CLEC5A is critical in Pseudomonas aeruginosa–induced NET formation and acute lung injury
Pei-Shan Sung, … , Cheng-Hsun Chiu, Shie-Liang Hsieh
Pei-Shan Sung, … , Cheng-Hsun Chiu, Shie-Liang Hsieh
Published September 1, 2022
Citation Information: JCI Insight. 2022;7(18):e156613. https://doi.org/10.1172/jci.insight.156613.
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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 3

P. aeruginosa upregulates CRAMP to promote NET formation via CLEC5A in vivo.

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P. aeruginosa upregulates CRAMP to promote NET formation via CLEC5A in ...
(A) WT and Clec5a–/– mice were intratracheally inoculated with median lethal dose (LD50) of P. aeruginosa (Xen41, PAO1 strain), and BALF was harvested at 15 hours after infection to detect Ly6G+ cells (mock: WT, n = 6; Clec5a–/–, n = 3; WT + DNase 1, n = 4; Xen41LD50: WT, n = 7; Clec5a–/–, n = 6; WT + DNase 1, n = 8). (B) To detect NET formation, lung tissue was collected at 24 hours after infection and fixed in 10% formamide and embedded in paraffin. Tissue sections were stained with antibodies to myeloperoxidase (MPO) (green), citrullinated histone H3 (Cit-H3) (red), and Hoechst 33342 (blue) for NET structure visualization. Scale bar: 200 μm. (C and D) The areas of MPO (C) and MPO/Cit-H3 colocalization (D) were analyzed using MetaMorph software (n = 6 for each group). (E) Expression levels of Cramp mRNA in tissues were determined by qPCR (mock: WT, n = 4; Clec5a–/–, n = 3; Xen41LD50: WT, n = 4; Clec5a–/–, n = 4). (F) CRAMP protein in BALF was measured by ELISA (mock: WT, n = 5; Clec5a–/–, n = 3; Xen41LD50: WT, n = 8; Clec5a–/–, n = 7) (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (2-way ANOVA). (G) Neutrophils from WT and Clec5a–/– mice were stimulated with CRAMP (50 μg/mL) at 37°C for 3 hours (mock: WT, n = 3; Clec5a–/–, n = 3; Xen41LD50: WT, n = 5; Clec5a–/–, n = 5). The level of NET formation was calculated from the area (μm2) of histone overlapping with MPO using MetaMorph software. Data are mean ± SEM from at least 3 independent experiments. The statistical significance was calculated with 2-way ANOVA for A and C and by unpaired and nonparametric Student’s t test with Mann-Whitney U test for D–G. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.