Alpha-linolenic acid pretreatment alleviates NETs-induced alveolar macrophage pyroptosis by inhibiting pyrin inflammasome activation in a mouse model of sepsis-induced ALI/ARDS

Background Neutrophil extracellular traps (NETs) can cause acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) by inducing macrophage pyroptosis. The purpose of this study was to find out whether pretreatment of alpha-linolenic acid (ALA) could inhibit NETs-induced macrophage pyroptosis in sepsis-induced ALI/ARDS, as well as to identify which inflammasome is involved in this process. Methods LPS was instilled into the trachea to establish sepsis-induced ALI/ARDS in a mouse model. Lung injury was assessed by microscopic examination of lung tissue after hematoxylin and eosin staining, pathology score, and bronchoalveolar lavage fluid (BALF) total protein concentration. The level of NETs in lung tissue was detected by MPO-DNA ELISA. Purified NETs, extracted from peritoneal neutrophils, induced macrophage pyroptosis in vitro. Expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in the lung tissue and bone marrow-derived macrophages (BMDMs) were determined by western blotting or ELISA. Specks of Pyrin/ASC were examined by confocal immunofluorescence microscopy. Mefv (Pyrin)-/- mice were used to study the role of Pyrin in the process of sepsis-induced ALI/ARDS. Results ALA alleviated LPS-induced lung injury. ALA reduced the level of NETs, pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC), and IL-1β in the lung tissue of sepsis mice. In vitro, NETs increased the expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β significantly in BMDMs. Pyrin protein was found to be higher and form the inflammasome with ASC in NETs challenged-BMDMs. Knockout of Mefv (Pyrin) gene fully restored the increased expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in vitro and in vivo. Lung injury was alleviated significantly in Mefv (Pyrin)-/- mice as well. ALA suppresses all the NETs-induced changes as mentioned above. Conclusion Our study is the first to demonstrate Pyrin inflammasome driving NETs-induced macrophage pyroptosis, and ALA may reduce ALI/ARDS by inhibiting the activation of the Pyrin inflammasome-driven macrophage pyroptosis.

[1]  Qifu Li,et al.  Neutrophil Extracellular Traps Induce Glomerular Endothelial Cell Dysfunction and Pyroptosis in Diabetic Kidney Disease. , 2022, Diabetes.

[2]  L. Ware,et al.  Acute Respiratory Distress Syndrome 2022 1 Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes , 2022 .

[3]  D. McAuley,et al.  Acute respiratory distress syndrome in adults: diagnosis, outcomes, long-term sequelae, and management , 2022, The Lancet.

[4]  Masafumi Takahashi,et al.  dsDNA-induced AIM2 pyroptosis halts aberrant inflammation during rhabdomyolysis-induced acute kidney injury. , 2022, Cell death and differentiation.

[5]  M. Gladwin,et al.  Liver to lung microembolic NETs promote Gasdermin-D-dependent inflammatory lung injury in Sickle Cell Disease. , 2022, Blood.

[6]  J. Lieberman,et al.  Inflammasome activation in infected macrophages drives COVID-19 pathology , 2021, Nature.

[7]  Simon C Watkins,et al.  Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates , 2022, PLoS pathogens.

[8]  Richard F Kraus,et al.  Neutrophils—From Bone Marrow to First-Line Defense of the Innate Immune System , 2021, Frontiers in Immunology.

[9]  T. Lüscher,et al.  Dietary alpha‐linolenic acid reduces platelet activation and collagen‐mediated cell adhesion in sickle cell disease mice , 2021, Journal of thrombosis and haemostasis : JTH.

[10]  Xueyin Shi,et al.  Exosomal miR-30d-5p of neutrophils induces M1 macrophage polarization and primes macrophage pyroptosis in sepsis-related acute lung injury , 2021, Critical Care.

[11]  Fan Xie,et al.  The review of alpha‐linolenic acid: Sources, metabolism, and pharmacology , 2021, Phytotherapy research : PTR.

[12]  P. Pan,et al.  Neutrophil Extracellular Traps Augmented Alveolar Macrophage Pyroptosis via AIM2 Inflammasome Activation in LPS-Induced ALI/ARDS , 2021, Journal of inflammation research.

[13]  G. Bowlin,et al.  Patients with COVID-19: in the dark-NETs of neutrophils , 2021, Cell Death & Differentiation.

[14]  T. Mak,et al.  GSDME-mediated pyroptosis promotes inflammation and fibrosis in obstructive nephropathy , 2021, Cell Death & Differentiation.

[15]  Robert A. Campbell,et al.  Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome , 2020, Blood.

[16]  A. Malik,et al.  IL-1β Suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury. , 2020, The Journal of clinical investigation.

[17]  B. Wang,et al.  Alpha-linolenic acid protects against lipopolysaccharide-induced acute lung injury through anti-inflammatory and anti-oxidative pathways. , 2020, Microbial pathogenesis.

[18]  Si Ming Man,et al.  Emerging Activators and Regulators of Inflammasomes and Pyroptosis. , 2019, Trends in immunology.

[19]  R. Rezaee,et al.  Alpha‐linolenic acid ameliorates bronchial asthma features in ovalbumin‐sensitized rats , 2019, The Journal of pharmacy and pharmacology.

[20]  H. Aukema,et al.  Anti-inflammatory effects of α-linolenic acid in M1-like macrophages are associated with enhanced production of oxylipins from α-linolenic and linoleic acid. , 2018, The Journal of nutritional biochemistry.

[21]  K. Tarte,et al.  Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS , 2018, European Respiratory Journal.

[22]  Zhengyu He,et al.  NLRP3/ASC-mediated alveolar macrophage pyroptosis enhances HMGB1 secretion in acute lung injury induced by cardiopulmonary bypass , 2018, Laboratory Investigation.

[23]  Jie Fan,et al.  Neutrophil extracellular traps promote macrophage pyroptosis in sepsis , 2018, Cell Death and Disease.

[24]  F. Shahidi,et al.  Omega-3 Polyunsaturated Fatty Acids and Their Health Benefits. , 2018, Annual review of food science and technology.

[25]  B. Mallavia,et al.  Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury. , 2018, JCI insight.

[26]  D. Kastner,et al.  Pyrin Inflammasome Activation and RhoA Signaling in the Autoinflammatory Diseases FMF and HIDS , 2016, Nature Immunology.

[27]  Si Ming Man,et al.  Converging roles of caspases in inflammasome activation, cell death and innate immunity , 2015, Nature Reviews Immunology.

[28]  T. Cai,et al.  Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death , 2015, Nature.

[29]  J. Spicer,et al.  Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. , 2015, Journal of visualized experiments : JoVE.

[30]  Hee-Yong Kim,et al.  Discovery of essential fatty acids , 2015, Journal of Lipid Research.

[31]  Z. Werb,et al.  Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury. , 2012, The Journal of clinical investigation.

[32]  A. Zychlinsky,et al.  Neutrophil Extracellular Traps Kill Bacteria , 2004, Science.

[33]  M. Dorf,et al.  Isolation of Mouse Neutrophils , 1997, Current protocols in immunology.

[34]  R. Chambers,et al.  Acute Respiratory Distress Syndrome , 2017, The New England journal of medicine.

[35]  M. Gage,et al.  Isolation, Culture, and Polarization of Murine Bone Marrow-Derived and Peritoneal Macrophages. , 2015, Methods in molecular biology.