title: Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy.
abstract: The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.
authors.affiliation: a Institute of Immunity, Infection and Inflammation ; University of Glasgow ; UK.
authors.forename: Majid Sakhi
authors.initials: MS
authors.lastname: Jabir
authors.forename: Lee
authors.initials: L
authors.lastname: Hopkins
authors.forename: Neil D
authors.initials: ND
authors.lastname: Ritchie
authors.forename: Ihsan
authors.initials: I
authors.lastname: Ullah
authors.forename: Hannah K
authors.initials: HK
authors.lastname: Bayes
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Tourlomousis
authors.forename: Alison
authors.initials: A
authors.lastname: Lupton
authors.forename: Daniel
authors.initials: D
authors.lastname: Puleston
authors.forename: Anna Katharina
authors.initials: AK
authors.lastname: Simon
authors.forename: Clare
authors.initials: C
authors.lastname: Bryant
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Evans
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D001854:Bone Marrow Cells / Q000473:pathology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism / Q000648:ultrastructure; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D063306:Mitophagy; D011485:Protein Binding; D011552:Pseudomonas Infections / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C540422:Aim2 protein, mouse; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D058847:Inflammasomes; C512299:Ipaf protein, mouse; D017382:Reactive Oxygen Species
keywords: AIM2, absent in melanoma 2; ATG, autophagy related; ATPIF1, ATPase inhibitory factor 1; BID, BH3 interacting domain death agonist; BMDM, bone marrow-derived macrophages; BrdU, 5-bromo-2-deoxyuridine; CASP, caspase; DNA detection; GFP, green fluorescent protein; IL1B, interleukin 1, β; LC3B, microtubule-associated protein 1 light chain 3 β; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MT-CO1, mitochondrially encoded cytochrome c oxidase I; Mito-TEMPO, (2-(2, 2, 6, 6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride; NAC, N-acetylcysteine; NAIP, NLR family apoptosis inhibitor; NGS, normal goat serum; NLR proteins; NLR, nucleotide-binding domain, leucine-rich repeat containing; NLRC4, NLR family, CARD domain containing 4; NLRP3, NLR family, pyrin domain containing 3; PBS, phosphate-buffered saline; PINK1, PTEN induced putative kinase 1; Rn18s, 18S rRNA; T3SS, type III secretion system; TNF, tumor necrosis factor; TUBB5, tubulin, β 5 class I; Three-MA, 3-methyladenine; Vav, vav 1 oncogene; infection; mitophagy; mtDNA, mitochondrial DNA; type III secretion system