Caspase-11–mediated endothelial pyroptosis underlies endotoxemia-induced lung injury
Kwong Tai Cheng, Shiqin Xiong, Zhiming Ye, Zhigang Hong, Anke Di, Kit Man Tsang, Xiaopei Gao, Shejuan An, Manish Mittal, Stephen M. Vogel, Edward A. Miao, Jalees Rehman, Asrar B. Malik
Kwong Tai Cheng, Shiqin Xiong, Zhiming Ye, Zhigang Hong, Anke Di, Kit Man Tsang, Xiaopei Gao, Shejuan An, Manish Mittal, Stephen M. Vogel, Edward A. Miao, Jalees Rehman, Asrar B. Malik
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Research Article Pulmonology Vascular biology

Caspase-11–mediated endothelial pyroptosis underlies endotoxemia-induced lung injury

Abstract

Acute lung injury is a leading cause of death in bacterial sepsis due to the wholesale destruction of the lung endothelial barrier, which results in protein-rich lung edema, influx of proinflammatory leukocytes, and intractable hypoxemia. Pyroptosis is a form of programmed lytic cell death that is triggered by inflammatory caspases, but little is known about its role in EC death and acute lung injury. Here, we show that systemic exposure to the bacterial endotoxin lipopolysaccharide (LPS) causes severe endothelial pyroptosis that is mediated by the inflammatory caspases, human caspases 4/5 in human ECs, or the murine homolog caspase-11 in mice in vivo. In caspase-11–deficient mice, BM transplantation with WT hematopoietic cells did not abrogate endotoxemia-induced acute lung injury, indicating a central role for nonhematopoietic caspase-11 in endotoxemia. Additionally, conditional deletion of caspase-11 in ECs reduced endotoxemia-induced lung edema, neutrophil accumulation, and death. These results establish the requisite role of endothelial pyroptosis in endotoxemic tissue injury and suggest that endothelial inflammatory caspases are an important therapeutic target for acute lung injury.

Authors

Kwong Tai Cheng, Shiqin Xiong, Zhiming Ye, Zhigang Hong, Anke Di, Kit Man Tsang, Xiaopei Gao, Shejuan An, Manish Mittal, Stephen M. Vogel, Edward A. Miao, Jalees Rehman, Asrar B. Malik

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Figure 5

Pyroptotic ECs generate MP in caspase-11–dependent manner.

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Pyroptotic ECs generate MP in caspase-11–dependent manner. (A and B) Flo...
(A and B) Flow cytometry showing size calibration and gate definition of MPs using fluorescent polystyrene bead standards of various sizes. MPs were gated by size gating (<2 μm) correlated with object area in the bright field channel and intensity of the dark field side scatter (SSC). (C) Pyroptotic ECs released CD31+ MPs. Supernatants were collected from either control hMVECs only incubated (I) with LPS or primed cells transfected with LPS (2 μg/ml) for 16 hours. Samples were then analyzed using imaging flow cytometer. (D) Representative images of MPs from control (gray), LPS incubation (magenta), and LPS transduction (T) (blue). (E) Pyroptotic ECs displayed increase in total MP counts as well as enhanced CD31+ expression on MPs. Representative flow cytometry dot blots (F) and quantification (G) of MPs measured in the plasma of WT, Casp11–/–, Casp11fl/fl, and Casp11EC–/–mice 6 hours after LPS challenge (40 mg/kg i.p.). Endothelial-specific deletion of caspase-11 prevented the generation of endothelial MPs similarly to global caspase-11 deletion, thus demonstrating that EC MP release was due to the activation of endothelial caspase-11. (H) Micrograph of MPs from mouse plasma, visualized by imaging flow cytometry, showing MPs of endothelial (CD31+CD41–) and platelet (CD31+CD41+) origins. Assessment of endothelial-derived MPs from plasma of healthy volunteers (control) and ARDS patients (described in Supplemental Table 1) in a representative flow cytometry dot plot (I) and a bar graph quantification (J) shows significant increases in endothelial MP release, consistent with endothelial pyroptosis during ALI/ARDS in patients. ** P < 0.01; *** P < 0.001 by ANOVA (E and G) and 2-tailed Student’s t test (J).