that the two NK-cell subsets may respond differently to excessive iNOS-derived NO• during neuroinflammation. CD56 dim NK cells may be more susceptible to NO•-induced oxidative stress, whereas CD56 bright NK cells may be relatively resistant because of their greater antioxidant capacity. Further studies are needed to determine how GSNO-mediated S-nitrosylation affects the survival, activation, and effector functions of these NK-cell subsets in MS and EAE.

Th9 cells are an emerging CD4 + T-cell subset implicated in MS pathogenesis. Increased frequencies of Th9 cells and elevated IL-9 levels are reported in patients with MS and in animals with EAE ( 202 ). Although initially associated with allergic inflammation, IL-9 is now recognized as an important regulator of autoimmune neuroinflammation. In EAE, IL-9 is reported to enhance the suppressive activity of Foxp3 + Treg cells while modulating Th17 responses, suggesting that Th9 cells function as immunomodulators rather than primary drivers of disease ( 203 ). Consistent with this concept, higher levels of IL-9 in the cerebrospinal fluid are associated with lower inflammatory activity and reduced disability progression in relapsing-remitting MS ( 202 ). Although GSNO-mediated regulation of Th9 cells remains largely unexplored, NO• is reported to promote Th9 differentiation through Mdm2-dependent S-nitrosylation, leading to activation of the p53–IL-2–STAT5–IRF4 signaling pathway ( 184 ). NO• is also reported to enhance IL-9 expression through HIF-1α-dependent metabolic reprogramming ( 204 ). Interestingly, HIF-1α and IRF4 are also involved in GSNO-mediated regulation of regulatory B cells ( 133 ), thus raising the possibility that similar redox-dependent mechanisms contribute to Th9 differentiation. Further studies are likely to delineate how GSNO-mediated S-nitrosylation regulates Th9-cell development and function in MS and EAE.

Collectively, accumulating evidence indicates that NO• regulates multiple immune cell populations beyond conventional CD4 + T cells and B cells, including CD8 + T cells, NK cells, and Th9 cells. Although direct evidence for GSNO-mediated S-nitrosylation in these immune subsets remains limited, the well-established role of NO• in controlling their activation, differentiation, and effector functions suggests that GSNO may similarly regulate these processes through shared redox-sensitive signaling pathways. Elucidating how GSNO influences these emerging immune populations should improve our understanding of immune regulation in MS and EAE and may also uncover additional mechanisms underlying the therapeutic benefits of GSNO and GSNOR inhibition.

S-nitrosylation as a regulator of vascular and CNS inflammation in MS and EAE

In MS and EAE, disruption of BBB integrity is an early and pivotal event that permits the infiltration of peripheral immune cells into the CNS ( 205 , 206 ). Among these infiltrating cells, circulating myeloid populations, particularly monocytes and neutrophils, play a central role in initiating and sustaining neuroinflammation. Once inside the CNS, these cells, together with resident microglia, interact with encephalitogenic T and B lymphocytes and differentiate into either proinflammatory (M1) or anti-inflammatory (M2) phenotypes, thereby shaping the local immune milieu and contributing to demyelination, neuronal injury, and overall disease progression ( 207 , 208 ). During this process, NO•, primarily produced by iNOS, exerts dual, context-dependent effects: while excessive NO• and reactive nitrogen species (RNS) formation can contribute to tissue damage, NO• can also mitigate inflammation by modulating leukocyte infiltration, cytokine signaling, and redox homeostasis ( 32 , 209 ). This section discusses the multifaceted roles of NO•, particularly through S-nitrosylation-mediated mechanisms, in modulating CNS inflammation in MS and EAE, with a focus on its impact on vascular integrity, immune cell infiltration and function, myeloid cell polarization, and regulation of proinflammatory gene expression.

S-nitrosylation-mediated regulation of vascular inflammation in MS and EAE

In MS, increased BBB permeability is a common pathological feature observed across different disease subtypes, including RR-MS, SP-MS, and PP-MS ( 210 , 211 ). BBB disruption is particularly prominent during active inflammatory phases and facilitates the entry of peripheral immune cells and blood-derived molecules into the CNS, contributing to sustained neuroinflammation and neurodegeneration ( 212 ). BBB breakdown is a dynamic and multifactorial process involving hemostatic imbalance (e.g., thrombin activation), oxidative stress, aberrant angiogenesis, and inflammatory signaling ( 212 ). Notably, emerging evidence indicates that early BBB disruption is at least partially reversible, suggesting that it may serve as a therapeutic window for early intervention ( 212 ). Early BBB damage is triggered by cytoskeletal changes involving the formation of F-actin stress fibers, which lead to endothelial cell deformation. Mechanistically, this process is driven by aberrant activation of RhoA, resulting in sustained phosphorylation of endothelial myosin light chain (MLC) ( 213 ). These alterations destabilize intercellular junctions, ultimately leading to the disassembly and matrix metalloproteinase (MMP)-mediated degradation of tight junction components, including occludin, claudin, JAMs, and ZO-1, which are hallmark features of early BBB disruption ( 214 – 216 ).

Studies have shown that eNOS-derived NO• plays a critical role in regulating endothelial cytoskeletal dynamics ( 217 ), but its effects vary depending on the surrounding redox environment. Under oxidative stress, NO• reacts with O 2 • - to form ONOO - , which activates RhoA via tyrosine N-nitration, promoting stress fiber formation and compromising the endothelial barrier ( 218 , 219 ). On the other hand, NO•, via the formation of GSNO, inhibits these processes through S-nitrosylation-mediated inhibition of RhoA ( 219 , 220 ) ( Figure 3A ). Kim et al. ( 219 ) reported that, in human brain endothelial cell cultures, GSNO treatment inhibited thrombin-induced F-actin stress fiber formation and endothelial barrier disruption by suppressing RhoA activation, thereby preserving MLC phosphatase (MLCP)-mediated MLC dephosphorylation ( 221 ), and by reducing intracellular Ca 2+ influx, which in turn limited activation of MLC kinase (MLCK) ( 222 ) ( Figure 3A ). Accordingly, GSNO treatment in EAE mice reduced BBB disruption, as indicated by decreased Evans blue dye extravasation into the CNS, and alleviated both inflammatory demyelination and clinical symptoms of EAE ( 219 ). These findings highlight the role of GSNO- mediated S-nitrosylation in maintaining BBB integrity throughout the course of MS and EAE.

Beyond structural disruption of the endothelial barrier, infiltration of peripheral immune cells into the CNS requires active engagement with the endothelium via cell adhesion molecules (CAMs) ( 223 ). In MS and EAE, pro-inflammatory mediators upregulate endothelial expression of key CAMs such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (or endothelial-leukocyte adhesion molecule-1 or ELAM-1). These molecules interact with their ligands, such as E-selectin ligand-1 (ESL-1), very late antigen-4 (VLA-4; integrin α4β1), and lymphocyte function-associated antigen-1 (LFA-1), on circulating leukocytes, particularly T cells, monocytes, and other myeloid cells, thereby facilitating firm adhesion and migration into the CNS parenchyma ( 224 , 225 ) ( Figure 3A ). Importantly, the temporal and spatial expression of these adhesion molecules correlates with leukocyte infiltration during disease progression ( 226 ). In fact, their increased expression often appears before any clinical symptoms develop in EAE, highlighting their key role in triggering CNS inflammation ( 227 ).

Previously, Prasad et al. ( 228 ) reported that GSNO treatment of brain endothelial cells in vitro reduced TNF-α-induced expression of ICAM-1, VCAM-1, and E-selectin, through S-nitrosylation-mediated inhibition of NF-κB p65. They proposed that TNF-α stimulation leads to denitrosylation of S-nitrosylated p65 in endothelial cells, thereby allowing the p65/p50 NF-κB complex to translocate to the nucleus and induce transcription of CAM genes ( Figures 3A, C ). However, GSNO treatment attenuates this TNFα-induced denitrosylation, limiting NF-κB binding to CAM gene promoters and thereby reducing their transcription ( 228 ). The downregulation of CAMs subsequently impairs endothelial cell-leukocyte (e.g., monocyte) interactions, leading to decreased leukocyte infiltration into the CNS in EAE models ( 228 ). These findings highlight the therapeutic potential of GSNO in reducing vascular inflammation and immune cell infiltration in MS and EAE.

Macrophage and microglial polarization: M1 vs. M2 in MS and EAE

During the effector phase of EAE, infiltrating monocytes differentiate into macrophages within the CNS, while resident microglia become activated. These changes are largely driven by infiltrating autoreactive T cells, particularly Th1 and Th17 subsets, which secrete proinflammatory cytokines such as TNF-α, IFN-γ, GM-CSF, and IL-17 that modulate the local inflammatory environment and contribute to CNS pathology ( 229 , 230 ). Within this inflammatory milieu, activated macrophages and microglia predominantly acquire a proinflammatory M1 phenotype. M1 polarization is promoted by cytokines such as TNF-α, IFN-γ, and IL-1β and is regulated by transcription factors including NF-κB, STAT1, and IRF5, which induce the expression of inflammatory mediators such as TNF-α, IL-1β, IL-6, and iNOS ( 231 – 235 ) ( Figure 3B ). M1 macrophages accumulate in demyelinated lesions in MS patients, where they contribute to neuroinflammation and demyelination ( 236 ). However, this proinflammatory state is not fixed; myeloid cells retain the capacity to undergo functional reprogramming toward an anti-inflammatory M2 phenotype. This shift is driven by cytokines such as IL-4, IL-13, IL-10, and TGF-β, which activate transcription factors including STAT6, PPARγ, and KLF4 ( 237 – 239 ) ( Figure 3B ). M2 polarization drives the upregulation of genes involved in anti-inflammatory and immunomodulatory responses (e.g., IL-10, TGF-β, CCL7, and CCL22), as well as pathways supporting tissue repair, remyelination, neuroprotection, and metabolic adaptation that promote sustained regulatory function ( 240 – 242 ) ( Figure 3B ).

M1 and M2 phenotypes of macrophages and microglia also differ in their NO• profiles: M1 cells express high levels of iNOS and produce abundant NO•, supporting pathogen and tumor cell elimination, whereas M2 cells express arginase-1, which channels arginine toward ornithine and subsequently proline, a major amino acid in collagen synthesis for tissue repair, while limiting NO• production to prevent further tissue damage ( 233 ). Interestingly, accumulating evidence suggests that iNOS-derived NO• may exert negative feedback on M1 polarization ( 243 ). In support, splenocytes from iNOS-deficient mice exhibit enhanced M1 macrophage polarization under pro-inflammatory conditions ( 83 ). Mechanistically, M1 polarization depends on the activation of transcription factors such as NF-κB and IRF5, which are known to be inhibited by S-nitrosylation and tyrosine nitration, respectively ( 83 , 243 ) ( Figure 3B ).

Beyond its direct effects on inflammatory signaling pathways, accumulating evidence suggests that NO• also regulates macrophage function through immunometabolic mechanisms. Recent studies have identified metabolic reprogramming as a key regulator of macrophage activation and polarization. In this context, itaconate, an anti-inflammatory immunometabolite produced from cis-aconitate by the enzyme encoded by immune-responsive gene 1 (IRG1, also known as ACOD1) ( 244 ), modulates inflammatory signaling and cellular metabolism partly through the electrophilic modification of redox-sensitive cysteine residues ( 245 , 246 ). In EAE, treatment with the cell-permeable itaconate derivative 4-octyl itaconate is reported to attenuate disease severity by suppressing pro-inflammatory microglial activation while promoting NRF2-dependent cytoprotective signaling ( 247 ). In complementary studies, electrophilic itaconate and fumarate derivatives inhibited both the priming and activation of the canonical NLRP3 inflammasome in macrophages and microglia ( 248 ).

Previous studies demonstrated that iNOS-derived NO• contributes to the regulation of intracellular itaconate metabolism during macrophage activation ( 249 , 250 ). Mechanistically, NO• inhibits mitochondrial aconitase 2 (ACO2), disrupting tricarboxylic acid (TCA) cycle flux at the citrate-to-isocitrate step. Although this metabolic remodeling initially promotes citrate accumulation, sustained ACO2 inhibition is believed to limit the availability of cis-aconitate for IRG1-mediated itaconate synthesis, thereby restricting excessive itaconate accumulation ( 249 , 250 ). More recently, an additional NO•-independent mechanism has been identified in which mitochondrial iNOS directly interacts with IRG1 in a conformation-dependent manner, thereby suppressing IRG1 enzymatic activity independently of NO• production ( 251 ). Collectively, these findings suggest that iNOS regulates macrophage immunometabolism through at least two mechanistically distinct pathways: a canonical NO•-dependent pathway that modulates redox-sensitive metabolic enzymes and inflammatory signaling, and a newly identified NO•-independent pathway that directly limits intracellular itaconate production through the inhibition of IRG1. Given that itaconate suppresses inflammatory activation while promoting cytoprotective and inflammation-resolving programs, the latter mechanism may indirectly influence macrophage polarization, although this possibility remains speculative at present. Because GSNO-mediated S-nitrosylation represents a major downstream signaling mechanism of NO• biology, it is conceivable that GSNO may also influence macrophage immunometabolism. However, whether the IRG1–itaconate axis contributes to macrophage polarization in MS and EAE, and whether this pathway is regulated by GSNO-mediated S-nitrosylation, remain unknown.

At present, the direct mechanisms by which GSNO-mediated S-nitrosylation regulates M2 polarization remain poorly defined. In vitro studies using iNOS-deficient macrophages showed markedly enhanced M1 polarization with little effect on M2 differentiation, suggesting that endogenous NO• plays a more prominent role in restraining M1 activation than in directly driving the M2 phenotype ( 83 ). In contrast, GSNO treatment reduced M2 macrophage abundance in a murine model of castration-resistant prostate cancer ( 252 ), indicating that the effects of GSNO on macrophage polarization are likely disease- and tissue-dependent. In EAE, pharmacological inhibition of GSNOR with N6022 increased CNS infiltration of Th2 and Treg cells ( 81 ), which produce IL-4, IL-13, and IL-10, cytokines known to favor M2 polarization ( 237 – 239 ) ( Figure 3B ). Collectively, these findings suggest that GSNO shapes macrophage polarization through both direct redox signaling and indirect cytokine-mediated effects, with their relative contributions and possible interplay with the IRG1–itaconate axis remaining to be determined in MS and EAE.

S-nitrosylation-mediated mechanisms for inflammatory gene expression

Inflammatory conditions lead to iNOS-derived NO• production, which exerts both cytotoxic and anti-inflammatory effects ( 32 ). Among its regulatory mechanisms, S-nitrosylation plays a pivotal role, as GSNOR inhibition elevates GSNO levels and reshapes S-nitrosylation patterns, particularly on transcription factors such as NF-κB, STAT3, HIF-1α, and Nrf2, thereby suppressing inflammatory gene expression ( 253 ). NF-κB is a master transcription factor central to the regulation of proinflammatory gene expression. It functions as a dimeric complex composed of five family members: p65 (RelA), RelB, c-Rel, p50, and p52, with the p65/p50 heterodimer being the principal form activated in response to inflammatory stimuli ( 254 ). In resting cells, p65/p50 dimers are retained in the cytoplasm in an inactive state by binding to inhibitory proteins known as inhibitors of κB (IκB) ( 255 ). Upon exposure to proinflammatory signals such as lipopolysaccharide (LPS) or TNF-α, IκB is phosphorylated by the IκB kinase (IKK) complex, comprising two catalytic subunits (IKKα and IKKβ) and a regulatory subunit (NEMO or IKKγ), leading to its ubiquitination and proteasomal degradation ( Figure 3C ). This process liberates the NF-κB dimers, allowing their translocation into the nucleus and activation of target genes ( 256 , 257 ).

Among the IKK components, IKKβ is recognized as the predominant kinase responsible for activating the p65/p50 pathway in response to proinflammatory cytokines ( 258 – 260 ). Notably, Cys179 within the activation loop of IKKβ is essential for its kinase activity and for phosphorylation of IκB ( 261 ). NO• has been shown to inhibit IKKβ through S-nitrosylation of this cysteine residue, thereby attenuating NF-κB-mediated proinflammatory gene expression ( 228 , 262 – 265 ). Furthermore, iNOS-derived NO• also directly inhibits NF-κB activity by S-nitrosylating Cys62 of the p50 subunit and Cys38 of the p65 subunit ( 266 ), both of which reside within the DNA-binding Rel homology domain (RHD) ( 266 , 267 ) ( Figure 3C ).

The STAT family comprises transcription factors, including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which play crucial roles in cytokine and growth factor signaling. In their inactive state, STAT proteins reside in the cytoplasm. Upon stimulation by cytokines or growth factors, specific cell surface receptors activate associated tyrosine kinases (TKs), particularly members of the Janus kinase (JAK) family: JAK1, JAK2, JAK3, and TYK2. These kinases phosphorylate a conserved tyrosine residue on STAT proteins, leading to their formation of either homodimers or heterodimers. The phosphorylated STAT dimers then translocate to the nucleus, where they bind to specific DNA sequences and regulate the transcription of target genes ( 268 – 271 ).

Among the STAT family, STAT1 and STAT3 are particularly important in mediating inflammatory and immune responses ( 272 ). STAT1 is predominantly activated by interferons, particularly IFN-γ through JAK1 and JAK2 and IFN-α/β through JAK1 and TYK2. Upon phosphorylation, STAT1 enters the nucleus and induces genes critical for antiviral defense and inflammation ( 273 ) ( Figure 3C ). Notably, IFN-γ-driven STAT1 activation leads to M1 macrophage polarization and the production of pro-inflammatory cytokines such as TNF-α and IL-12 ( 273 ). The precise role of NO• in the regulation of STAT1-mediated signaling remains incompletely understood. In macrophages, stimulation with lipopolysaccharide (LPS) and IFN-γ induces iNOS expression, resulting in elevated NO• production. This leads to tyrosine nitration of STAT1, which impairs phosphorylation at the critical Tyr701 residue and consequently inhibits downstream signaling ( 274 ). Additionally, other studies have reported that S-nitrosylating agents, such as SNAP and GSNO, suppress STAT1 phosphorylation and impair its binding to γ-interferon activation site (GAS) elements in macrophages and endothelial cells ( 243 , 275 ) ( Figure 3C ). However, the specific sites on STAT1 where tyrosine nitration or S-nitrosylation occurs, as well as the precise effects of these modifications on STAT1 activity and STAT1-mediated inflammatory gene expression in the context of MS and EAE, remain unknown.

STAT3 activation occurs in a cytokine-specific manner via JAK family kinases, including JAK1, JAK2, and TYK2. For instance, IL-6 engages a receptor complex composed of the IL-6Rα and the signal-transducing gp130 chain, which facilitates the recruitment and activation of JAK1 and JAK2, leading to STAT3 phosphorylation at Tyr705 and its subsequent activation ( 276 ) ( Figure 3C ). Activated STAT3 dimerizes and translocates to the nucleus, where it induces the transcription of genes that contribute to the pathogenesis of chronic inflammation ( 277 ). Kim et al. ( 91 ) previously demonstrated that, under inflammatory conditions, IL-6–induced STAT3 activation in microglia is suppressed by either iNOS-derived NO• or exogenous GSNO treatment. Notably, GSNO does not affect JAK activity or the gp130 receptor; rather, it inhibits STAT3 phosphorylation through S-nitrosylation of STAT3 at Cys259 ( 91 ) ( Figure 3C ). As STAT3 is pivotal in driving the proliferation and survival of inflammatory cells by regulating genes related to cell cycle progression and anti-apoptotic signaling during immune responses ( 278 ), GSNO-mediated inhibition of STAT3 leads to suppression of IL-6-induced microglial proliferation ( 91 ). Similar findings have been reported in head and neck squamous cell carcinoma and multiple myeloma, where S-nitrosylating agents inhibit survival and proliferation of cancer cells through STAT3 suppression ( 279 , 280 ). In addition to its role in inflammation and cancer, STAT3 also plays a crucial role in Th17 polarization and effector function ( 281 ). Nath et al. ( 80 ) demonstrated that GSNO treatment significantly reduced IL-17 production in MOG 35-55 - and PLP 139-151 -primed T cells by inhibiting STAT3 phosphorylation and RORγt expression, key regulators of Th17 differentiation. Consistently, in EAE models, both prophylactic and therapeutic oral administration of GSNO attenuated disease severity by suppressing IL-17-mediated neuroinflammation, underscoring the therapeutic potential of S-nitrosylation-dependent regulation of STAT3 in modulating Th17-driven autoimmunity ( 80 ).

S-nitrosylation-mediated regulation of oxidative stress and mitochondrial dysfunction in MS/EAE

MS is marked by disrupted redox homeostasis and mitochondrial dysfunction, which together contribute to inflammation, demyelination, and neurodegeneration. Activated microglia and infiltrating immune cells generate excessive levels of reactive oxygen and nitrogen species (ROS/RNS), resulting in widespread oxidative damage to DNA, proteins, and lipids, particularly within chronically demyelinated lesions ( 282 , 283 ). Mitochondria are not only damaged by oxidative stress but also amplify it, ultimately leading to energy failure and neuronal loss. In MS, although endogenous antioxidant defenses are upregulated in response to oxidative stress, they are often insufficient to counteract sustained oxidative stress ( 282 – 284 ). Recent studies have identified S-nitrosylation as a critical redox-sensitive regulatory mechanism in pathological conditions. By attenuating ROS/RNS-induced toxicity, preserving mitochondrial integrity, and restoring redox balance, S-nitrosylation may help mitigate oxidative damage. This section discusses the emerging role of S-nitrosylation in regulating oxidative stress and mitochondrial dysfunction in MS and its potential as a therapeutic target.

GSNO/S-nitrosylation-mediated regulation of NO•/RNS synthesis

During inflammation, NO• generated by iNOS not only promotes tissue damage through ONOO - formation but also engages in a self-limiting feedback loop by S-nitrosylating iNOS at Cys109 near the oxygenase domain. This modification disrupts the dimer–monomer equilibrium, destabilizing the active dimer, reducing catalytic efficiency, and lowering NO• output ( 285 – 287 ). Additionally, ONOO - -mediated tyrosine nitration has been reported as another negative regulatory mechanism of iNOS activity ( 288 , 289 ). Unlike other NOS isoforms, iNOS expression is markedly upregulated during inflammation and is subject to limited intrinsic regulatory control. Accordingly, these autoinhibitory feedback mechanisms are thought to play an important role in moderating NO• overproduction and reducing nitrosative stress in sepsis and other inflammatory conditions.

In contrast to iNOS, eNOS and nNOS are constitutively expressed and generate low, tightly regulated NO• levels that support vascular and neuronal function. These isoforms generally serve protective or regulatory roles; however, under pathological conditions, uncoupling from essential cofactors such as L-arginine and tetrahydrobiopterin (BH 4 ) can lead to O 2 • - production, shifting their role from physiological regulators to drivers of oxidative and nitrosative stress ( 290 ). In this context, S-nitrosylation-mediated feedback regulation of eNOS and nNOS may be critical for mitigating such pathological outcomes.

In endothelial cells, NO• induces S-nitrosylation of eNOS at Cys94 and Cys99, two critical residues at the homodimer interface required for proper dimer formation and enzymatic activation ( 291 , 292 ). Additional S-nitrosylation occurs at Cys661, Cys802, and Cys1114, which are located near the cofactor-binding domains for FMN, FAD, and NADPH; however, the precise functional roles of these sites remain incompletely understood ( 293 ). NO• can also regulate eNOS activity indirectly through S-nitrosylation of heat shock protein 90 (Hsp90), which impairs its ATPase activity and disrupts the Hsp90-eNOS complex. This disruption reduces eNOS phosphorylation at Ser1177, a modification required for full enzymatic activation ( 294 ).

Similarly, nNOS is subject to feedback inhibition via S-nitrosylation, predominantly at Cys331 and Cys336, which are located near the NADPH-binding and heme domains. Modification of these residues disrupts electron transfer and enzyme dimer stability, leading to autoinhibition of nNOS and limiting excess NO• production, thereby preventing nitrosative stress ( 295 , 296 ). Collectively, available evidence indicates that S-nitrosylation of eNOS and nNOS can attenuate their NO•-producing capacity. Nonetheless, it remains unclear whether NO• or GSNO modulates O 2 • - generation via S-nitrosylation under uncoupled conditions, as this has not yet been experimentally established. Further studies will be required to clarify this potential regulatory mechanism.

Overall, as summarized in Figure 4A , NO•/GSNO-mediated S-nitrosylation inhibits NOS activity and limits excessive NO•/RNS production. Under pathological conditions involving inflammation and oxidative stress, this regulation may function as a self-limiting feedback loop that protects cells and tissues from severe RNS-mediated injury.

S-nitrosylation-mediated regulation of oxidative stress by NADPH oxidase

NADPH oxidase (NOX) transfers electrons from NADPH to molecular oxygen, generating large amounts of O 2 • - in a process known as the oxidative burst. While this plays a critical role in eliminating invading pathogens, it can also contribute to tissue injury in pathological conditions such as atherosclerosis, hypertension, diabetes, and MS ( 297 , 298 ). The NOX family comprises seven known isoforms: NOX1, NOX2, NOX3, NOX4, NOX5, and the dual oxidases DUOX1 and DUOX2. Among them, NOX2, also known as phagocyte oxidase (phox), is the most extensively studied isoform and is considered the most pathologically significant, particularly due to its role in immune-mediated oxidative stress.

NOX2 activation requires the assembly of a multi-subunit enzyme complex. Its membrane-bound core consists of gp91 phox (the catalytic subunit, also known as cytochrome b 245 β chain, encoded by CYBB) and p22 phox , which together form flavocytochrome b 558 . Upon stimulation by pathogens or cytokines, the key regulatory subunit p47 phox orchestrates the translocation of cytosolic components, including p67 phox and p40 phox , to the membrane, thereby initiating assembly of the active NOX2 complex. Full activation also requires the small GTPase Rac (predominantly Rac2 in phagocytes and Rac1 in non-phagocytic cells) ( 299 ) ( Figure 4B ).

NO• is known to inhibit NOX2-derived O 2 • - production by S-nitrosylating the regulatory subunit p47 phox at Cys196. This modification alters its conformation and enhances its phosphorylation by casein kinase 2 (CK2), ultimately impairing p47phox’s ability to translocate to the membrane and assemble the functional NOX2 complex ( 300 , 301 ) ( Figure 4B ). Additionally, S-nitrosylation of p38 MAPK reduces its kinase activity, leading to decreased p47 phox activity. This indirectly impairs NOX2 complex assembly, adding another layer of redox-based control over O 2 • - production ( 297 , 302 ).

S-nitrosylation-mediated regulation of oxidative stress in mitochondria

In MS, ROS from inflammatory cells damage mitochondrial DNA (mtDNA), electron transport chain (ETC) proteins, and lipids ( 303 – 305 ). Dysfunctional mitochondria then generate additional ROS, amplifying oxidative stress in a vicious cycle ( 306 , 307 ). This further depletes ATP, particularly in demyelinated axons that have high energy demands ( 308 , 309 ), resulting in disturbed ion homeostasis, calcium accumulation, and activation of cell death pathways ( 310 , 311 ). Oxidative stress also hinders oligodendrocyte maturation and remyelination by suppressing key mitochondrial transcripts ( 312 , 313 ). Postmortem MS brains show reduced ETC activity, mtDNA deletions, and elevated markers of mitochondrial stress ( 308 , 310 , 314 , 315 ). Together, these findings highlight mitochondrial dysfunction as both a driver and consequence of neurodegeneration in MS.

Mechanistically, O 2 • - is generated in mitochondria through premature electron leakage from ETC complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and III (ubiquinol-cytochrome c reductase), resulting in incomplete one-electron reduction of O 2 ( 316 ). Under normal conditions, low levels of O 2 • - are efficiently dismuted to hydrogen peroxide (H 2 O 2 ) by manganese superoxide dismutase (MnSOD or SOD2) in the matrix and copper/zinc superoxide dismutase (CuZnSOD or SOD1) in the intermembrane space. However, under pathological conditions, O 2 • - production can exceed the neutralizing capacity of endogenous SODs, thus leading to the accumulation of ROS, mitochondrial damage, and the initiation of downstream cell death pathways ( 317 , 318 ) ( Figure 4C ).

NO• influences mitochondrial function through both physiological and pathological mechanisms. Under normal conditions, NO• is known to modulate respiration by reversible inhibition of ETC complexes I, III, and IV (cytochrome c oxidase, COX) ( 319 – 323 ). For example, NO• binds to the heme moiety at the catalytic center of complex IV, temporarily reducing its activity and thereby decreasing electron flux, oxygen consumption, and ATP synthesis ( 324 – 326 ). Under pathological conditions, however, when excessive mitochondrial O 2 • - production coincides with high NO• levels, the two radicals rapidly react to form ONOO - , at a rate greater than that of O 2 • - dismutation to H 2 O 2 ( 305 ). ONOO - induces further mitochondrial dysfunction by nitrating key proteins (e.g., NADH dehydrogenase and succinate dehydrogenase), oxidizing essential lipids (e.g., cardiolipin), and damaging mitochondrial DNA, ultimately exacerbating bioenergetic failure and promoting cell death ( 327 ).

In mitochondria, aberrant S-nitrosylation can disrupt protein folding and promote mitochondrial fragmentation, ultimately contributing to energy failure and cell death ( 328 ). However, emerging evidence also underscores the protective role of physiological S-nitrosylation, particularly in maintaining mitochondrial homeostasis by modulating ROS production and preserving redox balance. For example, physiological micromolar concentrations of GSNO markedly suppress mitochondrial O 2 • - and H 2 O 2 production arising from electron leakage at complexes I and III during succinate- or NAD-linked substrate-driven ETC activity ( 329 , 330 ). Similarly, S-nitroso-N-acetylpenicillamine (SNAP), a synthetic S-nitrosylating agent, has been shown to inhibit electron transfer at complex I through S-nitrosylation of Cys39 on the ND3 subunit of NADH dehydrogenase, resulting in reduced ROS production and enhanced protection against ischemic injury ( 331 ). Moreover, S-nitrosylation exerts a protective effect by suppressing the peroxidase activity of the cytochrome c-cardiolipin complex, thereby preventing cardiolipin oxidation under oxidative stress, an early event in mitochondria-mediated apoptosis ( 332 ).

Collectively, these findings suggest that S-nitrosylation at physiological concentrations is essential for preserving mitochondrial function and mitigating oxidative stress ( Figure 4C ). However, under pathological conditions, excessive levels of GSNO (in the millimolar range) or other S-nitrosylating agents have been reported to exert deleterious effects, including persistent complex I modification that enhances O 2 • - production ( 333 ), as well as Ca 2+ -induced opening of the mitochondrial permeability transition pore (MPTP) via modification of critical mitochondrial proteins such as adenine nucleotide translocase (ANT), voltage-dependent anion channel (VDAC), and ATP synthase ( 334 ). As S-nitrosylation confers protective effects at physiological levels but becomes detrimental when excessive, its precise regulation is crucial for maintaining mitochondrial integrity and function.

S-nitrosylation-mediated regulation of the cellular antioxidant system

O 2 • - is a key initiator of oxidative stress. While moderately reactive under physiological conditions, excess O 2 • - can disrupt mitochondrial Fe-S clusters (e.g., aconitase, succinate dehydrogenase), causing enzyme inactivation, ferrous iron (Fe 2+ ) release, and mitochondrial dysfunction ( 317 , 318 ). Normally, O 2 • - is converted to H 2 O 2 by SOD1 in the cytoplasm and intermembrane space and SOD2 in the mitochondrial matrix ( 335 ) ( Figure 4D ). Although less reactive than O 2 • - , H 2 O 2 accumulation, together with increased release of redox-active Fe 2+ from heme degradation under pathological conditions, can drive Fenton-type reactions, generating highly reactive hydroxyl radicals (HO•) ( 336 ). To limit this threat, cells activate antioxidant defenses that sequester free heme and Fe 2+ while degrading excess H 2 O 2 .

Under stress, free heme itself acts as a potent pro-oxidant but is degraded by heme oxygenase-1 (HO-1) into biliverdin, carbon monoxide (CO), and Fe 2+ ( 337 ). The released Fe 2+ is rapidly sequestered by ferritin, thereby preventing iron-driven Fenton chemistry and oxidative injury, while biliverdin is subsequently reduced to bilirubin; both bile pigments provide potent antioxidant protection ( 338 , 339 ) ( Figure 4D ). H 2 O 2 is further detoxified by catalase in peroxisomes, as well as by glutathione peroxidases (GPx1-GPx7) and peroxiredoxins (Prx1-Prx6) distributed across multiple cellular compartments, including the cytoplasm. GPxs use GSH to convert H 2 O 2 into water and detoxify lipid hydroperoxides by converting them into alcohols. In this process, GSH is oxidized to glutathione disulfide (GSSG), which is then converted back to GSH by glutathione reductase (GR) in a reaction that requires NADPH ( 340 , 341 ) ( Figure 4D ). Prxs catalyze similar reductions with thioredoxin (Trx) as the electron donor, which is regenerated by thioredoxin reductase (TrxR) in an NADPH-dependent process ( 342 , 343 ) ( Figure 4D ).

In MS and EAE, antioxidant enzymes such as SOD, catalase, GPx, and GR are upregulated; however, oxidative damage in the CNS persists due to limited enzyme expression, rapid GSH depletion by reactive aldehydes like acrolein, which also inhibits GR, and sustained ROS/RNS production from infiltrating immune cells ( 344 , 345 ). NO• has long been recognized not only as a key player in ROS/RNS chemistry but also as a crucial modulator of endogenous antioxidant defenses. For instance, NO• influences antioxidant enzymes through post-translational modifications. One notable example is the catalase, whose activity is modulated through S-nitrosylation. Silencing of GSNOR, which elevates intracellular levels of GSNO, enhances the S-nitrosylation of catalase at Cys401, Cys642, and Cys653, of which Cys401 plays a particularly critical role in regulating its enzymatic activity. This post-translational modification markedly enhances catalase function, thereby contributing to a reduction in oxidative stress ( 346 ) ( Figure 4D ).

Beyond post-translational regulation, NO• also modulates redox homeostasis at the transcriptional level by targeting key transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF-1α). Through S-nitrosylation, NO• can stabilize and enhance the transcriptional activity of these regulators, thereby promoting the expression of diverse antioxidant and cytoprotective genes ( Figure 4D ) ( 347 ).

Under basal conditions, Nrf2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1) and Cullin-3, which together form an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation ( 348 ). Upon oxidative or electrophilic stress, specific cysteine residues on Keap1 are modified, disrupting the Keap1-Cul3 complex and allowing Nrf2 to accumulate, translocate to the nucleus, and dimerize with small Maf proteins ( 349 , 350 ). This leads to the activation of antioxidant response element (ARE)-driven genes, including HO-1, NQO1, SOD2, catalase, Trx1, Grx1, GPx4, and GSTs ( 351 – 356 ). Importantly, NO• can potentiate this pathway by S-nitrosylating Keap1, thereby preventing its repressive interaction with Nrf2 and enhancing antioxidant gene transcription ( 353 , 354 , 357 ) ( Figure 4D ).

HIF-1α is primarily responsible for cellular adaptation to hypoxia but also contributes to redox regulation by inducing antioxidant enzymes such as SOD2, CAT, GPx4, Prx1, Prx5, and HO-1 ( 347 , 358 ), as well as by promoting glutathione biosynthesis ( 359 ). Under normoxia, HIF-1α is hydroxylated by prolyl hydroxylase domain protein 2 (PHD2), which targets it for ubiquitination and proteasomal degradation ( 167 – 169 ). NO• can stabilize HIF-1α even under normoxic conditions by S-nitrosylating both HIF-1α and PHD2, thereby preventing hydroxylation-dependent degradation and enabling sustained transcription of HIF-1α target genes ( 173 – 175 ) ( Figure 4D ).

Among the antioxidant genes regulated by Nrf2 and HIF-1α, GPx4 is particularly important because it uses GSH to detoxify lipid hydroperoxides and prevent membrane phospholipid peroxidation ( 360 ), thereby protecting cells against ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation In MS and EAE, ferroptosis contributes to oligodendrocyte loss, neuronal injury, and disease progression through GSH depletion, reduced GPx4 expression and activity, iron accumulation, and lipid peroxidation ( 361 , 362 ). Accordingly, inhibiting ferroptosis or restoring GPx4 activity attenuates neuroinflammation and demyelination in experimental models, supporting ferroptosis as a potential therapeutic target in MS ( 361 , 363 ).

The relationship between NO• and ferroptosis appears to be highly context-dependent. On the one hand, excessive NO• readily reacts with O 2 • - to generate ONOO - , a potent oxidant that promotes lipid peroxidation and may contribute to ferroptotic cell death under conditions of sustained nitrosative stress ( 364 ). On the other hand, emerging evidence suggests that GSNO-mediated S-nitrosylation may indirectly counteract ferroptosis through multiple mechanisms. As discussed above, S-nitrosylation of Keap1 activates the Nrf2 antioxidant pathway ( 353 , 354 , 357 ), whereas stabilization of HIF-1α further supports cellular antioxidant capacity by inducing antioxidant enzymes and glutathione biosynthesis ( 173 – 175 ). In addition, S-nitrosylation has been also proposed to modulate ferroptosis-associated signaling pathways, including the ASK1-P38/JNK MAPK cascade, although current evidence remains largely indirect and is derived primarily from cancer models ( 365 ). Consistent with an overall protective role of NO•, NO• donors suppress ferroptosis by terminating lipid peroxidation chain reactions independently of the canonical GPx4 pathway ( 366 ). Moreover, iNOS-derived NO• confers resistance to ferroptosis even under conditions of GPx4 deficiency ( 367 ). Whether GSNO-mediated S-nitrosylation directly regulates the core ferroptotic machinery in MS and EAE remains unknown and thus warrants further investigation.

In summary, S-nitrosylation is a critical mechanism by which NO• regulates cellular antioxidant defenses at both the enzymatic and transcriptional level. These mechanisms may also limit ferroptotic cell death by reducing oxidative stress, enhancing glutathione-dependent antioxidant capacity, and inducing cytoprotective gene expression, though whether GSNO-mediated S-nitrosylation directly regulates core ferroptotic machinery remains unknown. While antioxidants have shown promise in inflammatory and oxidative disease settings, upregulating antioxidant enzymes in peripheral compartments does not necessarily protect vulnerable CNS regions such as white matter and cortex in MS, and many antioxidant compounds show limited CNS bioavailability, underscoring the need for CNS-targeted strategies ( 368 ). Recent findings in mice suggest that systemic administration of the GSNOR inhibitor N91115 can elevate S-nitrosothiol levels within the CNS, potentially enhancing endogenous antioxidant responses in MS or EAE ( 53 ). However, these observations require further experimental validation to confirm their therapeutic relevance and efficacy.

S-nitrosylation-mediated metabolic reprogramming to coordinate energy production and redox balance

NADPH plays a central role in maintaining cellular redox homeostasis by providing reducing equivalents to GR and TrxR, which regenerate key antioxidants such as GSH and Trx. Simultaneously, it fuels NOX and NOS, thereby supporting the production of ROS and RNS. This dual functionality enables NADPH to mediate a fine balance between the generation of oxidative/nitrosative stress and antioxidant defense, allowing cells to dynamically regulate their redox state in response to fluctuating conditions ( 369 ).

In MS and EAE, redox imbalance is particularly evident in brain white matter, where reduced GSH levels have been detected in patients, reflecting impaired antioxidant defenses ( 370 , 371 ). Under these pathological conditions, a stable supply of NADPH is essential to counteract oxidative and nitrosative stress. NADPH is mainly produced through the oxidative pentose phosphate pathway (oxPPP), which competes with glycolysis for glucose as a common substrate ( Figure 4E ). Thus, increased demand for NADPH creates a metabolic trade-off: greater oxPPP flux may reduce glycolytic ATP production, and vice versa. As a result, glucose metabolism is reprogrammed to prioritize either energy generation or redox balance depending on cellular demands, a shift that becomes especially relevant in the inflammatory and degenerative environment of MS.

NO• is a key modulator of glucose metabolism. It promotes the expression and membrane localization of glucose transporters (GLUT1 and GLUT4) ( 372 – 374 ), thereby facilitating increased glucose uptake ( 372 , 375 , 376 ) ( Figure 4E ). Intriguingly, NO• exhibits a dose-dependent dual effect on glycolysis: at low concentrations, it enhances glycolysis, partly through activation of 5′-AMP-activated protein kinase (AMPK) by increasing AMP levels or promoting phosphorylation of AMPK-α1 at Thr172 ( 377 , 378 ). NO• also stabilizes HIF-1α, which enhances glycolysis by upregulating glucose transporters (GLUT1, GLUT3) and key glycolytic enzymes, including hexokinase 2 (HK2), phosphofructokinase (PFK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), thereby accelerating glucose metabolism through the glycolytic pathway ( 379 ).

However, in inflammatory states, excessive NO• produced by iNOS negatively impacts glycolytic flux. It inhibits key enzymes like GAPDH through S-nitrosylation at Cys149, which promotes its nuclear translocation and disrupts cytoplasmic glycolytic metabolism ( 380 – 384 ). Similarly, pyruvate kinase M2 (PKM2), essential for the final step in glycolysis in embryonic cells, activated macrophages, and CD4 + T cells, is inhibited by S-nitrosylation, which prevents the formation of its active tetramer ( 385 , 386 ) ( Figure 4E ). These findings suggest that, under pathological conditions, excessive NO• redirects glucose flux from glycolysis to the oxPPP, thereby increasing NADPH production and antioxidant capacity to buffer nitrosative stress ( 385 , 387 , 388 ).

Importantly, while glucose is indispensable for fueling the oxPPP for NADPH synthesis, ATP production can be supported by alternative fuels like ketone bodies and amino acids under conditions of glucose scarcity ( 389 ). Interestingly, NO• has also been shown to promote energy production through lipid metabolism. Specifically, it enhances the activity of very long-chain acyl-CoA dehydrogenase (VLCAD), a rate-limiting enzyme in mitochondrial fatty acid β-oxidation, via S-nitrosylation at Cys238, thus supporting acetyl-CoA and ATP production through this parallel route ( 390 ) ( Figure 4E ).

Metabolic reprogramming is now recognized as a major determinant of immune-cell differentiation and function. Accordingly, NO•-dependent metabolic changes may also influence adaptive immune responses relevant to MS pathogenesis. In MS, the balance between Th17 cells and Treg cells, which have opposing roles in immunity and inflammation, is crucial for immune homeostasis ( 391 ). Notably, Th17 cells rely mainly on aerobic glycolysis, whereas Treg cells depend on fatty acid oxidation and oxidative phosphorylation to meet their metabolic demands ( 392 – 394 ). Recent findings show that GSNO or reversible inhibition of GSNOR (e.g., with N6022 and N91115 ) suppresses Th17 differentiation and effector functions while enhancing those of Treg cells in EAE mice ( 53 , 80 , 81 , 134 , 163 ). These distinct metabolic requirements raise the possibility that GSNO-mediated signaling contributes to immune regulation by altering cellular metabolic programs. However, whether GSNO regulates Th17 and Treg cell polarization through metabolic reprogramming, potentially by modulating glycolysis and fatty acid β-oxidation, remains unclear and warrants further investigation.

Because NADPH-dependent redox pathways operate in virtually all metabolically active tissues, the metabolic effects of GSNOR inhibition are unlikely to be restricted to CNS-infiltrating immune cells. Systemic GSNOR inhibition may therefore influence redox homeostasis in peripheral tissues with high metabolic demand, including the liver, skeletal muscle, and erythrocytes, where NADPH-dependent antioxidant systems are essential for maintaining cellular function. Such systemic effects may influence not only cellular antioxidant defenses but also tissue-specific metabolic functions, highlighting the importance of evaluating potential off-target physiological consequences during chronic GSNOR inhibition. Consistent with this possibility, preclinical toxicology studies of the GSNOR inhibitor N6022 reported mild, redox-related hepatotoxicity at high doses, whereas lower doses were well tolerated ( 395 ). Although these findings suggest the existence of a therapeutic window, the systemic metabolic consequences of chronic GSNOR inhibition have not been systematically evaluated and remain an important consideration for long-term safety assessment.

In summary, metabolic reprogramming is fundamental to coordinating cellular energy production and redox homeostasis, with NADPH serving as a central hub linking oxidative stress to antioxidant defense. Through modulation of glycolysis, the oxidative pentose phosphate pathway, and fatty acid oxidation, GSNO-mediated S-nitrosylation may shape immune cell metabolism and function while maintaining redox balance. At the same time, because these metabolic pathways are broadly utilized across multiple organs, future studies should define the systemic metabolic consequences, therapeutic window, and tissue-specific safety profile of chronic GSNOR inhibition to maximize immunomodulatory efficacy while preserving physiological redox homeostasis.

Summary, conclusions, and future perspectives

MS is a chronic autoimmune disorder of the CNS that requires lifelong management. Although DMTs have substantially improved disease management, each therapeutic class possesses inherent limitations. Table 1 provides an overview of the mechanisms of action, major advantages, and limitations of currently available and emerging DMTs for MS. Biologic therapies, including anti-CD20 monoclonal antibodies, natalizumab, interferon-β, and glatiramer acetate, provide targeted immunomodulation and substantial clinical efficacy but generally require parenteral administration and may be associated with prolonged immune cell depletion, immunogenicity, or infusion-related complications ( 396 – 407 ). More recently, cytokine-targeted biologics, such as anti-IL-17/IL-23 therapies, have demonstrated remarkable efficacy in psoriasis and related autoimmune diseases; however, their clinical benefit in MS has remained limited, suggesting that selective blockade of the IL-17/Th17 pathway alone may be insufficient to control disease progression ( 72 , 408 ). In contrast, orally available small-molecule therapies, including S1P receptor modulators, fumarates, teriflunomide, and cladribine, offer greater convenience but frequently rely on broad immunosuppressive mechanisms that can cause lymphopenia, increased infection risk, and other systemic adverse effects ( 409 – 418 ). Emerging BTK inhibitors represent another promising class of oral therapeutics by targeting both adaptive and innate immune responses, although their long-term efficacy and safety remain under investigation ( 419 – 421 ).

Unlike these therapeutic approaches, GSNOR inhibitors represent a mechanistically distinct class of orally active small molecules that selectively enhance endogenous GSNO-mediated S-nitrosylation. By elevating intracellular GSNO, they may restore physiological NO• signaling, thereby regulating immune responses, reducing oxidative stress, and supporting mitochondrial function. Unlike many currently available biologic and oral small-molecule DMTs, which primarily rely on broad immunosuppressive or immune-depleting mechanisms, preclinical studies indicate that GSNOR inhibition reprograms immune responses by suppressing pathogenic T cells (Th1/Th17) as well as inflammatory B cells while promoting regulatory T- and B-cell populations, without inducing systemic lymphopenia ( 53 , 80 , 81 , 133 , 134 , 163 , 219 , 228 , 422 ) ( Table 1 ). In addition to its immunomodulatory effects, GSNOR inhibition may also confer neuroprotective benefits through complementary mechanisms, including limiting microglial and macrophage activation, preserving blood-brain barrier integrity, upregulating antioxidant enzymes, and activating transcriptional programs such as Nrf2 and HIF-1α. Together, these multimodal actions integrate selective immune modulation, redox/metabolic regulation, and neuroprotective mechanisms, as illustrated in Figure 5 , potentially extending the therapeutic scope beyond the primary immunomodulatory actions of most currently available DMTs.

Nevertheless, the biological effects of GSNO-mediated S-nitrosylation are highly concentration- and context-dependent. Physiological, low-micromolar concentrations of GSNO generally support antioxidant, cytoprotective, and immunomodulatory signaling, whereas excessive or sustained elevations in GSNO may lead to maladaptive S-nitrosylation of non-target proteins, impaired protein folding, mitochondrial dysfunction, and disruption of normal redox signaling networks ( 328 , 333 , 334 ). Because S-nitrosylation regulates numerous proteins involved in immune responses, mitochondrial respiration, apoptosis, and vascular homeostasis, prolonged or indiscriminate GSNOR inhibition may perturb systemic redox homeostasis beyond the intended therapeutic pathways. Consequently, the therapeutic benefit of GSNOR inhibition is likely to depend on achieving a balanced increase in GSNO rather than indiscriminate enhancement of global S-nitrosylation. These considerations highlight an important challenge for future clinical development. Although currently available GSNOR inhibitors have demonstrated favorable safety and tolerability profiles in preclinical studies and Phase I/II clinical trials for asthma and cystic fibrosis, the therapeutic window that optimally balances efficacy with preservation of physiological S-nitrosylation homeostasis remains to be defined. Future studies should therefore determine the optimal degree, duration, and tissue specificity of GSNOR inhibition, particularly in the context of chronic neuroinflammatory diseases such as MS.

Importantly, GSNOR inhibitors such as N91115 and N6022 are small molecules with favorable pharmacological and practical profiles. They are suitable for oral or parenteral administration, depending on the compound, avoid the logistical burdens associated with monoclonal antibody therapies, and demonstrate predictable pharmacokinetics, scalable production, and lower manufacturing costs. Both compounds have completed Phase I and II clinical trials in non-neurological diseases with favorable safety and tolerability, supporting the clinical feasibility of GSNOR inhibition in humans. However, evidence supporting the therapeutic efficacy of GSNOR inhibitors in MS remains limited to mechanistic studies, in vitro experiments, and EAE models, and no clinical trials have yet evaluated GSNO/GSNOR-targeted therapies in patients with MS. Therefore, well-designed clinical trials are required to determine whether the promising immunomodulatory and neuroprotective effects observed in preclinical studies can be translated into meaningful clinical benefit in patients with MS.

Taken together, current mechanistic and preclinical evidence supports the GSNO/GSNOR axis as a promising therapeutic target for MS by integrating selective immune modulation with neuroprotection. Although these conclusions are based predominantly on preclinical evidence, the favorable pharmacological properties and clinical safety profiles of GSNOR inhibitors support continued clinical development. If future clinical studies confirm these promising preclinical findings, GSNOR inhibition may represent a next-generation therapeutic strategy capable of providing durable immune reprogramming while minimizing the systemic immunosuppression associated with many current DMTs.

Funding Statement

The authors declared that financial support was received for this work and/or its publication. This work was supported in part by the U.S. Department of Veterans Affairs (BX002829).

Footnotes

Edited by: Roumen Balabanov , Northwestern Medicine, United States

Reviewed by: Javier Ramirez Jirano , Centro de Investigación Biomédica de Occidente (CIBO), Mexico

Giulio Papiri , Madonna del Soccorso Hospital, Italy

Author contributions

JW: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JK: Investigation, Writing – original draft. AS: Resources, Writing – review & editing. IS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The authors declared that generative AI was not used in the creation of this manuscript.

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References

1. Compston A, Coles A. Multiple sclerosis. Lancet. (2008) 372:1502–17. doi: 10.1016/s0140-6736(08)61620-7

2. Sadovnick AD, Baird PA. The familial nature of multiple sclerosis: Age-corrected empiric recurrence risks for children and siblings of patients. Neurology. (1988) 38:990–1. doi: 10.1212/wnl.38.6.990

3. Gourraud PA, Harbo HF, Hauser SL, Baranzini SE. The genetics of multiple sclerosis: An up-to-date review. Immunol Rev. (2012) 248:87–103. doi: 10.1111/j.1600-065X.2012.01134.x

4. Koch M, Kingwell E, Rieckmann P, Tremlett H. The natural history of primary progressive multiple sclerosis. Neurology. (2009) 73:1996–2002. doi: 10.1212/WNL.0b013e3181c5b47f

5. Pegoretti V, Swanson KA, Bethea JR, Probert L, Eisel ULM, Fischer R. Inflammation and oxidative stress in multiple sclerosis: Consequences for therapy development. Oxid Med Cell Longev. (2020) 2020:7191080. doi: 10.1155/2020/7191080

6. Mariottini A, Muraro PA, Lünemann JD. Antibody-mediated cell depletion therapies in multiple sclerosis. Front Immunol. (2022) 13:953649. doi: 10.3389/fimmu.2022.953649

7. Lambert CM, Hussain T, Peters J, Longbrake EE. Real-world infection risk in multiple sclerosis patients on long-term immunomodulatory treatments. Mult Scler Relat Disord. (2025) 94:106236. doi: 10.1016/j.msard.2024.106236

8. Klotz L, Havla J, Schwab N, Hohlfeld R, Barnett M, Reddel S, et al. Risks and risk management in modern multiple sclerosis immunotherapeutic treatment. Ther Adv Neurol Disord. (2019) 12:1756286419836571. doi: 10.1177/1756286419836571

9. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. (1994) 298:249–58. doi: 10.1042/bj2980249

10. Wang XH, Tong M, Dou D, Usha JR, Gao YS. Role of cgmp-dependent protein kinase in the cardiovascular system. Sheng Li Ke Xue Jin Zhan. (2005) 36:299–303.

11. Ivanova K, Hemmersbach R. Guanylyl cyclase-cgmp signaling pathway in melanocytes: Differential effects of altered gravity in non-metastatic and metastatic cells. Int J Mol Sci. (2020) 21. doi: 10.3390/ijms21031139

12. Francis SH, Busch JL, Corbin JD, Sibley D. Cgmp-dependent protein kinases and cgmp phosphodiesterases in nitric oxide and cgmp action. Pharmacol Rev. (2010) 62:525–63. doi: 10.1124/pr.110.002907

13. Stachon T, Latta L, Seitz B, Szentmáry N. Hypoxic stress increases nf-κb and inos mrna expression in normal, but not in keratoconus corneal fibroblasts. Graefes Arch Clin Exp Ophthalmol. (2021) 259:449–58. doi: 10.1007/s00417-020-04900-8

14. Morris SM, Jr., Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol. (1994) 266:E829–39. doi: 10.1152/ajpendo.1994.266.6.E829

15. Nathan C, Xie QW. Nitric oxide synthases: Roles, tolls, and controls. Cell. (1994) 78:915–8. doi: 10.1016/0092-8674(94)90266-6

16. Moncada S, Higgs A. The l-arginine-nitric oxide pathway. N Engl J Med. (1993) 329:2002–12. doi: 10.1056/nejm199312303292706

17. Stuehr DJ, Griffith OW. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Mol Biol. (1992) 65:287–346. doi: 10.1002/9780470123119.ch8

18. Jones ML, Ganopolsky JG, Labbé A, Wahl C, Prakash S. Antimicrobial properties of nitric oxide and its application in antimicrobial formulations and medical devices. Appl Microbiol Biotechnol. (2010) 88:401–7. doi: 10.1007/s00253-010-2733-x

19. Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med. (1998) 25:434–56. doi: 10.1016/s0891-5849(98)00092-6

20. Tengan CH, Moraes CT. No control of mitochondrial function in normal and transformed cells. Biochim Biophys Acta Bioenerg. (2017) 1858:573–81. doi: 10.1016/j.bbabio.2017.02.009

21. Roe ND, Ren J. Nitric oxide synthase uncoupling: A therapeutic target in cardiovascular diseases. Vascul Pharmacol. (2012) 57:168–72. doi: 10.1016/j.vph.2012.02.004

22. Hogg N. The biochemistry and physiology of s-nitrosothiols. Annu Rev Pharmacol Toxicol. (2002) 42:585–600. doi: 10.1146/annurev.pharmtox.42.092501.104328

23. Li S, Yu K, Wu G, Zhang Q, Wang P, Zheng J, et al. Pcysmod: Prediction of multiple cysteine modifications based on deep learning framework. Front Cell Dev Biol. (2021) 9:617366. doi: 10.3389/fcell.2021.617366

24. Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS. Protein s-nitrosylation: Purview and parameters. Nat Rev Mol Cell Biol. (2005) 6:150–66. doi: 10.1038/nrm1569

25. Fernando V, Zheng X, Walia Y, Sharma V, Letson J, Furuta S. S-nitrosylation: An emerging paradigm of redox signaling. Antioxidants (Basel). (2019) 8. doi: 10.3390/antiox8090404

26. Pollock JS, Förstermann U, Mitchell JA, Warner TD, Schmidt HH, Nakane M, et al. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci USA. (1991) 88:10480–4. doi: 10.1073/pnas.88.23.10480

27. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA. (1990) 87:682–5. doi: 10.1073/pnas.87.2.682

28. Huang PL. Enos and nnos in stroke. In: Lajtha A, Chan PH, editors. Handbook of Neurochemistry and Molecular Neurobiology: Acute Ischemic Injury and Repair in the Nervous System. Springer US, New York, NY: (2007). p. 47–63.

29. Gow AJ, Chen Q, Hess DT, Day BJ, Ischiropoulos H, Stamler JS. Basal and stimulated protein s-nitrosylation in multiple cell types and tissues. J Biol Chem. (2002) 277:9637–40. doi: 10.1074/jbc.C100746200

30. Stamler JS, Lamas S, Fang FC. Nitrosylation. The prototypic redox-based signaling mechanism. Cell. (2001) 106:675–83. doi: 10.1016/s0092-8674(01)00495-0

31. Hess DT, Stamler JS. Regulation by s-nitrosylation of protein post-translational modification. J Biol Chem. (2012) 287:4411–8. doi: 10.1074/jbc.R111.285742

32. Foster MW, Hess DT, Stamler JS. Protein s-nitrosylation in health and disease: A current perspective. Trends Mol Med. (2009) 15:391–404. doi: 10.1016/j.molmed.2009.06.007

33. Hogg N, Singh RJ, Kalyanaraman B. The role of glutathione in the transport and catabolism of nitric oxide. FEBS Lett. (1996) 382:223–8. doi: 10.1016/0014-5793(96)00086-5

34. DeMaster EG, Quast BJ, Redfern B, Nagasawa HT. Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide. Biochemistry. (1995) 34:11494–9. doi: 10.1021/bi00036a023

35. Liu X, Miller MJ, Joshi MS, Thomas DD, Lancaster JR., Jr. Accelerated reaction of nitric oxide with o2 within the hydrophobic interior of biological membranes. Proc Natl Acad Sci USA. (1998) 95:2175–9. doi: 10.1073/pnas.95.5.2175

36. Wink DA, Nims RW, Darbyshire JF, Christodoulou D, Hanbauer I, Cox GW, et al. Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral ph. Insights into the fate and physiological effects of intermediates generated in the no/o2 reaction. Chem Res Toxicol. (1994) 7:519–25. doi: 10.1021/tx00040a007

37. Goldstein S, Czapski G. Mechanism of the nitrosation of thiols and amines by oxygenated •no solutions: the nature of the nitrosating intermediates. J Am Chem Soc. (1996) 118:3419–25. doi: 10.1021/ja9536680

38. Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inflammatory diseases. Inflammopharmacology. (2007) 15:252–9. doi: 10.1007/s10787-007-0013-x

39. Panday A, Sahoo MK, Osorio D, Batra S. Nadph oxidases: An overview from structure to innate immunity-associated pathologies. Cell Mol Immunol. (2015) 12:5–23. doi: 10.1038/cmi.2014.89

40. Gaston B, Reilly J, Drazen JM, Fackler J, Ramdev P, Arnelle D, et al. Endogenous nitrogen oxides and bronchodilator s-nitrosothiols in human airways. Proc Natl Acad Sci USA. (1993) 90:10957–61. doi: 10.1073/pnas.90.23.10957

41. Eu JP, Liu L, Zeng M, Stamler JS. An apoptotic model for nitrosative stress. Biochemistry. (2000) 39:1040–7. doi: 10.1021/bi992046e

42. Hilliquin P, Borderie D, Hernvann A, Menkès CJ, Ekindjian OG. Nitric oxide as s-nitrosoproteins in rheumatoid arthritis. Arthritis Rheum. (1997) 40:1512–7. doi: 10.1002/art.1780400820

43. Mayer B, Pfeiffer S, Schrammel A, Koesling D, Schmidt K, Brunner F. A new pathway of nitric oxide/cyclic gmp signaling involving s-nitrosoglutathione. J Biol Chem. (1998) 273:3264–70. doi: 10.1074/jbc.273.6.3264

44. Barnett SD, Buxton ILO. The role of s-nitrosoglutathione reductase (gsnor) in human disease and therapy. Crit Rev Biochem Mol Biol. (2017) 52:340–54. doi: 10.1080/10409238.2017.1304353

45. Jahnová J, Luhová L, Petřivalský M. S-nitrosoglutathione reductase-the master regulator of protein s-nitrosation in plant no signaling. Plants (Basel). (2019) 8. doi: 10.3390/plants8020048

46. Brown-Steinke K, deRonde K, Yemen S, Palmer LA. Gender differences in s-nitrosoglutathione reductase activity in the lung. PloS One. (2010) 5:e14007. doi: 10.1371/journal.pone.0014007

47. Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS. A metabolic enzyme for s-nitrosothiol conserved from bacteria to humans. Nature. (2001) 410:490–4. doi: 10.1038/35068596

48. Sengupta R, Holmgren A. The role of thioredoxin in the regulation of cellular processes by s-nitrosylation. Biochim Biophys Acta. (2012) 1820:689–700. doi: 10.1016/j.bbagen.2011.08.012

49. Que LG, Yang Z, Stamler JS, Lugogo NL, Kraft M. S-nitrosoglutathione reductase: An important regulator in human asthma. Am J Respir Crit Care Med. (2009) 180:226–31. doi: 10.1164/rccm.200901-0158OC

50. Grasemann H, Gaston B, Fang K, Paul K, Ratjen F. Decreased levels of nitrosothiols in the lower airways of patients with cystic fibrosis and normal pulmonary function. J Pediatr. (1999) 135:770–2. doi: 10.1016/s0022-3476(99)70101-0

51. Liu G, Lu D, Wu J, Wang S, Duan A, Ren Y, et al. Enhancing S-nitrosoglutathione reductase decreases S-nitrosylation of Ero1α and reduces neuronal death in secondary traumatic brain injury. Nitric Oxide. (2025) 154:29–41. doi: 10.1016/j.niox.2024.11.005

52. Kim J, Qiao F, Singh AK, Won J, Singh I. Efficacies of S-nitrosoglutathione (GSNO) and GSNO reductase inhibitor in SARS-CoV-2 spike protein induced acute lung disease in mice. Front Pharmacol. (2023) 14:1304697. doi: 10.3389/fphar.2023.1304697

53. Singh I, Kim J, Touhidul Islam SM, Fei Q, Singh AK, Won J. The role of S-nitrosoglutathione reductase (GSNOR) in T cell-mediated immunopathology of experimental autoimmune encephalomyelitis (EAE). Neuroscience. (2025) 564:1–12. doi: 10.1016/j.neuroscience.2024.11.025

54. Green LS, Chun LE, Patton AK, Sun X, Rosenthal GJ, Richards JP. Mechanism of inhibition for N6022, a first-in-class drug targeting S-nitrosoglutathione reductase. Biochemistry. (2012) 51:2157–68. doi: 10.1021/bi201785u

55. Donaldson SH, Solomon GM, Zeitlin PL, Flume PA, Casey A, McCoy K, et al. Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508del-CFTR. J Cyst Fibros. (2017) 16:371–9. doi: 10.1016/j.jcf.2017.01.009

56. Nikitovic D, Holmgren A. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J Biol Chem. (1996) 271:19180–5. doi: 10.1074/jbc.271.32.19180

57. Mancuso C, Bonsignore A, Di Stasio E, Mordente A, Motterlini R. Bilirubin and S-nitrosothiols interaction: Evidence for a possible role of bilirubin as a scavenger of nitric oxide. Biochem Pharmacol. (2003) 66:2355–63. doi: 10.1016/j.bcp.2003.08.022

58. Cocks TM, Angus JA, Campbell JH, Campbell GR. Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J Cell Physiol. (1985) 123:310–20. doi: 10.1002/jcp.1041230304

59. Griffith TM, Edwards DH, Lewis MJ, Newby AC, Henderson AH. The nature of endothelium-derived vascular relaxant factor. Nature. (1984) 308:645–7. doi: 10.1038/308645a0

60. Lancaster JR., Jr. A tutorial on the diffusibility and reactivity of free nitric oxide. Nitric Oxide. (1997) 1:18–30. doi: 10.1006/niox.1996.0112

61. Lancaster JR., Jr. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc Natl Acad Sci USA. (1994) 91:8137–41. doi: 10.1073/pnas.91.17.8137

62. Broniowska KA, Diers AR, Hogg N. S-nitrosoglutathione. Biochim Biophys Acta. (2013) 1830:3173–81. doi: 10.1016/j.bbagen.2013.02.004

63. He W, Frost MC. Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors. Redox Biol. (2016) 9:1–14. doi: 10.1016/j.redox.2016.05.002

64. Chiueh CC. S-nitrosoglutathione (GSNO) mediates brain response to hypoxia. Pediatr Res. (2002) 51:414. doi: 10.1203/00006450-200204000-00002

65. Broniowska KA, Hogg N. The chemical biology of S-nitrosothiols. Antioxid Redox Signal. (2012) 17:969–80. doi: 10.1089/ars.2012.4590

66. Gaston BM, Carver J, Doctor A, Palmer LA. S-nitrosylation signaling in cell biology. Mol Interv. (2003) 3:253–63. doi: 10.1124/mi.3.5.253

67. Baecher-Allan C, Kaskow BJ, Weiner HL. Multiple sclerosis: Mechanisms and immunotherapy. Neuron. (2018) 97:742–68. doi: 10.1016/j.neuron.2018.01.021

68. Lassmann H. Pathogenic mechanisms associated with different clinical courses of multiple sclerosis. Front Immunol. (2018) 9:3116. doi: 10.3389/fimmu.2018.03116

69. Sawcer S, Hellenthal G, Pirinen M, Spencer CC, Patsopoulos NA, Moutsianas L, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature. (2011) 476:214–9. doi: 10.1038/nature10251

70. Lock C, Hermans G, Pedotti R, Brendolan A, SChadt E, Garren H, et al. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med. (2002) 8:500–8. doi: 10.1038/nm0502-500

71. Panitch HS, Hirsch RL, Schindler J, Johnson KP. Treatment of multiple sclerosis with gamma interferon: Exacerbations associated with activation of the immune system. Neurology. (1987) 37:1097–102. doi: 10.1212/wnl.37.7.1097

72. Havrdová E, Belova A, Goloborodko A, Tisserant A, Wright A, Wallstroem E, et al. Activity of secukinumab, an anti-IL-17A antibody, on brain lesions in RRMS: Results from a randomized, proof-of-concept study. J Neurol. (2016) 263:1287–95. doi: 10.1007/s00415-016-8128-x

73. Kunkl M, Frascolla S, Amormino C, Volpe E, Tuosto L. T helper cells: The modulators of inflammation in multiple sclerosis. Cells. (2020) 9. doi: 10.3390/cells9020482

74. Arellano G, Ottum PA, Reyes LI, Burgos PI, Naves R. Stage-specific role of interferon-gamma in experimental autoimmune encephalomyelitis and multiple sclerosis. Front Immunol. (2015) 6:492. doi: 10.3389/fimmu.2015.00492

75. Lees JR, Cross AH. A little stress is good: IFN-gamma, demyelination, and multiple sclerosis. J Clin Invest. (2007) 117:297–9. doi: 10.1172/jci31254

76. Smith BC, Tinkey RA, Brock OD, Mariam A, Habean ML, Dutta R, et al. Astrocyte interferon-gamma signaling dampens inflammation during chronic central nervous system autoimmunity via PD-L1. J Neuroinflamm. (2023) 20:234. doi: 10.1186/s12974-023-02917-4

77. Mills KHG. IL-17 and IL-17-producing cells in protection versus pathology. Nat Rev Immunol. (2023) 23:38–54. doi: 10.1038/s41577-022-00746-9

78. Peters A, Pitcher LA, Sullivan JM, Mitsdoerffer M, Acton SE, Franz B, et al. Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation. Immunity. (2011) 35:986–96. doi: 10.1016/j.immuni.2011.10.015

79. Niedbala W, Wei XQ, Campbell C, Thomson D, Komai-Koma M, Liew FY. Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor beta 2 expression via cGMP. Proc Natl Acad Sci USA. (2002) 99:16186–91. doi: 10.1073/pnas.252464599

80. Nath N, Morinaga O, Singh I. S-nitrosoglutathione a physiologic nitric oxide carrier attenuates experimental autoimmune encephalomyelitis. J Neuroimmune Pharmacol. (2010) 5:240–51. doi: 10.1007/s11481-009-9187-x

81. Saxena N, Won J, Choi S, Singh AK, Singh I. S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis. Free Radic Biol Med. (2018) 121:57–68. doi: 10.1016/j.freeradbiomed.2018.04.558

82. Huang FP, Niedbala W, Wei XQ, Xu D, Feng GJ, Robinson JH, et al. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages. Eur J Immunol. (1998) 28:4062–70. doi: 10.1002/(SICI)1521-4141(199812)28:12<4062::AID-IMMU4062>3.0.CO;2-K

83. Xiong H, Zhu C, Li F, Hegazi R, He K, Babyatsky M, et al. Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells. J Biol Chem. (2004) 279:10776–83. doi: 10.1074/jbc.M313416200

84. Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, et al. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature. (1995) 375:408–11. doi: 10.1038/375408a0

85. McInnes IB, Leung B, Wei XQ, Gemmell CC, Liew FY. Septic arthritis following Staphylococcus aureus infection in mice lacking inducible nitric oxide synthase. J Immunol. (1998) 160:308–15. doi: 10.4049/jimmunol.160.1.308

86. MacLean A, Wei XQ, Huang FP, Al-Alem UA, Chan WL, Liew FY. Mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced Th1 cell responses. J Gen Virol. (1998) 79:825–30. doi: 10.1099/0022-1317-79-4-825

87. Dalton DK, Wittmer S. Nitric-oxide-dependent and independent mechanisms of protection from CNS inflammation during Th1-mediated autoimmunity: Evidence from EAE in iNOS KO mice. J Neuroimmunol. (2005) 160:110–21. doi: 10.1016/j.jneuroim.2004.11.004

88. Jianjun Y, Zhang R, Lu G, Shen Y, Peng L, Zhu C, et al. T cell–derived inducible nitric oxide synthase switches off Th17 cell differentiation. J Exp Med. (2013) 210:1447–62. doi: 10.1084/jem.20122494

89. Niedbala W, Alves-Filho JC, Fukada SY, Vieira SM, Mitani A, Sonego F, et al. Regulation of type 17 helper T-cell function by nitric oxide during inflammation. Proc Natl Acad Sci USA. (2011) 108:9220–5. doi: 10.1073/pnas.1100667108

90. Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, et al. Fingolimod (FTY720): Discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov. (2010) 9:883–97. doi: 10.1038/nrd3248

91. Kim J, Won JS, Singh AK, Sharma AK, Singh I. STAT3 regulation by S-nitrosylation: Implication for inflammatory disease. Antioxid Redox Signal. (2014) 20:2514–27. doi: 10.1089/ars.2013.5223

92. Saura M, Zaragoza C, Herranz B, Griera M, Diez-Marques L, Rodriguez-Puyol D, et al. Nitric oxide regulates transforming growth factor-beta signaling in endothelial cells. Circ Res. (2005) 97:1115–23. doi: 10.1161/01.RES.0000191538.76771.66

93. Danikowski KM, Jayaraman S, Prabhakar BS. Regulatory T cells in multiple sclerosis and myasthenia gravis. J Neuroinflamm. (2017) 14:117. doi: 10.1186/s12974-017-0892-8

94. Kleinewietfeld M, Hafler DA. Regulatory T cells in autoimmune neuroinflammation. Immunol Rev. (2014) 259:231–44. doi: 10.1111/imr.12169

95. Stephens LA, Malpass KH, Anderton SM. Curing CNS autoimmune disease with myelin-reactive Foxp3+ Treg. Eur J Immunol. (2009) 39:1108–17. doi: 10.1002/eji.200839073

96. Fritzsching B, Haas J, König F, Kunz P, Fritzsching E, Pöschl J, et al. Intracerebral human regulatory T cells: Analysis of CD4+ CD25+ Foxp3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients. PloS One. (2011) 6:e17988. doi: 10.1371/journal.pone.0017988

97. Fritzsching B, Korporal M, Haas J, Krammer PH, Suri-Payer E, Wildemann B. Similar sensitivity of regulatory T cells towards CD95L-mediated apoptosis in patients with multiple sclerosis and healthy individuals. J Neurol Sci. (2006) 251:91–7. doi: 10.1016/j.jns.2006.09.006

98. Costantino CM, Baecher-Allan C, Hafler DA. Multiple sclerosis and regulatory T cells. J ClinImmunol. (2008) 28:697–706. doi: 10.1007/s10875-008-9236-x

99. Schmitt EG, Williams CB. Generation and function of induced regulatory T cells. Front Immunol. (2013) 4:152. doi: 10.3389/fimmu.2013.00152

100. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. (2003) 299:1057–61. doi: 10.1126/science.1079490

101. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, et al. A Cd4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. (1997) 389:737–42. doi: 10.1038/39614

102. Levings MK, Gregori S, Tresoldi E, Cazzaniga S, Bonini C, Roncarolo MG. Differentiation of Tr1 cells by immature dendritic cells requires Il-10 but not Cd25+Cd4+ Tr cells. Blood. (2005) 105:1162–9. doi: 10.1182/blood-2004-03-1211

103. Weiner HL. Oral tolerance: immune mechanisms and the generation of Th3-type Tgf-beta-secreting regulatory cells. Microbes Infect. (2001) 3:947–54. doi: 10.1016/s1286-4579(01)01456-3

104. Lee SW, Choi H, Eun SY, Fukuyama S, Croft M. Nitric oxide modulates Tgf-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development. J Immunol. (2011) 186:6972–80. doi: 10.4049/jimmunol.1100485

105. Brahmachari S, Pahan K. Myelin basic protein priming reduces the expression of Foxp3 in T cells via nitric oxide. J Immunol. (2010) 184:1799–809. doi: 10.4049/jimmunol.0804394

106. Niedbala W, Cai B, Liu H, Pitman N, Chang L, Liew FY. Nitric oxide induces Cd4+Cd25+ Foxp3 regulatory T cells from Cd4+Cd25 T cells via P53, Il-2, and Ox40. Proc Natl Acad Sci USA. (2007) 104:15478–83. doi: 10.1073/pnas.0703725104

107. Langley B, Gensert JM, Beal MF, Ratan RR. Remodeling chromatin and stress resistance in the central nervous system: histone deacetylase inhibitors as novel and broadly effective neuroprotective agents. Curr Drug Targets CNS Neurol Disord. (2005) 4:41–50. doi: 10.2174/1568007053005091

108. Saha RN, Pahan K. Hats and Hdacs in neurodegeneration: a tale of disconcerted acetylation homeostasis. Cell Death Differ. (2006) 13:539–50. doi: 10.1038/sj.cdd.4401769

109. Kazantsev AG, Thompson LM. Therapeutic application of histone deacetylase inhibitors for central nervous system disorders. Nat Rev Drug Discov. (2008) 7:854–68. doi: 10.1038/nrd2681

110. Lopez-Diego RS, Weiner HL. Novel therapeutic strategies for multiple sclerosis--a multifaceted adversary. Nat Rev Drug Discov. (2008) 7:909–25. doi: 10.1038/nrd2358

111. Nencioni A, Beck J, Werth D, Grünebach F, Patrone F, Ballestrero A, et al. Histone deacetylase inhibitors affect dendritic cell differentiation and immunogenicity. Clin Cancer Res. (2007) 13:3933–41. doi: 10.1158/1078-0432.Ccr-06-2903

112. Reddy P, Sun Y, Toubai T, Duran-Struuck R, Clouthier SG, Weisiger E, et al. Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase-dependent Dc functions and regulates experimental graft-versus-host disease in mice. J Clin Invest. (2008) 118:2562–73. doi: 10.1172/jci34712

113. Bosisio D, Vulcano M, Del Prete A, Sironi M, Salvi V, Salogni L, et al. Blocking Th17-polarizing cytokines by histone deacetylase inhibitors in vitro and in vivo . J Leukoc Biol. (2008) 84:1540–8. doi: 10.1189/jlb.0708401

114. Venken K, Hellings N, Liblau R, Stinissen P. Disturbed regulatory T cell homeostasis in multiple sclerosis. Trends Mol Med. (2010) 16:58–68. doi: 10.1016/j.molmed.2009.12.003

115. Kohm AP, Carpentier PA, Anger HA, Miller SD. Cutting edge: Cd4+Cd25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol. (2002) 169:4712–6. doi: 10.4049/jimmunol.169.9.4712

116. Zhang X, Koldzic DN, Izikson L, Reddy J, Nazareno RF, Sakaguchi S, et al. Il-10 is involved in the suppression of experimental autoimmune encephalomyelitis by Cd25+Cd4+ regulatory T cells. Int Immunol. (2004) 16:249–56. doi: 10.1093/intimm/dxh029

117. Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol. (2007) 8:191–7. doi: 10.1038/ni1428

118. Wang L, de Zoeten EF, Greene MI, Hancock WW. Immunomodulatory effects of deacetylase inhibitors: therapeutic targeting of Foxp3+ regulatory T cells. Nat Rev Drug Discov. (2009) 8:969–81. doi: 10.1038/nrd3031

119. Tao R, de Zoeten EF, Ozkaynak E, Chen C, Wang L, Porrett PM, et al. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nat Med. (2007) 13:1299–307. doi: 10.1038/nm1652

120. Akimova T, Ge G, Golovina T, Mikheeva T, Wang L, Riley JL, et al. Histone/protein deacetylase inhibitors increase suppressive functions of human Foxp3+ Tregs. Clin Immunol. (2010) 136:348–63. doi: 10.1016/j.clim.2010.04.018

121. van Loosdregt J, Vercoulen Y, Guichelaar T, Gent YY, Beekman JM, van Beekum O, et al. Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization. Blood. (2010) 115:965–74. doi: 10.1182/blood-2009-02-207118

122. Dasgupta S, Zhou Y, Jana M, Banik NL, Pahan K. Sodium phenylacetate inhibits adoptive transfer of experimental allergic encephalomyelitis in Sjl/J mice at multiple steps. J Immunol. (2003) 170:3874–82. doi: 10.4049/jimmunol.170.7.3874

123. Camelo S, Iglesias AH, Hwang D, Due B, Ryu H, Smith K, et al. Transcriptional therapy with the histone deacetylase inhibitor trichostatin a ameliorates experimental autoimmune encephalomyelitis. J Neuroimmunol. (2005) 164:10–21. doi: 10.1016/j.jneuroim.2005.02.022

124. Huang L, Baban B, Johnson BA, Mellor AL. Dendritic cells, indoleamine 2,3 dioxygenase and acquired immune privilege. Int Rev Immunol. (2010) 29:133–55. doi: 10.3109/08830180903349669

125. Munn DH, Mellor AL, Rossi M, Young JW. Dendritic cells have the option to express Ido-mediated suppression or not. Blood. (2005) 105:2618. doi: 10.1182/blood-2005-01-0122

126. Ryu H, Lee J, Olofsson BA, Mwidau A, Dedeoglu A, Escudero M, et al. Histone deacetylase inhibitors prevent oxidative neuronal death independent of expanded polyglutamine repeats via an Sp1-dependent pathway. Proc Natl Acad Sci USA. (2003) 100:4281–6. doi: 10.1073/pnas.0737363100

127. Rivieccio MA, Brochier C, Willis DE, Walker BA, D'Annibale MA, McLaughlin K, et al. Hdac6 is a target for protection and regeneration following injury in the nervous system. Proc Natl Acad Sci USA. (2009) 106:19599–604. doi: 10.1073/pnas.0907935106

128. Parmigiani RB, Xu WS, Venta-Perez G, Erdjument-Bromage H, Yaneva M, Tempst P, et al. Hdac6 is a specific deacetylase of peroxiredoxins and is involved in redox regulation. Proc Natl Acad Sci USA. (2008) 105:9633–8. doi: 10.1073/pnas.0803749105

129. Nott A, Nitarska J, Veenvliet JV, Schacke S, Derijck AA, Sirko P, et al. S-nitrosylation of Hdac2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration. Proc Natl Acad Sci USA. (2013) 110:3113–8. doi: 10.1073/pnas.1218126110

130. Colussi C, Mozzetta C, Gurtner A, Illi B, Rosati J, Straino S, et al. Hdac2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment. Proc Natl Acad Sci USA. (2008) 105:19183–7. doi: 10.1073/pnas.0805514105

131. Okuda K, Ito A, Uehara T. Regulation of histone deacetylase 6 activity via S-nitrosylation. Biol Pharm Bull. (2015) 38:1434–7. doi: 10.1248/bpb.b15-00364

132. Shinozaki S, Chang K, Sakai M, Shimizu N, Yamada M, Tanaka T, et al. Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase Sirt1 to increase acetylation and activation of P53 and P65. Sci Signal. (2014) 7:ra106. doi: 10.1126/scisignal.2005375

133. Kim J, Islam SMT, Qiao F, Singh AK, Khan M, Won J, et al. Regulation of B cell functions by S-nitrosoglutathione in the Eae model. Redox Biol. (2021) 45:102053. doi: 10.1016/j.redox.2021.102053

134. Singh I, Nath N, Saxena N, Singh AK, Won JS. Regulation of Il-10 and Il-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione. Immunobiology. (2018) 223:549–54. doi: 10.1016/j.imbio.2018.06.003

135. Hampe CS. B cell in autoimmune diseases. Scientifica (Cairo). (2012) 2012. doi: 10.6064/2012/215308

136. Matsushita T. Regulatory and effector B cells: friends or foes? J Dermatol Sci. (2019) 93:2–7. doi: 10.1016/j.jdermsci.2018.11.008

137. Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, et al. B cell depletion therapy ameliorates autoimmune disease through ablation of Il-6-producing B cells. J Exp Med. (2012) 209:1001–10. doi: 10.1084/jem.20111675

138. Molnarfi N, Schulze-Topphoff U, Weber MS, Patarroyo JC, Prod'homme T, Varrin-Doyer M, et al. Mhc class Ii-dependent B cell Apc function is required for induction of Cns autoimmunity independent of myelin-specific antibodies. J Exp Med. (2013) 210:2921–37. doi: 10.1084/jem.20130699

139. Florou D, Katsara M, Feehan J, Dardiotis E, Apostolopoulos V. Anti-Cd20 agents for multiple sclerosis: spotlight on ocrelizumab and ofatumumab. Brain Sci. (2020) 10. doi: 10.3390/brainsci10100758

140. Mok CC. Rituximab for the treatment of rheumatoid arthritis: an update. Drug Des Devel Ther. (2013) 8:87–100. doi: 10.2147/dddt.S41645

141. Ancau M, Berthele A, Hemmer B. Cd20 monoclonal antibodies for the treatment of multiple sclerosis: up-to-date. Expert Opin Biol Ther. (2019) 19:829–43. doi: 10.1080/14712598.2019.1611778

142. Merrill JT, Neuwelt CM, Wallace DJ, Shanahan JC, Latinis KM, Oates JC, et al. Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase Ii/Iii systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum. (2010) 62:222–33. doi: 10.1002/art.27233

143. Goetz M, Atreya R, Ghalibafian M, Galle PR, Neurath MF. Exacerbation of ulcerative colitis after rituximab salvage therapy. Inflammation Bowel Dis. (2007) 13:1365–8. doi: 10.1002/ibd.20215

144. Dass S, Vital EM, Emery P. Development of psoriasis after B cell depletion with rituximab. Arthritis Rheum. (2007) 56:2715–8. doi: 10.1002/art.22811

145. Staun-Ram E, Miller A. Effector and regulatory B cells in multiple sclerosis. Clin Immunol. (2017) 184:11–25. doi: 10.1016/j.clim.2017.04.014

146. Vasileiadis GK, Dardiotis E, Mavropoulos A, Tsouris Z, Tsimourtou V, Bogdanos DP, et al. Regulatory B and T lymphocytes in multiple sclerosis: friends or foes? Auto Immun Highlights. (2018) 9:9. doi: 10.1007/s13317-018-0109-x

147. Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, et al. Characterization of a rare Il-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood. (2011) 117:530–41. doi: 10.1182/blood-2010-07-294249

148. Blair PA, Norena LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, et al. Cd19(+)Cd24(Hi)Cd38(Hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients. Immunity. (2010) 32:129–40. doi: 10.1016/j.immuni.2009.11.009

149. Kalampokis I, Yoshizaki A, Tedder TF. Il-10-producing regulatory B cells (B10 cells) in autoimmune disease. Arthritis Res Ther. (2013) 15:S1. doi: 10.1186/ar3907

150. Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. Regulatory B cells inhibit Eae initiation in mice while other B cells promote disease progression. J Clin Invest. (2008) 118:3420–30. doi: 10.1172/JCI36030

151. Blair PA, Chavez-Rueda KA, Evans JG, Shlomchik MJ, Eddaoudi A, Isenberg DA, et al. Selective targeting of B cells with agonistic anti-cd40 is an efficacious strategy for the generation of induced regulatory t2-like B cells and for the suppression of lupus in mrl/lpr mice. J Immunol. (2009) 182:3492–502. doi: 10.4049/jimmunol.0803052

152. Fujimoto M. Regulatory B cells in skin and connective tissue diseases. J Dermatol Sci. (2010) 60:1–7. doi: 10.1016/j.jdermsci.2010.08.010

153. Matsushita T, Le Huu D, Kobayashi T, Hamaguchi Y, Hasegawa M, Naka K, et al. A novel splenic b1 regulatory cell subset suppresses allergic disease through phosphatidylinositol 3-kinase-akt pathway activation. J Allergy Clin Immunol. (2016) 138:1170–82:e9. doi: 10.1016/j.jaci.2015.12.1319

154. Wolf SD, Dittel BN, Hardardottir F, Janeway CA., Jr. Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice. J Exp Med. (1996) 184:2271–8. doi: 10.1084/jem.184.6.2271

155. Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of il-10. Nat Immunol. (2002) 3:944–50. doi: 10.1038/ni833

156. Tumurkhuu G, Koide N, Dagvadorj J, Noman AS, Khuda II, Naiki Y, et al. B1 cells produce nitric oxide in response to a series of toll-like receptor ligands. Cell Immunol. (2010) 261:122–7. doi: 10.1016/j.cellimm.2009.11.009

157. Bogdan C. Nitric oxide and the immune response. Nat Immunol. (2001) 2:907–16. doi: 10.1038/ni1001-907

158. Jayasekera JP, Vinuesa CG, Karupiah G, King NJC. Enhanced antiviral antibody secretion and attenuated immunopathology during influenza virus infection in nitric oxide synthase-2-deficient mice. J Gen Virol. (2006) 87:3361–71. doi: 10.1099/vir.0.82131-0

159. Tezuka H, Abe Y, Iwata M, Takeuchi H, Ishikawa H, Matsushita M, et al. Regulation of iga production by naturally occurring tnf/inos-producing dendritic cells. Nature. (2007) 448:929–33. doi: 10.1038/nature06033

160. Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and epstein-barr virus reactivation. Cell. (1994) 79:1137–46. doi: 10.1016/0092-8674(94)90005-1

161. Saini AS, Shenoy GN, Rath S, Bal V, George A. Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol. (2014) 15:275–82. doi: 10.1038/ni.2806

162. Li J, Billiar TR, Talanian RV, Kim YM. Nitric oxide reversibly inhibits seven members of the caspase family via s-nitrosylation. Biochem Biophys Res Commun. (1997) 240:419–24. doi: 10.1006/bbrc.1997.7672

163. Won J, Kim J, Qiao F, Singh AK, Singh I. Therapeutic potential of s-nitrosoglutathione reductase inhibitor in B cell-driven experimental autoimmune encephalomyelitis. J Neuroimmunol. (2025) 406:578673. doi: 10.1016/j.jneuroim.2025.578673

164. Olson N, van der Vliet A. Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease. Nitric Oxide. (2011) 25:125–37. doi: 10.1016/j.niox.2010.12.010

165. Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an o2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA. (1998) 95:7987–92. doi: 10.1073/pnas.95.14.7987

166. Palmer LA, Doctor A, Chhabra P, Sheram ML, Laubach VE, Karlinsey MZ, et al. S-nitrosothiols signal hypoxia-mimetic vascular pathology. J Clin Invest. (2007) 117:2592–601. doi: 10.1172/jci29444

167. Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify hif. Science. (2001) 294:1337–40. doi: 10.1126/science.1066373

168. Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von hippel-lindau tumor suppressor protein. J Biol Chem. (2000) 275:25733–41. doi: 10.1074/jbc.M002740200

169. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Activation of hif1alpha ubiquitination by a reconstituted von hippel-lindau (vhl) tumor suppressor complex. Proc Natl Acad Sci USA. (2000) 97:10430–5. doi: 10.1073/pnas.190332597

170. Flück K, Breves G, Fandrey J, Winning S. Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory t cells in murine colitis. Mucosal Immunol. (2016) 9:379–90. doi: 10.1038/mi.2015.67

171. Liu J, Qiu P, Qin J, Wu X, Wang X, Yang X, et al. Allogeneic adipose-derived stem cells promote ischemic muscle repair by inducing m2 macrophage polarization via the hif-1α/il-10 pathway. Stem Cells. (2020) 38:1307–20. doi: 10.1002/stem.3250

172. Meng X, Grotsch B, Luo Y, Knaup KX, Wiesener MS, Chen XX, et al. Hypoxia-inducible factor-1alpha is a critical transcription factor for il-10-producing B cells in autoimmune disease. Nat Commun. (2018) 9:251. doi: 10.1038/s41467-017-02683-x

173. Li F, Sonveaux P, Rabbani ZN, Liu S, Yan B, Huang Q, et al. Regulation of hif-1alpha stability through s-nitrosylation. Mol Cell. (2007) 26:63–74. doi: 10.1016/j.molcel.2007.02.024

174. Metzen E, Zhou J, Jelkmann W, Fandrey J, Brune B. Nitric oxide impairs normoxic degradation of hif-1alpha by inhibition of prolyl hydroxylases. Mol Biol Cell. (2003) 14:3470–81. doi: 10.1091/mbc.e02-12-0791

175. Chowdhury R, Flashman E, Mecinovic J, Kramer HB, Kessler BM, Frapart YM, et al. Studies on the reaction of nitric oxide with the hypoxia-inducible factor prolyl hydroxylase domain 2 (egln1). J Mol Biol. (2011) 410:268–79. doi: 10.1016/j.jmb.2011.04.075

176. Klein U, Casola S, Cattoretti G, Shen Q, Lia M, Mo T, et al. Transcription factor irf4 controls plasma cell differentiation and class-switch recombination. Nat Immunol. (2006) 7:773–82. doi: 10.1038/ni1357

177. Mittrücker HW, Matsuyama T, Grossman A, Kündig TM, Potter J, Shahinian A, et al. Requirement for the transcription factor lsirf/irf4 for mature B and T lymphocyte function. Science. (1997) 275:540–6. doi: 10.1126/science.275.5299.540

178. Sciammas R, Shaffer AL, Schatz JH, Zhao H, Staudt LM, Singh H. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity. (2006) 25:225–36. doi: 10.1016/j.immuni.2006.07.009

179. Simonetti G, Carette A, Silva K, Wang H, De Silva NS, Heise N, et al. Irf4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating notch2 expression and activity. J Exp Med. (2013) 210:2887–902. doi: 10.1084/jem.20131026

180. Matsumoto M, Baba A, Yokota T, Nishikawa H, Ohkawa Y, Kayama H, et al. Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation. Immunity. (2014) 41:1040–51. doi: 10.1016/j.immuni.2014.10.016

181. Suzuki-Yamazaki N, Yanobu-Takanashi R, Okamura T, Takaki S. Il-10 production in murine igm(+) cd138(hi) cells is driven by blimp-1 and downregulated in class-switched cells. Eur J Immunol. (2017) 47:493–503. doi: 10.1002/eji.201646549

182. Rangaswamy US, Speck SH. Murine gammaherpesvirus m2 protein induction of irf4 via the nfat pathway leads to il-10 expression in B cells. PloS Pathog. (2014) 10:e1003858. doi: 10.1371/journal.ppat.1003858

183. Mudter J, Amoussina L, Schenk M, Yu J, Brustle A, Weigmann B, et al. The transcription factor ifn regulatory factor-4 controls experimental colitis in mice via t cell-derived il-6. J Clin Invest. (2008) 118:2415–26. doi: 10.1172/JCI33227

184. Niedbala W, Besnard AG, Nascimento DC, Donate PB, Sonego F, Yip E, et al. Nitric oxide enhances th9 cell differentiation and airway inflammation. Nat Commun. (2014) 5:4575. doi: 10.1038/ncomms5575

185. Laidlaw BJ, Cyster JG. Transcriptional regulation of memory B cell differentiation. Nat Rev Immunol. (2021) 21:209–20. doi: 10.1038/s41577-020-00446-2

186. Numajiri N, Takasawa K, Nishiya T, Tanaka H, Ohno K, Hayakawa W, et al. On-off system for pi3-kinase-akt signaling through s-nitrosylation of phosphatase with sequence homology to tensin (pten). Proc Natl Acad Sci USA. (2011) 108:10349–54. doi: 10.1073/pnas.1103503108

187. Rahman MA, Senga T, Ito S, Hyodo T, Hasegawa H, Hamaguchi M. S-nitrosylation at cysteine 498 of c-src tyrosine kinase regulates nitric oxide-mediated cell invasion. J Biol Chem. (2010) 285:3806–14. doi: 10.1074/jbc.M109.059782

188. Wang L, Ning S. Interferon regulatory factor 4 is activated through c-src-mediated tyrosine phosphorylation in virus-transformed cells. J Virol. (2013) 87:9672–9. doi: 10.1128/JVI.01435-13

189. Mars LT, Saikali P, Liblau RS, Arbour N. Contribution of cd8 t lymphocytes to the immuno-pathogenesis of multiple sclerosis and its animal models. Biochim Biophys Acta. (2011) 1812:151–61. doi: 10.1016/j.bbadis.2010.07.006

190. Choy JC, Pober JS. Generation of no by bystander human cd8 t cells augments allogeneic responses by inhibiting cytokine deprivation-induced cell death. Am J Transplant. (2009) 9:2281–91. doi: 10.1111/j.1600-6143.2009.02771.x

191. Cunha PP, Bargiela D, Minogue E, Krause LCM, Barbieri L, Brombach C, et al. Infiltration of tumors is regulated by t cell-intrinsic nitric oxide synthesis. Cancer Immunol Res. (2023) 11:351–63. doi: 10.1158/2326-6066.Cir-22-0387

192. Niedbala W, Cai B, Liew FY. Role of nitric oxide in the regulation of t cell functions. Ann Rheum Dis. (2006) 65 Suppl 3:iii37–40. doi: 10.1136/ard.2006.058446

193. Gross CC, Schulte-Mecklenbeck A, Wiendl H, Marcenaro E, Kerlero de Rosbo N, Uccelli A, et al. Regulatory functions of natural killer cells in multiple sclerosis. Front Immunol. (2016) 7:606. doi: 10.3389/fimmu.2016.00606

194. Beliën J, Goris A, Matthys P. Natural killer cells in multiple sclerosis: entering the stage. Front Immunol. (2022) 13:869447. doi: 10.3389/fimmu.2022.869447

195. Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, et al. Human natural killer cells: a unique innate immunoregulatory role for the cd56(bright) subset. Blood. (2001) 97:3146–51. doi: 10.1182/blood.v97.10.3146

196. Laroni A, Armentani E, Kerlero de Rosbo N, Ivaldi F, Marcenaro E, Sivori S, et al. Dysregulation of regulatory cd56(bright) nk cells/t cells interactions in multiple sclerosis. J Autoimmun. (2016) 72:8–18. doi: 10.1016/j.jaut.2016.04.003

197. Hao J, Liu R, Piao W, Zhou Q, Vollmer TL, Campagnolo DI, et al. Central nervous system (cns)-resident natural killer cells suppress th17 responses and cns autoimmune pathology. J Exp Med. (2010) 207:1907–21. doi: 10.1084/jem.20092749

198. Abel AM, Yang C, Thakar MS, Malarkannan S. Natural killer cells: development, maturation, and clinical utilization. Front Immunol. (2018) 9:1869. doi: 10.3389/fimmu.2018.01869

199. Ding J, Yan X, Zhao C, Zhao D, Jia Y, Ren K, et al. The ratio of circulating cd56(dim) nk cells to follicular t helper cells as a promising predictor for disease activity of relapsing-remitting multiple sclerosis. Heliyon. (2024) 10:e31533. doi: 10.1016/j.heliyon.2024.e31533

200. Takabayashi A, Kawai Y, Iwata S, Kanai M, Denno R, Kawada K, et al. Nitric oxide induces a decrease in the mitochondrial membrane potential of peripheral blood lymphocytes, especially in natural killer cells. Antioxid Redox Signal. (2000) 2:673–80. doi: 10.1089/ars.2000.2.4-673

201. Thorén FB, Romero AI, Hermodsson S, Hellstrand K. The Cd16-/Cd56bright subset of Nk cells is resistant to oxidant-induced cell death. J Immunol. (2007) 179:781–5. doi: 10.4049/jimmunol.179.2.781

202. Ruocco G, Rossi S, Motta C, Macchiarulo G, Barbieri F, De Bardi M, et al. T helper 9 cells induced by plasmacytoid dendritic cells regulate interleukin-17 in multiple sclerosis. Clin Sci (Lond). (2015) 129:291–303. doi: 10.1042/cs20140608

203. Elyaman W, Bradshaw EM, Uyttenhove C, Dardalhon V, Awasthi A, Imitola J, et al. Il-9 induces differentiation of Th17 cells and enhances function of Foxp3+ natural regulatory T cells. Proc Natl Acad Sci USA. (2009) 106:12885–90. doi: 10.1073/pnas.0812530106

204. Roy S, Awasthi A. Atp triggers human Th9 cell differentiation via nitric oxide-mediated mtor-hif1α pathway. Front Immunol. (2019) 10:1120. doi: 10.3389/fimmu.2019.01120

205. Zierfuss B, Larochelle C, Prat A. Blood-brain barrier dysfunction in multiple sclerosis: Causes, consequences, and potential effects of therapies. Lancet Neurol. (2024) 23:95–109. doi: 10.1016/s1474-4422(23)00377-0

206. Mainero C, Louapre C. Meningeal inflammation in multiple sclerosis: The key to the origin of cortical lesions? Neurology. (2015) 85:12–3. doi: 10.1212/wnl.0000000000001586

207. Rawji KS, Yong VW. The benefits and detriments of macrophages/microglia in models of multiple sclerosis. Clin Dev Immunol. (2013) 2013:948976. doi: 10.1155/2013/948976

208. Cherry JD, Olschowka JA, O'Banion MK. Neuroinflammation and M2 microglia: The good, the bad, and the inflamed. J Neuroinflamm. (2014) 11:98. doi: 10.1186/1742-2094-11-98

209. Fang FC. Perspectives series: Host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. J Clin Invest. (1997) 99:2818–25. doi: 10.1172/jci119473

210. Minagar A, Alexander JS. Blood-brain barrier disruption in multiple sclerosis. Mult Scler. (2003) 9:540–9. doi: 10.1191/1352458503ms965oa

211. Kermode AG, Thompson AJ, Tofts P, MacManus DG, Kendall BE, Kingsley DP, et al. Breakdown of the blood-brain barrier precedes symptoms and other mri signs of new lesions in multiple sclerosis. Pathogenetic and clinical implications. Brain. (1990) 113:1477–89. doi: 10.1093/brain/113.5.1477

212. Obermeier B, Daneman R, Ransohoff RM. Development, maintenance and disruption of the blood-brain barrier. Nat Med. (2013) 19:1584–96. doi: 10.1038/nm.3407

213. Hirano M, Hirano K. Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption. J Physiol Sci. (2022) 72:32. doi: 10.1186/s12576-022-00857-x

214. Shi Y, Zhang L, Pu H, Mao L, Hu X, Jiang X, et al. Rapid endothelial cytoskeletal reorganization enables early blood-brain barrier disruption and long-term ischaemic reperfusion brain injury. Nat Commun. (2016) 7:10523. doi: 10.1038/ncomms10523

215. Erickson MA, Dohi K, Banks WA. Neuroinflammation: A common pathway in cns diseases as mediated at the blood-brain barrier. Neuroimmunomodulation. (2012) 19:121–30. doi: 10.1159/000330247

216. Wojciak-Stothard B, Ridley AJ. Rho gtpases and the regulation of endothelial permeability. Vascul Pharmacol. (2002) 39:187–99. doi: 10.1016/s1537-1891(03)00008-9

217. Di Lorenzo A, Lin MI, Murata T, Landskroner-Eiger S, Schleicher M, Kothiya M, et al. Enos-derived nitric oxide regulates endothelial barrier function through ve-cadherin and rho gtpases. J Cell Sci. (2013) 126:5541–52. doi: 10.1242/jcs.115972

218. Rafikov R, Dimitropoulou C, Aggarwal S, Kangath A, Gross C, Pardo D, et al. Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of Rhoa. J Biol Chem. (2014) 289:4710–22. doi: 10.1074/jbc.M114.547596

219. Choi S, Saxena N, Dhammu T, Khan M, Singh AK, Singh I, et al. Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries. Nitric Oxide. (2019) 83:51–64. doi: 10.1016/j.niox.2018.12.007

220. Lin L, Xu C, Carraway MS, Piantadosi CA, Whorton AR, Li S. Rhoa inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling. Nitric Oxide. (2018) 74:56–64. doi: 10.1016/j.niox.2018.01.007

221. van Nieuw Amerongen GP, van Delft S, Vermeer MA, Collard JG, van Hinsbergh VW. Activation of Rhoa by thrombin in endothelial hyperpermeability: Role of rho kinase and protein tyrosine kinases. Circ Res. (2000) 87:335–40. doi: 10.1161/01.res.87.4.335

222. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. (2001) 53:245–82. doi: 10.1016/s0031-6997(24)01493-5

223. Andjelkovic AV, Pachter JS. Central nervous system endothelium in neuroinflammatory, neuroinfectious, and neurodegenerative disease. J Neurosci Res. (1998) 51:423–30. doi: 10.1002/(sici)1097-4547(19980215)51:4<423::Aid-jnr2>3.0.Co;2-e

224. Archelos JJ, Hartung HP. The role of adhesion molecules in multiple sclerosis: Biology, pathogenesis and therapeutic implications. Mol Med Today. (1997) 3:310–21. doi: 10.1016/s1357-4310(97)01066-6

225. Lee SJ, Benveniste EN. Adhesion molecule expression and regulation on cells of the central nervous system. J Neuroimmunol. (1999) 98:77–88. doi: 10.1016/s0165-5728(99)00084-3

226. Washington R, Burton J, Todd RF, Newman W, Dragovic L, Dore-Duffy P. Expression of immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis. Ann Neurol. (1994) 35:89–97. doi: 10.1002/ana.410350114

227. Dopp JM, Breneman SM, Olschowka JA. Expression of Icam-1, Vcam-1, L-selectin, and leukosialin in the mouse central nervous system during the induction and remission stages of experimental allergic encephalomyelitis. J Neuroimmunol. (1994) 54:129–44. doi: 10.1016/0165-5728(94)90239-9

228. Prasad R, Giri S, Nath N, Singh I, Singh AK. Gsno attenuates eae disease by S-nitrosylation-mediated modulation of endothelial-monocyte interactions. Glia. (2007) 55:65–77. doi: 10.1002/glia.20436

229. Domingues HS, Mues M, Lassmann H, Wekerle H, Krishnamoorthy G. Functional and pathogenic differences of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. PloS One. (2010) 5:e15531. doi: 10.1371/journal.pone.0015531

230. Furtado GC, Marcondes MC, Latkowski JA, Tsai J, Wensky A, Lafaille JJ. Swift entry of myelin-specific T lymphocytes into the central nervous system in spontaneous autoimmune encephalomyelitis. J Immunol. (2008) 181:4648–55. doi: 10.4049/jimmunol.181.7.4648

231. Li H, Jiang T, Li MQ, Zheng XL, Zhao GJ. Transcriptional regulation of macrophages polarization by micrornas. Front Immunol. (2018) 9:1175. doi: 10.3389/fimmu.2018.01175

232. Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, et al. Irf5 promotes inflammatory macrophage polarization and Th1-Th17 responses. Nat Immunol. (2011) 12:231–8. doi: 10.1038/ni.1990

233. Mills CD. M1 and M2 macrophages: Oracles of health and disease. Crit Rev Immunol. (2012) 32:463–88. doi: 10.1615/critrevimmunol.v32.i6.10

234. Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, et al. Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med. (2014) 211:1533–49. doi: 10.1084/jem.20132477

235. Vogel DY, Vereyken EJ, Glim JE, Heijnen PD, Moeton M, van der Valk P, et al. Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status. J Neuroinflamm. (2013) 10:35. doi: 10.1186/1742-2094-10-35

236. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mörk S, Bö L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med. (1998) 338:278–85. doi: 10.1056/nejm199801293380502

237. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: Time for reassessment. F1000Prime Rep. (2014) 6:13. doi: 10.12703/p6-13

238. Bouhlel MA, Derudas B, Rigamonti E, Dièvart R, Brozek J, Haulon S, et al. Ppargamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. (2007) 6:137–43. doi: 10.1016/j.cmet.2007.06.010

239. Liao X, Sharma N, Kapadia F, Zhou G, Lu Y, Hong H, et al. Krüppel-like factor 4 regulates macrophage polarization. J Clin Invest. (2011) 121:2736–49. doi: 10.1172/jci45444

240. Yunna C, Mengru H, Lei W, Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. (2020) 877:173090. doi: 10.1016/j.ejphar.2020.173090

241. Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, et al. M2 microglia and macrophages drive oligodendrocyte differentiation during cns remyelination. Nat Neurosci. (2013) 16:1211–8. doi: 10.1038/nn.3469

242. Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: Phenotypical vs. functional differentiation. Front Immunol. (2014) 5:514. doi: 10.3389/fimmu.2014.00514

243. Lu G, Zhang R, Geng S, Peng L, Jayaraman P, Chen C, et al. Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization. Nat Commun. (2015) 6:6676. doi: 10.1038/ncomms7676

244. Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci USA. (2013) 110:7820–5. doi: 10.1073/pnas.1218599110

245. Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of Keap1. Nature. (2018) 556:113–7. doi: 10.1038/nature25986

246. Bambouskova M, Gorvel L, Lampropoulou V, Sergushichev A, Loginicheva E, Johnson K, et al. Electrophilic properties of itaconate and derivatives regulate the iκbζ-atf3 inflammatory axis. Nature. (2018) 556:501–4. doi: 10.1038/s41586-018-0052-z

247. Zhao N, Yi M, Zhang LJ, Zhang QX, Yang L. 4-octyl itaconate attenuates neuroinflammation in experimental autoimmune encephalomyelitis via regulating microglia. Inflammation. (2025) 48:151–64. doi: 10.1007/s10753-024-02050-1

248. Hoyle C, Green JP, Allan SM, Brough D, Lemarchand E. Itaconate and fumarate derivatives inhibit priming and activation of the canonical Nlrp3 inflammasome in macrophages. Immunology. (2022) 165:460–80. doi: 10.1111/imm.13454

249. Palmieri EM, Gonzalez-Cotto M, Baseler WA, Davies LC, Ghesquière B, Maio N, et al. Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase. Nat Commun. (2020) 11:698. doi: 10.1038/s41467-020-14433-7

250. Bailey JD, Diotallevi M, Nicol T, McNeill E, Shaw A, Chuaiphichai S, et al. Nitric oxide modulates metabolic remodeling in inflammatory macrophages through tca cycle regulation and itaconate accumulation. Cell Rep. (2019) 28:218–230.e7. doi: 10.1016/j.celrep.2019.06.018

251. Diotallevi M, Outeiral C, Patel P, Purvis GSD, Chuaiphichai S, Nicol T, et al. Inos modulates inflammatory responses in an no-independent manner through direct interaction with irg1 in mitochondria. Nat Metab. (2026) 8:855–68. doi: 10.1038/s42255-026-01492-1

252. Arora H, Panara K, Kuchakulla M, Kulandavelu S, Burnstein KL, Schally AV, et al. Alterations of tumor microenvironment by nitric oxide impedes castration-resistant prostate cancer growth. Proc Natl Acad Sci USA. (2018) 115:11298–303. doi: 10.1073/pnas.1812704115

253. Foster MW, Yang Z, Gooden DM, Thompson JW, Ball CH, Turner ME, et al. Proteomic characterization of the cellular response to nitrosative stress mediated by s-nitrosoglutathione reductase inhibition. J Proteome Res. (2012) 11:2480–91. doi: 10.1021/pr201180m

254. Zhang Q, Lenardo MJ, Baltimore D. 30 years of nf-kappab: a blossoming of relevance to human pathobiology. Cell. (2017) 168:37–57. doi: 10.1016/j.cell.2016.12.012

255. Giuliani C, Bucci I, Napolitano G. The role of the transcription factor nuclear factor-kappa b in thyroid autoimmunity and cancer. Front Endocrinol (Lausanne). (2018) 9:471. doi: 10.3389/fendo.2018.00471

256. Israel A. The ikk complex, a central regulator of nf-kappab activation. Cold Spring Harb Perspect Biol. (2010) 2:a000158. doi: 10.1101/cshperspect.a000158

257. Karin M, Yamamoto Y, Wang QM. The ikk nf-kappa b system: a treasure trove for drug development. Nat Rev Drug Discov. (2004) 3:17–26. doi: 10.1038/nrd1279

258. Gamble C, McIntosh K, Scott R, Ho KH, Plevin R, Paul A. Inhibitory kappa b kinases as targets for pharmacological regulation. Br J Pharmacol. (2012) 165:802–19. doi: 10.1111/j.1476-5381.2011.01608.x

259. Kwon Y, Choi SK, Byeon S, Lee YH. Involvement of inhibitor kappa b kinase 2 (ikk2) in the regulation of vascular tone. Lab Invest. (2018) 98:1311–9. doi: 10.1038/s41374-018-0061-4

260. Aupperle K, Bennett B, Han Z, Boyle D, Manning A, Firestein G. Nf-kappa b regulation by i kappa b kinase-2 in rheumatoid arthritis synoviocytes. J Immunol. (2001) 166:2705–11. doi: 10.4049/jimmunol.166.4.2705

261. Gupta SC, Prasad S, Reuter S, Kannappan R, Yadav VR, Ravindran J, et al. Modification of cysteine 179 of ikappabalpha kinase by nimbolide leads to down-regulation of nf-kappab-regulated cell survival and proliferative proteins and sensitization of tumor cells to chemotherapeutic agents. J Biol Chem. (2010) 285:35406–17. doi: 10.1074/jbc.M110.161984

262. Katsuyama K, Shichiri M, Marumo F, Hirata Y. No inhibits cytokine-induced inos expression and nf-kappab activation by interfering with phosphorylation and degradation of ikappab-alpha. Arterioscler Thromb Vasc Biol. (1998) 18:1796–802. doi: 10.1161/01.atv.18.11.1796

263. Khan M, Sekhon B, Giri S, Jatana M, Gilg AG, Ayasolla K, et al. S-nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke. J Cereb Blood Flow Metab. (2005) 25:177–92. doi: 10.1038/sj.jcbfm.9600012

264. D'Acquisto F, Maiuri MC, de Cristofaro F, Carnuccio R. Nitric oxide prevents inducible cyclooxygenase expression by inhibiting nuclear factor-kappa b and nuclear factor-interleukin-6 activation. Naunyn Schmiedebergs Arch Pharmacol. (2001) 364:157–65. doi: 10.1007/s002100100435

265. Fortenberry JD, Owens ML, Chen NX, Brown LA. S-nitrosoglutathione inhibits tnf-alpha-induced nfkappab activation in neutrophils. Inflammation Res. (2001) 50:89–95. doi: 10.1007/s000110050729

266. Kelleher ZT, Matsumoto A, Stamler JS, Marshall HE. Nos2 regulation of nf-kappab by s-nitrosylation of p65. J Biol Chem. (2007) 282:30667–72. doi: 10.1074/jbc.M705929200

267. Marshall HE, Stamler JS. Inhibition of nf-kappa b by s-nitrosylation. Biochemistry. (2001) 40:1688–93. doi: 10.1021/bi002239y

268. Darnell JE., Jr. Stats and gene regulation. Science. (1997) 277:1630–5. doi: 10.1126/science.277.5332.1630

269. Levy DE, Darnell JE., Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. (2002) 3:651–62. doi: 10.1038/nrm909

270. Bowman T, Garcia R, Turkson J, Jove R. Stats in oncogenesis. Oncogene. (2000) 19:2474–88. doi: 10.1038/sj.onc.1203527

271. Bromberg JF. Activation of stat proteins and growth control. Bioessays. (2001) 23:161–9. doi: 10.1002/1521-1878(200102)23:2<161::AID-BIES1023>3.0.CO;2-0

272. Copeland NG, Gilbert DJ, Schindler C, Zhong Z, Wen Z, Darnell JE, Jr., et al. Distribution of the mammalian stat gene family in mouse chromosomes. Genomics. (1995) 29:225–8. doi: 10.1006/geno.1995.1235

273. Ivashkiv LB, Donlin LT. Regulation of type i interferon responses. Nat Rev Immunol. (2014) 14:36–49. doi: 10.1038/nri3581

274. Llovera M, Pearson JD, Moreno C, Riveros-Moreno V. Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of stat1 by endogenous nitric oxide. Br J Pharmacol. (2001) 132:419–26. doi: 10.1038/sj.bjp.0703838

275. Won JS, Kim J, Annamalai B, Shunmugavel A, Singh I, Singh AK. Protective role of s-nitrosoglutathione (gsno) against cognitive impairment in rat model of chronic cerebral hypoperfusion. J Alzheimers Dis. (2013) 34:621–35. doi: 10.3233/JAD-121786

276. O'Shea JJ, Holland SM, Staudt LM. Jaks and stats in immunity, immunodeficiency, and cancer. N Engl J Med. (2013) 368:161–70. doi: 10.1056/NEJMra1202117

277. Hillmer EJ, Zhang H, Li HS, Watowich SS. Stat3 signaling in immunity. Cytokine Growth Factor Rev. (2016) 31:1–15. doi: 10.1016/j.cytogfr.2016.05.001

278. Kasembeli MM, Bharadwaj U, Robinson P, Tweardy DJ. Contribution of stat3 to inflammatory and fibrotic diseases and prospects for its targeting for treatment. Int J Mol Sci. (2018) 19. doi: 10.3390/ijms19082299

279. Kim J, Choi S, Saxena N, Singh AK, Singh I, Won JS. Regulation of stat3 and nf-κb activations by s-nitrosylation in multiple myeloma. Free Radic Biol Med. (2017) 106:245–53. doi: 10.1016/j.freeradbiomed.2017.02.039

280. Kaliyaperumal K, Sharma AK, McDonald DG, Dhindsa JS, Yount C, Singh AK, et al. S-nitrosoglutathione-mediated stat3 regulation in efficacy of radiotherapy and cisplatin therapy in head and neck squamous cell carcinoma. Redox Biol. (2015) 6:41–50. doi: 10.1016/j.redox.2015.07.001

281. Poholek CH, Raphael I, Wu D, Revu S, Rittenhouse N, Uche UU, et al. Noncanonical stat3 activity sustains pathogenic th17 proliferation and cytokine response to antigen. J Exp Med. (2020) 217. doi: 10.1084/jem.20191761

282. Tanaka M, Vécsei L. Monitoring the redox status in multiple sclerosis. Biomedicines. (2020) 8. doi: 10.3390/biomedicines8100406

283. Carvalho AN, Firuzi O, Gama MJ, Horssen V, Saso L. Oxidative stress and antioxidants in neurological diseases: is there still hope? Curr Drug Targets. (2017) 18:705–18. doi: 10.2174/1389450117666160401120514

284. Kuntić M, Hahad O, Münzel T, Daiber A. Crosstalk between oxidative stress and inflammation caused by noise and air pollution-implications for neurodegenerative diseases. Antioxidants (Basel). (2024) 13. doi: 10.3390/antiox13030266

285. Rosenfeld RJ, Bonaventura J, Szymczyna BR, MacCoss MJ, Arvai AS, Yates JR, et al. Nitric-oxide synthase forms n-no-pterin and s-no-cys: implications for activity, allostery, and regulation. J Biol Chem. (2010) 285:31581–9. doi: 10.1074/jbc.M109.072496

286. Chen Y, Panda K, Stuehr DJ. Control of nitric oxide synthase dimer assembly by a heme-no-dependent mechanism. Biochemistry. (2002) 41:4618–25. doi: 10.1021/bi011877t

287. Wynia-Smith SL, Smith BC. Nitrosothiol formation and s-nitrosation signaling through nitric oxide synthases. Nitric Oxide. (2017) 63:52–60. doi: 10.1016/j.niox.2016.10.001

288. Robinson VK, Sato E, Nelson DK, Camhi SL, Robbins RA, Hoyt JC. Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro . Free Radic Biol Med. (2001) 30:986–91. doi: 10.1016/s0891-5849(01)00489-0

289. Lanone S, Manivet P, Callebert J, Launay JM, Payen D, Aubier M, et al. Inducible nitric oxide synthase (nos2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity. Biochem J. (2002) 366:399–404. doi: 10.1042/bj20020339

290. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. (2012) 33:829–37. doi: 10.1093/eurheartj/ehr304

291. Erwin PA, Lin AJ, Golan DE, Michel T. Receptor-regulated dynamic s-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells. J Biol Chem. (2005) 280:19888–94. doi: 10.1074/jbc.M413058200

292. Ravi K, Brennan LA, Levic S, Ross PA, Black SM. S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity. Proc Natl Acad Sci USA. (2004) 101:2619–24. doi: 10.1073/pnas.0300464101

293. Tummala M, Ryzhov V, Ravi K, Black SM. Identification of the cysteine nitrosylation sites in human endothelial nitric oxide synthase. DNA Cell Biol. (2008) 27:25–33. doi: 10.1089/dna.2007.0655

294. Martínez-Ruiz A, Villanueva L, González de Orduña C, López-Ferrer D, Higueras MA, Tarín C, et al. S-nitrosylation of hsp90 promotes the inhibition of its atpase and endothelial nitric oxide synthase regulatory activities. Proc Natl Acad Sci USA. (2005) 102:8525–30. doi: 10.1073/pnas.0407294102

295. Martínez-Ruiz A, Araújo IM, Izquierdo-Álvarez A, Hernansanz-Agustín P, Lamas S, Serrador JM. Specificity in s-nitrosylation: a short-range mechanism for no signaling? Antioxid Redox Signal. (2013) 19:1220–35. doi: 10.1089/ars.2012.5066

296. Cinelli MA, Do HT, Miley GP, Silverman RB. Inducible nitric oxide synthase: regulation, structure, and inhibition. Med Res Rev. (2020) 40:158–89. doi: 10.1002/med.21599

297. Tarek B, Gougerot-Pocidalo M-A, Hayem G, Ciappelloni S, Raad H, Derkawi R, et al. The prolyl isomerase pin1 acts as a novel molecular switch for tnf- -induced priming of the nadph oxidase in human neutrophils. Blood. (2010) 116:5795–802. doi: 10.1182/blood-2010-03-273094

298. Fischer MT, Sharma R, Lim JL, Haider L, Frischer JM, Drexhage J, et al. Nadph oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain. (2012) 135:886–99. doi: 10.1093/brain/aws012

299. Bedard K, Krause KH. The nox family of ros-generating nadph oxidases: physiology and pathophysiology. Physiol Rev. (2007) 87:245–313. doi: 10.1152/physrev.00044.2005

300. Selemidis S, Dusting GJ, Peshavariya H, Kemp-Harper BK, Drummond GR. Nitric oxide suppresses nadph oxidase-dependent superoxide production by s-nitrosylation in human endothelial cells. Cardiovasc Res. (2007) 75:349–58. doi: 10.1016/j.cardiores.2007.03.030

301. Kil IS, Lee JH, Yoon SH, Bae YS, Kim S, Shin SW, et al. S-nitrosylation of P47(Phox) enhances phosphorylation by casein kinase 2. Redox Rep. (2015) 20:228–33. doi: 10.1179/1351000215Y.0000000014

302. Qi SH, Hao LY, Yue J, Zong YY, Zhang GY. Exogenous nitric oxide negatively regulates the S-nitrosylation p38 mitogen-activated protein kinase activation during cerebral ischaemia and reperfusion. Neuropathol Appl Neurobiol. (2013) 39:284–97. doi: 10.1111/j.1365-2990.2012.01284.x

303. Friese MA, Schattling B, Fugger L. Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. Nat Rev Neurol. (2014) 10:225–38. doi: 10.1038/nrneurol.2014.37

304. Heidker RM, Emerson MR, LeVine SM. Metabolic pathways as possible therapeutic targets for progressive multiple sclerosis. Neural Regener Res. (2017) 12:1262–7. doi: 10.4103/1673-5374.213542

305. Mahad DH, Trapp BD, Lassmann H. Pathological mechanisms in progressive multiple sclerosis. Lancet Neurol. (2015) 14:183–93. doi: 10.1016/s1474-4422(14)70256-x

306. Patergnani S, Fossati V, Bonora M, Giorgi C, Marchi S, Missiroli S, et al. Mitochondria in multiple sclerosis: molecular mechanisms of pathogenesis. Int Rev Cell Mol Biol. (2017) 328:49–103. doi: 10.1016/bs.ircmb.2016.08.003

307. Kozin MS, Kulakova OG, Favorova OO. Involvement of mitochondria in neurodegeneration in multiple sclerosis. Biochem (Mosc). (2018) 83:813–30. doi: 10.1134/s0006297918070052

308. Dutta R, McDonough J, Yin X, Peterson J, Chang A, Torres T, et al. Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann Neurol. (2006) 59:478–89. doi: 10.1002/ana.20736

309. Trapp BD, Stys PK. Virtual hypoxia and chronic necrosis of demyelinated axons in multiple sclerosis. Lancet Neurol. (2009) 8:280–91. doi: 10.1016/s1474-4422(09)70043-2

310. Witte ME, Nijland PG, Drexhage JA, Gerritsen W, Geerts D, van Het Hof B, et al. Reduced expression of Pgc-1α partly underlies mitochondrial changes and correlates with neuronal loss in multiple sclerosis cortex. Acta Neuropathol. (2013) 125:231–43. doi: 10.1007/s00401-012-1052-y

311. Dziedzic T, Metz I, Dallenga T, König FB, Müller S, Stadelmann C, et al. Wallerian degeneration: a major component of early axonal pathology in multiple sclerosis. Brain Pathol. (2010) 20:976–85. doi: 10.1111/j.1750-3639.2010.00401.x

312. French HM, Reid M, Mamontov P, Simmons RA, Grinspan JB. Oxidative stress disrupts oligodendrocyte maturation. J Neurosci Res. (2009) 87:3076–87. doi: 10.1002/jnr.22139

313. Schoenfeld R, Wong A, Silva J, Li M, Itoh A, Horiuchi M, et al. Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation. Mitochondrion. (2010) 10:143–50. doi: 10.1016/j.mito.2009.12.141

314. Campbell GR, Ziabreva I, Reeve AK, Krishnan KJ, Reynolds R, Howell O, et al. Mitochondrial DNA deletions and neurodegeneration in multiple sclerosis. Ann Neurol. (2011) 69:481–92. doi: 10.1002/ana.22109

315. Witte ME, Bø L, Rodenburg RJ, Belien JA, Musters R, Hazes T, et al. Enhanced number and activity of mitochondria in multiple sclerosis lesions. J Pathol. (2009) 219:193–204. doi: 10.1002/path.2582

316. Wong HS, Dighe PA, Mezera V, Monternier PA, Brand MD. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. J Biol Chem. (2017) 292:16804–9. doi: 10.1074/jbc.R117.789271

317. Fridovich I. Fundamental aspects of reactive oxygen species, or what's the matter with oxygen? Ann N Y Acad Sci. (1999) 893:13–8. doi: 10.1111/j.1749-6632.1999.tb07814.x

318. Muscoli C, Cuzzocrea S, Riley DP, Zweier JL, Thiemermann C, Wang ZQ, et al. On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. Br J Pharmacol. (2003) 140:445–60. doi: 10.1038/sj.bjp.0705430

319. Clementi E, Brown GC, Feelisch M, Moncada S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci USA. (1998) 95:7631–6. doi: 10.1073/pnas.95.13.7631

320. Riobó NA, Clementi E, Melani M, Boveris A, Cadenas E, Moncada S, et al. Nitric oxide inhibits mitochondrial Nadh:ubiquinone reductase activity through peroxynitrite formation. Biochem J. (2001) 359:139–45. doi: 10.1042/0264-6021:3590139

321. Cadenas E. Mitochondrial free radical production and cell signaling. Mol Aspects Med. (2004) 25:17–26. doi: 10.1016/j.mam.2004.02.005

322. Brown GC, Cooper CE. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. (1994) 356:295–8. doi: 10.1016/0014-5793(94)01290-3

323. Cleeter MW, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AH. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. (1994) 345:50–4. doi: 10.1016/0014-5793(94)00424-2

324. Taylor CT, Moncada S. Nitric oxide, cytochrome c oxidase, and the cellular response to hypoxia. Arterioscler Thromb Vasc Biol. (2010) 30:643–7. doi: 10.1161/atvbaha.108.181628

325. Giulivi C. Characterization and function of mitochondrial nitric-oxide synthase. Free Radic Biol Med. (2003) 34:397–408. doi: 10.1016/s0891-5849(02)01298-4

326. Ghafourifar P, Cadenas E. Mitochondrial nitric oxide synthase. Trends Pharmacol Sci. (2005) 26:190–5. doi: 10.1016/j.tips.2005.02.005

327. Radi R. Peroxynitrite, a stealthy biological oxidant. J Biol Chem. (2013) 288:26464–72. doi: 10.1074/jbc.R113.472936

328. Qiu F, Liu Y, Liu Z. The role of protein S-nitrosylation in mitochondrial quality control in central nervous system diseases. Aging Dis. (2024) 16:2641–2658. doi: 10.14336/ad.2024.0099

329. Steffen M, Sarkela TM, Gybina AA, Steele TW, Trasseth NJ, Kuehl D, et al. Metabolism of S-nitrosoglutathione in intact mitochondria. Biochem J. (2001) 356:395–402. doi: 10.1042/0264-6021:3560395

330. Tessari F, Bortolami S, Zoccarato F, Alexandre A, Cavallini L. Different effects of Snp and Gsno on mitochondrial O2 .- /H 2o2 production. J Bioenerg Biomembr. (2011) 43:267–74. doi: 10.1007/s10863-011-9352-9

331. Chouchani ET, Methner C, Nadtochiy SM, Logan A, Pell VR, Ding S, et al. Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I. Nat Med. (2013) 19:753–9. doi: 10.1038/nm.3212

332. Vlasova II, Tyurin VA, Kapralov AA, Kurnikov IV, Osipov AN, Potapovich MV, et al. Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation. J Biol Chem. (2006) 281:14554–62. doi: 10.1074/jbc.M509507200

333. Dahm CC, Moore K, Murphy MP. Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria. J Biol Chem. (2006) 281:10056–65. doi: 10.1074/jbc.M512203200

334. Chang AH, Sancheti H, Garcia J, Kaplowitz N, Cadenas E, Han D. Respiratory substrates regulate S-nitrosylation of mitochondrial proteins through a thiol-dependent pathway. Chem Res Toxicol. (2014) 27:794–804. doi: 10.1021/tx400462r

335. Wang Y, Branicky R, Noë A, Hekimi S. Superoxide dismutases: dual roles in controlling Ros damage and regulating Ros signaling. J Cell Biol. (2018) 217:1915–28. doi: 10.1083/jcb.201708007

336. Prakash O, Tripathy P, Panchal D, Sharma A, Pal S. Chapter 16 - Advance oxidation processes for remediation of antibiotics from wastewater. In: Singh P, Sillanpää M, editors. Degradation of Antibiotics and Antibiotic-Resistant Bacteria From Various Sources. London, United Kingdom: Academic Press; (2023). p. 369–88.

337. Gozzelino R, Jeney V, Soares MP. Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol. (2010) 50:323–54. doi: 10.1146/annurev.pharmtox.010909.105600

338. Chiabrando D, Vinchi F, Fiorito V, Mercurio S, Tolosano E. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes. Front Pharmacol. (2014) 5:61. doi: 10.3389/fphar.2014.00061

339. Wilson K, Detsika MG, Poulaki E, Gakiopoulou H, Lianos EA. Morphometric effects of Ho-1 deficiency and overexpression in rat glomeruli and podocytes. J Clin Anat Pathol (JCAP). (2021) 6:1. doi: 10.47275/2332-4864-118

340. Muthukumar K, Rajakumar S, Sarkar MN, Nachiappan V. Glutathione peroxidase3 of Saccharomyces cerevisiae protects phospholipids during cadmium-induced oxidative stress. Antonie van Leeuwenhoek. (2011) 99:761–71. doi: 10.1007/s10482-011-9550-9

341. Mannervik B. The enzymes of glutathione metabolism: an overview. Biochem Soc Trans. (1987) 15:717–8. doi: 10.1042/bst0150717

342. Jeong SJ, Park JG, Oh GT. Peroxiredoxins as potential targets for cardiovascular disease. Antioxidants (Basel). (2021) 10. doi: 10.3390/antiox10081244

343. Yoshida T, Nakamura H, Masutani H, Yodoi J. The involvement of thioredoxin and thioredoxin binding protein-2 on cellular proliferation and aging process. Ann N Y Acad Sci. (2005) 1055:1–12. doi: 10.1196/annals.1323.002

344. Moezzi D, Dong Y, Jain RW, Lozinski BM, Ghorbani S, D'Mello C, et al. Expression of antioxidant enzymes in lesions of multiple sclerosis and its models. Sci Rep. (2022) 12:12761. doi: 10.1038/s41598-022-16840-w

345. Alfarhan M, Jafari E, Narayanan SP. Acrolein: a potential mediator of oxidative damage in diabetic retinopathy. Biomolecules. (2020) 10. doi: 10.3390/biom10111579

346. Liu R, Zhu T, Chen X, Wang Z, Yang Z, Ren A, et al. Gsnor regulates ganoderic acid content in Ganoderma lucidum under heat stress through S-nitrosylation of catalase. Commun Biol. (2022) 5:32. doi: 10.1038/s42003-021-02988-0

347. Bae T, Hallis SP, Kwak MK. Hypoxia, oxidative stress, and the interplay of Hifs and Nrf2 signaling in cancer. Exp Mol Med. (2024) 56:501–14. doi: 10.1038/s12276-024-01180-8

348. Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. (1999) 13:76–86. doi: 10.1101/gad.13.1.76

349. Yamamoto T, Suzuki T, Kobayashi A, Wakabayashi J, Maher J, Motohashi H, et al. Physiological significance of reactive cysteine residues of Keap1 in determining Nrf2 activity. Mol Cell Biol. (2008) 28:2758–70. doi: 10.1128/mcb.01704-07

350. Sekhar KR, Rachakonda G, Freeman ML. Cysteine-based regulation of the Cul3 adaptor protein Keap1. Toxicol Appl Pharmacol. (2010) 244:21–6. doi: 10.1016/j.taap.2009.06.016

351. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, et al. An Nrf2/Small Maf heterodimer mediates the induction of phase ii detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. (1997) 236:313–22. doi: 10.1006/bbrc.1997.6943

352. Hayes JD, Chanas SA, Henderson CJ, McMahon M, Sun C, Moffat GJ, et al. The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem Soc Trans. (2000) 28:33–41. doi: 10.1042/bst0280033

353. Liu X, Ward K, Xavier C, Jann J, Clark AF, Pang IH, et al. The novel triterpenoid Rta 408 protects human retinal pigment epithelial cells against H2o2-induced cell injury via Nf-E2-related factor 2 (Nrf2) activation. Redox Biol. (2016) 8:98–109. doi: 10.1016/j.redox.2015.12.005

354. Ma Q. Role of Nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. (2013) 53:401–26. doi: 10.1146/annurev-pharmtox-011112-140320

355. Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and C-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of Nad(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci USA. (1996) 93:14960–5. doi: 10.1073/pnas.93.25.14960

356. Dodson M, Castro-Portuguez R, Zhang DD. Nrf2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. (2019) 23:101107. doi: 10.1016/j.redox.2019.101107

357. Um HC, Jang JH, Kim DH, Lee C, Surh YJ. Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in Pc12 cells. Nitric Oxide. (2011) 25:161–8. doi: 10.1016/j.niox.2011.06.001

358. Hu W, Cai Y, Cai D, Chen Z, Huang S, Zhang S, et al. Hif-1α alleviates ferroptosis in ulcerative colitis by regulation of Gpx4. Cell Death Dis. (2025) 16:542. doi: 10.1038/s41419-025-07883-8

359. Lu H, Samanta D, Xiang L, Zhang H, Hu H, Chen I, et al. Chemotherapy triggers Hif-1-dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype. Proc Natl Acad Sci USA. (2015) 112:E4600–9. doi: 10.1073/pnas.1513433112

360. Conrad M, Friedmann Angeli JP. Glutathione peroxidase 4 (Gpx4) and ferroptosis: What's so special about it? Mol Cell Oncol. (2015) 2:e995047. doi: 10.4161/23723556.2014.995047

361. Jhelum P, Zandee S, Ryan F, Zarruk JG, Michalke B, Venkataramani V, et al. Ferroptosis induces detrimental effects in chronic Eae and its implications for progressive Ms. Acta Neuropathol Commun. (2023) 11:121. doi: 10.1186/s40478-023-01617-7

362. Li X, Chu Y, Ma R, Dou M, Li S, Song Y, et al. Ferroptosis as a mechanism of oligodendrocyte loss and demyelination in experimental autoimmune encephalomyelitis. J Neuroimmunol. (2022) 373:577995. doi: 10.1016/j.jneuroim.2022.577995

363. Luoqian J, Yang W, Ding X, Tuo QZ, Xiang Z, Zheng Z, et al. Ferroptosis promotes T-cell activation-induced neurodegeneration in multiple sclerosis. Cell Mol Immunol. (2022) 19:913–24. doi: 10.1038/s41423-022-00883-0

364. Wang F, Yuan Q, Chen F, Pang J, Pan C, Xu F, et al. Fundamental mechanisms of the cell death caused by nitrosative stress. Front Cell Dev Biol. (2021) 9:742483. doi: 10.3389/fcell.2021.742483

365. He Q, Qu M, Xu C, Shi W, Hussain M, Jin G, et al. The emerging roles of nitric oxide in ferroptosis and pyroptosis of tumor cells. Life Sci. (2022) 290:120257. doi: 10.1016/j.lfs.2021.120257

366. Homma T, Kobayashi S, Conrad M, Konno H, Yokoyama C, Fujii J. Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction. Nitric Oxide. (2021) 115:34–43. doi: 10.1016/j.niox.2021.07.003

367. Dar HH, Anthonymuthu TS, Ponomareva LA, Souryavong AB, Shurin GV, Kapralov AO, et al. A new thiol-independent mechanism of epithelial host defense against Pseudomonas aeruginosa: Inos/No(•) sabotage of theft-ferroptosis. Redox Biol. (2021) 45:102045. doi: 10.1016/j.redox.2021.102045

368. Feng J, Zheng Y, Guo M, Ares I, Martínez M, Lopez-Torres B, et al. Oxidative stress, the blood-brain barrier and neurodegenerative diseases: The critical beneficial role of dietary antioxidants. Acta Pharm Sin B. (2023) 13:3988–4024. doi: 10.1016/j.apsb.2023.07.010

369. Yang T, Peleli M, Zollbrecht C, Giulietti A, Terrando N, Lundberg JO, et al. Inorganic nitrite attenuates Nadph oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism. Free Radic Biol Med. (2015) 83:159–66. doi: 10.1016/j.freeradbiomed.2015.02.016

370. Al-Ayadhi LY. Oxidative stress and neurodegenerative disease. Neurosci (Riyadh). (2004) 9:19–23. doi: 10.17712/1658-3183.1242

371. Ferreira B, Mendes F, Osório N, Caseiro A, Gabriel A, Valado A. Glutathione in multiple sclerosis. Br J BioMed Sci. (2013) 70:75–9. doi: 10.1080/09674845.2013.11669939

372. Rogacka D, Audzeyenka I, Rachubik P, Szrejder M, Typiak M, Angielski S, et al. Involvement of nitric oxide synthase/nitric oxide pathway in the regulation of Sirt1-Ampk crosstalk in podocytes: Impact on glucose uptake. Arch Biochem Biophys. (2021) 709:108985. doi: 10.1016/j.abb.2021.108985

373. Jiang H, Torregrossa AC, Potts A, Pierini D, Aranke M, Garg HK, et al. Dietary nitrite improves insulin signaling through Glut4 translocation. Free Radic Biol Med. (2014) 67:51–7. doi: 10.1016/j.freeradbiomed.2013.10.809

374. Tian Y, Heng D, Xu K, Liu W, Weng X, Hu X, et al. Cgmp/Pkg-I pathway-mediated Glut1/4 regulation by No in female rat granulosa cells. Endocrinology. (2018) 159:1147–58. doi: 10.1210/en.2017-00863

375. Yeo CT, Kropp EM, Hansen PA, Pereckas M, Oleson BJ, Naatz A, et al. Beta-cell-selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake. J Biol Chem. (2023) 299:102994. doi: 10.1016/j.jbc.2023.102994

376. Higaki Y, Hirshman MF, Fujii N, Goodyear LJ. Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Diabetes. (2001) 50:241–7. doi: 10.2337/diabetes.50.2.241

377. Almeida A, Moncada S, Bolaños JP. Nitric oxide switches on glycolysis through the Amp protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol. (2004) 6:45–51. doi: 10.1038/ncb1080

378. Cidad P, Almeida A, Bolaños JP. Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective Glut3-mediated glucose uptake through 5'-Amp-activated protein kinase. Biochem J. (2004) 384:629–36. doi: 10.1042/bj20040886

379. Kierans SJ, Taylor CT. Regulation of glycolysis by the hypoxia-inducible factor (Hif): Implications for cellular physiology. J Physiol. (2021) 599:23–37. doi: 10.1113/jp280572

380. Padgett CM, Whorton AR. S-nitrosoglutathione reversibly inhibits Gapdh by S-nitrosylation. Am J Physiol. (1995) 269:C739–49. doi: 10.1152/ajpcell.1995.269.3.C739

381. Mateo RB, Reichner JS, MastroFrancesco B, Kraft-Stolar D, Albina JE. Impact of nitric oxide on macrophage glucose metabolism and glyceraldehyde-3-phosphate dehydrogenase activity. Am J Physiol. (1995) 268:C669–75. doi: 10.1152/ajpcell.1995.268.3.C669

382. Mohr S, Stamler JS, Brune B. Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent Nadh attachment. J Biol Chem. (1996) 271:4209–14. doi: 10.1074/jbc.271.8.4209

383. Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y, et al. S-nitrosylated Gapdh initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol. (2005) 7:665–74. doi: 10.1038/ncb1268

384. Hara MR, Snyder SH. Nitric oxide-Gapdh-Siah: A novel cell death cascade. Cell Mol Neurobiol. (2006) 26:527–38. doi: 10.1007/s10571-006-9011-6

385. Lowenstein CJ. Metabolism reprogrammed by the nitric oxide signalling molecule. Nature. (2019) 565:33–4. doi: 10.1038/d41586-018-07457-z

386. Siragusa M, Thöle J, Bibli SI, Luck B, Loot AE, de Silva K, et al. Nitric oxide maintains endothelial redox homeostasis through Pkm2 inhibition. EMBO J. (2019) 38:e100938. doi: 10.15252/embj.2018100938

387. Xiao W, Loscalzo J. Metabolic responses to reductive stress. Antioxid Redox Signal. (2020) 32:1330–47. doi: 10.1089/ars.2019.7803

388. Baardman J, Verberk SGS, Prange KHM, van Weeghel M, van der Velden S, Ryan DG, et al. A defective pentose phosphate pathway reduces inflammatory macrophage responses during hypercholesterolemia. Cell Rep. (2018) 25:2044–52. doi: 10.1016/j.celrep.2018.10.092

389. Cherkas A, Holota S, Mdzinarashvili T, Gabbianelli R, Zarkovic N. Glucose as a major antioxidant: When, what for and why it fails? Antioxidants (Basel). (2020) 9. doi: 10.3390/antiox9020140

390. Doulias PT, Tenopoulou M, Greene JL, Raju K, Ischiropoulos H. Nitric oxide regulates mitochondrial fatty acid metabolism through reversible protein S-nitrosylation. Sci Signal. (2013) 6:rs1. doi: 10.1126/scisignal.2003252

391. Lee GR. The balance of Th17 versus Treg cells in autoimmunity. Int J Mol Sci. (2018) 19. doi: 10.3390/ijms19030730

392. Cluxton D, Petrasca A, Moran B, Fletcher JM. Differential regulation of human Treg and Th17 cells by fatty acid synthesis and glycolysis. Front Immunol. (2019) 10:115. doi: 10.3389/fimmu.2019.00115

393. Geltink RIK, Kyle RL, Pearce EL. Unraveling the complex interplay between T cell metabolism and function. Annu Rev Immunol. (2018) 36:461–88. doi: 10.1146/annurev-immunol-042617-053019

394. O'Neill LA, Kishton RJ, Rathmell J. A guide to immunometabolism for immunologists. Nat Rev Immunol. (2016) 16:553–65. doi: 10.1038/nri.2016.70

395. Colagiovanni DB, Drolet DW, Langlois-Forget E, Piché MP, Looker D, Rosenthal GJ. A nonclinical safety and pharmacokinetic evaluation of N6022: A first-in-class S-nitrosoglutathione reductase inhibitor for the treatment of asthma. Regul Toxicol Pharmacol. (2012) 62:115–24. doi: 10.1016/j.yrtph.2011.12.012

396. Hauser SL, Bar-Or A, Comi G, Giovannoni G, Hartung HP, Hemmer B, et al. Ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis. N Engl J Med. (2017) 376:221–34. doi: 10.1056/NEJMoa1601277

397. Montalban X, Hauser SL, Kappos L, Arnold DL, Bar-Or A, Comi G, et al. Ocrelizumab versus placebo in primary progressive multiple sclerosis. N Engl J Med. (2017) 376:209–20. doi: 10.1056/NEJMoa1606468

398. Hauser SL, Kappos L, Montalban X, Craveiro L, Chognot C, Hughes R, et al. Safety of ocrelizumab in patients with relapsing and primary progressive multiple sclerosis. Neurology. (2021) 97:e1546–59. doi: 10.1212/wnl.0000000000012700

399. Hauser SL, Zielman R, Das Gupta A, Xi J, Stoneman D, Karlsson G, et al. Efficacy and safety of four-year ofatumumab treatment in relapsing multiple sclerosis: The Alithios open-label extension. Mult Scler. (2023) 29:1452–64. doi: 10.1177/13524585231195346

400. Montague T, Cheung E, Kang Y, Venkatesha V, Zeme K, Ferrier S, et al. Real-world safety and effectiveness of ofatumumab in multiple sclerosis: A clinic-based study in Australia. Mult Scler J Exp Transl Clin. (2026) 12:20552173251414528. doi: 10.1177/20552173251414528

401. Polman CH, O'Connor PW, Havrdova E, Hutchinson M, Kappos L, Miller DH, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med. (2006) 354:899–910. doi: 10.1056/NEJMoa044397

402. Bloomgren G, Richman S, Hotermans C, Subramanyam M, Goelz S, Natarajan A, et al. Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N Engl J Med. (2012) 366:1870–80. doi: 10.1056/NEJMoa1107829

403. Ebers GC. Randomised double-blind placebo-controlled study of interferon β-1a in relapsing/remitting multiple sclerosis. Lancet. (1998) 352:1498–504. doi: 10.1016/S0140-6736(98)03334-0

404. Goodin DS, Reder AT, Ebers GC, Cutter G, Kremenchutzky M, Oger J, et al. Survival in ms: a randomized cohort study 21 years after the start of the pivotal ifnβ-1b trial. Neurology. (2012) 78:1315–22. doi: 10.1212/WNL.0b013e3182535cf6

405. Boster AL, Ford CC, Neudorfer O, Gilgun-Sherki Y. Glatiramer acetate: long-term safety and efficacy in relapsing-remitting multiple sclerosis. Expert Rev Neurother. (2015) 15:575–86. doi: 10.1586/14737175.2015.1040768

406. Johnson KP, Brooks BR, Cohen JA, Ford CC, Goldstein J, Lisak RP, et al. Copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase iii multicenter, double-blind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group. Neurology. (1995) 45:1268–76. doi: 10.1212/wnl.45.7.1268

407. Khan O, Rieckmann P, Boyko A, Selmaj K, Zivadinov R. Three times weekly glatiramer acetate in relapsing-remitting multiple sclerosis. Ann Neurol. (2013) 73:705–13. doi: 10.1002/ana.23938

408. Longbrake EE, Racke MK. Why did il-12/il-23 antibody therapy fail in multiple sclerosis? Expert Rev Neurother. (2009) 9:319–21. doi: 10.1586/14737175.9.3.319

409. Kappos L, Radue EW, O'Connor P, Polman C, Hohlfeld R, Calabresi P, et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med. (2010) 362:387–401. doi: 10.1056/NEJMoa0909494

410. Kappos L, Antel J, Comi G, Montalban X, O'Connor P, Polman CH, et al. Oral fingolimod (fty720) for relapsing multiple sclerosis. N Engl J Med. (2006) 355:1124–40. doi: 10.1056/NEJMoa052643

411. Hatcher SE, Waubant E, Nourbakhsh B, Crabtree-Hartman E, Graves JS. Rebound syndrome in patients with multiple sclerosis after cessation of fingolimod treatment. JAMA Neurol. (2016) 73:790–4. doi: 10.1001/jamaneurol.2016.0826

412. Gold R, Kappos L, Arnold DL, Bar-Or A, Giovannoni G, Selmaj K, et al. Placebo-controlled phase 3 study of oral bg-12 for relapsing multiple sclerosis. N Engl J Med. (2012) 367:1098–107. doi: 10.1056/NEJMoa1114287

413. Gold R, Arnold DL, Bar-Or A, Fox RJ, Kappos L, Chen C, et al. Safety and efficacy of delayed-release dimethyl fumarate in patients with relapsing-remitting multiple sclerosis: 9 years' follow-up of define, confirm, and endorse. Ther Adv Neurol Disord. (2020) 13:1756286420915005. doi: 10.1177/1756286420915005

414. O'Connor P, Wolinsky JS, Confavreux C, Comi G, Kappos L, Olsson TP, et al. Randomized trial of oral teriflunomide for relapsing multiple sclerosis. N Engl J Med. (2011) 365:1293–303. doi: 10.1056/NEJMoa1014656

415. Glass JD. Calming the excitement in als. Neurology. (2016) 86:1468–9. doi: 10.1212/wnl.0000000000002506

416. Kieseier BC, Benamor M. Pregnancy outcomes following maternal and paternal exposure to teriflunomide during treatment for relapsing-remitting multiple sclerosis. Neurol Ther. (2014) 3:133–8. doi: 10.1007/s40120-014-0020-y

417. Giovannoni G, Comi G, Cook S, Rammohan K, Rieckmann P, Soelberg Sørensen P, et al. A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis. N Engl J Med. (2010) 362:416–26. doi: 10.1056/NEJMoa0902533

418. Giovannoni G, Soelberg Sorensen P, Cook S, Rammohan K, Rieckmann P, Comi G, et al. Safety and efficacy of cladribine tablets in patients with relapsing-remitting multiple sclerosis: results from the randomized extension trial of the clarity study. Mult Scler. (2018) 24:1594–604. doi: 10.1177/1352458517727603

419. Reich DS, Arnold DL, Vermersch P, Bar-Or A, Fox RJ, Matta A, et al. Safety and efficacy of tolebrutinib, an oral brain-penetrant btk inhibitor, in relapsing multiple sclerosis: a phase 2b, randomised, double-blind, placebo-controlled trial. Lancet Neurol. (2021) 20:729–38. doi: 10.1016/s1474-4422(21)00237-4

420. Krämer J, Bar-Or A, Turner TJ, Wiendl H. Bruton tyrosine kinase inhibitors for multiple sclerosis. Nat Rev Neurol. (2023) 19:289–304. doi: 10.1038/s41582-023-00800-7

421. Bar-Or A, Dufek M, Budincevic H, Drulovic J, Habek M, Hua LH, et al. Safety and efficacy of fenebrutinib in relapsing multiple sclerosis (fenopta): a multicentre, double-blind, randomised, placebo-controlled, phase 2 trial and open-label extension study. Lancet Neurol. (2025) 24:656–66. doi: 10.1016/s1474-4422(25)00174-7

422. Won J, Kim J, Islam SMT, Qiao F, Singh AK, Singh I. The potential role of s-nitrosoglutathione reductase in astrocyte baff expression and b cell activation in experimental autoimmune encephalomyelitis. J Neuroimmunol. (2026) 412:578847. doi: 10.1016/j.jneuroim.2025.578847

423. Naismith RT, Wundes A, Ziemssen T, Jasinska E, Freedman MS, Lembo AJ, et al. Diroximel fumarate demonstrates an improved gastrointestinal tolerability profile compared with dimethyl fumarate in patients with relapsing-remitting multiple sclerosis: results from the randomized, double-blind, phase iii evolve-ms-2 study. CNS Drugs. (2020) 34:185–96. doi: 10.1007/s40263-020-00700-0

424. Singer BA, Arnold DL, Drulovic J, Freedman MS, Gold R, Gudesblatt M, et al. Diroximel fumarate in patients with relapsing-remitting multiple sclerosis: final safety and efficacy results from the phase 3 evolve-ms-1 study. Mult Scler. (2023) 29:1795–807. doi: 10.1177/13524585231205708