Elimination of Pseudomonas aeruginosa through Efferocytosis upon Binding to Apoptotic Cells
Fig 5
Expression of dominant negative Rac1 and preincubation with unlabeled Annexin V inhibit P. aeruginosa internalization.
(A) Proportion of internalized apoptotic material (Intracellular/Total cell-associated apoptotic material). (B) Proportion of internalized P. aeruginosa (Internalized/Total cell-associated bacteria). (A and B) Apoptotic cells generated by UV irradiation were stained with Annexin V-Alexa 647 and added to MDCK-Rac1-N17 filter-grown monolayers and infected with PAK-GFP. The cell-associated apoptotic material and cell-associated bacteria were quantified by image analysis as described in M&M. Dox+ (control) Dox- (dominant negative Rac1). Data were normalized to control. (C) Proportion of internalized apoptotic material after pre-incubating CellTrace-labeled apoptotic cells with unlabeled Annexin V or with binding buffer alone (control) and adding them to transwell-grown lifeact-GFP MDCK monolayers. (D) Proportion of internalized P. aeruginosa after pre-incubating transwell-grown lifeact-GFP MDCK monolayers with unlabeled Annexin V for 15 min in binding buffer. (E) Transwell-grown MDCK monolayers were infected with P. aeruginosa and internalization was measured by standard antibiotic protection assays. When indicated, apoptotic cells from overgrown cultures, pre-incubated with binding buffer alone or with unlabeled-Annexin V, were added to monolayers right before infection. The mean of colony forming units (CFUs) ± SEM was calculated. Data were normalized to control. *p<0.05 vs. control, one-sample t-test.