The GSDMD-mediated pyroptosis is activated in psoriasis.

a) Expression of GSDMD in patients with psoriasis patients and healthy people from GSE178197, GSE161683 and GSE153007.

b) Representative immunohistochemical staining of GSDMD in sections of skin tissue from healthy individuals and patients with psoriasis (n=3-5, each group). Scale bar =100µm.

c) Schematic representation of the IMQ-induced psoriasis mouse model.

d) Representative images and statistical analysis of western blot analysis showing the expression of GSDMD and its N-terminal fragments in dorsal skin of WT mice treated with vehicle or IMQ at day 4 (n=4).

e) Representative images and statistical analysis of western blot analysis showing the expression level of pro-caspase1 and caspase1 in dorsal skin of WT mice treated with vehicle or IMQ at day 4.

psn, psoriasis non-lesional skin; ps, psoriasis skin; con, control; HD, healthy donor; GPP, generalized pustular psoriasis; IMQ, imiquimod; WT, wild-type. Error bars show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P ≤ 0.0001. Data are representative of three independent experiments for (b, d, e).

GSDMD deficiency mitigates psoriasis-like inflammation in mice.

a) Macroscopic phenotypic representation of the dorsal skin in WT and GSDMD-KO mice treated with IMQ at day 4.

b) Disease severity of psoriasis induced by IMQ in mice as assessed by PASI score (n=10-12).

c) Representative images and statistical analysis of hematoxylin and eosin staining of the dorsal skin in WT and GSDMD-KO mice treated with IMQ at day 4 (n=5). Scale bar =100µm.

d) Representative images and statistical analysis of western blot analysis showing the expression level of GSDMD and caspase1 in dorsal skin of WT and GSDMD-KO mice treated with IMQ at day 4 (n=4).

e) Quantitative PCR analysis of the relative mRNA expression of proinflammatory cytokines in the dorsal skin of WT and GSDMD-KO mice treated with IMQ at day 4 (n = 4). Data were normalized to a reference gene, Gapdh.

f) ELISA analysis of IL-6 and IL-1β per 1 mg of the dorsal skin from WT and GSDMD-KO mice treated with IMQ at day 4 (n=5).

IMQ, imiquimod; WT, wild-type; KO, knockout; PASI, psoriasis Area and Severity Index. Error bars show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of three independent experiments for (a, c, d).

Neutrophils undergo GSDMD mediated pyroptosis in psoriasis.

a) Expression of GSDMD in neutrophils in psoriasis patients and healthy people from GSE123785.

b) Representative immunohistochemical staining of CD66b and GSDMD in two consecutive sections of skin tissue from patients with psoriasis vulgaris or generalized pustular psoriasis (left); representative immunohistochemical staining of CD66b (brown) and GSDMD (red) in sections of skin tissue from patients with psoriasis vulgaris (n=3-5, each group). Scale bar =100µm.

c) Dot plot of Gsdmd expression in each cell type in the skin of IMQ-induced psoriasis-like mice (GSE165021).

d) Representative immunofluorescence of GSDMD (red), Ly6G (green) and nuclear (blue) in dorsal skin of WT mice treated with vehicle, WT mice treated with IMQ and GSDMD-KO mice treated with IMQ at day 5. Scale bar =50µm.

e) ssGSEA analysis showed the expression of pyroptosis-related genes in neutrophils infiltrating in control and IMQ-induced psoriasis-like tissue (GSE165021).

f) Representative and statistical graphs of the mean fluorescence intensity of propidium iodide in neutrophils from skin of WT mice as detected by flow cytometry(n=3).

ps, psoriasis skin; con, control; PV, psoriasis vulgaris; GPP, generalized pustular psoriasis; IMQ, imiquimod; WT, wild-type; MFI, mean fluorescence intensity; PI, propidium iodide; MFO, Fluorescence Minus One. Error bars show mean ± SEM. *P < 0.05, ****P < 0.0001. Data are representative of three independent experiments for (b, d-e).

GSDMD depletion in neutrophils attenuates the development of skin inflammation in psoriasis.

a) Representative images of western blot showing the expression of GSDMD in bone marrow-derived neutrophils of cKO and isotype control mice.

b) Macroscopic phenotypic representation of the dorsal skin in control and GSDMD-cKO mice treated with IMQ at day 4.

c) Disease severity of psoriasis induced by IMQ in mice as assessed by PASI score (n=7).

d) Representative images and statistical analysis of hematoxylin and eosin staining of the dorsal skin in control and GSDMD-cKO mice treated with IMQ at day 4 (n=5). Scale bar =100µm.

e) Quantitative PCR analysis of the relative mRNA expression of proinflammatory cytokines in the dorsal skin of control and GSDMD-cKO mice treated with IMQ at day 4 (n = 4). Data were normalized to a reference gene, Gapdh.

f) ELISA analysis of IL-6 and IL-1β per 1 mg of the dorsal skin from control and GSDMD-cKO mice treated with IMQ at day 4(n=5).

g) Representative images and statistical analysis of western blot analysis showing the expression level of GSDMD and its N-terminal fragments in dorsal skin of WT, cKO and Gsdmd-/- mice treated with IMQ at day 4 (n=4).

h) Schematic representation of the use of anti-Ly6G antibody in the IMQ-induced psoriasis mouse model.

i) Macroscopic phenotypic representation and PASI score of cKO mice and control mice treated with IMQ and Ly6G antibody.

cKO, conditional knockout. Error bars show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Data are representative of three independent experiments for (a, b, d, g, i).