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Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

ABSTRACT: NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1.

SUBMITTER: Jessop F

PROVIDER: S-EPMC5326503 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

Jessop Forrest F Hamilton Raymond F RF Rhoderick Joseph F JF Fletcher Paige P Holian Andrij A

Toxicology and applied pharmacology 20170124

NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanot ...[more]

PMID: 28126413

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Project description:1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

Project description:NOD-like receptor protein 3 (NLRP3) inflammasome is closely related to silica particle‑induced chronic lung inflammation but its role in epithelial remodeling, repair and regeneration in the distal lung during development of silicosis remains to be elucidated. The present study aimed to determine the effects of the NLRP3 inflammasome on epithelial remodeling and cellular regeneration and potential mechanisms in the distal lung of silica‑treated mice at three time points. Pulmonary function assessment, inflammatory cell counting, enzyme‑linked immunosorbent assay, histological and immunological analyses, hydroxyproline assay and western blotting were used in the study. Single intratracheal instillation of a silica suspension caused sustained NLRP3 inflammasome activation in the distal lung. Moreover, a time‑dependent increase in airway resistance and a decrease in lung compliance accompanied progression of pulmonary fibrosis. In the terminal bronchiole, lung remodeling including pyroptosis (membrane‑distributed GSDMD+), excessive proliferation (Ki67+), mucus overproduction (mucin 5 subtype AC and B) and epithelial‑mesenchymal transition (decreased E‑Cadherin+ and increased Vimentin+), was observed by immunofluorescence analysis. Notably, aberrant spatiotemporal expression of the embryonic lung stem/progenitor cell markers SOX2 and SOX9 and ectopic distribution of bronchioalveolar stem cells were observed in the distal lung only on the 7th day after silica instillation (the early inflammatory phase of silicosis). Western blotting revealed that the Sonic hedgehog/Glioma‑associated oncogene (Shh/Gli) and Wnt/β‑catenin pathways were involved in NLRP3 inflammasome activation‑mediated epithelial remodeling and dysregulated regeneration during the inflammatory and fibrotic phases. Overall, sustained NLRP3 inflammasome activation led to epithelial remodeling in the distal lung of mice. Moreover, understanding the spatiotemporal profile of dysregulated epithelial repair and regeneration may provide a novel therapeutic strategy for inhalable particle‑related chronic inflammatory and fibrotic lung disease.

Dataset Information

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

Publications

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

Similar Datasets

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