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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 is required for inflammasome activation in vitroHuman THP-1 macrophages were differentiated and primed with PMA (0.5 μM) and treated with either scramble (CT) or vimentin (KD) siRNA and subsequently treated with saline, MSU (150 μg/mL), or asbestos (40 μg/cm2) for 6 h. IL-1β, vimentin, and actin levels in cell lysates were assessed by Western blot (A) whereas levels of IL-1β (B) and caspase-1 (C) in supernatants were assessed by ELISA. (D) Confocal microscopy of BMDMs isolated from WT and Vim-/- mice, primed with LPS as above, and activated with MSU (150 μg/mL) for 3 h or left untreated. Cells were then labeled for active caspase-1 (green) according to manufacturer's instructions, DNA (blue), and vimentin (red). Scale bar, 10 μm. Data shown are means ± SD, *P < 0.05, ***P<0.0001, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
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Figure 7: Vimentin is required for inflammasome activation in vitroHuman THP-1 macrophages were differentiated and primed with PMA (0.5 μM) and treated with either scramble (CT) or vimentin (KD) siRNA and subsequently treated with saline, MSU (150 μg/mL), or asbestos (40 μg/cm2) for 6 h. IL-1β, vimentin, and actin levels in cell lysates were assessed by Western blot (A) whereas levels of IL-1β (B) and caspase-1 (C) in supernatants were assessed by ELISA. (D) Confocal microscopy of BMDMs isolated from WT and Vim-/- mice, primed with LPS as above, and activated with MSU (150 μg/mL) for 3 h or left untreated. Cells were then labeled for active caspase-1 (green) according to manufacturer's instructions, DNA (blue), and vimentin (red). Scale bar, 10 μm. Data shown are means ± SD, *P < 0.05, ***P<0.0001, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

Mentions: Uric acid released from cells exposed to bleomycin triggers NLRP3 inflammasome assembly leading to IL-1β maturation and inflammation in the lungs19,40. In an in vitro model, we examined whether silencing vimentin would impair NLRP3 inflammasome activation in response to MSU. A human macrophage-like cell line was treated with silencing RNA (siRNA) targeted to vimentin (VimKD) or a scrambled siRNA (CT) (Figure 7A; full blots are presented in Supplemental Figure 8) and subsequently activated with MSU. No differences were observed in the levels of pro-IL-1β between CT and VimKD cells (Figure 7A), suggesting that vimentin deficiency does not impair signal 1. NLRP3 inflammasome activation was repressed in VimKD cells as assessed by IL-1β and caspase-1 levels in cell supernatants (Figure 7B and 7C). Similar results were obtained when CT and VimKD cells were activated with asbestos (Figure 7B and 7C). In addition, bone marrow–derived macrophages (BMDMs) were isolated from WT and Vim-/- mice, primed with LPS, and activated with MSU. Caspase-1 activation was assessed using a specific fluorescent probe, FAM-YVAD-FMK 41. WT BMDMs showed robust caspase-1 activation following treatment with MSU, with caspase-1 forming aggregates throughout the cytoplasm. However, caspase-1 activation was severely reduced in BMDMs isolated from Vim-/- mice (Figure 7D). No caspase-1 activation was observed in untreated BMDMs isolated from either WT or Vim-/- mice.


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 is required for inflammasome activation in vitroHuman THP-1 macrophages were differentiated and primed with PMA (0.5 μM) and treated with either scramble (CT) or vimentin (KD) siRNA and subsequently treated with saline, MSU (150 μg/mL), or asbestos (40 μg/cm2) for 6 h. IL-1β, vimentin, and actin levels in cell lysates were assessed by Western blot (A) whereas levels of IL-1β (B) and caspase-1 (C) in supernatants were assessed by ELISA. (D) Confocal microscopy of BMDMs isolated from WT and Vim-/- mice, primed with LPS as above, and activated with MSU (150 μg/mL) for 3 h or left untreated. Cells were then labeled for active caspase-1 (green) according to manufacturer's instructions, DNA (blue), and vimentin (red). Scale bar, 10 μm. Data shown are means ± SD, *P < 0.05, ***P<0.0001, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
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Related In: Results  -  Collection

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Figure 7: Vimentin is required for inflammasome activation in vitroHuman THP-1 macrophages were differentiated and primed with PMA (0.5 μM) and treated with either scramble (CT) or vimentin (KD) siRNA and subsequently treated with saline, MSU (150 μg/mL), or asbestos (40 μg/cm2) for 6 h. IL-1β, vimentin, and actin levels in cell lysates were assessed by Western blot (A) whereas levels of IL-1β (B) and caspase-1 (C) in supernatants were assessed by ELISA. (D) Confocal microscopy of BMDMs isolated from WT and Vim-/- mice, primed with LPS as above, and activated with MSU (150 μg/mL) for 3 h or left untreated. Cells were then labeled for active caspase-1 (green) according to manufacturer's instructions, DNA (blue), and vimentin (red). Scale bar, 10 μm. Data shown are means ± SD, *P < 0.05, ***P<0.0001, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
Mentions: Uric acid released from cells exposed to bleomycin triggers NLRP3 inflammasome assembly leading to IL-1β maturation and inflammation in the lungs19,40. In an in vitro model, we examined whether silencing vimentin would impair NLRP3 inflammasome activation in response to MSU. A human macrophage-like cell line was treated with silencing RNA (siRNA) targeted to vimentin (VimKD) or a scrambled siRNA (CT) (Figure 7A; full blots are presented in Supplemental Figure 8) and subsequently activated with MSU. No differences were observed in the levels of pro-IL-1β between CT and VimKD cells (Figure 7A), suggesting that vimentin deficiency does not impair signal 1. NLRP3 inflammasome activation was repressed in VimKD cells as assessed by IL-1β and caspase-1 levels in cell supernatants (Figure 7B and 7C). Similar results were obtained when CT and VimKD cells were activated with asbestos (Figure 7B and 7C). In addition, bone marrow–derived macrophages (BMDMs) were isolated from WT and Vim-/- mice, primed with LPS, and activated with MSU. Caspase-1 activation was assessed using a specific fluorescent probe, FAM-YVAD-FMK 41. WT BMDMs showed robust caspase-1 activation following treatment with MSU, with caspase-1 forming aggregates throughout the cytoplasm. However, caspase-1 activation was severely reduced in BMDMs isolated from Vim-/- mice (Figure 7D). No caspase-1 activation was observed in untreated BMDMs isolated from either WT or Vim-/- mice.

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