Mitoxantrone Elicits the Crosstalk Between ROS-Dependent Elimination of Dysfunctional Mitochondria and Xenophagy Responses to Eliminate Intracellular Mycobacteria in Human Macrophages

Abstract

The rise of drug-resistant Mycobacterium tuberculosis has emerged as a global health emergency. Host-directed therapies that elevates innate immune responses offer a viable alternative. In this study, we investigated the potential of Mitoxantrone (MTX) as a host modulating agent with anti-mycobacterial activity. The cell viability assay confirmed that 1 µM MTX is non-toxic to dTHP-1 cells. Time kinetics experiments identified 12hrs treatment period as optimal for eliciting autophagic responses. Pharmacological blockade of autophagy using 3-MA significantly reduced MTX-induced autophagy, while inhibition of lysosomal degradation with bafilomycin-A1 resulted in LC3-II accumulation, confirming that MTX enhances functional autophagic flux rather than causing autophagosome arrest. In macrophages infected with mycobacteria, MTX treatment markedly increased autophagic activity and significantly limited intracellular bacterial proliferation. Mechanistic investigations revealed that MTX disrupts mitochondrial homeostasis by reducing cellular ATP production, altering mitochondrial membrane protein integrity, and elevating mitochondrial reactive oxygen species levels. These mitochondrial disturbances promoted phosphorylation of Drp1, leading to enhanced mitochondrial fragmentation. Parallelly, MTX accelerated the selective removal of damaged mitochondria through mitophagy. The induction of mitophagy was closely associated with improved intracellular clearance of mycobacteria. Collectively, our findings demonstrate that MTX exerts anti-mycobacterial effects by inducing mitochondrial dysfunction that activates coordinated autophagic and mitophagic pathways. These host mediated responses restrict mycobacterial survival and promote pathogen elimination, supporting the potential repositioning of MTX as a HDT candidate for tuberculosis management.