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Vimentin regulates activation of the NLRP3 inflammasome.

dos Santos G, Rogel MR, Baker MA, Troken JR, Urich D, Morales-Nebreda L, Sennello JA, Kutuzov MA, Sitikov A, Davis JM, Lam AP, Cheresh P, Kamp D, Shumaker DK, Budinger GR, Ridge KM - Nat Commun (2015)

Bottom Line: Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis.Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages.This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

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Vimentin interacts with components of the NLRP3 inflammasomeDifferentiated THP-1 cells primed with LPS (100 ng/mL) and activated with ATP (2.5 mM) (A) or MSU (150 μg/mL) (B) were immunoprecipitated. The presence of vimentin, actin, and inflammasome proteins in immunoprecipitates was assessed by Western blotting. In activated macrophages vimentin co-immunoprecipitates with both NLRP3 and caspase-1 whereas actin does not. (C) The association between NLRP3 and vimentin was determined using a bio-layer interferometer (BLI). Various concentrations of vimentin were tested and representative association and dissociation curves are shown: 6 μM, purple; 1.8 μM, orange; 180 nM, pink; and 18 nM, green. (D) Vimentin (red) colocalizes with NLRP3 (green) in activated J774.1 macrophages treated with control shRNA, as assessed by confocal microscopy. Primed macrophages were either activated with ATP or left untreated prior to fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.
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Figure 8: Vimentin interacts with components of the NLRP3 inflammasomeDifferentiated THP-1 cells primed with LPS (100 ng/mL) and activated with ATP (2.5 mM) (A) or MSU (150 μg/mL) (B) were immunoprecipitated. The presence of vimentin, actin, and inflammasome proteins in immunoprecipitates was assessed by Western blotting. In activated macrophages vimentin co-immunoprecipitates with both NLRP3 and caspase-1 whereas actin does not. (C) The association between NLRP3 and vimentin was determined using a bio-layer interferometer (BLI). Various concentrations of vimentin were tested and representative association and dissociation curves are shown: 6 μM, purple; 1.8 μM, orange; 180 nM, pink; and 18 nM, green. (D) Vimentin (red) colocalizes with NLRP3 (green) in activated J774.1 macrophages treated with control shRNA, as assessed by confocal microscopy. Primed macrophages were either activated with ATP or left untreated prior to fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.

Mentions: Inflammasome activation involves the physical interaction of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-128, but the mechanism by which the inflammasome assembles is not clear. If the vimentin network serves as a protein scaffold upon which the NLRP3 inflammasome components are assembled, then vimentin should interact with inflammasome proteins. Immunoprecipitation of caspase-1 and NLRP3 from human macrophage lysates resulted in a robust co-precipitation of vimentin, which was enhanced following activation of cells with ATP (Figure 8A; full blots are presented in Supplemental Figure 9). These interactions were specific for vimentin, as no actin was detectable in immunoprecipitates.


Vimentin regulates activation of the NLRP3 inflammasome.

dos Santos G, Rogel MR, Baker MA, Troken JR, Urich D, Morales-Nebreda L, Sennello JA, Kutuzov MA, Sitikov A, Davis JM, Lam AP, Cheresh P, Kamp D, Shumaker DK, Budinger GR, Ridge KM - Nat Commun (2015)

Vimentin interacts with components of the NLRP3 inflammasomeDifferentiated THP-1 cells primed with LPS (100 ng/mL) and activated with ATP (2.5 mM) (A) or MSU (150 μg/mL) (B) were immunoprecipitated. The presence of vimentin, actin, and inflammasome proteins in immunoprecipitates was assessed by Western blotting. In activated macrophages vimentin co-immunoprecipitates with both NLRP3 and caspase-1 whereas actin does not. (C) The association between NLRP3 and vimentin was determined using a bio-layer interferometer (BLI). Various concentrations of vimentin were tested and representative association and dissociation curves are shown: 6 μM, purple; 1.8 μM, orange; 180 nM, pink; and 18 nM, green. (D) Vimentin (red) colocalizes with NLRP3 (green) in activated J774.1 macrophages treated with control shRNA, as assessed by confocal microscopy. Primed macrophages were either activated with ATP or left untreated prior to fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: Vimentin interacts with components of the NLRP3 inflammasomeDifferentiated THP-1 cells primed with LPS (100 ng/mL) and activated with ATP (2.5 mM) (A) or MSU (150 μg/mL) (B) were immunoprecipitated. The presence of vimentin, actin, and inflammasome proteins in immunoprecipitates was assessed by Western blotting. In activated macrophages vimentin co-immunoprecipitates with both NLRP3 and caspase-1 whereas actin does not. (C) The association between NLRP3 and vimentin was determined using a bio-layer interferometer (BLI). Various concentrations of vimentin were tested and representative association and dissociation curves are shown: 6 μM, purple; 1.8 μM, orange; 180 nM, pink; and 18 nM, green. (D) Vimentin (red) colocalizes with NLRP3 (green) in activated J774.1 macrophages treated with control shRNA, as assessed by confocal microscopy. Primed macrophages were either activated with ATP or left untreated prior to fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.
Mentions: Inflammasome activation involves the physical interaction of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-128, but the mechanism by which the inflammasome assembles is not clear. If the vimentin network serves as a protein scaffold upon which the NLRP3 inflammasome components are assembled, then vimentin should interact with inflammasome proteins. Immunoprecipitation of caspase-1 and NLRP3 from human macrophage lysates resulted in a robust co-precipitation of vimentin, which was enhanced following activation of cells with ATP (Figure 8A; full blots are presented in Supplemental Figure 9). These interactions were specific for vimentin, as no actin was detectable in immunoprecipitates.

Bottom Line: Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis.Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages.This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Activation of the NLRP3 inflammasome and subsequent maturation of IL-1β have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1β levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1β levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

Show MeSH
Related in: MedlinePlus