Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: Allografts transplanted into GSDMD −/− mice reduced pyroptosis and have prolonged survival. (A) PCR‐based genotyping of WT, GSDMD +/− , and GSDMD −/− mice. The WT allele was detected at 550 bp, and the knockout allele was detected at 423 bp. (B) Schematic illustration of BALB/c donor hearts transplanted into WT or Gsdmd−/− recipients on a C57BL/6 background. (C) Kaplan–Meier survival curves of cardiac grafts. n = 6 mice per group. (D) qPCR analysis of Il1b, Il18, Il6 and Tnfα mRNA expression in Day 5 grafts. n = 4 biologically independent samples per group. (E and F) Representative immunohistochemical staining and quantification of GSDMD, N‐GSDMD, IL‐1β and TNF‐α in Day 5 grafts. n = 4 biologically independent samples per group. (G–I) Representative flow cytometry plots and quantification of infiltrating CD45 + leukocytes in Day 5 grafts. n = 5 biologically independent samples per group. (J–L) Representative flow cytometry plots and quantification of infiltrating CD8 + T cells in Day 5 grafts. n = 5 biologically independent samples per group. (M–O) Representative flow cytometry plots and quantification of infiltrating macrophages in Day 5 grafts. n = 5 biologically independent samples per group. Data are presented as mean ± SEM. ns, not significant; * p < .05; ** p < .01; *** p < .001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Knock-Out, Expressing, Immunohistochemical staining, Staining, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: GSDMD‐deficient macrophages attenuated CD8 + T cell recruitment and activation through IL‐1β. (A) Schematic illustration of the macrophage–CD8 + T cell Transwell co‐culture assay. BALB/c donor hearts were transplanted into GSDMD‐WT or GSDMD‐CKO recipients. On post‐operative Day 5, CD68 + macrophage‐enriched cells were isolated from cardiac grafts and spleens. CD8 + T cells were isolated from WT C57BL/6 spleens and activated with anti‐CD3/CD28 before co‐culture. Activated CD8 + T cells were seeded in the upper chamber, and macrophages with TNF‐α and IL‐6 stimulation were placed in the lower chamber. (B) Representative flow cytometry plots showing the percentage of CD8 + T cells among cells collected from the lower chamber after Transwell co‐culture. (C) Representative flow cytometry plots showing TNF‐α + CD8 + T cells among cells collected from the lower chamber after Transwell co‐culture. (D) Quantification of the number of migrated CD8 + T cells in the lower chamber. n = 6 biologically independent samples per group. (E) Quantification of TNF‐α + CD8 + T cells in the lower chamber. n = 6 biologically independent samples per group. Data are presented as mean ± SEM. *** p < .001; **** p < .0001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Activation Assay, Co-culture Assay, Isolation, Co-Culture Assay, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: TNF‐α/IL‐6 induces GSDMD upregulation in macrophages via NF‐κB/STAT3 during acute rejection. (A) Volcano plot showing differentially expressed genes in macrophages between Day 1 and Day 5 after transplantation. (B) Pathway enrichment analysis showing activation of inflammatory pathways, including interferon responses, allograft rejection, TNF‐α signalling via NF‐κB, and IL‐6/JAK/STAT3 signalling. (C) Gene ontology analysis showing enrichment of biological processes related to type II interferon response, cellular response to cytokine stimulus, and positive regulation of cytokine production. (D and E) qPCR analysis of Stat3, Nfkb, Gsdmd, Il1b, Ifna and Ifng mRNA expression in RAW264.7 macrophages stimulated with TNF‐α or IL‐6. n = 3 independent experiments per group. (F) Western blot analysis of STAT3, p‐STAT3, p‐NF‐κB, GSDMD, N‐GSDMD, IFN‐α and IFN‐γ expression in RAW264.7 macrophages after TNF‐α or IL‐6 stimulation. (G and H) qPCR analysis of Gsdmd, Il1b, Ifna and Ifng mRNA expression in Day 5 allogeneic heart grafts after treatment with BAY 11–7082 or Stattic. n = 4 biologically independent samples per group. (I and J) Dual‐luciferase reporter assay showing STAT3‐induced GSDMD promoter activity and dose‐dependent activation of the GSDMD promoter by STAT3. n = 3 independent experiments per group. (K and L) Dual‐luciferase reporter assay showing NF‐κB‐induced GSDMD promoter activity and dose‐dependent activation of the GSDMD promoter by NF‐κB. n = 3 independent experiments per group. Data are presented as mean ± SEM. ** p < .01; *** p < .001; **** p < .0001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Transplantation Assay, Activation Assay, Expressing, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: Proposed working model of macrophage GSDMD‐mediated pyroptosis in acute cardiac allograft rejection. After heart transplantation, recipient‐derived macrophages and CD8 + T cells progressively infiltrate the cardiac graft during the acute rejection stage. Recipient‐derived macrophages in acute rejection undergo M1‐like polarization under the influence of interferon signalling, IL‐6, and TNF‐α. These inflammatory signals activate NF‐κB/STAT3 signalling, leading to increased GSDMD and IL‐1β expression, GSDMD cleavage, N‐GSDMD pore formation, and IL‐1β release. NU6300 and disulfiram inhibit GSDMD‐mediated pyroptosis. Macrophage‐derived IL‐1β further enhances CD8 + T‐cell recruitment and activation, promoting immune crosstalk between macrophages and T cells and contributing to acute cardiac allograft rejection.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Transplantation Assay, Derivative Assay, Expressing, Activation Assay