DataSheet1_Protease-activated receptor 2 promotes clearance of Pseudomonas aeruginosa infection by inducing cAMP-Rac1 signaling in alveolar macrophages.pdf

Efficient phagocytosis of pathogens by the innate immune system during infectious injury is vital for restoring tissue integrity. Impaired phagocytosis, such as in the case of infection with Pseudomonas aeruginosa, a broad-spectrum antibiotic-resistant Gram-negative bacterium, can lead to a life threatening lung disorder, acute lung injury (ALI). Evidence indicates that loss of protease-activated receptor 2 (PAR2) impaired Pseudomonas aeruginosa clearance leading to non-resolvable ALI, but the mechanism remains unclear. Here, we focused on the alveolar macrophages (AMs), the predominant population of lung-resident macrophages involved in sensing bacteria, to understand their role in PAR2-mediated phagocytosis of Pseudomonas aeruginosa. We found that upon binding Pseudomonas aeruginosa, PAR2-expressing but not PAR2-null AMs had increased cAMP levels, which activated Rac1 through protein kinase A. Activated Rac1 increased actin-rich protrusions to augment the phagocytosis of Pseudomonas aeruginosa. Administration of liposomes containing constitutively active Rac1 into PAR2-null mice lungs rescued phagocytosis and enhanced the survival of PAR2-null mice from pneumonia. These studies showed that PAR2 drives the cAMP-Rac1 signaling cascade that activates Pseudomonas aeruginosa phagocytosis in AMs, thereby preventing death from bacterial pneumonia.</p

ROS generation in U937 cells upon treatment with pLLD (150 μg/ml).

<p>Flow cytometric analysis of ROS by DCFDA showed the degrees of scavenging effect in time dependent manner (A). Evaluation of mitochondrial membrane potential using JC-1 showed time dependent changes (B). Assessment of activation of caspases 9 and 3 with inhibitors and of DNA fragmentation by pLLD was done in a time dependent analysis by ELISA (C, D, E and F, respectively). Expression of pro- and anti-apoptotic molecules was assessed by western blot (G). Analysis of cytochrome C activation upon pLLD treatment was performed by confocal microscopy (H). The data are reported as the mean ± SEM of triplicate experiments (*P>0.05; ** P>0.01; *** P>0.001).</p

TLC profile of pLLD (A). Growth inhibitory effect of pLLD on different cancer cells including U937, K562, HL-60 and MOLT-4 at different concentrations (0, 25, 50, 150, 200 μg/ml) at 24 h; viability was measured by MTT assay (B). The data are represented as mean ± SEM from triplicate independent experiments. (*P>0.05; ** P>0.01) Viability of U937 cells was measured in time dependent manner by flow cytometry after treatment with pLLD at the concentration 150 μg/ml (C). Morphological and nuclear changes observed in U937 cells after treatment of 150 μg/ml pLLD for 24 h (D). Morphological changes were seen under light microscope, and nuclear changes (Right to left) under fluorescence microscope after DAPI and A.O./EtBr staining respectively.

<p>TLC profile of pLLD (A). Growth inhibitory effect of pLLD on different cancer cells including U937, K562, HL-60 and MOLT-4 at different concentrations (0, 25, 50, 150, 200 μg/ml) at 24 h; viability was measured by MTT assay (B). The data are represented as mean ± SEM from triplicate independent experiments. (*P>0.05; ** P>0.01) Viability of U937 cells was measured in time dependent manner by flow cytometry after treatment with pLLD at the concentration 150 μg/ml (C). Morphological and nuclear changes observed in U937 cells after treatment of 150 μg/ml pLLD for 24 h (D). Morphological changes were seen under light microscope, and nuclear changes (Right to left) under fluorescence microscope after DAPI and A.O./EtBr staining respectively.</p

Analysis of apoptosis and cell cycle arrest in U937 cells by flow cytometry.

<p>Cells were treated with 150 μg/ml of pLLD and binding of Annexin-V/FITC to phosphatidyl serine was measured by flow cytometry to determine the percentages of apoptotic cells in time dependent manner (A). Study of cell cycle arrest in U937 cells was carried out by propidium iodide staining. Percentage of G0/G1 cell population increases after treatment of 150 μg/ml of pLLD in time dependent manner (B). The data are represented as mean ± SEM from triplicate independent experiments (*P>0.05; ** P>0.01).</p

The effect of pLLD on MAPK pathway in U937 cells.

<p>Representative western blot results show phospho-ERK1/2/ERK1/2, phospho-JNK/JNK and phospho-p38/p38 expressions in U937 cells after incubation with pLLD (A). AP-1 expression in U937 cells 24 h after treatment of pLLD, evaluated by confocal microscopy (B) (*P>0.05; ** P>0.01; *** P>0.001).</p