Abstract T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis. Keywords: Peroxynitrite, Nitration, IL-2R, Experimental autoimmune encephalomyelitis, Multiple sclerosis Graphical abstract [43]Image 1 [44]Open in a new tab 1. Introduction Multiple sclerosis (MS) is an inflammatory autoimmune disease characterized by focal demyelination, axonal, and neuronal damage in the central nervous system (CNS) [[45][1], [46][2], [47][3]]. Both peripheral and myelin immune cells play pivotal roles in MS pathology. Peripheral myelin-specific CD4^+ T cells can infiltrate the CNS and participate in innate and adaptive immune responses [[48][4], [49][5], [50][6]]. T-helper cells and Forkhead box P3^+ (FoxP3^+) regulatory T cells (Treg) are two essential regulators in MS pathology. Activated T-helper 17 (Th17) cells and T-helper 1 (Th1) cells exacerbate CNS damage in experimental autoimmune encephalomyelitis (EAE), a commonly used MS animal model [[51]7]. In contrast, Tregs maintain peripheral immune tolerance and inhibit autoimmune responses by suppressing effector CD4^+ T cell subsets [[52]8]. Th17 cells secrete IL-17 A and chemokines to attract other immune cells to the CNS, while Tregs produce anti-inflammatory cytokines such as IL-10 and TGF-β to suppress immune cells [[53]9]. Tregs can control Th17 cells expressing IL-10 receptor through interleukin-10 (IL-10) and STAT3 phosphorylation [[54]10,[55]11]. Impaired Treg functions contribute to the dysregulation of effector T cell responses, exacerbating MS severity [[56][12], [57][13], [58][14], [59][15]]. The disrupted Treg/Th17 balance, which shifts toward an inflammatory phenotype, aggravates symptoms in relapsed MS patients [[60]16] and other autoimmune diseases [[61][17], [62][18], [63][19]]. Therefore, restoring Treg/Th17 balance could maintain immune homeostasis and reduce MS severity. Redox balance is highly associated with the Treg/Th17 balance. Physiologically, oxidative stress and antioxidant capacity could be maintained at a homeostasis condition for redox balance. Excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) affect Treg/Th17 and cytokine production to mediate inflammatory and immune dysfunctions in MS/EAE pathology [[64]18,[65][20], [66][21], [67][22], [68][23], [69][24], [70][25]]. Nitric oxide (·NO) suppresses Treg cells via affecting soluble guanylyl cyclase pathway [[71]26]. Homeostasis of S-nitrosoglutathione (GSNO), an endogenous ·NO carrier, selectively inhibited Th1/Th17 subsets CD4^+ cells and regulated Treg cells [[72]27]. With interferon (IFN)-beta 1 b treatment, the increased superoxide dismutase-1 promoted Treg differentiation with the increased Foxp3-exon 2 expression in MS patients, indicating the inhibitory effects of superoxide (O[2]^.-) on Treg cells [[73]28]. Inhibition of mitochondrial ROS attenuated peripheral Treg death in active EAE mice [[74]29,[75]30]. Notably, ·NO can rapidly react with O[2]^.- to form peroxynitrite (ONOO^−) that mediates protein nitration and produce 3-nitrotyrosine (3-NT) [[76]31]. The levels of 3-NT in both plasma and MS lesions were correlated with the MS severity [[77]32]. The roles of peroxynitrite in mediating axonal damage and disease progression have been well documented in active EAE mice [[78][33], [79][34], [80][35]]. Both peroxynitrite scavenger uric acid and peroxynitrite decomposition catalyst (PDC) revealed to ameliorate CNS inflammation and tissue damage in chronic relapsing EAE mice via modulating peripheral redox balance [[81][36], [82][37], [83][38]]. However, whether peroxynitrite could modulate CD4^+ cells subsets (Th1, Th17 and Treg cells) and affect neuroinflammation remains unknown yet. If so, modulating peripheral Treg dysfunction using peroxynitrite scavengers would create an opportunity to attenuate CNS inflammation and demyelination in active EAE/MS pathology. In the present study, we tested the hypothesis that peroxynitrite could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS/EAE pathology by inducing IL-2R nitration and down-regulating RAS/JNK-AP-1 signalling pathway, Subsequently, we verified that targeting peroxynitrite could modulate peripheral Treg population and Treg/Th17 balance, and ameliorate MS/EAE pathology. Furthermore, the PDC-treated autologous Tregs isolated from EAE mice revealed to attenuate the EAE/MS pathology. Therefore, peroxynitrite scavengers could restore the Treg/Th17 balance, inhibit CD4^+ T cell infiltration and demyelination, and ameliorate active MS/EAE pathology. 2. Results 2.1. Increased 3-nitrotyrosine in peripheral T cells is coincided with disease severity and progression in EAE mice and MS patients Our previous study reported the correlation of serum peroxynitrite level with disease progression in mouse model of active experimental autoimmune encephalomyelitis (EAE) [[84]34]. Recent progress documented that T cells play crucial roles in the neuroinflammation and neuropathology in both MS patients and EAE animal model [[85][4], [86][5], [87][6]]. To elucidate the impacts of peroxynitrite production on T cell function, we firstly investigated the dynamic changes of 3-nitrotyrosine (3-NT), a biomarker for peroxynitrite [[88]39], in peripheral CD4^+ T cells at different phases of active EAE mice. CD4^+ T cells were isolated and purified from the lymph nodes of the EAE mice at 0, 11, 18, and 30 days of post-immunization (dpi), corresponding to the early onset, peak, and chronic stages of EAE, respectively. Notably, the elevated 3-NT level in CD4^+ T cells was coincided with the severities of clinical score and disease progression, peaking at 18 dpi and decreasing at 30 dpi ([89]Fig. 1A–C). There was a positive correlation between the T cell's 3-NT level and the disease severity ([90]Fig. 1D). Moreover, we also compared serum 3-NT level in healthy donors and the MS patients at both active and remission phases clinically. Consistently, active phase MS patients (within 1.5 months) had significantly higher serum 3-NT levels than the healthy donor group. While, the remission phase MS patients had significantly lower serum 3-NT levels than the active phase patients ([91]Fig. 1E). Those data suggest that the serum 3-NT level could be correlated with the severity of MS. To confirm this idea, we extensively analysed public human datasets that supported the involvement of reactive nitrogen species (RNS) in MS patients. We compared the gene sets between relapsing-remitting MS patients (108 individuals) and health donors (60 individuals) by using the [92]GSE136411 dataset [[93]40]. The differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) of the MS patients and healthy donors were analysed that annotated various molecular functions and biological processes ([94]Figs. S1A–B). The DEGs data revealed an enriched oxidation-reduction system and an activated immune response. Gene set enrichment analysis (GSEA) showed that the PBMCs from healthy donors were predominantly associated with the negative regulation of nitrogen compound metabolic processes ([95]Fig. S1C). Therefore, our study indicates that peroxynitrite could be a critical player in peripheral immune responses during MS/EAE pathogenesis. Fig. 1. [96]Fig. 1 [97]Open in a new tab Expression of 3-nitrotyrosine (3-NT) in T cells was coincident with severities of inflammation, demyelination, and neurological deficits in active EAE mice, which was attenuated by PDC treatment A. Time-dependent responses of 3-NT levels in purified T cells of immunized EAE mice at different disease phases (0, 11, 18 and 30 dpi). B. Immunofluorescent staining of 3-NT in purified T cell of immunized EAE mice (18 dpi). Scale bars, 10 μm. Immunostaining of non-adherent T cells were performed after adhesion of cells onto microscope slides by cytocentrifugation. C. Quantification analysis of 3NT level analysed by flow cytometry. (means ± SEM, n = 6–8; *p < 0.05, ***p < 0.001 compared to Ctl group) D. Correlations between disease clinical scores and grey values of 3-NT level in EAE mice. Correlation Coefficient = 0.7813. E. 3-NT level in serum from healthy donor, active (within 1.5 month) and remission phase of MS patients was detected by ELISA kit. (n = 8–20 per group; mean ± SEM; **p < 0.01, ****p < 0.0001 compared to active MS group) F–H. Representative flow cytometric profiles and quantification analysis showed the expression of 3-NT in Th17 cells (CD4^+IL-17^+) and Th1 cells (CD4^+IFNγ^+) in LNs from EAE induced mice (Ctl −0 dpi, onset −11 dpi, peak −18 dpi, and chronic −30 dpi phase). I-J. Representative flow cytometric profiles and quantification analysis showed the expression of 3-NT in Treg cells (CD4^+CD25^+Foxp3^+) in LNs from EAE induced mice. Th1 and Th17 cells are leading immunological effectors through releasing IFN-γ and IL-17, respectively [[98]41,[99]42]. Treg/Th17 axis crucially controls neuroinflammation and autoimmunity during MS pathogenesis [[100]43]. The suppressive regulation of Treg cells, defined as CD4^+CD25^+Foxp3^+, on Th17 and Th1 cells is impaired in the MS pathogenesis [[101]44,[102]45]. The disruption of Treg/Th17 balance toward inflammatory phenotype was correlated with the severity of symptoms in relapsed MS patients [[103]16]. To elucidate the production of peroxynitrite in T cells, we investigated the expression of 3-NT in self-antigen specific T cell subsets including Th1, Th17 and Treg cells. The subsets of CD4^+Th cells were isolated from the lymph nodes and lumbar spinal cord (LSC) in the active EAE mice at 0, 11, 18, and 30 dpi, corresponding to the stages of normal control, onset, peak, and chronic phage, respectively. The expression levels of 3-NT in all types of Th1, Th17 and Treg cells were significantly increased at the peak time of the disease severities in the EAE mice ([104]Fig. 1F–J). Those results suggest that the production of peroxynitrite in the Th1, Th17 and Treg cells could be correlated with the disease severity in EAE/MS pathogenesis. 2.2. FeTMPyP, a representative PDC, inhibits 3-NT formation in Th1, Th17 and Treg cells and alleviates neuroinflammation, demyelination and neurological deficit in active EAE mice We then explored the impacts of peroxynitrite productions in Th1, Th17 and Treg cells on the neurological deficit scores, neuroinflammation and demyelination in active EAE mice. As a representative PDC, FeTMPyP was intraperitoneally administered to the EAE mice at the dosage of 10 mg/kg/day. The PDC treatment significantly alleviated the neurological deficit scores in the active EAE mice ([105]Fig. 2A–B). We then investigated the effects of PDC treatment on neuroinflammation and demyelination in the lumbar spinal cord lesions of the EAE mice at 18 dpi, the peak time of EAE. Treatment of PDC remarkably reduced the inflammatory foci in the white matter of lumbar spinal cord of the EAE mice ([106]Fig. 2C–D). Furthermore, LFB staining revealed that the PDC treatment attenuated the demyelination ([107]Fig. 2E–F) and inhibited CD4^+ T cell infiltration in the spinal cords of active EAE mice ([108]Fig. 2G–H). Simultaneously, the PDC treatment significantly inhibited the expression of 3-NT in the Th1, Th17 and Treg cells ([109]Fig. 2I–K). These results suggest that the production of peroxynitrite in the Th1, Th17 and Treg cells aggravates neuroinflammation, demyelination and neurological deficits in active EAE mice. Fig. 2. [110]Fig. 2 [111]Open in a new tab Inhibition of peroxynitrite decreased 3-NT level in Th17, Treg and Th17 in active EAE mice. A-B. Clinical scores in vehicle EAE and PDC-treated EAE mice (10 mg/kg/day). PDC treatment was started from 11 dpi. C. H&E staining showed PDC reduced inflammatory infiltrations in the white matter of lumbar spinal cord lesion (L4-L6). Samples were obtained from normal and EAE mice treated with vehicle or PDC at 18 dpi. (scale bar, 100 μm) D. Pathology scores of inflammations for H&E images. (means ± SEM, n = 6; ****p < 0.0001 compared to EAE + Vehicle group) E. Luxol Fast Blue (LFB) staining for the effects of PDC on attenuating the demyelination in the lumbar spinal cords (L4-L6) of the active EAE mice at 30 dpi. (scale bar, 100 μm) F. Pathology scores of demyelination for LFB images. (means ± SEM, n = 6; ***p < 0.001, ****p < 0.0001 compared to EAE + Vehicle group) G-H. Representative co-staining of CD4 (green) and DAPI (blue) in LSC of active EAE and PDC treated EAE mice (18dpi). (means ± SEM, n = 6; ***p < 0.001 compared to EAE + Vehicle group) I–K. Expression levels of 3-NT in Th17 cells, Th1 cells and Treg cells from the vehicle-treated EAE mice and PDC-treated mice (18 dpi) analysed by flow cytometry. (n = 5–6; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to EAE + Vehicle group). (For interpretation of the references to color in this figure legend, the reader is referred