B cell-mediated immunomodulation by S-nitrosoglutathione (GSNO) in experimental autoimmune encephalomyelitis

Background Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model for human multiple sclerosis (MS), a demyelinating autoimmune disease mediated by T and B lymphocytes. The aim of the present study was to investigate the role of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in regulation of effector or regulatory B cell function as IL-6 and IL-10 expressions and thus the potential role of GSNO in targeting B cell-mediated immunopathogenesis in MS using EAE model. Methods To this purpose, the in vivo EAE mouse model, generated by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, or in vitro model of cultured B cells stimulated with lipopolysaccharide or anti-IgM antibody were treated with exogenous GSNO or N6022, an inhibitor of GSNO reductase (GSNOR; GSNO degrading enzyme) to increase endogenous GSNO, and then analyzed for B cell specic IL-6 and IL-10 expression.

GSNO/N6022 treatments increased the number of IL-10+ B cells but decreased the number of IL-6+ B cells in the CNS and spleen. Accordingly, GSNO/N6022 treatments increased the expression of IL-10 while reducing the IL-6 expression in the blood. Similar observations were also made in in vitro B cell culture model where GSNO treatment increased the IL-10+ B cells but decreased the IL-6+ B cells under BCR or TLR4 stimulatory conditions and under CD40 and BAFF co-stimulatory conditions. Accordingly, GSNO treatment increased the B cell production of IL-10 but reduced the IL-6 production under both stimulatory and co-stimulatory conditions. In vitro stimulation and co-stimulation of cultured naïve B cells increased two major distinct B cell populations; CD1dlow CD5high and CD1dhigh CD5high. In both populations, GSNO treatment increased the number of IL-10+ cells but decreased the IL-6+ cells.
Conclusion These data document, for the rst time, that cellular GSNO homeostasis is a critical target for the regulation of IL-10+ B cells vs. IL-6+ B cells mediated immune balance under auto-immune disease conditions.

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However, the latest manuscript can be downloaded and accessed as a PDF. Figure 1 Effect of GSNOR inhibitor N6022 on EAE disease. C57BL/6 mice were immunized with MOG35-55 for the induction of EAE. EAE mice received daily doses of saline or N6022 (1 mg/kg/day/ip) starting before the onset of the disease (day 9 post-immunization). Daily clinical scores (A-i), area under each curve as a measure of quantitative clinical disease (A-ii), daily body weight changes (B-i), and statistic analysis of body weight changes at the day 21 postimmunization (B-ii) of control mice, EAE mice (treated with saline), and EAE mice treated with N6022 were analyzed. Data are expressed as mean ± standard deviation (SD). ** p ≤ 0.01, *** p ≤ 0.001 vs. control mice and + p ≤ 0.05, ++ p ≤ 0.01 vs. saline-treated EAE mice. At day 21 postimmunization, spinal lumbar cords were analyzed for mononuclear cell in ltration (H&E staining; C-i) and immunostaining for B220 (B cell marker; C-ii). AMF=anterior median ssure, GM=grey matter, SAS=subarachnoid space, WM=white matter. The n's represent the number of animals in each group.

Figure 2
Effect of N6022 treatment on the B cell subsets expressing IL-10 and IL-6 in the CNS and spleen of EAE animals. C57BL/6 mice were immunized with MOG35-55 and receive daily doses of saline or N6022 (1 mg/kg/day/ip) starting on day 9 post-immunization. On the day 21 post-immunization, mononuclear and splenic cells were isolated from the CNS (A) and spleens (C) and the number of B220+ total B cells (A-i and C-i), IL-10+ B220+ B cells (A-ii and C-ii), IL-6+ B220+ B cells (A-iii and C-iii), the ratio of IL-10+ vs. IL-6+ B cells (A-iv and C-iv), and IL-10 and IL6 double-positive B220+ B cells (A-v and C-v) were analyzed by uorescence ow cytometric analysis. The blood was collected on the same day and analyzed for blood levels of IL-10 (B-i) and IL-6 (B-ii). Data are expressed as mean ± standard deviation (SD); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control mice; + p ≤ 0.05, ++ p ≤ 0.01 vs. saline-treated EAE mice. N.S.=not signi cant. The n's represent the number of samples in each group.  Effect of GSNO treatment on the B cell subsets expressing IL-10 and IL-6 under in vitro culture conditions. Naïve B cells were puri ed from C57BL/6 mice and stimulated with anti-IgM mAb (@IgM) or lipopolysaccharide (LPS) and co-stimulated (CS) with anti-CD40 mAb and BAFF as indicated and incubated for 48 hrs. One hour before the stimulation/co-stimulation, the cells were treated with vehicle or GSNO (50 μM).  Effect of GSNO treatment on the B cell production of IL-10 and IL-6 under in vitro culture conditions. Naïve B cells were puri ed from C57BL/6 mice and stimulated with anti-IgM mAb (@IgM) or lipopolysaccharide (LPS) and co-stimulated (CS) with anti-CD40 mAb and BAFF as indicated and incubated for 48 hrs. One hour before the stimulation/co-stimulation, the cells were treated with vehicle or GSNO (50 μM). Following the incubation, B cell production of IL-10 (A-i and B-i) and IL-6 (A-ii and B-ii) was analyzed by ELISA using the culture media. Data are expressed as mean ± standard deviation (SD); ** p ≤ 0.01, *** p ≤ 0.001 vs. control; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs. as indicated. N.S.=not signi cant. The n's represent the number of samples in each group. The cell culture experiments performed once using three different samples.

Figure 6
Effect of GSNO treatment on the expansion of CD1dhigh CD5high and CD1dlow CD5high B cell subsets under in vitro culture conditions stimulated with anti-IgM mAb. Naïve B cells were puri ed from C57BL/6 mice and stimulated with anti-IgM mAb (@IgM) and costimulated (CS) with anti-CD40 mAb and BAFF as indicated and incubated for 48 hrs. One hour before the stimulation/co-stimulation, the cells were treated with vehicle or GSNO (50 μM). Following the incubation, B cells were stained with antibodies speci c to B220, CD1d, and CD5 and followed by uorescence ow cytometric analysis. Following the gating of B220+ cells, distribution of CD1dlow CD5low, CD1dhigh CD5high, and CD1dlow CD5high B cells was represented by two-parametric scatter plots (A). In addition, percent of CD1dlow CD5low (B-i), CD1dhigh CD5high (Bii), and CD1dlow CD5high (B-iii) B cell numbers were represented by a bar graph. Data are expressed as mean ± standard deviation (SD); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control; +++ p ≤ 0.001 vs. as indicated. N.S.=not signi cant. The relative proportion of CD1dlow CD5low, CD1dhigh CD5high, and CD1dlow CD5high B cells was represented by a stacked bar graph (C). The n's represent the number of samples in each group. The cell culture experiments performed once using three different samples.

Figure 7
Effect of GSNO treatment on the expansion of CD1dhigh CD5high and CD1dlow CD5high B cell subsets under in vitro culture conditions stimulated with LPS. Naïve B cells were puri ed from C57BL/6 mice and stimulated with lipopolysaccharide (LPS) and co-stimulated (CS) with anti-CD40 mAb and BAFF as indicated and incubated for 48 hrs. One hour before the stimulation/co-stimulation, the cells were treated with vehicle or GSNO (50 μM). Following the incubation, B cells were stained with antibodies speci c to B220, CD1d, and CD5 and followed by uorescence ow cytometric analysis. Following the gating of B220+ cells, distribution of CD1dlow CD5low, CD1dhigh CD5high, and CD1dlow CD5high B cells was represented by two-parametric scatter plots (A). In addition, percent of CD1dlow CD5low (B-i), CD1dhigh CD5high (B-ii), and CD1dlow CD5high (B-iii) B cell numbers were represented by a bar graph. Data are expressed as mean ± standard deviation (SD); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control; +++ p ≤ 0.001 vs. as indicated. N.S.=not signi cant. The relative proportion of CD1dlow CD5low, CD1dhigh CD5high, and CD1dlow CD5high B cells was represented by a stacked bar graph (C). The n's represent the number of samples in each group. The cell culture experiments performed once using three different samples.

Figure 8
Effect of GSNO treatment on the expression of IL-10 and IL-6 by CD1dhigh CD5high and CD1dlow CD5high B cell subsets under in vitro culture conditions. Naïve B cells were puri ed from C57BL/6 mice and stimulated with anti-IgM mAb (@IgM) (A and C) or lipopolysaccharide (LPS) (B and D) and costimulated (CS) with anti-CD40 mAb and BAFF as indicated and incubated for 48 hrs. One hour before the stimulation/co-stimulation, the cells were treated with vehicle or GSNO (50 μM). Following the incubation, B cells were stained with antibodies speci c to B220, CD1d, CD5, IL-10, and IL-6 and followed by uorescence ow cytometric analysis. Following the serial gating of B220 and then CD1d/CD5, the proportion of IL10+ CD1dhigh CD5high (A-i and B-i), IL-6+ CD1dhigh CD5high (A-ii and B-ii), IL-10+ IL-6+ CD1dhigh CD5high (A-iii and B-iii), IL-10+ CD1dlow CD5high (C-i and D-i), IL-6+ CD1dlow CD5high (C-ii and D-ii), and IL-10+ IL-6+ CD1dlow CD5high (C-iii and D-iii) were represented bar graphs. In addition, their relative proportions were also represented by a stacked bar graph (A-iv, B-iv, C-iv, and D-iv). Data are expressed as mean ± standard deviation (SD); * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs. as indicated. N.S.=not signi cant. The n's represent the number of samples in each group. The cell culture experiments performed once using three different samples.