Pierre-André Jarrot ^1,2 Jiyoun Kim ^1,2 William Chan ^1,2 Lukas Heger ^1,2,3 Nicolas Schommer ^1,2 Pierre Cunin ⁴ Camila M S Silva ^1,2 Stephane Robert ⁵ Peter A Nigrovic ⁴ Bruce Ewenstein ^1,2 Denisa Wagner ^1,2*

• ¹ Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, United States
• ² Departments of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States
• ³ Department of Cardiology and Angiology I, Heart Center, University of Freiburg, Freiburg, Germany
• ⁴ Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States
• ⁵ Centre de recherche en CardioVasculaire et Nutrition, Faculté des Sciences Médicales et Paramédicales, Aix-Marseille Université, Marseille, Provence-Alpes-Côte d'Azur, France

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.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 apoptosisassociated 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.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.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.