CLEC5A is critical in Pseudomonas aeruginosa–induced NET formation and acute lung injury
Pei-Shan Sung, Yu-Chun Peng, Shao-Ping Yang, Cheng-Hsun Chiu, Shie-Liang Hsieh
Pei-Shan Sung, Yu-Chun Peng, Shao-Ping Yang, Cheng-Hsun Chiu, Shie-Liang Hsieh
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Research Article Infectious disease Inflammation

CLEC5A is critical in Pseudomonas aeruginosa–induced NET formation and acute lung injury

Abstract

Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and it frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa–induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with LPS of P. aeruginosa and is responsible for P. aeruginosa–induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1–dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa–induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa–induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa–induced pneumonia.

Authors

Pei-Shan Sung, Yu-Chun Peng, Shao-Ping Yang, Cheng-Hsun Chiu, Shie-Liang Hsieh

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Figure 2

P. aeruginosa induces cytokine production and caspase-1–dependent GSDMD cleavage in BM-derived macrophages.

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P. aeruginosa induces cytokine production and caspase-1–dependent GSDMD...
(A and B) BMDMs from WT (n = 6), Clec5a–/– (n = 6), Tlr2–/– (n = 4), and Tlr4–/– (n = 4) mice were stimulated with LPS from E.coli, Klebsiella pneumoniae (K.p), and PAO1 (100 ng/mL) (A), or they were incubated with live or UV-killed P. aeruginosa PAO1 strain (MOI = 3 or 10) (B) at 37°C for 1 hour. After washing, with serum-free RPMI containing 10 μg/mL gentamicin, cells were cultured in fresh RPMI containing 10% (v/v) FBS for 24 hours at 37°C. The levels of cytokine were measured by ELISA. Data were collected from 6 independent experiments. (C) BMDMs from WT mice were primed with Pam3CsK4 (1 μg/mL) for 4 hours at 37°C, followed by incubation with UV-killed or live P. aeruginosa PAO1 strain (MOI = 3 or 10) or transfected (T) with LPS from E. coli (2.5 μg/mL) for 15 hours at 37°C. Western blots were incubated with anti-GSDMD Ab, anti–caspase-11 (CASP11) Ab, anti–caspase-1 (CASP1) Ab, anti–caspase-3 (CASP3) Ab, or anti-GAPDH Ab. (D and E) BMDMs from WT and Clec5a–/– mice were incubated with live P. aeruginosa PAO1 strain (MOI = 10) for 15 hours at 37°C. Western blots of cell lysates were probed with anti-GSDMD Ab, anti–caspase-1 Ab, or anti-GAPDH Ab. Cleaved caspase-1 (cleaved CASP1) and cleaved GSDMD are shown as fold change compared with WT. LPS (T), transfection of E. coli LPS. Representative immunoblot of 4 independent experiments. Data are mean ± SEM, and statistical analysis for A and B were performed with 2-way ANOVA and by unpaired and nonparametric Student’s t test with Mann-Whitney U test for E. *P < 0.05, **P < 0.01, ***P < 0.001.