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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 bleomycin-induced lung injuryWT and Vim-/- mice were treated with either bleomycin (0.07 U/mL,intratracheally) or saline and assessed on day 5. Bleomycin-induced lung injury was assessed by histological examination of H&E staining of lung tissue (A) (original magnification ×10; scale bars, 200 μm), lung wet-to-dry weight ratio (B), and protein levels in BALF (C). Inflammasome activation was assessed by ELISA for levels of caspase-1 (D) and IL-1β (E) in BALF.Levels of IL-6 (F) and TGF-β1 (G) in BALF were assessed by ELISA. Inflammasome-independent cytokines MCP-1 (H) and TNF-α (I) were measured in WT and Vim-/- mice. Images in (A) are representative of 4-6 animals per condition. Data in (B-J) are means ± SD from 2 independent experiments n= 4-6 animals per group. *P < 0.05, **P < 0.001, ***P<0.0001, relative to WT versus Vim-/-, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
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Figure 3: Vimentin is required for bleomycin-induced lung injuryWT and Vim-/- mice were treated with either bleomycin (0.07 U/mL,intratracheally) or saline and assessed on day 5. Bleomycin-induced lung injury was assessed by histological examination of H&E staining of lung tissue (A) (original magnification ×10; scale bars, 200 μm), lung wet-to-dry weight ratio (B), and protein levels in BALF (C). Inflammasome activation was assessed by ELISA for levels of caspase-1 (D) and IL-1β (E) in BALF.Levels of IL-6 (F) and TGF-β1 (G) in BALF were assessed by ELISA. Inflammasome-independent cytokines MCP-1 (H) and TNF-α (I) were measured in WT and Vim-/- mice. Images in (A) are representative of 4-6 animals per condition. Data in (B-J) are means ± SD from 2 independent experiments n= 4-6 animals per group. *P < 0.05, **P < 0.001, ***P<0.0001, relative to WT versus Vim-/-, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.

Mentions: Bleomycin is one of the best characterized rodent models of pulmonary fibrosis33; intratracheal delivery of the drug causes severe ALI, which is followed by clearance of alveolar inflammatory cells, fibroblast proliferation, and increased collagen deposition34. Moreover, bleomycin-induced fibrosis has been shown to be dependent on the NLRP3 inflammasome35. WT and Vim-/- mice were exposed to bleomycin for 5 d. WT mice displayed intense lung inflammation as assessed by H&E staining (Figure 3A), increased wet-to-dry lung weight ratio (Figure 3B), and increased protein concentration in BALF (Figure 3C). All of these endpoints were significantly attenuated in Vim-/- mice (Figure 3A–3C). Exposure to bleomycin resulted in a large increase in immune cells in the airspace of both WT and Vim-/- mice as assessed by flow cytometric analysis of whole lung lysates (Supplemental Figure 3A). Vim-/- mice failed to exhibit an increase in caspase-1 (Figure 3D) and IL-1β (Figure 3E) in BALF following exposure to bleomycin; in contrast, WT mice showed a robust activation of caspase-1 and production of mature IL-1β (Figure 3D and 3E). As a pro-inflammatory cytokine, macrophage-produced IL-1β is known to induce production of interleukin 6 (IL-6). We observed that WT mice, but not Vim-/- mice, had increased levels of IL-6 following exposure to bleomycin (Figure 3F). TGF-β1 is a critical mediator of remodeling and fibrotic responses in the lung. Total TGF-β1 was detected after activation in BALF from WT mice 5 d following exposure to bleomycin, but remained at the baseline level in BALF from Vim-/- mice (Figure 3G). No significant difference was observed between WT and Vim-/- mice in NLRP3 inflammasome-independent cytokines TNF-α and MCP-1 (Figure 3H and 3I). Interestingly, IL-1β mRNA levels were increased in the lungs of both WT and Vim-/- mice following exposure to bleomycin (Supplemental Figure 3B).


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 bleomycin-induced lung injuryWT and Vim-/- mice were treated with either bleomycin (0.07 U/mL,intratracheally) or saline and assessed on day 5. Bleomycin-induced lung injury was assessed by histological examination of H&E staining of lung tissue (A) (original magnification ×10; scale bars, 200 μm), lung wet-to-dry weight ratio (B), and protein levels in BALF (C). Inflammasome activation was assessed by ELISA for levels of caspase-1 (D) and IL-1β (E) in BALF.Levels of IL-6 (F) and TGF-β1 (G) in BALF were assessed by ELISA. Inflammasome-independent cytokines MCP-1 (H) and TNF-α (I) were measured in WT and Vim-/- mice. Images in (A) are representative of 4-6 animals per condition. Data in (B-J) are means ± SD from 2 independent experiments n= 4-6 animals per group. *P < 0.05, **P < 0.001, ***P<0.0001, relative to WT versus Vim-/-, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
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Figure 3: Vimentin is required for bleomycin-induced lung injuryWT and Vim-/- mice were treated with either bleomycin (0.07 U/mL,intratracheally) or saline and assessed on day 5. Bleomycin-induced lung injury was assessed by histological examination of H&E staining of lung tissue (A) (original magnification ×10; scale bars, 200 μm), lung wet-to-dry weight ratio (B), and protein levels in BALF (C). Inflammasome activation was assessed by ELISA for levels of caspase-1 (D) and IL-1β (E) in BALF.Levels of IL-6 (F) and TGF-β1 (G) in BALF were assessed by ELISA. Inflammasome-independent cytokines MCP-1 (H) and TNF-α (I) were measured in WT and Vim-/- mice. Images in (A) are representative of 4-6 animals per condition. Data in (B-J) are means ± SD from 2 independent experiments n= 4-6 animals per group. *P < 0.05, **P < 0.001, ***P<0.0001, relative to WT versus Vim-/-, by one way ANOVA with a correction provided by the Bonferroni multiple comparisons test.
Mentions: Bleomycin is one of the best characterized rodent models of pulmonary fibrosis33; intratracheal delivery of the drug causes severe ALI, which is followed by clearance of alveolar inflammatory cells, fibroblast proliferation, and increased collagen deposition34. Moreover, bleomycin-induced fibrosis has been shown to be dependent on the NLRP3 inflammasome35. WT and Vim-/- mice were exposed to bleomycin for 5 d. WT mice displayed intense lung inflammation as assessed by H&E staining (Figure 3A), increased wet-to-dry lung weight ratio (Figure 3B), and increased protein concentration in BALF (Figure 3C). All of these endpoints were significantly attenuated in Vim-/- mice (Figure 3A–3C). Exposure to bleomycin resulted in a large increase in immune cells in the airspace of both WT and Vim-/- mice as assessed by flow cytometric analysis of whole lung lysates (Supplemental Figure 3A). Vim-/- mice failed to exhibit an increase in caspase-1 (Figure 3D) and IL-1β (Figure 3E) in BALF following exposure to bleomycin; in contrast, WT mice showed a robust activation of caspase-1 and production of mature IL-1β (Figure 3D and 3E). As a pro-inflammatory cytokine, macrophage-produced IL-1β is known to induce production of interleukin 6 (IL-6). We observed that WT mice, but not Vim-/- mice, had increased levels of IL-6 following exposure to bleomycin (Figure 3F). TGF-β1 is a critical mediator of remodeling and fibrotic responses in the lung. Total TGF-β1 was detected after activation in BALF from WT mice 5 d following exposure to bleomycin, but remained at the baseline level in BALF from Vim-/- mice (Figure 3G). No significant difference was observed between WT and Vim-/- mice in NLRP3 inflammasome-independent cytokines TNF-α and MCP-1 (Figure 3H and 3I). Interestingly, IL-1β mRNA levels were increased in the lungs of both WT and Vim-/- mice following exposure to bleomycin (Supplemental Figure 3B).

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