Mitoxantrone Induces ROS-Dependent Elimination of Dysfunctional Mitochondria Via Mitophagy to Regulate the Survival of Intracellular Mycobacteria in dTHP-1 cells

Abstract

Host-directed therapy is regarded as an innovative anti-tuberculosis approach aimed at tackling the tuberculosis burden by inducing autophagy by various agents to inhibit the proliferation of intracellular pathogen. For the treatment of drug-resistant tuberculosis, autophagy serves as a crucial defense mechanism employed by the body to eliminate the bug. This resilient and dangerous bacterium may be effectively countered by pharmacological induction of autophagy. This discovery represents intriguing therapeutic alternatives. This work investigates the anti-mycobacterial properties of the anthracene-dione compound Mitoxantrone (MTX) through the activation of autophagy in differentiated THP-1 cells. We checked the anti-mycobacterial effect of MTX by conducting CFU assay and the expression of major autophagy genes in MTX treated and infected samples by western blotting. To understand the mechanism behind autophagy induction we checked mitochondrial ROS by flow cytometry and extracellular ATP release by fluorescence. MTX induced fission of dysfunctional mitochondria was observed via confocal microscopy through co-localization studies. MTX augmented mitophagy was revealed through mt-Mkeima plasmid transfection followed by microscopic visualization and also by checking mitophagy markers through qRT-PCR and western blotting analysis. The non-cytotoxic dose of MTX reduced the intracellular viability of mycobacteria compared to the control cells, and inhibition of autophagy reversed the effect of MTX on intracellular bacterial burden. Through multi-parametric approaches, our investigation established the effect of MTX on mitochondria, the principal source of endogenous reactive oxygen species (ROS), acting as essential signal transducers that promote autophagy. Further, we have demonstrated that MTX decreased ATP production, which caused disruption of mitochondrial membrane proteins and increased mitochondrial ROS generation, resulting in mitochondrial fission and accelerating the initiation of mitophagy, leading to the elimination of intracellular mycobacteria. Our findings collectively indicated that MTX-induced mitochondrial dysfunction initiated an interaction between two specific autophagic responses, reducing mycobacterial infection and facilitating its clearance.