hil 15  (R&D Systems)

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: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Intercellular communication analysis predicts IL-1β as a candidate upstream signal potentially associated with neutrophils. ( A ) Chord diagram showing predicted ligand–target interactions between sender cell populations and receiver SSPCs in the NonU microenvironment. Immune cell populations contributed prominently to the inferred SSPC-directed signaling network. ( B ) Dot plot showing the top predicted ligands received by SSPCs and their expression patterns across potential sender cell populations. Dot size indicates the percentage of ligand-expressing cells, and color indicates average expression. ( C ) Heatmap showing log-fold changes in prioritized ligands in their corresponding sender populations when comparing NonU with Frac conditions. ( D ) Heatmap showing the predicted regulatory potential of prioritized ligands on SSPC target genes. Candidate transcription factors are indicated. ( E ) GO enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis-related terms are highlighted. ( F ) KEGG enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis and fatty acid metabolism pathways are highlighted. Red arrows and red-colored labels indicate representative ligands, cell populations, target genes, transcription factors, or enriched terms highlighted and discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: IL-1β induces ferroptotic stress and impairs SSPC function in vitro. ( A ) Schematic overview of SSPC isolation by fluorescence-activated cell sorting and subsequent in vitro validation experiments. ( B ) Flow cytometry gating strategy for isolating Lin − /DPP4 + SSPCs from mouse callus tissue at 7 days post-fracture. ( C ) Representative Western blot images showing the expression of ACSL4, COX2, 4-HNE, NF-κB p65, and EPAS1 in SSPCs treated with vehicle, erastin, or IL-1β. β-actin was used as the loading control. ( D ) Quantification of Western blot protein expression levels shown in panel C ( n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Oxidized lipids are indicated by increased green fluorescence and reduced red fluorescence. Scale bar = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs. Scale bar = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining and the relative FerroOrange fluorescence intensity ( n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of colony-forming unit efficiency and cell viability measured by CCK-8 assay ( n = 3). ( I ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression ( n = 3). ( J ) Representative images of tri-lineage differentiation assays, including Alizarin Red S staining for osteogenesis, Alcian Blue staining for chondrogenesis, and Oil Red O staining for adipogenesis. Scale bar = 200 μm. ( K ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression, including Runx2, Opn, Acan, Col2a1, Pparg, and Cebpa ( n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: In Vitro, Isolation, Fluorescence, FACS, Biomarker Discovery, Flow Cytometry, Western Blot, Expressing, Control, Staining, CCK-8 Assay, Quantitative RT-PCR, Marker, Gene Expression, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Pharmacological EPAS1 inhibition attenuates IL-1β-induced ferroptotic stress and differentiation impairment in primary SSPCs. ( A ) Representative images of Alcian Blue and Alizarin Red S staining in primary SSPCs treated with vehicle control, IL-1β, IL-1β plus PT2385, or PT2385 alone. Scale bars = 200 μm. ( B ) Representative Western blot images showing ACSL4, 4-HNE, EPAS1, and NF-κB p65 protein levels in SSPCs under the indicated treatments. GAPDH was used as the loading control. ( C ) qRT-PCR analysis of differentiation-related marker genes, including the chondrogenic markers Sox9 and Acan and the osteogenic markers Runx2 and Opn (n = 3). ( D ) Quantification of Western blot protein expression levels shown in panel ( B ) (n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Red fluorescence indicates non-oxidized lipid signal, and green fluorescence indicates oxidized lipid signal. Scale bars = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs under the indicated treatments. Scale bars = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining (n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of relative FerroOrange fluorescence intensity (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, Staining, Control, Western Blot, Quantitative RT-PCR, Marker, Expressing, Fluorescence, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: EPAS1 inhibition attenuates IL-1β-impaired bone regeneration in vivo. ( A ) Representative micro-CT three-dimensional reconstructions and micro-CT sectional images of fractured mouse femurs from vehicle control, IL-1β, IL-1β plus PT2385, and PT2385 alone groups at 28 days post-fracture. Scale bars = 1 mm. ( B ) Quantitative micro-CT analysis of bone volume (BV) and bone volume fraction (BV/TV) in the fracture callus region. ( C ) Quantitative histomorphometric analysis of bone area and callus area among the four treatment groups. ( D ) Representative Safranin O/Fast Green staining images of the fracture callus region showing callus organization, cartilage matrix, and newly formed bone tissue under the indicated treatments. Scale bars = 500 μm. Data are presented as mean ± SD; n = 6 mice per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, In Vivo, Micro-CT, Control, Staining, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Mendelian randomization provides exploratory genetic support linking IL-1β and EPAS1 to human bone nonunion risk. ( A ) Forest plot summarizing inverse-variance weighted Mendelian randomization estimates for the tested exposures and bone nonunion risk. Odds ratios and 95% confidence intervals are shown. ( B ) Scatter plots showing the associations between SNP effects on EPAS1 expression or IL-1β levels and SNP effects on bone nonunion risk. Lines indicate estimates from different Mendelian randomization methods. ( C ) Single-SNP forest plots showing individual SNP estimates for EPAS1 and IL-1β. The combined inverse-variance weighted estimates are shown at the bottom. ( D ) Leave-one-out analyses for EPAS1 and IL-1β, performed by sequentially excluding one SNP at a time. ( E ) Funnel plots used to assess potential directional pleiotropy in the Mendelian randomization analyses for EPAS1 and IL-1β. Red colors, red lines, and numbers indicate representative annotations used to highlight key features discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing