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  • We previously evaluated the immuno modulatory efficacy

    2021-10-15

    We previously evaluated the immuno-modulatory efficacy of GSNO in different EAE models and reported prophylactic and therapeutic efficacy of GSNO against the clinical disease of EAE [22], [36]. GSNO inhibited the IL-6-induced STAT3 activation (Tyr705 phosphorylation) by S-nitrosylation of the STAT3 protein on Cys259 [47] and also downregulated the IL-6 and TGF-β induced expression of RORγt, a TH17 cell specific transcription factor [22]. Additionally, GSNO treatment also inhibited the IL-6/TGF-β and IL-23 induced TH17 cell polarization and their effector function under in vitro cell culture conditions but without affecting TH1 (IFN-γ) and TH2 (IL-4) immune responses [22]. Moreover, GSNO was reported to modulates the activities of proinflammatory transcription factors, such as NF-κB, AP-1, and STAT3 [35], [36], [48] and thus modulates gene expression for various proinflammatory effectors, such as ICAM-1, and VCAM-1 [35], [36], [43], [48], [49], as well as endothelial recruitment of peripheral immune cells for their CNS infiltration [36]. Therefore, it is important to understand the role of NO in immune and CNS disease process for MS and EAE. Cellular GSNO homeostasis is regulated by its synthesis by reaction between NO and GSH and its catabolism by GSNO reductase (GSNOR/ADH5), an enzyme in the alcohol dehydrogenase (ADH) family [26]. In this study, we assessed the role of exogenous vs. endogenous GSNO in modulation of pro-inflammatory (TH1 and TH17) and anti-inflammatory (TH2 and Treg) CD4+ T cells by treating EAE mice with exogenous GSNO as compared to inhibitor of GSNOR (N6022) for endogenous GSNO under EAE conditions. N6022 is a first-in-class very potent, specific, and reversible inhibitor of GSNOR [50]. N6022 has been found to be beneficial in animal models of experimental ruthenium red australia [51], allergic airway inflammation [52] and endothelial vasodilatory dysfunction [53]. In addition, safety of N6022 for human use is proven by Phase I and II studies for asthma and cystic fibrosis (ClinicalTrials.gov) [50]. In this study, we observed that overall effects of exogenously supplemented GSNO vs. endogenous generated GSNO by N6022-mediated inhibition of its degradation were similar in terms of attenuation of EAE disease with greater efficacy with N6022 than GSNO treatment. While both GSNO and N6022 inhibit TH17 cells for expression of IL-17 and induced CD4+ CD25+ FOXP3- Treg for expression of IL-10, N6022 also inhibits TH1 for expression of IFN-γ and induces CD4+ CD25+ FOXP3+ Treg for expression of IL-10. These data suggest that cellular GSNO homeostasis is important for differentiation and ruthenium red australia effector function of proinflammatory (TH1 and TH17) and anti-inflammatory (TH2 and Treg) CD4+ T cells. Moreover, this study documents that GSNOR inhibitor (N6022) is also a novel therapeutic approach for targeting NO metabolome in cells expressing NOS and GSNOR for selective modulation of CD4+ subsets (TH1/TH17 vs. TH2/Treg) and thus attenuation of autoimmune disease of MS/EAE without causing a deleterious lymphopenic effect.
    Materials and methods
    Results
    Discussion Current drugs approved for MS treatment are generally non-specific immune-modulating agents that can cause a global immune suppression as well as various adverse effects [10]. Therefore, development of new approach that modulates of disease specific autoimmune responses while minimizing the global immune suppression is critical for MS therapeutics. This study reports the efficacy of a drug targeting GSNO mediated mechanisms (GSNOR inhibitor N6022) on subset specific modulation of pro-inflammatory (TH1/TH17) vs. anti-inflammatory CD4+ T cells (TH2/Treg) as well as neuroprotection in EAE animal model and thus its therapeutic potential for MS. We previously reported that GSNO treatment attenuated the EAE disease by inhibiting TH17 signaling pathways (STAT3/RORγt) [22]. In this study, we evaluated the role of GSNO mediated mechanism in induction or inhibition of subset specific CD4+ T cells under EAE conditions. Consistent with earlier study [22], treatment of progressive EAE mice (immunized with MOG peptide) with exogenous GSNO attenuated the clinical disease of EAE (Fig. 1A). In addition, GSNO treatment selectively inhibited EAE-induced polarization and CNS effector function of proinflammatory TH17 cells but without affecting polarization and CNS effector function of TH1 and TH2 (Figs. 1C and 2B) [22]. Similar observation was also made earlier by Yang et al. [21] that S-nitroso-N-acetylpenicillamine (SNAP), another S-NO donor, selectively inhibited TH17 without affecting TH1 and TH2. At present, the mechanism underlying GSNO and SNAP mediated inhibition of TH17 is not well understood but we reproted that GSNO or S-nitroso-N-acetylcysteine (SNAC, a S-NO donor) inhibits IL-6-induced activation of STAT3 (phosphorylation on tyrosine705) via S-nitrosylation of cysteine259 of STAT3 [49], [60], [61]. Activated STAT3 by IL-6 and IL-23 plays a pivotal role in polarization and effector function of TH17 cells [62], and thus documenting that GSNO mediated mechanism inhibit polarization and effector function of TH17 via inactivation of STAT3.