IADR Abstract Archives
Porphyromonas gingivalis skews efferocytic uptake of apoptotic neutrophils
Objectives: Efferocytosis is a process by which macrophages phagocytose apoptotic cells. Recognition of specific ligands displayed on apoptotic cell membranes by macrophage efferocytic receptors is essential for efficient uptake. We recently showed that neutrophils intracellularly process complement protein C3 into iC3b during physiological apoptosis. Recognition of iC3b on apoptotic neutrophils (ANs) by macrophage complement receptor 3 (CR3) positively regulates efferocytic uptake of ANs, and activates signaling pathways essential for digestion of ingested ANs and other resolution pathways. Bacterial proteases can dysregulate efferocytosis by cleaving efferocytic ligands and receptors, leading to bacterial persistence and chronic inflammation. We determined whether gingipains, produced by the periodontal pathogen Porphyromonas gingivalis, dysregulated iC3b mediated efferocytic pathways.
Methods: iC3b expression in ANs and gingipain treated ANs (gANs) was measured by flow cytometry, confocal microscopy, and uptake determined by efferocyosis assays. Cell surface expression of efferocytic ligands, and receptors was determined by flow cytometry. Macrophage reprogramming on AN or gAN uptake was assessed by qRT-PCR and multiplex-cytokine arrays.
Results: We found that gingipain treatment did not alter the expression of phosphotidylserine or the ‘don’t-eat me receptors CD31 and CD47, but significantly upregulated iC3b expression. gANs were phagocytosed at significantly higher rates compared to ANs and blocked LPS induced proinflammatory cytokine production by macrophages.
Conclusions: Our data show that P. gingivaliscan manipulate efferocytosis by enhancing expression of iC3b in apoptotic neutrophils. Enhanced uptake blunted proinflammatory cytokine production, and skewed macrophages towards an M2 phenotype. Ongoing studies will determine whether gingipain mediated conversion of C3 to iC3b occurs within specific endocytic compartments within neutrophils or extracellularly at the plasma membrane. While the role of C3 in complement cascade is well described, we are only beginning to understand its role in immune regulation. Thus, we propose that P. gingivalisexploits host efferocytic pathways to dampen macrophage inflammatory responses.
Methods: iC3b expression in ANs and gingipain treated ANs (gANs) was measured by flow cytometry, confocal microscopy, and uptake determined by efferocyosis assays. Cell surface expression of efferocytic ligands, and receptors was determined by flow cytometry. Macrophage reprogramming on AN or gAN uptake was assessed by qRT-PCR and multiplex-cytokine arrays.
Results: We found that gingipain treatment did not alter the expression of phosphotidylserine or the ‘don’t-eat me receptors CD31 and CD47, but significantly upregulated iC3b expression. gANs were phagocytosed at significantly higher rates compared to ANs and blocked LPS induced proinflammatory cytokine production by macrophages.
Conclusions: Our data show that P. gingivaliscan manipulate efferocytosis by enhancing expression of iC3b in apoptotic neutrophils. Enhanced uptake blunted proinflammatory cytokine production, and skewed macrophages towards an M2 phenotype. Ongoing studies will determine whether gingipain mediated conversion of C3 to iC3b occurs within specific endocytic compartments within neutrophils or extracellularly at the plasma membrane. While the role of C3 in complement cascade is well described, we are only beginning to understand its role in immune regulation. Thus, we propose that P. gingivalisexploits host efferocytic pathways to dampen macrophage inflammatory responses.