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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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Activation of the inflammasome in murine cells is dependent on vimentinPrimary WT and Vim-/- alveolar macrophages were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 1 h. Caspase-1 (A) and IL-1β (B) levels in supernatants were assessed by ELISA and by Western blot (C). J744.1 cells expressing either control shRNA (Ctrl) or shRNA against vimentin (KD, two distinct clones) were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 25 min. Vimentin, actin, and caspase-1 protein levels were assessed by Western blot analysis (D). Mature caspase-1 levels were assessed in WT and KD cells activated with ATP for 25, 50, or 85 min (E). Data shown are means ± SD. of three replicates. **P < 0.001, by Student's t- test. SN, supernatant; TCL, total cell lysate.
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Figure 6: Activation of the inflammasome in murine cells is dependent on vimentinPrimary WT and Vim-/- alveolar macrophages were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 1 h. Caspase-1 (A) and IL-1β (B) levels in supernatants were assessed by ELISA and by Western blot (C). J744.1 cells expressing either control shRNA (Ctrl) or shRNA against vimentin (KD, two distinct clones) were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 25 min. Vimentin, actin, and caspase-1 protein levels were assessed by Western blot analysis (D). Mature caspase-1 levels were assessed in WT and KD cells activated with ATP for 25, 50, or 85 min (E). Data shown are means ± SD. of three replicates. **P < 0.001, by Student's t- test. SN, supernatant; TCL, total cell lysate.

Mentions: As stated above, NLRP3 inflammasome activation requires two distinct stimuli. An inflammatory stimulus, such as LPS, primes cells to transcribe and synthesize pro-IL-1β (signal 1), and then another stimulus such as extracellular adenosine triphosphate (ATP), monosodium urate (MSU), or asbestos (signal 2) is necessary for inflammasome complex formation, caspase-1 activation, and IL-1β maturation19,22,39. To further investigate the possibility that NLRP3 inflammasome activation requires vimentin, we isolated primary alveolar macrophages from WT and Vim-/- mice. Macrophages were primed with LPS and stimulated with ATP as previously described39. Levels of mature caspase-1 and IL-1β were significantly increased in the supernatants collected from WT macrophages following LSP and ATP treatment compared with saline treatment, but in the supernatant collected from Vim-/- macrophages there was only a slight increase (Figure 6A–6B). There was no difference in pro-IL-1β levels in total cell lysates between activated WT and Vim-/- alveolar macrophages (Figure 6C; full blots are presented in Supplemental Figure 7). As an independent approach, we expressed a small hairpin RNA (shRNA) targeted to vimentin (VimKD) or a scrambled shRNA (Ctrl) in J774a.1 macrophages and treated the cells with ATP. Caspase-1 is required to cleave pro-IL-1β into its biologically active form and like other caspases is proteolytically activated from a pro-enzyme to produce a tetramer of its two active subunits, p20 and p1028. The levels of mature caspase-1 p20, but not pro-caspase-1, were considerably reduced in VimKD cells (Figure 6D and 6E; full blots are presented in Supplemental Figure 7) compared with Ctrl cells treated with ATP, further supporting the hypothesis that vimentin deficiency prevents NLRP3 inflammasome activation.


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)

Activation of the inflammasome in murine cells is dependent on vimentinPrimary WT and Vim-/- alveolar macrophages were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 1 h. Caspase-1 (A) and IL-1β (B) levels in supernatants were assessed by ELISA and by Western blot (C). J744.1 cells expressing either control shRNA (Ctrl) or shRNA against vimentin (KD, two distinct clones) were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 25 min. Vimentin, actin, and caspase-1 protein levels were assessed by Western blot analysis (D). Mature caspase-1 levels were assessed in WT and KD cells activated with ATP for 25, 50, or 85 min (E). Data shown are means ± SD. of three replicates. **P < 0.001, by Student's t- test. SN, supernatant; TCL, total cell lysate.
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Related In: Results  -  Collection

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Figure 6: Activation of the inflammasome in murine cells is dependent on vimentinPrimary WT and Vim-/- alveolar macrophages were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 1 h. Caspase-1 (A) and IL-1β (B) levels in supernatants were assessed by ELISA and by Western blot (C). J744.1 cells expressing either control shRNA (Ctrl) or shRNA against vimentin (KD, two distinct clones) were primed with LPS (100 ng/mL) and treated with ATP (1 mM) or saline for 25 min. Vimentin, actin, and caspase-1 protein levels were assessed by Western blot analysis (D). Mature caspase-1 levels were assessed in WT and KD cells activated with ATP for 25, 50, or 85 min (E). Data shown are means ± SD. of three replicates. **P < 0.001, by Student's t- test. SN, supernatant; TCL, total cell lysate.
Mentions: As stated above, NLRP3 inflammasome activation requires two distinct stimuli. An inflammatory stimulus, such as LPS, primes cells to transcribe and synthesize pro-IL-1β (signal 1), and then another stimulus such as extracellular adenosine triphosphate (ATP), monosodium urate (MSU), or asbestos (signal 2) is necessary for inflammasome complex formation, caspase-1 activation, and IL-1β maturation19,22,39. To further investigate the possibility that NLRP3 inflammasome activation requires vimentin, we isolated primary alveolar macrophages from WT and Vim-/- mice. Macrophages were primed with LPS and stimulated with ATP as previously described39. Levels of mature caspase-1 and IL-1β were significantly increased in the supernatants collected from WT macrophages following LSP and ATP treatment compared with saline treatment, but in the supernatant collected from Vim-/- macrophages there was only a slight increase (Figure 6A–6B). There was no difference in pro-IL-1β levels in total cell lysates between activated WT and Vim-/- alveolar macrophages (Figure 6C; full blots are presented in Supplemental Figure 7). As an independent approach, we expressed a small hairpin RNA (shRNA) targeted to vimentin (VimKD) or a scrambled shRNA (Ctrl) in J774a.1 macrophages and treated the cells with ATP. Caspase-1 is required to cleave pro-IL-1β into its biologically active form and like other caspases is proteolytically activated from a pro-enzyme to produce a tetramer of its two active subunits, p20 and p1028. The levels of mature caspase-1 p20, but not pro-caspase-1, were considerably reduced in VimKD cells (Figure 6D and 6E; full blots are presented in Supplemental Figure 7) compared with Ctrl cells treated with ATP, further supporting the hypothesis that vimentin deficiency prevents NLRP3 inflammasome activation.

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