Sex-specific NLRP3 activation in neutrophils promotes neutrophil recruitment and NETosis in the murine model of diffuse alveolar hemorrhage

Abstract

Objectives: Diffuse alveolar hemorrhage (DAH) is a life-threatening complication of systemic lupus erythematosus and small vessel vasculitis. We previously showed that neutrophil extracellular traps (NETs) were associated with the pathogenesis of pristane-induced DAH and demonstrated that neutrophil NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome assembly participated in NET generation under sterile stimulation. We investigated whether NLRP3 inflammasome assembly in neutrophils may drive pulmonary NETosis in a mouse model of pristane-induced DAH.

Methods: C57BL/6J mice received a single intraperitoneal injection of 0.5mL of pristane. Neutrophil NLRP3 inflammasome assembly and NETs were characterized by immunofluorescence staining of apoptosis-associated speck-like protein a CARD (ASC), co-staining of DNA, and citrullinated histones, respectively. Clinical status of mice was assessed 11 days after pristane injection by measurement of arterial oxygen saturation and of weight loss; severity of lung injury was determined using a quantification score from hematoxylin-eosin-stained slides.

Results: Pristane induced ASC speck formation in neutrophils and we confirmed that NLRP3 inflammasome was involved in NET generation after pristane stimulation in vitro. NLRP3 deficiency reduced the severity of pristane-induced DAH in female, but not male mice. Interestingly, NLRP3 deficiency reduced the number of neutrophils and NETs in the lungs of females compared to males.

Conclusions: Our results suggest a link between female sex-specific NLRP3 inflammasome activation and subsequent pulmonary NETosis in the development of pristane-induced DAH. Therefore, we identified NLRP3 inflammasome as a potential new therapeutic target in this severe complication of pro-female autoimmune disease for which specific inhibitors of NLRP3 are currently developed.

Keywords: NLRP3 inflammasome; diffuse alveolar hemorrhage; murine model; neutrophil extracellular traps; sexspecific.