Research ArticleImmunology

IRF2 transcriptionally induces GSDMD expression for pyroptosis

See allHide authors and affiliations

Science Signaling  21 May 2019:
Vol. 12, Issue 582, eaax4917
DOI: 10.1126/scisignal.aax4917
  • Fig. 1 A forward genetic screening in mice identifies an Irf2 mutation that abolishes inflammasome signaling.

    (A) Screening of G3 offspring from ENU-treated C57BL/6 mice. The graph indicates the amount of IL-1β released from Pam3CSK4-primed BMDMs after culture for 2 hours with ATP or medium alone (cont). The green dot represents IGL03671 G3 12 (fig. S1, A and B). The gray dots represent other G3 mice in the same batch from multiple pedigrees. (B) WT and IGL03671 Irf2 genes. The exon 5 coding sequence is in uppercase, and the SNV mutation is in green and highlighted with an asterisk. Gray boxes represent exons. (C) RT-PCR analysis of full-length and truncated splicing variant Irf2 transcripts in BMDMs from the indicated mice. Rpl19, control housekeeping gene. (D) Western blotting analysis of IRF2 in extracts of BMDMs from the indicated mice. (E) Analysis of the amounts of IL-1β (top) and LDH (bottom) released from unprimed or Pam3CSK4-primed BMDMs that were untreated (cont), stimulated with ATP (for 4 hours), transfected with LPS (16 hours), or stimulated with FasL (16 hours). Data are means (bars) of at least three individual replicates (circles) from two mice per genotype. Data in (C) to (E) are representative of at least three independent experiments.

  • Fig. 2 IRF2 is essential for Gsdmd transcript expression in iMACs.

    (A) Measurement of LDH release from the indicated unprimed or Pam3CSK4-primed iMACs stimulated by transfection with LPS or treated with FasL for 16 hours. Luc, Luciferase. Data are means (bars) of three individual replicates (circles). (B) Volcano plot of RNA-seq data. Data are values (dots) of differentially expressed genes (log2 of the fold change was <−1 or >+1; −log10 P value: >1.3) between control Luc gRNA iMACs and IRF2 gRNA2 KO iMACs highlighted in red and blue. Data are from three individual replicates per line. (C) Measurement of the relative amounts of Gsdmd, Casp11, and Casp1 mRNAs (expressed as fold change) in the indicated iMACs stimulated with Pam3CSK4 for 6 hours. Data are means (bars) of four individual replicates (circles). (D) Western blotting analysis of IRF2, GSDMD, caspase-11, and caspase-1 in extracts from the indicated iMACs that were stimulated with Pam3CSK4 for 6 hours. (E) Analysis of cell death in the indicated Pam3CSK4-primed (LPS) or unprimed (FasL) iMACs as determined by measurement of the percentage of YOYO-1+ cells in a live-cell imaging analysis after Amaxa-based electroporation with LPS or stimulation with FasL for the indicated times. Data are means (circles) ± SD (shaded area) of three individual replicates. (F) Western blotting analysis of IRF2 and caspase-11 in the supernatants (sup) and extracts (ext) of the indicated iMACs 2 hours after they were electroporated with LPS. Data in (A) and (C) to (F) are representative of at least three independent experiments.

  • Fig. 3 IRF2 directly drives GSDMD mRNA expression by binding to a consensus site proximal to the GSDMD TSS.

    (A) Schematics of the GSDME (top) and GSDMD (bottom) loci with IRF2 ChIP-seq peaks. The IRF2-binding consensus sequence is in green, and the GSDMD TSS is underlined in the genomic sequence. (B) Analysis of GSDMD promoter activity in a reporter assay and schematics of the IRF2 motif mutations. HEK 293T cells were cotransfected with a Gsdmd promoter luciferase reporter gene with the indicated amounts of mock plasmid or of plasmid expressing IRF2. Reporter gene activation was measured by luciferase activity. Data are means (bars) of three individual replicates (circles). (C) CRISPR tiling screen to identify functional Gsdmd promoter regions. Each dot represents the fold enrichment in gRNA after electroporation with LPS. Data are from three individual replicates. (D) Analysis of the relative amounts of Gsdmd, Casp11, and Irf2 mRNAs in the indicated knock-in iMAC clones (fig. S6A) or parental cells stimulated with Pam3CSK4 for 6 hours. Data are means (bars) of four individual replicates (circles). (E) Cell death (percentage cytotoxicity as measured by CellTiter-Glo) of iMACs 16 hours after electroporation with LPS or Cyto-c. Data are means (bars) of three individual replicates (circles). Data in (B), (D), and (E) are representative of at least three independent experiments.

  • Fig. 4 IRF2 is indispensable for GSDMD expression and pyroptosis in BMDMs.

    (A) Left: Western blotting analysis of GSDMD, IRF2, caspase-11, and caspase-1 in WT, Irf2−/−, and Gsdmd−/− BMDMs stimulated with Pam3CSK4 for 6 hours. Right: Quantified GSDMD band intensity values are means ± SEM from three independent experiments. ****P < 0.0001 by paired Student’s t test. (B) Analysis of the relative amounts of Gsdmd, Casp11, and Il1b mRNAs in BMDMs from the indicated mice treated with Pam3CSK4 for 6 hours. N.D., not detectable. Data are means (bars) of four individual replicates (circles) of two mice per genotype. (C) Measurement of IL-1β secretion and LDH release from the indicated BMDMs stimulated with ATP (4 hours) or after Amaxa-mediated electroporation with LPS (4 hours) or Cyto-c (16 hours). Data are means (bars) of at least three individual replicates (circles) from two mice per genotype. (D) Analysis of cell death in Pam3CSK4-primed BMDMs from the indicated mice as determined by measuring the percentage of YOYO-1+ cells in a live-cell imaging analysis after Amaxa-mediated electroporation with LPS or Cyto-c. Data are means (circles) ± SD (shaded area) of three individual replicates. Data in (A) to (D) are representative of at least three independent experiments.

  • Fig. 5 IRF1 plays a compensatory role in the absence of IRF2.

    (A) Gsdmd promoter activity as determined by luciferase reporter assay. HEK 293T cells were transfected with the indicated amounts of IRF-expressing plasmid or mock plasmid. Reporter gene activation was measured by luciferase assay. Data are means (bars) of three individual replicates (circles). (B) Left: Western blotting analysis of GSDMD, IRF1, IRF2, and caspase-4 in the indicated human EA.hy926 cells. Right: Quantified GSDMD band intensity values are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. (C) Analysis of cell death as determined by measurement of the percentage of YOYO-1+ EA.hy926 cells in a live-cell imaging time-course analysis of the cells after transfection with LPS or treatment with etoposide. Data are means (circles) ± SD (shaded area) of three individual replicates. Data in (A) to (C) are representative of at least three independent experiments.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/582/eaax4917/DC1

    Fig. S1. Homozygosity for the Irf2 point mutation correlates with low responsiveness to ATP.

    Fig. S2. Irf2splice is a hypomorphic mis-splice mutation.

    Fig. S3. Characterization of Irf2 gRNA KO iMACs.

    Fig. S4. IFNs are dispensable for the attenuation of GSDMD expression.

    Fig. S5. The GSDMD TSS-proximal IRF2 consensus motif is conserved across species.

    Fig. S6. IRF2 motif mutant alleles.

    Fig. S7. Characterization of Irf2−/− BMDMs and mice.

    Fig. S8. Expression of IRF1.

    Table S1. Bioinformatic analysis of ENU-induced SNVs present in IGL03671 pedigree.

  • This PDF file includes:

    • Fig. S1. Homozygosity for the Irf2 point mutation correlates with low responsiveness to ATP.
    • Fig. S2. Irf2splice is a hypomorphic mis-splice mutation.
    • Fig. S3. Characterization of Irf2 gRNA KO iMACs.
    • Fig. S4. IFNs are dispensable for the attenuation of GSDMD expression.
    • Fig. S5. The GSDMD TSS-proximal IRF2 consensus motif is conserved across species.
    • Fig. S6. IRF2 motif mutant alleles.
    • Fig. S7. Characterization of Irf2−/− BMDMs and mice.
    • Fig. S8. Expression of IRF1.
    • Table S1. Bioinformatic analysis of ENU-induced SNVs present in IGL03671 pedigree.

    [Download PDF]

Stay Connected to Science Signaling

Navigate This Article