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.

Figure 1

Pristane-induced NLRP3 inflammasome activation promotes NET formation in vitro (A) Representative IF images of ASC speck formation in isolated murine WT neutrophils, WT neutrophils + MCC950 and NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine or with β-cyclodextrin-pristane (BCD pristane): staining of DNA (blue) and ASC (green). White arrows show cells with ASC speck. Stimulation of neutrophils with ionomycine was a positive control for inflammasome formation. Bar 20µm. (B) Quantification of ASC speck formation in WT neutrophils, WT neutrophils + MCC950 and NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine, with BCD or with BCD pristane. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.01^**, p < 0.001^***). (C) Representative IF images of NETs (colocalization of DNA and extracellular citrullinated histones (H3-Cit) in WT neutrophils, WT neutrophils + MCC950 and NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine or BCD pristane: staining of DNA (blue) and H3-Cit (green). Bar 75µm. (D) Quantification of NETs released in WT neutrophils, WT neutrophils + MCC950 and NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine, with BCD or with BCD pristane. NETs were identified as neutrophils releasing DNA fibers with H3-Cit co-staining. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.001^***) (E) Quantification of ASC speck formation in neutrophils obtained from male or female mice as indicated in the figure: WT neutrophils, WT neutrophils + MCC950 and NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine, with BCD or with BCD pristane. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.01^** p < 0.001^***). (F) Quantification of NETs released in neutrophils obtained from male or female mice as indicated in the figure: WT neutrophils, WT neutrophils + MCC950 or NLRP3-/- neutrophils either unstimulated or stimulated with 4µM of ionomycine, with BCD or with BCD pristane. NETs were identified as neutrophils releasing DNA fibers with H3-Cit co-staining. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.001^***).

Figure 2

NLRP3 deficiency reduces severity of pristane-induced DAH in female, but not in male mice (A–C) Left column shows results with male mice (♂), right with female mice (♀). (A) Prevalence of pristane-induced DAH in WT and NLRP3 -/- mice, Mann Whitney test was used for statistical analysis to compare the two groups (ns: non-significant) (B) Oxygen-saturated hemoglobin level (%) in mice with pristane-induced DAH WT and NLRP3 -/- mice. WT and NLRP3-/- mice challenged with PBS IP injection were used as negative control. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.05^*, < 0.01^**) (C) DAH score from pristane-induced DAH WT and NLRP3 -/- mice. Results represent median ± interquartile range, Mann Whitney test was used for statistical analysis to compare the two groups (ns: non-significant, p < 0.05^*). Each experiment had n=3-6 mice/PBS group and n=7-10 mice/pristane group.

Figure 3

NLRP3 deficiency reduces severity of pristane-induced DAH in female, but not in male mice, representative macroscopic and microscopic views of lungs. (A, B) Left column shows results with male mice (♂), right with female mice (♀). (A) Macroscopic view of the left lung lobe from PBS group and pristane-induced DAH group. (B) Representative H&E-stained section of left lung lobe from pristane-induced DAH WT and NLRP3 -/- mice. Bar 100µm. WT and NLRP3-/- mice challenged with PBS IP injection served as negative control.

Figure 4

NLRP3 deficiency reduces number of neutrophils and amount of NETs in lungs of pristane-induced DAH female, but not in male mice (A–C) Left column shows results with male mice (♂), right with female mice (♀). (A) Flow-cytometry analysis of BAL from pristane-induced DAH WT and NLRP3 -/- male and female mice. WT and NLRP3 -/- males and females challenged with PBS IP injection were used as negative control. All cells were CD45+, neutrophils were identified as Ly6C+/Ly6G+, macrophages were identified as Ly6C+/Ly6G-. Quantification of neutrophils in BAL from pristane-induced DAH WT and NLRP3 -/- male. WT and NLRP3 -/- male challenged with PBS IP injection were used as negative control. Results represent median +/- interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p < 0.05^*, p < 0.01^**). (B) Lung tissue cryosections were obtained from pristane-induced DAH WT and NLRP3 -/- mice. WT and NLRP3 -/- mice challenged with PBS IP injection were a negative control: cryosections were immunostained for DNA (blue), Ly6G (white) and H3Cit (green). Bar 25µm. (C) Quantification of neutrophils in lungs from pristane-induced DAH WT and NLRP3 -/- mice. WT and NLRP3 -/- mice challenged with PBS IP injection were a negative control. Results represent median ± interquartile range, ANOVA multiple comparison test was used for statistical analysis between groups (ns: non-significant, p< 0.001^***). Quantification of H3Cit in lungs from pristane-induced DAH WT and NLRP3 -/- mice. WT and NLRP3 -/- mice challenged with PBS IP injection were a negative control. Results represent median ± interquartile range, ANOVA multiple comparison test was applied for statistical analysis between groups (ns: non-significant, p< 0.01^**, < 0.001^***). Each experiment had n=3-6 mice/PBS group and n=7-10 mice/pristane group.