Background/Purpose: Systemic lupus erythematosus (SLE) is characterized by glutathione depletion and oxidative stress in T cells which lead to abnormal lineage development and dysfunction. In turn, treatment with N-acetylcysteine (NAC), which is an amino acid precursor of glutathione, reversed T cell dysfunction, reduced anti-DNA production, and disease activity in a double-blind randomized placebo-controlled clinical trial of SLE patients. Nephritis in Chronic graft versus host disease (cGvHD) has been used as a mouse model of lupus. However,  NAC worsened GvHD in a recent clinical trial. Given the nuanced role of oxidative stress in the immune system, we examined the impact of transaldolase (TAL) deficiency, which causes glutathione depletion, and its reversal by NAC on cGVHD.

Methods: Age and gender matched C57BL/6 wild-type (TAL+/+) and TAL-deficient (TAL-/-) mice were fed with drinking water without (TAL+/+ n=18; TAL-/- n=17) or with 10 g/L NAC (TAL+/+ n=8; TAL-/- n=5). 10⁸ MHC-mismatched bm12 splenocytes were injected intraperitoneally. Urine and serum were collected at baseline and every 4 weeks until study completion at 23 weeks. Anti-nuclear antibodies (ANA) and proteinuria were measured and expressed relative to day 0, preceding injection of bm12 cells, and standardized at 1.0 for each mouse. CD3, CD19, CD4, CD8, FoxP3, CD25, Helios, mTOR, and glutathione were measured by flow cytometry of splenocytes. Severity of glomerulonephritis was blindly scored by an experienced renal pathologist. Statistical analysis was done by t-tests or ANOVA using GraphPad 5.0 software; with p<0.05 considered significant.

Results: Proteinuria was significantly increased in TAL+/+ mice (5.19 ±3.12) relative to TAL-/- mice (0.86 ±0.06; p<0.01) at 23-weeks. NAC further increased proteinuria in TAL+/+ mice (10.91 ± 3.45; p<0.05) but not in TAL-/- mice. By 12 weeks, TAL+/+ mice on normal water (1.81 ±0.22 ; p=0.02) or NAC  (7.54 ±3.25; p=0.01) as well as TAL-/- mice on NAC  had significantly increased ANA production relative to baseline (4.07 ± 0.22; p=0.009). In contrast, TAL-/- mice on normal water showed no increase in ANA during the study. Kidney pathology revealed reduced glomerulosclerosis in TAL-/- mice (0.3125 ± 0.1197) relative to TAL+/+ mice (0.7778 ± 0.1726; p=0.04). GSH levels were reduced in both T and B cells of TAL-/- mice, while NAC treatment enhanced GSH in both cell types of TAL+/+ and TAL-/- mice.  Flow cytometry revealed an expansion of CD8 T cells in TAL-/- mice on normal water (21.6 ±1.3%) relative to TAL+/+ mice on normal (15.2 ±1.0%; p=0.0003) and NAC-containing water (14.4 ± 2.0%; p=0.009). CD4⁺FoxP3⁺Helios⁺ Tregs were depleted from 10.7% in TAL+/+ mice to 5.1% in TAL-/- mice (p=0.001). NAC expanded Tregs both in TAL+/+ and TAL-/- mice. B cells were depleted in TAL-/- mice on normal water (48.2 ± 1.9%) relative to all other groups (TAL+/+ mice on normal water: 58.3 ± 1.6%, p=0.0003; TAL+/+ mice on NAC: 60.6 ± 3.0%, p=0.003; TAL-/- mice on NAC: 60.7 ± 4.4%, p=0.045).

Conclusion: Oxidative stress and Treg depletion protect against ANA production and nephritis via the expansion of CD8 T cells and contraction of B cells in cGvHD.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/oxidative-stress-protects-against-nephritis-induced-by-chronic-graft-versus-host-disease/