Studies with GSNOR knockout mice
Studies with GSNOR knockout mice reported decreased CD4+ concentration formula and increased CD19+ cells (B-cell) in the blood  as well as demyelination, neurodegeneration, and neuropathic pain . To understand the basis of these differences in immune function of GSNOR knockout mice and GSNO treated mice, we studied the relative efficacy of GSNO vs. sphingosine 1-phosphate receptor agonist FTY720 known to cause lymphopenia in EAE disease mechanisms. FTY720 is a first-in-class orally bioavailable compound for the treatment of MS. The proposed mechanism of action for FTY720 in MS is a reversible retention of T-cells and B-cells in lymph nodes which thereby reduces the number of inflammatory cells in circulation and thus their access to the CNS. Therefore, FTY720-mediated immuno-suppression is nonspecific and thus can cause a lymphopenia, a condition of low level of lymphocytes in the blood . In addition, FTY720 is also known to inhibit Treg and thus abrogate their anti-inflammatory and immune suppressor functions . Our data show that N6022 or GSNO treatment do not cause any obvious lymphopenic effects whereas FTY720 reduces the number of lymphocytes in blood (Figs. 2A and 6A). Moreover, N6022 treatment selectively inhibited EAE-induced pro-inflammatoryTH1 and TH17 immune responses while elevating the EAE-induced compromises in TH2 and Treg immune responses (Fig. 5B and C and Fig. 6B and C). Secondly, GSNOR knockout mice were reported to develop demyelination and neurodegeneration. For it we investigated the status of myelin in EAE animals treated with GSNOR inhibitor N6022. Our data shows that N6022 treatment provides protection rather than degeneration in EAE animals. At present, we do not fully understand the basis for opposing results from GSNOR knockout mice and GSNOR inhibitor studies. However, the different degree of inhibition of GSNOR activities under conditions with pharmacological inhibitor (N6022) vs. genetic elimination of GSNOR (GSNOR knockout mice) may account, at least in part, for these observed differences. Secondly, GSNOR degrades a number of other compounds in addition to GSNO  and this may also account for these observed differences.
In summary, this study describes the efficacies of exogenous GSNO and endogenous N6022 (GSNOR inhibitor) in EAE disease. Both drugs attenuated the clinical disease of EAE with inhibition of TH17 cells and induction of Treg cells in their differentiations and their effector functions but without producing any global lymphopenia effect, like FTY720. N6022 treatment was also effective on the inhibition of TH1 cells and induction of TH2 cells with greater efficacy compared to the same dose of GSNO against the clinical disease of EAE. It is of interest that both exogenous GSNO as well as endogenous GSNO by inhibition of GSNOR with N6022 induce inhibition of TH17 and expression of IL-10 in the spinal cord derived T cells, one of the potential mechanism for immunomodulation and CNS protection ,  and thus neuroprotective efficacy against EAE. Overall, these studies document that cellular GSNO homeostasis plays a critical role in differentiation and effector function of proinflammatory (TH1 and TH17) and anti-inflammatory (TH2 and Treg) CD4+ T cells. Moreover, this study also reports that GSNOR inhibitor (N6022) is a novel therapeutic approach for targetting NO metabolome in NOS/GSNOR expressing cell types for selective modulation of CD4+ subsets (TH1/TH17 vs. TH2/Treg) and thus greater efficacy against autoimmune disease of EAE as compared to treatment with exogenous GSNO. Secondly, the observed increased expression of IL-10 in the CNS of EAE animals treated with GSNO as well as N6022 indicates that these drugs may potentially promote neurorepair in EAE and MS.
Acknowledgments We acknowledge Ms. Joyce Bryan for her help in procurement of animals and supplies. This work was supported in part by U.S. Department of Veterans Affairs (BX002829) and National Institutes of Health (NS037766).