Epithelial Atg5 Deficiency Intensifies Caspase-11 Activation, Fueling Extracellular mtDNA Release to Activate cGAS–STING–NLRP3 Axis in Macrophages During Pseudomonas Infection

Abstract

Pseudomonas aeruginosa (P. aeruginosa) infections pose a significant threat to public health, underscoring the need for deeper insights into host cellular defenses. This study explores the critical role of autophagy-related protein 5 (ATG5) in lung epithelial cells during P. aeruginosa infection. Single-cell RNA transcriptomics revealed a pronounced enrichment of autophagy pathways in type II alveolar epithelial cells (AEC2). Using a conditional Atg5 knockout murine model, we demonstrated that ATG5 deficiency in AEC2 compromises survival, hampers bacterial clearance, and increases pathogen dissemination. Additionally, the loss of ATG5 exacerbated inflammatory responses, notably through the activation of the AKT/PI3K/NF-κB axis and pyroptosis, which culminated in severe lung injury and epithelial barrier disruption. Mechanistically, the absence of ATG5 disrupted mitophagy, leading to intensified mitochondrial damage. This exacerbated condition coupled with the activation of gasdermin D (GSDMD) by the noncanonical caspase-11, enhancing the release of mitochondrial DNA (mtDNA), which in turn activated cGAS–STING–NLRP3 signaling in macrophages. These findings highlight the essential role of ATG5 in modulating immune responses and suggest potential therapeutic targets for managing P. aeruginosa-induced pulmonary infections.

Keywords

ATG5 / caspase-11 / cGAS/STING / epithelial cells / mitochondrial DNA / Pseudomonas aeruginosa

References

[1] S. Qin, W. Xiao, C. Zhou, et al., “Pseudomonas aeruginosa: Pathogenesis, Virulence Factors, Antibiotic Resistance, Interaction With Host, Technology Advances and Emerging Therapeutics,” Signal Transduction and Targeted Therapy 7, no. 1 (2022): 199.

[2] D. M. P. De Oliveira, B. M. Forde, T. J. Kidd, et al., “Antimicrobial Resistance in ESKAPE Pathogens,” Clinical Microbiology Reviews 33, no. 3 (2020): e00181-19.

[3] E. Tacconelli, E. Carrara, A. Savoldi, et al., “Discovery, Research, and Development of New Antibiotics: The WHO Priority List of Antibiotic-Resistant Bacteria and Tuberculosis,” Lancet Infectious Diseases 18, no. 3 (2018): 318-327.

[4] N. Mizushima and B. Levine, “Autophagy in Human Diseases,” New England Journal of Medicine 383, no. 16 (2020): 1564-1576.

[5] J. Cahoon, D. Yang, and P. Wang, “The Noncanonical Inflammasome in Health and Disease,” Infectious Medicine (Beijing) 1, no. 3 (2022): 208-216.

[6] B. Aylan, E. M. Bernard, E. Pellegrino, et al., “ATG7 and ATG14 Restrict Cytosolic and Phagosomal Mycobacterium tuberculosis Replication in Human Macrophages,” Nature Microbiology 8, no. 5 (2023): 803-818.

[7] Q. Pu, C. Gan, R. Li, et al., “Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis,” Journal of Immunology 198, no. 8 (2017): 3205-3213.

[8] V. L. Campodonico, M. Gadjeva, C. Paradis-Bleau, A. Uluer, and G. B. Pier, “Airway Epithelial Control of Pseudomonas aeruginosa Infection in Cystic Fibrosis,” Trends in Molecular Medicine 14, no. 3 (2008): 120-133.

[9] J. Z. Cui, Z. H. Chew, and L. H. K. Lim, “New Insights Into Nucleic Acid Sensor AIM2: The Potential Benefit in Targeted Therapy for Cancer,” Pharmacological Research 200 (2024): 107079.

[10] X. Jin, Y. Ma, D. Liu, and Y. Huang, “Role of Pyroptosis in the Pathogenesis and Treatment of Diseases,” MedComm 4, no. 3 (2023): e249.

[11] G. V. Raghuram, B. K. Tripathy, K. Avadhani, et al., “Cell-Free Chromatin Particles Released From Dying Cells Inflict Mitochondrial Damage and ROS Production in Living Cells,” Cell Death Discovery 10, no. 1 (2024): 30.

[12] S. Wang, H. Long, L. Hou, et al., “The Mitophagy Pathway and Its Implications in human Diseases,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 304.

[13] J. Liu, J. Wang, A. Xiong, et al., “Mitochondrial Quality Control in Lung Diseases: Current Research and Future Directions,” Frontiers in Physiology 14 (2023): 1236651.

[14] L. Chen, M. Zhou, H. Li, et al., “Mitochondrial Heterogeneity in Diseases,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 311.

[15] C. de Torre-Minguela, A. I. Gomez, I. Couillin, and P. Pelegrin, “Gasdermins Mediate Cellular Release of Mitochondrial DNA During Pyroptosis and Apoptosis,” FASEB Journal 35, no. 8 ( 2021): e21757.

[16] W. Zhang, G. Li, R. Luo, et al., “Cytosolic Escape of Mitochondrial DNA Triggers cGAS-STING-NLRP3 Axis-Dependent Nucleus Pulposus Cell Pyroptosis,” Experimental & Molecular Medicine 54, no. 2 (2022): 129-142.

[17] Y. Wu, C. Hao, G. Han, et al., “SS-31 Ameliorates Hepatic Injury in Rats Subjected to Severe Burns plus Delayed Resuscitation via Inhibiting the mtDNA/STING Pathway in Kupffer Cells,” Biochemical and Biophysical Research Communications 546 (2021): 138-144.

[18] J. Vincent, C. Adura, P. Gao, et al., “Small Molecule Inhibition of cGAS Reduces Interferon Expression in Primary Macrophages From Autoimmune Mice,” Nature Communications 8, no. 1 (2017): 750.

[19] C. G. Zou, Y. C. Ma, L. L. Dai, and K. Q. Zhang, “Autophagy Protects C. elegans Against Necrosis During Pseudomonas aeruginosa Infection,” PNAS 111, no. 34 (2014): 12480-12485.

[20] Q. Deng, Y. Wang, Y. Zhang, et al., “Pseudomonas aeruginosa Triggers Macrophage Autophagy to Escape Intracellular Killing by Activation of the NLRP3 Inflammasome,” Infection and Immunity 84, no. 1 (2016): 56-66.

[21] P. Zhu, H. Bu, S. Tan, et al., “A Novel Cochlioquinone Derivative, CoB,” Journal of Immunology 1, no. 5 (2020): 1293-1305.

[22] V. Mohankumar, S. Ramalingam, G. P. Chidambaranathan, and L. Prajna, “Autophagy Induced by Type III Secretion System Toxins Enhances Clearance of Pseudomonas aeruginosa From human Corneal Epithelial Cells,” Biochemical and Biophysical Research Communications 503, no. 3 (2018): 1510-1515.

[23] X. G. Guo, T. X. Ji, Y. Xia, and Y. Y. Ma, “Autophagy Protects Type II Alveolar Epithelial Cells From Mycobacterium tuberculosis Infection,” Biochemical and Biophysical Research Communications 432, no. 2 (2013): 308.

[24] P. Lapaquette, A. Ducreux, L. Basmaciyan, et al., “Membrane Protective Role of Autophagic Machinery During Infection of Epithelial Cells by Candida albicans,” Gut Microbes 14, no. 1 (2022): 2004798.

[25] A. Shroff and K. V. R. Reddy, “Autophagy Gene ATG5 Knockdown Upregulates Apoptotic Cell Death During Candida albicans Infection in Human Vaginal Epithelial Cells,” American Journal of Reproductive Immunology 80, no. 6 (2018): e13056.

[26] M. Li, J. Li, R. Zeng, et al., “Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis,” Journal of Virology 92, no. 8 (2018): e02193-17.

[27] R. H. Zhang, H. L. Zhang, P. Y. Li, et al., “Autophagy Is Involved in the Replication of H9N2 Influenza Virus via the Regulation of Oxidative Stress in Alveolar Epithelial Cells,” Virology Journal 18, no. 1 (2021): 22.

[28] Q. Tan, Q. Ai, Y. He, F. Li, and J. Yu, “P. aeruginosa Biofilm Activates the NLRP3 Inflammasomes In Vitro,” Microbial Pathogenesis 164 (2022): 105379.

[29] M. S. Minns, K. Liboro, T. S. Lima, et al., “NLRP3 Selectively Drives IL-1Beta Secretion by Pseudomonas aeruginosa Infected Neutrophils and Regulates Corneal Disease Severity,” Nature Communications 14, no. 1 (2023): 5832.

[30] X. Li, Y. Ye, X. Zhou, C. Huang, and M. Wu, “Atg7 Enhances Host Defense Against Infection via Downregulation of Superoxide but Upregulation of Nitric Oxide,” Journal of Immunology 194, no. 3 (2015): 1112-1121.

[31] N. Kayagaki, I. B. Stowe, B. L. Lee, et al., “Caspase-11 Cleaves Gasdermin D for Non-Canonical Inflammasome Signalling,” Nature 526, no. 7575 (2015): 666-671.

[32] J. Wang, M. Sahoo, L. Lantier, et al., “Caspase-11-Dependent Pyroptosis of Lung Epithelial Cells Protects From Melioidosis While Caspase-1 Mediates Macrophage Pyroptosis and Production of IL-18,” PLoS Pathogens 14, no. 5 (2018): e1007105.

[33] J. Shi, Y. Zhao, K. Wang, et al., “Cleavage of GSDMD by Inflammatory Caspases Determines Pyroptotic Cell Death,” Nature 526, no. 7575 (2015): 660-665.

[34] X. Wan, J. Li, Y. Wang, et al., “H7N9 Virus Infection Triggers Lethal Cytokine Storm by Activating Gasdermin E-Mediated Pyroptosis of Lung Alveolar Epithelial Cells,” National Science Review 9, no. 1 (2022): nwab137.

[35] Z. Liu, M. Wang, X. Wang, et al., “XBP1 Deficiency Promotes Hepatocyte Pyroptosis by Impairing Mitophagy to Activate mtDNA-cGAS-STING Signaling in Macrophages During Acute Liver Injury,” Redox Biology 52 (2022): 102305.

[36] M. S. Jabir, L. Hopkins, N. D. Ritchie, et al., “Mitochondrial Damage Contributes to Pseudomonas aeruginosa Activation of the Inflammasome and Is Downregulated by Autophagy,” Autophagy 11, no. 1 (2015): 166-182.

[37] M. Onishi, K. Yamano, M. Sato, N. Matsuda, and K. Okamoto, “Molecular Mechanisms and Physiological Functions of Mitophagy,” EMBO Journal 40, no. 3 (2021): e104705.

[38] R. A. Williams, T. K. Smith, B. Cull, J. C. Mottram, and G. H. Coombs, “ATG5 is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major,” PLoS Pathogens 8, no. 5 (2012): e1002695.

[39] X. Li, J. Wu, X. Sun, et al., “Autophagy Reprograms Alveolar Progenitor Cell Metabolism in Response to Lung Injury,” Stem Cell Reports 14, no. 3 (2020): 420-432.

[40] L. Gui, H. Qian, K. A. Rocco, L. Grecu, and L. E. Niklason, “Efficient Intratracheal Delivery of Airway Epithelial Cells in Mice and Pigs,” American Journal of Physiology Lung Cellular and Molecular Physiology 308, no. 2 (2015): L221-L228.

[41] X. Jia, B. Liu, L. Bao, et al., “Delayed Oseltamivir Plus Sirolimus Treatment Attenuates H1N1 Virus-Induced Severe Lung Injury Correlated With Repressed NLRP3 Inflammasome Activation and Inflammatory Cell Infiltration,” PLoS Pathogens 14, no. 11 (2018): e1007428.

[42] H. Xian, K. Watari, E. Sanchez-Lopez, et al., “Oxidized DNA Fragments Exit Mitochondria via mPTP- and VDAC-Dependent Channels to Activate NLRP3 Inflammasome and Interferon Signaling,” Immunity 55, no. 8 (2022): 1370-1385.

[43] S. Hanzelmann, R. Castelo, and J. Guinney, “GSVA: Gene Set Variation Analysis for Microarray and RNA-seq Data,” BMC Bioinformatics [Electronic Resource] 14 (2013): 7.

[44] T. Stuart, A. Butler, P. Hoffman, et al., “Comprehensive Integration of Single-Cell Data,” Cell 177, no. 7 (2019): 1888-1902 e21.

[45] J. Wang, M. Jiang, A. Xiong, et al., “Integrated Analysis of Single-Cell and Bulk RNA Sequencing Reveals Pro-Fibrotic PLA2G7(High) Macrophages in Pulmonary Fibrosis,” Pharmacological Research 182 (2022): 106286.

[46] G. Finak, A. McDavid, M. Yajima, et al., “MAST: A Flexible Statistical Framework for Assessing Transcriptional Changes and Characterizing Heterogeneity in Single-Cell RNA Sequencing Data,” Genome Biology 16 (2015): 278.

[47] S. Aibar, C. B. Gonzalez-Blas, T. Moerman, et al., “SCENIC: Single-Cell Regulatory Network Inference and Clustering,” Nature Methods 14, no. 11 (2017): 1083-1086.

[48] J. Wang, L. Zhang, L. Luo, et al., “Characterizing Cellular Heterogeneity in Fibrotic Hypersensitivity Pneumonitis by Single-Cell Transcriptional Analysis,” Cell Death Discovery 8, no. 1 (2022): 38.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.