Abstract
Presentation Description :
Rationale: In marked contrast to macrophages, neutrophils do not undergo inflammatory pyroptotic cell death following activation of the NLRP3 inflammasome even though Gasdermin D (GSDMD) is cleaved (Karmakar, Pearlman et al, Nat Comm 2020). Macrophages also undergo pyroptosis following activation of the NLRC4 inflammasome by P. aeruginosa. We examined the role of NLRP3 and NLRC4 in IL-1β secretion and pyroptosis by neutrophils compared with macrophages infected with Pseudomonas aeruginosa strain PAO1 that expresses the Type III secretion system (T3SS) effectors ExoS and ExoT (strain PAO1). We also examined the role of NLRP3 and NLRC4 in P. aeruginosa keratitis.
Methods: For in vitro studies, bone marrow neutrophils and BM derived macrophages from WT C57BL/6 and gene knockout strains were infected with P. aeruginosa strain PAO1 or T3SS mutants and IL-1β secretion and pyroptosis (LDH release) were quantified. The role for NLRP3 vs. NLRC4 was assessed in a murine model of P. aeruginosa keratitis using either inflammasome gene knockout mice or the NLRP3 inhibitor MCC950.
Results: In infected macrophages and neutrophils, IL-1β secretion was dependent on the needle structural proteins and GSDMD; however, only macrophages exhibited and pyroptotic cell death even though GSDMD was cleaved. IL-1β secretion in macrophages was completely dependent on NLRC4, whereas in ExoS ADPRT expressing PAO1, IL-1β secretion was dependent on NLRP3. In vivo, bacterial (PAO1) killing was impaired in NLRP3-/- mice, in MCC950 treated C57BL/6, and in caspase-1-/-, but not in NLRC4-/- mice.
Conclusions: We identified an unexpected role for ExoS ADPRT in mediating NLRP3 rather than NLRC4 in neutrophils, though not macrophages. We also identified NLRP3 as a critical mediator of P. aeruginosa keratitis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.