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

Abstract

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.

Quorum-Sensing Signaling Molecule 2-Aminoacetophenone Mediates the Persistence of Pseudomonas aeruginosa in Macrophages by Interference with Autophagy through Epigenetic Regulation of Lipid Biosynthesis

“…Loss of the protease-activated receptor 2 (PAR2) impairs the clearance of this pathogen in alveolar macrophages. PAR2 promotes P. aeruginosa clearance by inducing cAMP levels, showing that the cAMP-Rac1 signaling cascade activates the phagocytosis of this pathogen in alveolar macrophages ( 53 ). The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARg) also comes into play in P. aeruginosa clearance in mice lungs as it induces paraoxonase, an enzyme involved in hydrolysis and degradation of the QS homoserine lactones ( 54 ).…”

RGS2 is an innate immune checkpoint for TLR4 and Gαq-mediated IFNγ generation and lung injury

“…Femur and tibia were flushed with RPMI-1640 media (Gibco), containing 25 ng/ml M-CSF, 10% FBS and 1% antibiotic/anti-mycotic as described previously (Joshi et al, 2020; Rayees et al, 2022; Rayees et al, 2019; Rochford et al, 2021). On 5 th -day, bone marrow-derived macrophages were incubated in 0.1% FBS containing RPMI for one hour before adding LPS (1.0 µg/ml) with 1% FBS containing media.…”

“…Lipopolysaccharide, LPS, a cell wall component of all gram-negative bacteria, activates AM innate response through pattern recognition receptors such as toll-like receptors 4 (TLR4) and co-receptor CD14 (Joshi et al, 2020; Lu et al, 2015; Rayees et al, 2022; Rayees et al, 2020). Combining LPS with IFNγ boosted the massive release of proinflammatory and cytotoxic factors such as TNFα, IL6, and nitric oxide (NO) (Saleh et al, 2021).…”

Endothelial ERG programs neutrophil transcriptome for sustained anti-inflammatory vascular niche

“…Human lungs from de-identified autopsied patients dying of ARDS or non-lung related diseases were obtained from UIC biorepository. The sections were then immunostained for anti-ERG, anti-Myeloperoxidase, anti-Ly6G antibody and DAPI, followed by immunofluorescent imaging using Zeiss LSM 880 confocal laser scanning microscope as described earlier [58] Cell Culture Human pulmonary endothelial cells (HPAECs) were cultured in a 0.1% gelatin-coated flask using EBM TM -2 Endothelial Cell Growth Basal Medium-2 supplemented with growth factors (Lonza # 00190860) and 15% Fetal Bovine Serum (FBS) (Thermo Fisher, # A5256701) [56,57]. HL60 cells (Human promyelocytic leukemia cells) were grown in suspension with RPMI-1640 medium containing L-glutamine and 25 mM HEPES (Thermo Fisher #72400047), supplemented with 15% FBS (Thermo Fisher #A4766801) and penicillin-streptomycinamphotericin B (Thermo Fisher #15240096), at 37°C, 5% CO2.…”

“…HPAECs were transfected with either empty vector or human A20 wild-type or mutated promoter luciferase reporter followed by stimulation with LPS (1 µg/ml). The Gaussia luciferase (GLuc) and secreted alkaline phosphatase (SEAP) activity were determined as described previously [58]. GLuc activity was normalized taking SEAP activity as an internal control.…”