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Updated: Aug 8, 2025
Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli
Published on: November 4, 2022
Neutrophil Extracellular Traps Induce Alveolar Macrophage Pyroptosis by Regulating NLRP3 Deubiquitination,
Yamei Cui1, Ying Yang1, Wenqiang Tao1
1Department of Critical Care Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, People's Republic of China.
Background:
Uncontrolled inflammation is a typical feature of sepsis-related lung injury. The key event in the progression of lung injury is Caspase-1-dependent alveolar macrophage (AM) pyroptosis. Similarly, neutrophils are stimulated to release neutrophil extracellular traps (NETs) to participate in the innate immune response. This study aims to illustrate the specific mechanisms by which NETs activate AM at the post-translational level and maintain lung inflammation.
Methods:
We established a septic lung injury model by caecal ligation and puncture. We found elevated NETs and interleukin-1b (IL-1β) levels in the lung tissues of septic mice. Western blot and immunofluorescence analyses was utilized to determine whether NETs promote AM pyroptosis and whether degrading NETs or targeting the NLRP3 inflammasome had protective effects on AM pyroptosis and lung injury. Flow cytometric and co-immunoprecipitation analyses verified intracellular reactive oxygen species (ROS) levels and the binding of NLRP3 and ubiquitin (UB) molecules, respectively.
Results:
Increased NETs production and IL-1β release in septic mice were correlated with the degree of lung injury. NETs upregulated the level of NLRP3, followed by NLRP3 inflammasome assembly and caspase-1 activation, leading to AM pyroptosis executed by the activated fragment of full-length gasdermin D (FH-GSDMD). However, the opposite effect was observed in the context of NETs degradation. Furthermore, NETs markedly elicited an increase in ROS, which facilitated the activation of NLRP3 deubiquitination and the subsequent pyroptosis pathway in AM. Removal of ROS could promote the binding of NLRP3 and ubiquitin, inhibit NLRP3 binding to apoptosis-associated spotted proteins (ASC) and further alleviate the inflammatory changes in the lungs.
Conclusion:
In summary, these findings indicate that NETs prime ROS generation, which promotes NLRP3 inflammasome activation at the post-translational level to mediate AM pyroptosis and sustain lung injury in septic mice.
Insights
Neutrophil extracellular traps (NETs) drive sepsis-induced lung injury by activating alveolar macrophage (AM) pyroptosis. NETs promote reactive oxygen species (ROS) generation, leading to NLRP3 inflammasome activation and sustained inflammation.
Area of Science:
- Immunology
- Cell Biology
- Pathology
Background:
- Sepsis-induced lung injury features uncontrolled inflammation.
- Caspase-1-dependent alveolar macrophage (AM) pyroptosis is a key event in sepsis-related lung injury.
- Neutrophil extracellular traps (NETs) are involved in the innate immune response during sepsis.
Purpose of the Study:
- To elucidate the post-translational mechanisms by which NETs activate AM.
- To understand how NETs contribute to sustained lung inflammation in sepsis.
- To investigate the role of NETs in pyroptosis and lung injury progression.
Main Methods:
- Established a murine model of septic lung injury via cecal ligation and puncture.
- Utilized Western blot, immunofluorescence, and flow cytometry to analyze NETs, IL-1β, ROS, and inflammasome components.
- Performed co-immunoprecipitation to assess NLRP3 and ubiquitin binding.
Main Results:
- Elevated NETs and IL-1β levels correlated with lung injury severity in septic mice.
- NETs promoted NLRP3 inflammasome assembly and caspase-1 activation, leading to AM pyroptosis.
- NETs-induced reactive oxygen species (ROS) facilitated NLRP3 deubiquitination, enhancing pyroptosis and lung inflammation.
Conclusions:
- NETs initiate ROS generation, which drives NLRP3 inflammasome activation at the post-translational level.
- This NETs-ROS-NLRP3 axis mediates AM pyroptosis, sustaining lung injury in sepsis.
- Targeting NETs or ROS may offer therapeutic strategies for sepsis-related lung injury.
