Vimentin regulates activation of the NLRP3 inflammasome

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.

Figure 1. Vimentin is required for ALI and inflammasome activation in response to LPS.

(a) Survival of WT and Vim^−/− mice subjected to a lethal dose of LPS (80 mg kg⁻¹, intraperitoneally) was measured and compared. The survival curves were analysed using the log rank test, which calculates the chi-square (χ²) for each event time for each group and sums the results. The summed results for each group were added to derive the ultimate chi-square to compare the full curves of each group. The log rank test for the entire data set was P=0.001. (b–f) WT and Vim^−/− mice were challenged with a sublethal dose of LPS and markers of ALI were measured after 48 h, as assessed by H&E staining (scale bar, 200 μm) (b); by wet-to-dry lung weight ratio (c); and by protein content in the BALF (d). Inflammasome activation was measured by ELISA for caspase-1 (e) or IL-1β (f) in BALF from Vim^−/− and WT mice. Data in b–e are from three independent experiments of n=6–8 animals per group and presented as mean±s.d. **P<0.001, ***P<0.0001 relative to WT versus Vim^−/−, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 2. Vimentin deficiency prevents asbestos-induced lung injury and fibrosis.

WT and Vim^−/− mice were treated with PBS containing either 200 μg of asbestos crocidolite or the control particle titanium dioxide (TiO₂), intratracheally. Markers of inflammation and fibrosis were assessed 3 and 6 weeks after instillation, respectively. (a) Total cell count (macrophages, neutrophils, eosinophils, erythrocytes and lymphocytes ) and (b) protein levels were assessed in BALF collected from Vim^−/− mice 3 weeks after asbestos administration. Inflammasome activation in WT and Vim^−/− mice was measured by ELISA for caspase-1 (c) and IL-1β (d) levels in BALF at the same time point. Collagen deposition was evaluated in asbestos-treated Vim^−/− and WT mice by Masson’s trichrome and Picrosirius red staining of lung slices (e; scale bar, 200 μm) and by measuring total collagen content by Sircol assay (f). Data are from two independent experiments n=6–8 animals per group, and presented as mean±s.d. *P<0.05, **P<0.001, relative to WT versus Vim^−/−, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 3. Vimentin is required for bleomycin-induced lung injury.

WT and Vim^−/− mice were treated with either bleomycin (0.07 U ml⁻¹, intratracheally) or saline and assessed on day 5. (a) Bleomycin-induced lung injury was assessed by histological examination of H&E staining of lung tissue (original magnification × 10; scale bar, 200 μm), (b) lung wet-to-dry weight ratio and (c) protein levels in BALF. 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 four to six animals per condition. Data in b–i are means±s.d. from two independent experiments n=4–6 animals per group. **P<0.001, ***P<0.0001, relative to WT versus Vim^−/−, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 4. Bleomycin-induced lung injury and fibrosis are prevented in the absence of vimentin.

Shown is the histology of mice lungs 21 days following intratracheal instillation of bleomycin or PBS. Masson trichrome (a) and Picrosirius Red (b) stains for collagen. Original magnification for overview mosaics, × 5. Enlargements were taken with a × 40 objective (scale bar, 200 μm). (c) Representative elastographs from AFM micro-indentation of lung tissue from WT and Vim^−/− animals. The colour bar indicates elastic modulus, E. The darkest red corresponds to elastic modulus values of 50 kPa and above. The data represent 256 indentations per region, with at least two regions per tissue section from at least three mice per group. (d) Collagen content in lungs from WT and Vim^−/− mice assessed by Sircol assay. (e) Mice were ventilated 21 days after intratracheal instillation of bleomycin or PBS. Shown are quasi-static compliance measurements of WT and Vim^−/− lungs. (f) Elastic modulus frequency plot obtained from live, unfixed lung tissue of saline and bleomycin-treated WT and Vim^−/− animals. Microindentation data were fit using the Hertz model to acquire the elastic modulus of regions of lung tissue. Images in a–b represent data obtained from at least three animals per group; data shown in d–e are mean±s.d. of at least five animals. **P<0.001, ***P<0.0001 relative to WT versus Vim^−/−, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 5. Lung injury and fibrosis are attenuated in Vim^−/− bone marrow chimeric mice.

(a) Treatment protocol: CD45.1⁺ WT mice were lethally irradiated (6 Gy) and two sets of mice were transplanted either with 1 × 10⁶ CD45.2⁺ WT or Vim^−/− BM cells to generate chimeric mice. After 6.5 weeks (w), when >90% of AM were of donor (WT and Vim^−/−) phenotype, chimeric mice were treated with saline or bleomycin and subjected to analyses. Bleomycin-induced lung injury was assessed by protein levels (b), and ELISA for IL-1β (c), IL-6 (d) and TGF-β (e) in BALF, 5 days (d5) after instillation. Bleomycin-induced fibrosis was assessed 21 days (d21) after instillation by measuring collagen content in lungs by Sircol assay (f) and by histological examination of lung tissue with H&E staining (g). Original magnification × 40; scale bar, 200 μm. Data in b are means±s.e., c–f are means±s.d. from two independent experiments, each with at least three animals per experiment. *P<0.05, **P<0.001, ***P<0.0001, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 6. Activation of the inflammasome in murine cells is dependent on vimentin.

Primary WT and Vim^−/− alveolar macrophages were primed with LPS (100 ng ml⁻¹) 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⁻¹) 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±s.d. of three replicates. **P<0.001, by Student’s t-test. SN, supernatant; TCL, total cell lysate.

Figure 7. Vimentin is required for inflammasome activation in vitro.

Human 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⁻¹) or asbestos (40 μg cm⁻²) 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⁻¹) for 3 h or left untreated. Cells were then labelled for active caspase-1 (green) according to manufacturer’s instructions, DNA (blue) and vimentin (red). Scale bar, 10 μm. Data shown are means±s.d., *P<0.05, ***P<0.0001, by one-way analysis of variance with a correction provided by the Bonferroni multiple comparisons test.

Figure 8. Vimentin interacts with components of the NLRP3 inflammasome.

Differentiated THP-1 cells primed with LPS (100 ng ml⁻¹) and activated with ATP (2.5 mM) (a) or MSU (150 μg ml⁻¹) (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) co-localizes 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 before fixing and staining with anti-vimentin and anti-NLRP3 antibodies. Confocal images are representative of at least three independent experiments. Scale bar, 10 μm.