DIFFERENTIAL UPTAKE OF APOPTOTIC MATERIAL DURING PSEUDOMONAS AERUGINOSA INFECTION

TRIBULATTI, MARIA VIRGINIA; ARIAS, PAULA*; KIERBEL, ARLINET; PEPE, MARÍA VICTORIA

Mendoza

Congreso; SAIB - 58 Annual Meeting; 2022

Macrophages are cells specialized in the destruction of pathogens through phagocytosis, but they also play a role in the elimination of dead cells and cellular debris through efferocytosis. Since infection sites are usually full of pathogens and dead cells, macrophages encounter both stimuli simultaneously. However, depending on the perceived stimuli, macrophages are thought to acquire either a pathogen-killing or an efferocytic/healing phenotype, previously considered to be mutually exclusive. Besides, we have previously found that the bacterial pathogen Pseudomonas aeruginosa adheres to apoptotic cells. Therefore, we aimed to study whether pathogens and apoptotic cells can be internalized and processed jointly by macrophages and the modulation of efferocytosis during inflammation. We exposed primary bone marrow-derived macrophages (BMDM) to P. aeruginosa, apoptotic cells, and P. aeruginosa-laden apoptotic cells, to study the response of macrophages under different stimuli. To achieve this, we measured phagocytic and efferocytic efficiencies by confocal microscopy image analysis, and changes in cytokine expression levels by RT-PCR. We found that BMDMs are very efficient in engulfing both P. aeruginosa and apoptotic cells, with a high bactericidal capacity not affected by the concomitant presence of apoptotic material. Furthermore, after phagocytizing and processing P. aeruginosa, through IL-6 production, macrophages increase their efferocytic capacity. Thus, our results show that the inflammatory response generated by bacterial processing enhances the ability of these macrophages to control inflammation. We further described that, when macrophages are exposed to apoptotic cells, they engulf mainly intact cells that subsequently fragment within the phagocyte. In contrast, apoptotic cells loaded with bacteria are internalized into multiple fragments, preceding the formation of multiple efferosomes. We also found that apoptotic material and bacteria localize into separate LAMP-1 positive vesicles. Since macrophages produce an immune response only after phagocytosis, we hypothesize that a differential processing or trafficking of cargo must occur. Regulation of vesicle formation, trafficking, and maturation, involves the Rab family of small GTPases. Therefore, we plan to study the role of Rab GTPases in the sorting and trafficking of this differential cargo and the biological implications this may have.