Glutathione Redox Metabolomic Dysregulation Precedes TNF-α Elevation and Predicts Severity of Gvhd in Experimental Transplantation

Graft-versus-host disease (GVHD) is characterized by cytokine and chemokine dysregulation that predicts and also contributes to disease severity. Intermediates in sulfur amino acid (SAA) metabolism are known to modulate cytokines implicated in GVHD, such as TNF-a and IL-2. In this first report, plasma and hepatic SAAs and other amino acid metabolite concentrations were sequentially profiled using redox metabolomics assay, in experimental allogeneic bone marrow transplantation (Allo-BMT) models and compared to syngeneic (Syn) BMT. At day +4, prior to increase in plasma TNF-a and subsequent GVHD histopathological changes, a significant decline in both plasma and hepatic glutathione (GSH) concentrations occurred resulting in a more oxidized redox potential in Allo BMT (Balb/C® B6), relative to Syn BMT (B6Thy1.1® B6) mice. The plasma free GSH concentrations in allogeneic mice were significantly lower (~65%; p<0.005) when compared to Syn BMT controls and were accompanied by ~ 3 fold increase (p=0.012) in plasma GSSG. Paradoxically, the plasma concentration of total cysteine, which is the rate-limiting substrate for GSH synthesis, was significantly higher in Allo BMT versus Syn BMT mice at day +10 suggesting that loss in GSH was not due to substrate limitation. Significant difference in the GSSG/GSH ratio was also noted in Allo / GVHD+ compared to Allo/GVHD- in un-irradiated paternal to F1 hybrid transplantation model (B6 ® B6D2F1), indicating that GSH depletion was a metabolic event associated with GVHD, and not a consequence of conditioning regimen (0.3150 ± 0.06007 vs 17.00 ± 4.907, respectively, p=0.0145). At day+10, metabolomic profile was able to segregate Allo/GVHD+ from Allo/GVHD- based on hepatic total GSH, free GSH and total cystinylglycine concentration. The plasma TNF-a elevation lagged behind redox changes with no significant difference detected in Syn-BMT and Allo-BMT group at day +4. Hepatic GCLC mRNA abundance, regulated by Nrf2 transcription factor, was decreased by ~50% (P<0.0001) at day +4 and further declined ~80% by Day +10 (P<0.0004), in Allo BMT compared to Syn BMT. Interestingly, hepatic nuclear Nrf2 and TNF-a concentrations at Day +10 were inversely correlated (r²=0.8, p=0.01) in Allo-BMT. Modulation of GSH in mixed lymphocyte culture using GSH depleting agents, buthionine sulfoximine and diethyl maleate, increased TNF-a expression in CD4 T cells compared to untreated controls (p<0.001). These experiments show the utility of redox metabolomics to identify novel putative biomarkers in GVHD.

*Equal contribution