Glutathionylation of Pyruvate Dehydrogenase Complex E2 and Inflammatory Cytokine Production During Acute Inflammation Are Magnified By Mitochondrial Oxidative Stress

Abstract

ABSTRACTLipopolysaccharide (LPS) is a known inducer of inflammatory signaling which triggers generation of reactive oxygen species (ROS) and cell death in responsive cells like THP-1 promonocytes and freshly isolated human monocytes. A key LPS-responsive metabolic pivot point is the 9 megadalton mitochondrial pyruvate dehydrogenase complex (PDC), which provides pyruvate dehydrogenase (E1), lipoamide-linked transacetylase (E2) and lipoamide dehydrogenase (E3) activities to produce acetyl-CoA from pyruvate. While phosphorylation-dependent decreases in PDC activity following LPS treatment or sepsis have been deeply investigated, redox-linked processes have received less attention. Data presented here demonstrate that LPS-induced reversible oxidation within PDC occurs in PDCE2 in both THP-1 cells and primary human monocytes. Knockout of PDCE2 by CRISPR and expression of FLAG-tagged PDCE2 in THP-1 cells demonstrated that LPS-induced glutathionylation is associated with wild type PDCE2 but not mutant protein lacking the lipoamide-linking lysine residues. Moreover, the mitochondrially-targeted electrophile MitoCDNB, which impairs both glutathione-and thioredoxin-based reductase systems, elevates ROS similar to LPS but does not cause PDCE2 glutathionylation. However, LPS and MitoCDNB together are highly synergistic for PDCE2 glutathionylation, ROS production, and cell death. Surprisingly, the two treatments together had differential effects on cytokine production; pro-inflammatory IL-1β production was enhanced by the co-treatment, while IL-10, an important anti-inflammatory cytokine, dropped precipitously compared to LPS treatment alone. This new information may expand opportunities to understand and modulate PDC redox status and activity and improve the outcomes of pathological inflammation.HighlightsPDCE2 is glutathionylated (-SSG) during acute inflammation in monocytesLipopolysaccharide-induced PDCE2-SSG occurs in THP1 cells and human monocytes.Lipoamide-deficient PDCE2 lowers LPS-induced PDCE2-SSG and ROS production.MitoCDNB leads to ROS production without PDCE2-SSG, but is synergistic with LPSMitoCDNB causes the LPS-stimulated cytokine profile to become more proinflammatoryGraphical Abstract