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.
View: Text | PDF
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

×

Figure 6

CLEC5A is a potential therapeutic target in P. aeruginosa–induced cell infiltration and lethality.

Options: View larger image (or click on image) Download as PowerPoint
CLEC5A is a potential therapeutic target in P. aeruginosa–induced cell i...
WT mice were intratracheally injected with a sublethal dose (LD90) of P. aeruginosa Xen41 (1 × 106 PFU) and injected with isotype control (mIgG1, 100 μg per mouse, i.v.), anti-mCLEC5A mAb (clone 4A12D5, 100 μg per mouse, i.v.), ciprofloxacin (Cip, 4 mg/kg, i.p.), or anti-mCLEC5A mAb (i.v.) combined with ciprofloxacin (i.p.) at 15 hours after infection. Ciprofloxacin was further administered every 12 hours. (A) Survival rate was monitored up to 120 hours after infection. (B) Lung tissues were collected, and formalin-fixed paraffin-embedded sections were prepared. Cellular infiltration was visualized by H&E staining. Scale bar: 200 m. (C) Tissue sections were stained with anti-CD64 Ab (green), anti-CD11b Ab (red), anti–Siglec-F Ab (yellow), and anti-F4/80 Ab (light blue); fluorescence images were captured using a Leica confocal microscope with white light laser system (TCS-SP8-MP-SMD) and analyzed using Leica Application Suite X software. Scale bar: 200 μm. (D and E) The cell population was analyzed with MetaMorph software to determine the percentages of interstitial macrophages (interstitial MФ, CD64+CD11b+Siglec-F–F4/80+), alveolar macrophages (AMФ, CD64+CD11b–Siglec-F+F4/80+), eosinophils (Eos, CD64–CD11b+Siglec-F+F4/80+), and neutrophils/DCs (PMN/DC, CD11b+Siglec-F–F4/80–CD64–) (ciprofloxacin, n = 11; ciprofloxacin + anti-mCLEC5A mAb, n = 10, from 4 independent experiments) (F) Lung cell suspensions were incubated with BV650-conjugated anti-Ly6G Ab, BUV805-conjugated anti-CD45 Ab, BUV395-conjugated anti-CD11b Ab, BV480-conjugated anti-CD11c Ab, BV605-conjugated anti-CD64 Ab, PE-Cy5-conjugated anti-CD25 Ab, and BUV496-conjugated anti-IA/IE Ab to analyze the percentage of neutrophil (PMN), alveolar macrophage (AMФ), interstitial macrophage (IMФ), and eosinophils after P. aeruginosa inoculation (ciprofloxacin, n = 4; ciprofloxacin + anti-mCLEC5A mAb, n = 4, from 3 independent experiments). Survival rate was assessed using the log-rank (Mantel-Cox) test with GraphPad Prism software. ***P < 0.005. Statistical analysis for D and E was calculated with unpaired and nonparametric Student’s t test with Mann-Whitney U test. *P < 0.05.