[The mechanism by which NLRP3 inflammasome activation promotes the formation of neutrophil extracellular traps and affects the prognosis of sepsis].

OBJECTIVE: To explore the intrinsic relationship between NOD-like receptor protein 3 (NLRP3) inflammasome activation and neutrophil extracellular traps (NETs) formation, as well as their mechanism of action in the pathological process of sepsis.

METHODS: Wild-type (WT) and NLRP3 knockout (NLRP3-/-) mice were randomly divided into sham operation (Sham) group and cecal ligation and puncture (CLP) group, namely WT-Sham group, NLRP3-/--Sham group, WT-CLP group and NLRP3-/--CLP group, with 6 mice in each group. At 24 hours after operation, hematoxylin-eosin (HE) staining was used to observe histopathological changes in the liver and lung tissues; enzyme linked immunosorbent assay (ELISA) was performed to detect the levels of blood cytokines, citrullinated histone H3 (Cit-H3), and myeloperoxidase-DNA (MPO-DNA) complexes; Western blotting was used to measure the protein expressions of peptidylarginine deiminase 4 (PAD4) and Cit-H3 in lung tissue. Meanwhile, colony counts in peripheral blood and peritoneal lavage fluid (PLF) were determined in each group. The 7-day survival of mice in each group was observed postoperatively.

RESULTS: Compared with the corresponding Sham group, the survival rate of WT and NLRP3-/- mice was decreased after CLP, while the serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) were increased, pathological injury in the liver and lung tissues was aggravated, bacterial burden in peripheral blood and PLF was elevated, protein expressions of PAD4 and Cit-H3 in lung tissue were up-regulated, and serum levels of Cit-H3 and MPO-DNA complexes were increased (all P<0.05). Compared with the WT-CLP group, the NLRP3-/--CLP group showed a lower 7-day survival rate (16.7% vs. 55.6%, Log-rank test: χ 2=4.35, P<0.05), the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were decreased [IL-1β (ng/L): 136.30±56.16 vs. 243.50±55.29, IL-6 (ng/L): 411.30±114.50 vs. 669.70±222.60, TNF-α (ng/L): 672.80±16.43 vs. 717.30±30.16], whereas the anti-inflammatory cytokine IL-10 was increased (ng/L: 1 111.00±223.20 vs. 833.70±154.90), pathological injury in the liver and lung tissues was alleviated (liver injury score: 2.50±0.55 vs. 3.67±0.52, lung injury score: 2.33±0.52 vs. 3.67±0.52), bacterial clearance was markedly improved [peripheral blood colony count (×105 CFU/L): 12.16±7.31 vs. 36.00±13.80, PLF colony count (×107 CFU/L): 113.33±79.69 vs. 277.00±77.23], protein expressions of PAD4 and Cit-H3 in lung tissue were reduced (PAD4/β-actin: 0.17±0.05 vs. 0.29±0.05, Cit-H3/β-actin: 1.36±0.23 vs. 1.96±0.26), and serum levels of Cit-H3 and MPO-DNA complexes were also decreased [Cit-H3 (ng/L): 132.80±20.68 vs. 182.70±13.43, MPO-DNA (ng/L): 47.67±34.96 vs. 177.20±43.95], all differences were statistically significant (all P<0.05).

CONCLUSIONS: The NLRP3 inflammasome may participate in inflammatory amplification and organ injury by positively regulating NET formation, thereby influencing the course and outcome of sepsis. NLRP3 deficiency can suppress aberrant NET formation, alleviate inflammatory responses and organ damage, enhance bacterial clearance, and thus improve the prognosis of septic mice, suggesting that the NLRP3-NET axis may serve as a potential intervention target for sepsis.