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

Figures

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  FIGURE 1. GSDMD‐mediated pyroptosis was upregulated during the acute immune rejection stage of mouse cardiac transplantation. (A) Schematic showing reanalysis of the previously published scRNA‐seq dataset of mouse cardiac grafts collected at Day 0, Day 1, Day 3 and Day 5 post‐transplantation (GSA dataset: CRA005239). (B) Differential expression analysis showing changes in pyroptosis‐related genes, including Gsdmd and Il1b, during rejection progression. (C) Schematic illustration of sampling time points. Days 1–3 was considered the late ischemia/reperfusion stage, whereas Day 5 was defined as the acute immune rejection stage. (D and E) qPCR analysis of Il1b and Gsdmd mRNA expression in naïve hearts, syngeneic grafts, and allogeneic grafts. (F and G) Representative western blot images and quantification of GSDMD, N‐GSDMD, and IL‐1β expression in allogeneic grafts. (H and I) Representative immunohistochemical staining and quantification of GSDMD, N‐GSDMD and IL‐1β in naïve hearts and allogeneic grafts. For D, E, G and I, n = 4 biologically independent samples per group. Data are presented as mean ± SEM. ns, not significant; **p < .01; ***p < .001; ****p < .0001.

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  FIGURE 2. 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…

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  FIGURE 3. Increased M1 macrophage expression and its GSDMD‐mediated pyroptosis during acute cardiac rejection. (A) Dot plot showing the average expression levels of representative marker genes used for macrophage subset annotation, including M1‐like macrophages (Nos2, Il1b, Cxcl9, Cxcl10, Cd40, H2‐Aa, H2‐Ab1, Stat1 and Irf1), M2‐like macrophages (Arg1, Mrc1, Spp1 and Cxcl3), and resident macrophages (Folr2, C1qa, C1qb and C1qc). Cd68 was used as a macrophage‐associated marker to support macrophage identity. (B) UMAP visualization of M1‐like, M2‐like, and resident macrophage populations in cardiac grafts. (C) UMAP visualization of macrophages coloured by sampling time points. (D) UMAP plots showing macrophage subset distribution at Day 0, Day 1, Day 3 and Day 5 post‐transplantation. (E) Proportional distribution of macrophage subsets across different time points. (F) Feature plots showing Nos2, Gsdmd and Il1b expression in macrophages at the indicated time points. (G) qPCR analysis of Gsdmd, Il1b, Il18 and Tnfα mRNA expression in sorted macrophage‐enriched cells from nave hearts and allogeneic grafts. n = 4 biologically independent samples per group. (H and I) Representative western blot…

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  FIGURE 4. The majority of macrophages are derived from the transplant recipient during acute cardiac rejection. (A) UMAP visualization showing donor‐derived, recipient‐derived, and unknown‐origin cells in cardiac grafts. (B) UMAP plots showing donor–recipient cell‐origin distribution at Day 0, Day 1, Day 3 and Day 5 post‐transplantation. (C) Proportional distribution of major cell populations among donor‐derived, recipient‐derived, and unknown‐origin cells. (D) Proportional analysis of donor‐derived, recipient‐derived, and unknown‐origin cells within each major cell type across different time points. (E) Schematic illustration of BALB/c donor hearts transplanted into CAG‐EGFP C57BL/6 recipient mice, followed by graft collection on Days 3 and 5 for flow cytometry and immunofluorescence analysis. (F) Representative flow cytometry gating strategy for identifying GFP+ macrophages among live CD45+CD11b+F4/80+ cells. (G) Representative flow cytometry plots showing GFP+ cells among graft‐infiltrating macrophages in negative control, positive control and Day 5 allografts. (H) Quantification of GFP+ cells among graft‐infiltrating macrophages. n = 3 for negative control, positive controls and Day 5…

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  FIGURE 5. Recipient‐derived GSDMD‐deficient macrophages can prolong cardiac graft survival. (A) Schematic illustration of macrophage‐specific GSDMD knockout mice and PCR‐based genotyping of Lyz2Cre and floxed Gsdmd alleles. For Lyz2Cre genotyping, the mutant allele was detected at 700 bp and the WT allele at 350 bp. For floxed Gsdmd genotyping, the target allele was detected at 393 bp and the WT allele at 210 bp. (B) Survival analysis of cardiac allografts transplanted into GSDMD‐WT or GSDMD‐CKO recipients. n = 6 mice per group. (C) Representative flow cytometry plots showing infiltrating CD45+ leukocytes, CD8+T cells, and macrophages in Day 5 grafts from GSDMD‐WT and GSDMD‐CKO recipients. (D–F) Quantification of the percentages of CD45+ leucocytes, CD8+T cells, and macrophages in Day 5 grafts. n = 6 biologically independent samples per group. (G–I) Quantification of the absolute numbers of CD45+ leucocytes, CD8+T cells, and macrophages in Day 5 grafts. n = 6 biologically independent samples per group. (J and K) Quantification of the percentages of TNF‐α+ and IL‐1β+ macrophages in Day 5 grafts. n = 6 biologically independent samples per group. (L and M) Quantification of the absolute…

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  FIGURE 6. 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.

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  FIGURE 7. 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…

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  FIGURE 8. IL‐1β can reverse the survival extension of cardiac grafts mediated by macrophage GSDMD deficiency. (A) PCR‐based genotyping of WT and Il1r1−/− mice. The WT allele was detected at 310 bp, and the mutant allele was detected at 150 bp using allele‐specific PCR. (B) Schematic illustration and survival analysis of WT or Il1r1−/− C57BL/6 donor hearts transplanted into BALB/c recipients. n = 6 mice per group. (C) Schematic illustration and survival analysis of BALB/c donor hearts transplanted into WT or Il1r1−/− C57BL/6 recipients. n = 6 mice per group. (D) Schematic illustration of recombinant IL‐1β administration in GSDMD‐CKO recipients and survival analysis of cardiac allografts. Recombinant mouse IL‐1β was administered intraperitoneally at 100 µg/kg every other day after transplantation. n = 6 mice per group. (E and F) Representative immunohistochemical staining and quantification of CD8 and F4/80 in Day 5 grafts from GSDMD‐WT, GSDMD‐CKO and GSDMD‐CKO + IL‐1β groups. n = 4 biologically independent samples per group. (G) Representative flow cytometry density plots showing infiltrating CD45+ leukocytes, CD8+T cells and F4/80+ macrophages in Day 5 grafts from GSDMD‐WT, GSDMD‐CKO and…

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  FIGURE 9. GSDMD inhibitors improved cardiac transplant survival and reduces immune cell infiltration. (A) Schematic illustration of NU6300 or disulfiram administration after cardiac transplantation. NU6300 or disulfiram was administered intraperitoneally every other day after transplantation. (B) Survival analysis of cardiac allografts in vehicle‐ and NU6300‐treated recipients. n = 6 mice per group. (C) Survival analysis of cardiac allografts in vehicle‐ and disulfiram‐treated recipients. n = 6 mice per group. (D, E) Representative western blot images and quantification of GSDMD and N‐GSDMD expression in Day 5 grafts from vehicle‐ and NU6300‐treated recipients. n = 4 biologically independent samples per group. (F, G) Representative western blot images and quantification of GSDMD and N‐GSDMD expression in Day 5 grafts from vehicle‐ and disulfiram‐treated recipients. n = 4 biologically independent samples per group. (H) Western blot analysis of GSDMD and N‐GSDMD expression in Day 5 allogeneic grafts treated with increasing doses of NU6300. (I) Western blot analysis of GSDMD and N‐GSDMD expression in Day 5 allogeneic grafts treated with increasing doses of disulfiram. (J and K) Representative…

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  FIGURE 10. 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.