Eradication of HCV by direct antiviral agents restores mitochondrial function and energy homeostasis in peripheral blood mononuclear cells

Abstract

Hepatitis C virus (HCV) adopts several immune evasion mechanisms such as interfering with innate immunity or promoting T-cell exhaustion. However, the recent direct-antiviral agents (DAAs) rapidly eliminate the virus, and the repercussions in terms of immune system balance are unknown. Here we compared the PBMCs transcriptomic profile of patients with HCV chronic infection at baseline (T0) and 12 weeks after the end of the therapy (SVR12) with DAAs. 3862 genes were differently modulated, identifying oxidative phosphorylation as the top canonical pathway differentially activated. Therefore, we dissected PBMCs bioenergetic profile by analyzing mitochondrial respiration and glycolysis at 4 timepoints: T0, 4 weeks of therapy, end of therapy (EoT), and SVR12. Maximal and reserve respiratory capacity considerably increased at EoT, persisting until SVR12. Notably, over time a significant increase was observed in respiratory chain (RC) complexes protein levels and the enzymatic activity of complexes I, II, and IV. Mitochondrial-DNA integrity improved over time, and the expression of mitochondrial biogenesis key regulators such as TFAM, Nrf-1, and PPARGC1A significantly increased at SVR12; hence, RC complexes synthesis and mitochondrial respiration were supported after treatment. HCV clearance with DAAS profoundly changed PBMCs bioenergetic profile, suggesting the immunometabolism study as a new approach to the understanding of viral immune evasion mechanisms and host adaptations during infections and therapies.

Metabolic reprogramming in inflammatory microglia indicates a potential way of targeting inflammation in Alzheimer's disease

“…Equal amounts of RNA were reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. Real-time PCR was performed, using Sso Advanced universal SYBR green supermix on a Bio-Rad CFX96 Real-Time system as previously reported [ 13 , 28 ]. The cycle threshold (Ct) was determined and the relative gene expression was calculated with the ΔΔCT method.…”

“…The mitochondria-enriched fractions from cell suspension were prepared as previously reported [ 13 , [28] , [29] , [30] ] with minor adjustments. Briefly, cells were washed with ice-cold phosphate-buffered saline and centrifuged at 3500 rpm for 6 min at 4 °C.…”

“…Complex I enzymatic activity was measured spectrophotometrically at 600 nm as previously reported [ 13 , 28 , 29 ]. Briefly, 40 μg of each enzyme solution was mixed with complex I buffer (25 mM/L potassium phosphate, 3.5 g/L fatty acid free BSA), 60 μM/L 2,6-Dichlorophenolindophenol (DCPIP), 70 μM/L decylubiquinone (DBH), 1.0 μM/L antimycin-A) and incubated for 3 min at 37 °C.…”

“…Complex II enzymatic activity was measured spectrophotometrically at 600 nm as previously reported [ 13 , 28 , 31 ]. Briefly, 40 μg of each cell suspension was incubated with complex II buffer (25 mM KH 2 PO4 (pH 7.8), 2 mM EDTA, 1 mg/mL fatty acid free BSA), 10 mM succinate, 50uM DBH, 1 mM KCN, 4 μM rotenone, and 10 μM antimycin A, 0.2 mM ATP) for 10 min.…”

“…It is conventionally known that activated M1 macrophages rely on glycolytic metabolism to supply the pro-inflammatory activity, while oxidative phosphorylation (OXPHOS) would represent the main energy source of resting and regulatory phenotypes (e.g. M2 macrophages or Treg lymphocytes) [ [13] , [14] , [15] ]. Since the TLR4-mediated activation of microglia reflects an important player in AD, we investigated the role of microglia immunometabolism by dissecting cellular bioenergetics in LPS-activated human microglia, and studying potential applications in a well-established murine model of AD.…”