Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD(+) ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30µM EFV and submaximal effects at 50µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD(+) ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical.

Adult T-cell leukemia/lymphoma (ATL) cells express TNF receptor type-2 (TNFR2) on their surface and shed its soluble form (sTNFR2). We previously reported that sTNFR2 levels were highly elevated in the plasma of patients with acute ATL. To investigate whether its quantitation would be helpful for the diagnosis or prediction of the onset of acute ATL, we examined the plasma levels of sTNFR2 in a large number of specimens obtained from a cohort of ATL patients and asymptomatic human T-cell leukemia virus type 1 (HTLV-1) carriers (ACs) and compared them to those of other candidate ATL biomarkers (sCD25, sOX40, and IL-10) by enzyme-linked immunosorbent assays (ELISA) and HTLV-1 proviral loads. We observed that sTNFR2 levels were significantly elevated in acute ATL patients compared to ACs and patients with other types of ATL (chronic, smoldering, and lymphoma). Importantly, sTNFR2 levels were significantly correlated with those of sCD25, sOX40, and IL-10, as well as proviral loads. Thus, the present study confirmed that an increase in plasma sTNFR2 levels is a biomarker for the diagnosis of acute ATL. Examination of plasma sTNFR2 alone or in combination with other ATL biomarkers may be helpful for the diagnosis of acute ATL.

Since forkhead box P3 (FOXP3) is a hallmark of regulatory T (Treg) cells, the expansion of FOXP3⁺ adult T-cell leukemia/lymphoma (ATL) cells is believed to contribute to immune suppression and the pathogenesis of ATL. However, the mechanisms underlying the expansion of FOXP3⁺ ATL cells remain unclear. OX40, a co-stimulatory molecule, is expressed in ATL cells, and OX40 signaling has been shown to promote the differentiation and proliferation of Treg cells in mouse models. To investigate the mechanisms driving the expansion of FOXP3⁺ ATL cells, we examined the expression of OX40 and its ligand, OX40L. Our findings revealed that OX40 expression was elevated in patients with ATL and with a high frequency of FOXP3⁺ ATL cells. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from patients with acute ATL cultured for 18 h demonstrated that FOXP3⁻ and FOXP3⁺ cells predominantly expressed OX40L and OX40, respectively. Furthermore, small interfering RNA-mediated FOXP3 knockdown in HTLV-1-infected cell lines increased OX40L expression. These results suggest that interactions between FOXP3⁻ OX40L⁺ cells and FOXP3⁺ OX40⁺ cells may promote the proliferation of FOXP3⁺ ATL cells.