Targeting host mitochondrial respiration: artemisinin derivative potentiates macrophage bacterial clearance by up-regulating antibacterial autophagy via immunometabolic mechanism

To investigate the effect and mechanism of a novel artemisinin derivative, disuccinate artemisinin (DA), in enhancing bacterial clearance by upregulating antibacterial autophagy in macrophages through targeting host mitochondrial respiration.

The experiment utilized E. colistrains ATCC 35218 and FITC-labeled K12 to infect the mouse macrophage cell line RAW264.7. Phagocytosis of bacteria by macrophages under different time points and doses was assessed via fluorescence imaging to determine the optimal drug dosage and time point. The impact of the drug on antibacterial autophagy in macrophages was evaluated using colony counting, Western blotting and immunofluorescence co-localization assay. Autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 were employed to examine the effect of autophagy suppression on drug efficacy through colony counting and immunofluorescence co-localization. Glucose deprivation was induced using low-glucose medium, and its influence on drug action was observed via fluorescent bacterial counting and immunofluorescence co-localization. Glycolytic intervention was investigated using the glycolytic activator oligomycin A and inhibitor 2-deoxy-D-glucose (2-DG), with effects monitored through fluorescent bacterial counting and immunofluorescence co-localization. Mitochondrial respiration was modulated with the activator carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) and inhibitor rotenone, and its impact on drug action was assessed via fluorescent bacterial counting and immunofluorescence co-localization. The effect of the drug on mitochondrial respiration in macrophages was determined by measuring the oxygen consumption rate (OCR) using cellular energy metabolism assays.

DA concentration-dependently enhanced macrophage phagocytosis and clearance of both drug-resistant ATCC35218 and sensitive K12 E. coli (P<0.05). Immunofluorescence and immunoblotting demonstrated DA upregulated antibacterial autophagy (P<0.01), remaining effective when autophagy initiation was blocked (P<0.01) but losing efficacy upon autophagic flux inhibition (P<0.01). Under glucose deprivation, DA retained its autophagy-enhancing effect (P<0.01). Both activation and inhibition of glycolysis limited DA’s efficacy (P<0.01). Maximizing mitochondrial respiration with FCCP abolished DA’s pro-phagocytic and autophagy- enhancing effects (P<0.01), whereas respiratory blockade with rotenone preserved them (P<0.01). Metabolic analysis showed DA restored infection-impaired maximal mitochondrial respiration, spare respiratory capacity, and ATP production (P<0.01).

DA enhances bacterial clearance by upregulating antibacterial autophagy through restoration of mitochondrial respiration in infected macrophages, representing a host-directed anti-infective candidate targeting mitochondrial metabolism.