Research ArticleIMMUNOLOGY

Gasdermin D in macrophages restrains colitis by controlling cGAS-mediated inflammation

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Science Advances  20 May 2020:
Vol. 6, no. 21, eaaz6717
DOI: 10.1126/sciadv.aaz6717
  • Fig. 1 GSDMD is activated in the intestine of DSS-induced colitis mice and protects against DSS-induced colitis.

    (A) Immunoblot analysis of GSDMD expression in various organs, including heart, liver, kidney, lung, mLNs, spleen, small intestine (SI), colon, cortex, midbrain, and cerebellum from GSDMD−/− and wild-type (WT) mice. (B) Immunofluorescence labeling of GSDMD (red), 4′,6-diamidino-2-phenylindole (DAPI) (blue) in colon sections from WT mice, and labeling of GSDMD (green), Cx3cr1 (red), and DAPI (blue) in colon sections from Cx3cr1 reporter mice. The merging of GSDMD with E-cadherin or Cx3cr1 is indicated by arrows. Scale bar, 200 μm. (C) Immunoblot analysis of full-length and cleaved GSDMD and caspase-11 in colon from control or DSS-treated WT mice on day 9. (D) Immunoblot analysis of full-length and cleaved GSDMD in IECs and myeloid cells sorted from colon of DSS-treated WT mice on day 9. (E and F) Age-matched male WT (n = 12) and GSDMD−/− (n = 12) mice were given 2.5% DSS in their drinking water for 6 days and then normal water for three further days. Weight changes (E) and DAI (F) were monitored daily. (G) Gross morphology images of colons and colon lengths of WT and GSDMD−/− mice on day 9 after DSS treatment. (H) Representative H&E staining of distal colon sections and histology scores of WT and GSDMD−/− mice on day 9 after DSS treatment. Scale bars, 100×, 100 μm; 200×, 200 μm. (I and J) Daily weight changes (H) and DAI (I) of WT (n = 12) and ASC−/− (n = 12) mice treated with 2.5% DSS. (K) Gross morphology images of colons and colon lengths of WT and ASC−/− mice on day 9 after DSS treatment. (L) Representative H&E staining of distal colon sections and histology scores of WT and ASC−/− mice sampled on day 9 after DSS treatment. Scale bars, 100×, 100 μm; 200×, 200 μm. Data are representative of three independent experiments (A and B). Data are pooled from three independent experiments for (E) to (L). Error bars show means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test for weight changes and DAI and two-tailed unpaired Student’s t test for colon length and histology scores. Photo credit: (G) and (K) were taken by D.Y. (Department of Immunology, Nanjing Medical University).

  • Fig. 2 GSDMD deficiency exacerbates colonic inflammation but does not impair the production of AMPs and mucus secretion.

    (A) RT-qPCR analysis of the relative mRNA expression of Il6, Tnf, and Ccl2 mRNA levels in colonic tissues of WT and GSDMD−/− mice (n = 10 mice per group) on day 9 after DSS treatment. (B) ELISA of IL-6, TNF-α, CCL2, IL-1β, and IL-18 protein levels in supernatant of colonic explants of WT and GSDMD−/− mice (n = 10 mice per group) on day 9 after DSS treatment. (C and D) Flow cytometric analysis of colon-infiltrated immune cells of WT and GSDMD−/− mice (n = 6 mice per group) on day 5 after DSS treatment. Data are presented as representative plots (C) and summary graphs of quantified percentages and absolute cell numbers (D). SSC, side scatter. (E) Flow cytometric analysis of PI+ cells gated on the CD45+CD11b+ F4/80+ in colons of WT and GSDMD−/− mice on day 5 after DSS treatment (n = 3 mice per group). Data are presented as a representative plot (left) and quantified percentages (right). (F) RT-qPCR analysis of the relative mRNA expression of AMPs (Reg3b, Reg3g, Defn5, Lysozyme1, and Muc2) and Il22 in colons of WT (n = 9) and GSDMD−/− (n = 9) mice on day 9 after DSS treatment. (G) PAS staining of distal colon sections of WT and GSDMD−/− mice on day 5 after DSS treatment. Scale bars, 200×, 200 μm; 400×, 400 μm. (H) Immunofluorescence labeling of MUC2 (green) and DAPI (blue) in WT and GSDMD−/− mice on day 5 after DSS treatment. Scale bar, 200 μm. Data are pooled from three independent experiments (A to D and F to H) or from two independent experiments (E). Error bars show means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant. Two-tailed unpaired Student’s t test.

  • Fig. 3 Macrophage-intrinsic GSDMD controls DSS-induced colitis.

    (A and B) Weight changes (A) and DAI (B) of GSDMDfl/flVillin-Cre and littermate control GSDMDfl/fl mice (n = 10 mice per group) after colitis induction. (C) Colon lengths of GSDMDfl/flVillin-Cre and littermate control GSDMDfl/fl mice on day 9 after DSS treatment. (D) Representative H&E staining of distal colon sections and histology scores of GSDMDfl/flVillin-Cre and littermate control GSDMDfl/fl mice sampled on day 9 after DSS treatment. Scale bars, 100×, 100 μm; 200×, 200 μm. (E and F) Weight changes (E) and DAI (F) of GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice (n = 8 mice per group) after colitis induction. (G) Colon lengths of GSDMDfl/flLysM-Cre and GSDMDfl/fl mice on day 9 after DSS treatment. (H) Representative H&E staining of distal colon sections and histology scores of GSDMDfl/flLysM-Cre and GSDMDfl/fl mice on day 9 after DSS treatment. Scale bars, 100×, 100 μm; 200×, 200 μm. (I to K) GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice (n = 6 mice per group) were intravenously injected with anti-Ly6g antibody to delete CD11b+Ly6g+ neutrophils during DSS treatment. Weight changes (I), DAI (J), and colon lengths (K) of DSS-treated GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice that were deleted of neutrophils. (L and M) Flow cytometric analysis of colon-infiltrated immune cells of DSS-treated GSDMDfl/flLysM-Cre and littermate control GSDMDfl/fl mice that were deleted of neutrophils. Data are presented as representative plots (L) and summary graphs of absolute cell numbers (M). Data are pooled from three independent experiments. Error bars show means ± SEM. *P < 0.05,, **P < 0.01, and ***P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test for weight changes and DAI and two-tailed unpaired Student’s t test for colon length, histology scores, and FACS.

  • Fig. 4 GSDMD deficiency enhanced cytosolic DNA-sensing pathway in colonic macrophages during colitis.

    (A) FACS gating strategy for sorting colonic macrophages. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width. (B) KEGG analysis of the most significantly enriched signaling pathways in colonic macrophages sorted from WT and GSDMD−/− mice on day 5 after DSS treatment. ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase. (C) GSEA for the genes associated with “Cytosolic DNA-sensing pathway” in colonic macrophages sorted from mice in (B). Nominal P < 0.001. (D) Gene Network Analysis of the genes associated with cytosolic DNA-sensing pathway in colonic macrophages sorted from mice in (B). Node colors increasing from white to red indicate expression-level changes in GSDMD−/− cells relative with WTs. (E) Heatmaps of genes with adjusted P < 0.05 and log2 fold change >1.2 from RNA-seq analysis of colonic macrophages sorted from mice in (B). (F) RT-qPCR analysis of the indicated genes in colonic macrophages sorted from mice in (B). Data were normalized to a reference gene, HPRT. (G) UPLC/MS analysis of cGAMP levels in colonic tissues from WT and GSDMD−/− mice (n = 4 mice per group) on day 9 after DSS treatment. (H) Immunoblot analysis of phosphorylated (p-) STING, STING, phosphorylated (p-) TBK1, TBK1, phosphorylated (p-) IRF3, IRF3, GSDMD, and β-actin (loading control) in colons from DSS-treated WT and GSDMD−/− mice on day 9. Data are representative of two independent experiments (H). Error bars show means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-tailed unpaired Student’s t test.

  • Fig. 5 GSDMD deficiency enhanced cGAS-dependent inflammation in colonic macrophages.

    (A and B) CD45+CD11b+F4/80+ colonic macrophages were sorted from the LP of WT and GSDMD−/− mice and then were transfected with gDNA from Salmonella Typhimurium (ST-DNA) for 4 hours. The mRNA expression or protein production of the indicated cytokines and chemokines was analyzed by RT-qPCR (C) or ELISA (D), respectively. (C and D) CD45+CD11b+F4/80+ colonic macrophages from LP of WT mice and GSDMD−/− mice were treated with cGAS inhibitor RU.521 (20 μg/ml) and then were transfected with ST-DNA for 4 hours. The mRNA expression or protein production of indicated cytokines and chemokines was analyzed by RT-qPCR (E) or ELISA (F), respectively. Data are pooled from three independent experiments. Error bars show means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test.

  • Fig. 6 The administration of cGAS inhibitor restricts colitis in GSDMD−/− mice.

    (A to C) Age-matched male WT (n = 8) and GSDMD−/− (n = 8) mice were intraperitoneally injected with cGAS inhibitor RU.521 at a dose of 10 mg/kg daily after DSS treatment. Weight changes (A), DAI (B), and colon lengths (C) of DSS-treated WT and GSDMD−/− mice that were injected with cGAS inhibitor. (D and E) Representative H&E staining section of colons (D) and histology score (E) of mice in (A). Scale bars, 100×, 100 μm; 200×, 200 μm. (F) ELISA of cGAMP levels in colonic tissue homogenates of mice on day 9 after DSS treatment (n = 4 mice per group). (G) Immunoblot analysis of phosphorylated STING, STING, phosphorylated TBK1, TBK1, phosphorylated IRF3, IRF3, GSDMD, and β-actin (loading control) in colons from WT and GSDMD−/− mice that were administrated with cGAS inhibitor RU.521. Data are pooled from three independent experiments (A to E) or are representative of two independent experiments (G). Error bars show means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test for weight changes and DAI and two-tailed unpaired Student’s t test for colon length and histology scores. Photo credit: (C) was taken by C.M. (Department of Immunology, Nanjing Medical University).

  • Reg3bSAs
    5-AATGGAGGTGGATGGGAATG-35-CGGTCTAAGGCAGTAGATGGGT-3
    Reg3gSAs
    5-AACAGAGGTGGATGGGAGTGG-35-CACAGTGATTGCCTGAGGAAGA-3
    defn5SAs
    5-GGCTGATCCTATCCACAAAACA-35-AGACCCTTCTTGGCCTCCA-3
    lysozyme 1SAs
    5-ACTCTGGGACTCCTCCTGCTT-35-CGGTCTCCACGGTTGTAGTTT3-3
    Il22SAs
    5-CCCTTATGGGGACTTTGGC-35-GGTGCGGTTGACGATGTATG-3
    Muc2SAs
    5-TTGCTCTGCTGTCTCCGTCA-35-ACACTGGTCTTCTCCTCCTTGC-3
    Cxcl10SAs
    5-AAGTGCTGCCGTCATTTTCTG-35-TTCCCTATGGCCCTCATTCTC-3
    Il-6SAS
    5-CTTGGGACTGATGCTGGTGAC-35-GCCATTGCACAACTCTTTTCTC-3
    TnfSAS
    5-TACTGAACTTCGGGGTGATCG-35-TCCTCCACTTGGTGGTTTGC-3
    Ccl5SAS
    5-GACACCACTCCCTGCTGCTT-35-ACACTTGGCGGTTCCTTCG-3
    Irf7SAs
    5-CAGCACAGGGCGTTTTATCTT-35-TCTTCCCTATTTTCCGTGGC-3
    IfnbSAs
    CCCTATGGAGATGA CGGAGA5-CCCAGTGCTGGAGAAATTGT-3
    Irf3SAs
    5-CTACGGCAGGACGCACAGAT-35-TCAGCAGCTAACCGCAACAC-3
    Ddx41SAS
    5-AGTCCGCCAAGGAAAAGCAA-35-CTCAGACATGCTCAGGACATAAC-3
    Ifit1SAS
    5-TGCTTTGCGAAGGCTCTGA-35-AATCTTGGCGATAGGCTACGAC-3
    Ccl20SAS
    5-TGTACGAGAGGCAACAGTCG-35-TCTGCTCTTCCTTGCTTTGG-3
    Ccl4SAS
    5-GCTCTGTGCAAACCTAACCC-35-GAAACAGCAGGAAGTGGGAG-3
    Cc12SAS
    5-CTGTGCTGACCCCAAGAAGG-35-TTGAGGTGGTTGTGGAAAAGG-3
    Polr2cSAS
    5-CGTGGTTCTCGGAGAGTTTGG-35-CCAGCGTGTCTTCATCCATTT-3
    Polr2eSAS
    5-GGTGGGCATCAAGACCATCAA-35-TCAGGGACTAGCTCGTGCTC-3
    Cxcl1SAS
    5-CACCCAAACCGAAGTCATAGC-35-TTGGGGACACCTTTTAGCATCT-3
    AscSAS
    5-TCTGGAGTCGTATGGCTTGGA-35-AGTGCTTGCCTGTGCTGGTC-3
    GsdmdSAS
    5-ATCCTGGCATTCCGAGTGG-35-CTCTGGCCCACTGCTTTTCT-3
    HprtSAS
    5-GTCCCAGCGTCGTGATTAGC-35-TGGCCTCCCATCTCCTTCA-3

Supplementary Materials

  • Supplementary Materials

    Gasdermin D in macrophages restrains colitis by controlling cGAS-mediated inflammation

    Chunmei Ma, Dongxue Yang, Bingwei Wang, Chunyan Wu, Yuqing Wu, Sheng Li, Xue Liu, Kara Lassen, Lue Dai, Shuo Yang

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