We previously reported that S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, attenuated TH17-mediated immune responses in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Cellular GSNO homeostasis is regulated via its synthesis by reaction between nitric oxide and glutathione and its enzymatic catabolism by GSNO reductase (GSNOR). In this study, we evaluated potential of reversible inhibitor of GSNOR (N6022) in comparison with exogenous GSNO in immunopathogenesis of EAE. Daily treatment of EAE mice with N6022 or exogenous GSNO significantly attenuated the clinical disease of EAE, but N6022 treatment showed greater efficacy than GSNO. Both N6022 and exogenous GSNO treatments increased the spleen levels of GSNO, as documented by increased protein-associated S-nitrosothiols, and inhibited polarization and CNS effector function of proinflammatory TH17 cells while inducing the polarization and CNS effector function of anti-inflammatory CD4+ CD25+ FOXP3- regulatory T (Treg) cells. Moreover, N6022 further attenuated TH1 while inducing TH2 and CD4+ CD25+ FOXP3+ Treg in their polarization and CNS effector functions. Similar to GSNO, the N6022 treatment protected against the EAE disease induced demyelination. However, neither exogenous GSNO nor N6022 treatment did not cause significant systemic lymphopenic effect as compared to FTY720. Taken together, these data document that optimization of cellular GSNO homeostasis by GSNOR inhibitor (N6022) in NO metabolizing cells attenuates EAE disease via selective inhibition of pro-inflammatory subsets of CD4+ cells (TH1/TH17) while upregulating anti-inflammatory subsets of CD4+ cells (TH2/Treg) without causing lymphopenic effects and thus offers a potential treatment option for MS/EAE.

B cells play both protective and pathogenic roles in T cell-mediated autoimmune diseases by releasing regulatory vs. pathogenic cytokines. B cell-depleting therapy has been attempted in various autoimmune diseases but its efficacy varies and can even worsen symptoms due to depletion of B cells releasing regulatory cytokines along with B cells releasing pathogenic cytokines. Here, we report that S-nitrosoglutathione (GSNO) and GSNO-reductase (GSNOR) inhibitor N6022 drive upregulation of regulatory cytokine (IL-10) and downregulation of pathogenic effector cytokine (IL-6) in B cells and protected against the neuroinflammatory disease of experimental autoimmune encephalomyelitis (EAE). In human and mouse B cells, the GSNO/N6022-mediated regulation of IL-10 vs. IL-6 was not limited to regulatory B cells but also to a broad range of B cell subsets and antibody-secreting cells. Adoptive transfer of B cells from N6022 treated EAE mice or EAE mice deficient in the GSNOR gene also regulated T cell balance (Treg > Th17) and reduced clinical disease in the recipient EAE mice. The data presented here provide evidence of the role of GSNO in shifting B cell immune balance (IL-10 > IL-6) and the preclinical relevance of N6022, a first-in-class drug targeting GSNOR with proven human safety, as therapeutics for autoimmune disorders including multiple sclerosis.

Nitric oxide (NO) synthesized by eNOS plays a key role in regulation of endothelial barrier integrity but underlying cell signaling pathway is not fully understood at present. Here, we report opposing roles of two different redox-dependent NO metabolites; peroxynitrite (ONOO-) vs. S-nitrosoglutathione (GSNO), in cell signaling pathways for endothelial barrier disruption. In cultured human brain microvessel endothelial cells (hBMVECs), thrombin induced F-actin stress fiber formation causes barrier disruption via activating eNOS. Thrombin induced eNOS activity participated in cell signaling (e.g. RhoA and calcium influx mediated phosphorylation of myosin light chain) for F-actin stress fiber formation by increasing ONOO- levels. On the other hand, thrombin had no effect on intracellular levels of S-nitrosoglutathione (GSNO), another cellular NO metabolite. However, exogenous GSNO treatment attenuated the thrombin-induced cell signaling pathways for endothelial barrier disruption, thus suggesting the role of a shift of NO metabolism (GSNO vs. ONOO-) toward ONOO- synthesis in cell signaling for endothelial barrier disruption. Consistent with these in vitro studies, in animal models of traumatic brain injury and experimental autoimmune encephalomyelitis (EAE), ONOO- scavenger treatment as well as GSNO treatment were effective for attenuation of BBB leakage, edema formation, and CNS infiltration of mononuclear cells. Taken together, these data document that eNOS-mediated NO production and following redox-dependent NO metabolites (ONOO- vs. GSNO) are potential therapeutic target for CNS microvascular disease (traumatic and inflammatory) pathologies.

Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR-/- mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR-/- mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential.