THE INFLAMMATORY RESPONSE INDUCED BY Pseudomonas aeruginosa IN MACROPHAGES ENHACES APOPTOTIC CELL REMOVAL

PAULA ARIAS; ADRIANA JAGER; MARIA VIRGINIA TRIBULATTI; MARCELA BROCCO ; MARÍA VICTORIA PEPE; ARLINET KIERBEL

virtual

Congreso; Congreso Conjunto SAIB-SAMIGE 2021; 2021

SAIB SAMIGE

Macrophages are the first line of defense against pathogens (phagocytosis) and they respond efficiently to tissue injury by removing dead cells and cellular debris (efferocytosis). According to the stimuli perceived, macrophages are thought to acquire either a microbicidal/pathogen killing phenotype or an efferocytic/healing one, which were classically considered mutually exclusive. However, macrophages functions have shown to be more complex than that. We aimed to understand the modulation of macrophage efferocytosis during an inflammatory stimulus. In order to investigate the phagocytic and efferocytic efficiencies, we exposed primary bone marrow-derived macrophages (BMDM) to apoptotic cells, bacteria and bacteria-laden apoptotic cells and examined their internalization (independently or in conjuction) by confocal microscopy and subsequent image analysis. To study bacterial clearance, we measured intracellular survival over time through a standard internalization assay. Also, we measured changes in cytokine expression levels by real-time RT-PCR. In addition, we assessed the effect of the cytokine IL-6 on macrophages efferocytic capacity by pre-stimulating them with recombinant IL-6. Next, we analyzed the contribution of this cytokine to the efferocytic capabilities using a specific antibody to neutralize the potential effect after bacterial stimuli of the macrophage. We found that BMDM are very efficient in engulfing both the bacterial pathogen Pseudomonas aeruginosa and apoptotic cells. Also, BMDM showed a high bactericidal capacity unaffected by the concomitant presence of apoptotic material. We further showed that, after phagocyting and processing P. aeruginosa, macrophages highly increase their efferocytic capacity without affecting their phagocytic function. Moreover, we demonstrated IL-6 mediates this increased efferocytic capacity of bacterial-stimulated BMDM. Our results show that the inflammatory response generated by the bacterial processing, enhances these macrophages capacity to control inflammation through an increased efferocytosis. This means plasticity in macrophage programming, in response to changing environmental cues, may modulate this effect. In this scenario, macrophages reconcile two opposing functions: clearing the pathogen and repairing the damage suffered by the host tissue. Indeed, macrophages can be the initiators of the inflammatory response and participate in its resolution in a second step, through the regulation of their own profile. We are currently analyzing efferocytosis using a pH-sensitive probe, which emits fluorescence at low pH. We are interested in comparing two different methods to measure efferocytosis: microscopy and flow cytometry.