Supporting Materials and Methods.

(DOCX)</p

Apoptosis caspases are required for silica-induced pyroptosis.

a, Cell survival of primed Nlrp3-/- BMDMs pretreated with inhibitor mixture as indicated and stimulated with silica. b, Cell survival of silica-stimulated Nlrp3-/- macrophages pretreated with indicated caspase inhibitors. c, Cell survival of primed Caspase-1-/- BMDMs pretreated with inhibitor mixture as indicated and stimulated with silica. d, Cell survival of silica-stimulated Caspase-1-/- macrophages pretreated with indicated caspase inhibitors. a-d, The cell viability was measured through extracellular LDH release assay. e, BMDMs derived from WT, Caspase-1-/- and Gsdmd-/- mice on chambered coverslips were stimulated with silica and monitored for morphological changes over time by differential interference contrast (DIC) and fluorescence microscopy. Loss of membrane integrity was indicated by PI (red) staining of nuclear DNA. Scale bar represents 100 μm. z-VAD-FMK, pan-caspase inhibitor; C1i, VX765 (Caspase-1 inhibitor); C2i, z-VDVAD-FMK (Caspase-2 inhibitor); C3i, z-DEVD-FMK (Caspase-3 inhibitor); C6i, z-VEID-FMK (Caspase-6 inhibitor); C8i, z-IETD-FMK (Caspase-8 inhibitor); C9i, z-LEHD-FMK (Caspase-9 inhibitor); C10i, z-AEVD-FMK (Caspase-10 inhibitor); C12i, z-ATAD-FMK (Caspase-12 inhibitor). CIMix represents the mixture of all the indicated caspase inhibitors, while CIMix-C1i means lack of Caspase-1 specific inhibitor and the rest can be deduced by analogy. Results are expressed as mean ± SD from three independent experiments. *PPP (TIF)</p

Silicosis is associated with GSDME, GSDMD and caspases cleavage.

a GSDMD, GSDME, CASP1, CASP3, CASP6, CASP8, IL1B and IL18 cleavage in controls (#1~#6) and silicosis patients (#7~#14) lung tissues. b Grayscale analysis of (a). c Relative protein cleavage in the BALF of controls (207, 209, 223, 233 and 257) and silicosis patients (356, 387, 392, 398, 194897 and cf). d Grayscale analysis of (c). e Gsdmd, Gsdme, Caspase-1, Caspase-3, Caspase-6, Caspase-8, IL-1β and IL-18 cleavage in mouse BALF, n = 4. f Grayscale analysis of (e). Human data were presented as the median ± 95% CI, and analyzed with Mann-Whitney U test. e was expressed as mean ± SD. NS, not significant; *PPP<0.001.</p

<i>In vivo</i> administration of caspase inhibitors upon silica stimulation reduces immune cell infiltration and silicosis pathology.

a, Caspase inhibitors intervene strategy. b, Western blot of Gsdmd, Gsdme, Caspase-1, Caspase-3, Caspase-6, Caspase-8, IL-1β and IL-18 activation in collected BALF from mice treated as indicated, n = 2. c, Relative number of macrophages, monocytes and neutrophils in the whole lungs of mice 14 days after instillation of PBS, silica and the indicated inhibitors, n = 5. d, The number of macrophages, monocytes and neutrophils in lung tissue of mice that installed with PBS, silica and indicated inhibitors, n = 5. e, Immunofluorescence images of macrophages (CD11b+F480+), neutrophils (CD11b+Ly6G+) and monocytes (CD11b+Ly6C+) in lung tissues of mice. Arrowheads indicate the infiltrated immune cells that labelled with antibodies against CD11b (green), F4/80 (red) and Ly6G/6C (red). DAPI (grey) localizes with the nuclei. The scale bar represents 50 μm. f, H&E (left) and Masson (right) staining of the indicated mouse lung sections 14 days after the initial silica challenge. The scale bar represents 100 μm. z-VAD, z-VAD-FMK; 3is, VX765+z-DEVD-FMK+z-IETD-FMK. z-VAD-FMK, pan-caspase inhibitor; VX765, Caspase-1 inhibitor; z-DEVD-FMK, Caspase-3 inhibitor; z-IETD-FMK, Caspase-8 inhibitor. The total dosage of injected caspase inhibitor(s) was 0.25 mg per mouse administered once. Results are expressed as mean ± SD from three independent experiments. NS, not significant; *PPP<0.001.</p

Inactivation of Gsdmd and Gsdme by dimethyl fumarate rescue mouse silicosis.

a The activation of Gsdmd, Gsdme and Caspase-1 was measured through immunoblot analysis, both in WT and Gsdmd-/- BMDMs. b, c The release of IL-1β and IL-18 in WT and Gsdmd-/- BMDMs culture medium. d Immunofluorescence images of macrophages (CD11b+F480+), neutrophils (CD11b+Ly6G+) and monocytes (CD11b+Ly6C+) in lung tissues of mice. Arrowheads indicate the infiltrated immune cells that labelled with antibodies against CD11b (green), F4/80 (red) and Ly6G/6C (red). DAPI (grey) localizes with the nuclei. The scale bar represents 50 μm. e The levels of IL-1β and IL-18 in BALF of mice, n = 3. f H&E (upper) and Masson (lower) staining of the indicated mouse lung sections 14 days after the initial silica challenge. The scale bar represents 100 μm. g The activation of Gsdmd, Gsdme and Caspase-1 was measured through immunoblot analysis. Results are expressed as mean ± SD from three independent experiments. NS, not significant; **PP<0.001.</p

<i>In vivo</i> treatment of caspase inhibitors after silica exposure alleviates immune cells infiltration and silicosis pathology.

Related to Fig 6. a, Caspase inhibitors treatment strategy. b, The levels of IL-1β and IL-18 in BALF of mice, n = 3. c, Relative number of macrophages, monocytes and neutrophils in lung tissue of mice that installed with PBS, silica and indicated inhibitors, n = 3. d, The number of macrophages, monocytes and neutrophils in the whole lungs of mice 14 days after instillation of PBS, silica and the indicated inhibitors, n = 3. e, Immunofluorescence images of macrophages (CD11b+F480+), neutrophils (CD11b+Ly6G+) and monocytes (CD11b+Ly6C+) in lung tissues of mice. Arrowheads indicate the infiltrated immune cells that labelled with antibodies against CD11b (green), F4/80 (red) and Ly6G/6C (red). DAPI (grey) localizes with the nuclei. The scale bar represents 50 μm. f, H&E (upper) and Masson (lower) staining of the indicated mouse lung sections 14 days after the initial silica challenge. The scale bar represents 100 μm, n = 3. g, The Szapiel scores of the H&E staining and the Ashcroft scores of Masson staining. Results are expressed as median ± 95% CI. z-VAD, z-VAD-FMK, pan-caspase inhibitor; 3is, VX765+z-DEVD-FMK+z-IETD-FMK. VX765, Caspase-1 inhibitor; z-DEVD-FMK, Caspase-3 inhibitor; z-IETD-FMK, Caspase-8 inhibitor. The total dosage of injected caspase inhibitor(s) was 0.25 mg per mouse administered once. a-f Results are expressed as mean ± SD from three independent experiments. NS, not significant; *PPP (TIF)</p

Silencing of Caspase-3, Caspase-6 and Caspase8 reduced silica-induced cell death.

Related to Fig 4. a, Immunoblotting analysis of the siRNA efficacy targeting Caspase-3, Caspase-6 and Caspase-8 in WT, Gsdme-/-, Caspase-1-/-, Gsdmd-/- and Nlrp3-/- BMDMs. b, Images of Caspase-3, Caspase-6 or Caspase-8 downregulated-BMDMs that stimulated with silica or ATP. Arrowheads indicate pyroptotic cells. Scale bar represents 100 μm (data representative of three independent experiments). C, Gsdmd, Gsdme, Caspase-1, Caspase-3, Caspase-6, Caspase-8, IL-1β and IL-18 activation and release in both whole cell lysate and the supernatant of WT, Gsdmd-/-, Gsdme-/-, Caspase-1-/- and Nlrp3-/- BMDMs transfected with siRNA as indicated after silica or ATP treatment. * represents non-specific bands. (TIF)</p

Inhibition of Gsdmd and Gsdme-dependent pyroptosis impairs immune cells infiltration in lung.

a, Immunofluorescence images of neutrophils (CD11b+Ly6G+) and monocytes (CD11b+Ly6C+) in lung tissues of WT, Nlrp3-/-, Caspase-1-/-, Gsdmd-/-, Gsdme-/- and Gsdmd-/-Gsdme-/- mice 14 days after installation of PBS or silica, n = 3. Indicated cells were labelled with antibodies against CD11b (green), Ly6G/6C (red). DAPI (grey) localizes with the nuclei. Arrowheads indicate infiltrated immune cells. Scale bar represents 50 μm. b, Heat map of expression levels of proteins in Fig 1A, 1c and 1E, IL-18 release in the BALF of WT, Nlrp3-/-, Caspase-1-/-, Gsdmd-/-, Gsdme-/- and Gsdmd-/-Gsdme-/- mice 14 days after exposure to PBS or silica, n = 3. d,The Szapiel scores and the Ashcroft scores of lung tissues. Results are expressed as median ± 95% CI. e,f, LDH release (e) and PI positive cells (f) of WT and Gsdmd-/-Gsdme-/- BMDMs stimulated with silica for 1 h, 2 h, 4 h, 6 h and 8 h. g, Quantitative analysis of PI positive cells in f. (n = 3). Results are expressed as mean ± SD from three independent experiments. NS, not significant; *PPP (TIF)</p

Schematic of silica-induced pyroptosis and inflammation.

Schematic of silica-induced pyroptosis and inflammation.</p

Flow cytometry measurement of infiltrating immune cells in lung of mice.

a The relative number of macrophages, monocytes and neutrophils in lung tissue of mice that installed with PBS or silica, n = 3. (TIF)</p