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

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA inhibits the secretion of inflammatory factors and glycolysis in macrophages (A and B) Schematic diagram of THP-1 cell (A) and BMDMs (B) activation into pro-inflammatory macrophages. Created with BioRender.com. (C) The concentrations of IL-6, TNF-α, and IL-1β in THP-1 cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (D) The concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with different concentrations of 3-HPA (0, 0.625, 1.25, 5 mM) followed by LPS stimulation. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (E) The concentrations of IL-6, TNF-α, and IL-1β in BMDM cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL) or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Bubble plot of KEGG pathway enrichment analysis for differentially expressed genes between LPS (100 ng/mL) and LPS+3-HPA (5 mM) treated in THP-1 cells. The size of each bubble represents the number of differentially expressed genes, and the color indicates the enrichment factor. (G and H) Pyruvate and lactate levels in THP-1 cells (G) and BMDMs (H) treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM). (I) Immunoblots of protein expression levels of HK, GAPDH, PKM, and LDHA in THP-1 cells treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM), and quantitative results of GAPDH. (J) The mRNA levels of GAPDH in THP-1 cells treated with LPS (100 ng/mL) or LPS+3-HPA (5 mM). (K) GAPDH activity assay in THP-1 cells and BMDMs treated with PBS, LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data in (G–K) are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Mouse IL-6 Precoated ELISA Kit , Dakewe , 1210602.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA enhances the metabolite content of the TCA cycle and mitochondrial oxidation (A) Schematic diagram of THP-1 with 2-DG treatment. Created with BioRender.com. (B) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS, LPS+3-HPA, LPS+2-DG, or LPS+3-HPA+2-DG. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (C) Schematic diagram of THP-1 with high glucose treatment. Created with BioRender.com. (D) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS+3-HPA, LPS+3-HPA+glucose. Data are the means ± SD and n = 3 per group. Statistical significance was determined using unpaired Student’s t test with ∗∗∗p < 0.001; ns, not significant. (E) KEGG pathway enrichment analysis of metabolic pathways in BMDM cells treated with LPS or LPS+3-HPA. (F) Relative abundance of metabolite (ornithine, citrulline, L-malate, succinic acid, trans-aconitic acid, cis-aconitic acid) in BMDM cells treated with LPS or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using unpaired Student’s t test with ∗p < 0.05; ∗∗p < 0.01. (G) Correlation network of metabolites and genes in the metabolic pathway. Nodes represent metabolites (blue squares) and genes (colored circles). Gray edges indicate pairwise correlations between metabolites and genes. The colors similarly represent expression levels, with red typically indicating higher expression and blue indicating lower expression compared to the mean. (H) Schematic diagram of arginine metabolism and the TCA cycle. (I) Heatmap of mitochondrial oxidation-related gene expression associated with differentially expressed metabolites. Red indicates relatively high gene expression, while blue indicates relatively low gene expression within each row. (J) Schematic diagram of the catalytic function of the GAPDH enzyme. (K) Concentrations of the NAD + /NADH ratio in THP-1 cells and BMDM cells treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (L) ATP concentrations in THP-1 cells and BMDMs treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, not significant. (M) Representative images and mitochondrial analysis of BMDM cells treated with PBS, LPS, or LPS+3-HPA. Scale bars, 1 μm (upper) and 0.25 μm (lower). For mitochondrial number, n = 8–12 ( n = 12 for PBS, n = 8 for LPS, n = 9 for LPS+3-HPA group).

Article Snippet: Mouse IL-6 Precoated ELISA Kit , Dakewe , 1210602.

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH carboxyethylation inhibited macrophage glycolysis and the release of inflammatory factors (A) Schematic workflow illustrating the strategy of silencing endogenous GAPDH via 3′UTR-targeting siRNA and overexpressing exogenous GAPDH. (B) Immunoblot and quantitative analysis of GAPDH protein in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01. (C) Relative mRNA expression of GAPDH in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗∗∗p < 0.0001. (D) Immunoblot analysis of FLAG-tagged exogenous GAPDH(E) and GAPDH in 293 T cells. Knockdown of endogenous GAPDH with siRNA followed by the overexpression of GAPDH (E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test with ∗p < 0.05; ns, not significant. (E) Relative mRNA expression of GAPDH in 293 T cells knockdowned endogenous GAPDH (siGAPDH) and overexpressed GAPDH (C) and GAPDH (E), respectively. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (F) GAPDH activity assay in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. (G) Concentrations of lactate and pyruvate in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (H) Relative mRNA expression of IL-6 , TNF-α , and IL-1β in THP-1 cells which transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (I) The concentration of TNF-α in THP-1 cells that overexpressed GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Mouse IL-6 Precoated ELISA Kit , Dakewe , 1210602.

Techniques: Western Blot, Transfection, Expressing, Knockdown, Over Expression, Activity Assay, Concentration Assay