Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: CD177 is elevated in patients with NLRP3-AID. ( A ) Venn diagram shows the results of the transcriptome sequencing analysis. ( B ) Heatmap shows differentially expressed genes. ( C ) Flow cytometry detects the expression of CD177 in peripheral blood of NLRP3 mutant patient (PT) and healthy controls (H). ( D ) GEPIA analysis of the expression of CD177 in different immune cells in peripheral blood. ( E ) qPCR assay detects CD177 expression in peripheral blood neutrophils. ( F , G ) qPCR assay detects the expression of CD177 in neutrophils from two healthy controls (H3 and H4) treated with NLRP3 agonist (Nigericin) or NLRP3 inhibitor (CY-09). ( H – K ) qPCR assay detects the expression of IL-1β, IL-6, TNFα and IL-18 in neutrophils from HCs treated with Nigericin or CY-09. ( L ) GEPIA analysis of the expression correlation between CD177 and IL-1β. ( M ) qPCR assay examines the expression of CD177 in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. ( N ) Western blot analysis of CD177 protein levels in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. ( O ) The mRNA levels of IL-1β, IL-6, TNFα and IL-18 in MCF-7 cells transfected with CD177 siRNA or negative control siRNA. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as * p < 0.05; *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Sequencing, Flow Cytometry, Expressing, Mutagenesis, Transfection, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: NLRP3 mutant mice exhibit phenotypic heterogeneity despite identical genotypes. ( A ) Images of wild-type ( Nlrp3 +/+ ) mice and NLRP3 mutant ( Nlrp3 L573W/+ ) mice with different inflammatory states. Notably, both mild and severe phenotypic groups are confirmed to be heterozygous for the p.L573W mutation. ( B ) Body weight of wild-type mice, NLRP3 mutant (Nlrp3 L573W/+) mice with different inflammatory states (weight measured starting from day 12, every other day). ( C ) Flow cytometry analysis of neutrophils in mouse peripheral blood ( n = 6 per group). ( D – F ) Histopathological detection of mouse tissues (skin, liver, spleen) showing varying degrees of inflammatory infiltration, Scale bars: 400 μm, 100 μm. ( G – I ) Immunohistochemical detection of mouse tissues (skin, liver, spleen) showing differences in neutrophil marker Ly6G expression, Scale bars: 200 μm, 50 μm. ( J ) Cytokine microarray in serum of wild-type mice and NLRP3 mutant mice with different inflammatory states. ( K , L ) Flow cytometry detection of IL-1β and IL-6 protein expression in mouse peripheral blood cells ( n = 6 per group). ( M – O ) qPCR detection of IL-1β, IL-6, and TNFα mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as ** p < 0.01; *** p < 0.001; ns, not significant. CD177 expression is elevated in NLRP3 mutant mice and correlates with disease severity.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Mutagenesis, Flow Cytometry, Immunohistochemical staining, Marker, Expressing, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeted IL-6 therapy is ineffective for NLRP3 mutation-induced AID. ( A – G ) Flow cytometry detection of neutrophil, IL-1β, IL-6 and CD177 protein expression in peripheral blood of NLRP3 mutant severe mice treated with or without IL-6 antibody ( n = 7). ( H – K ) qPCR detection of IL-1β, IL-6, TNFα and CD177 mRNA expression levels in skin, liver, and spleen tissues of NLRP3 mutant severe mice treated with or without IL-6 antibody ( n = 3). ( L – N ) Histopathological detection of mouse tissues (skin, liver, spleen), Scale bars: 400 μm, 100 μm. ( O – Q ) Immunohistochemical detection of Ly6G levels in mouse skin, liver, spleen tissues, Scale bars:200 μm, 50 μm. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Mutagenesis, Flow Cytometry, Expressing, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeting CD177 reverses the inflammatory phenotype of NLRP3 -AID mice. ( A , B ) Flow cytometry detection of CD177 protein expression in peripheral blood of mice treated with either IL-1β antibody or CD177 siRNA ( n = 6). ( C ) qPCR detection of CD177 mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). ( D ) Treatment was initiated on Day 14. Body weights were recorded every two days to evaluate systemic recovery. Data are presented as mean ± SEM ( n = 6 per group). ( E ) Flow cytometry detection of neutrophils in mouse peripheral blood after treatment with either IL-1β antibody or CD177 siRNA ( n = 6). ( F – H ) Histopathological detection of mouse tissues (skin, liver, spleen), Scale bars: 400 μm, 100 μm. ( I – K ) Immunohistochemical detection of Ly6G protein expression in mouse tissues (skin, liver, spleen), Scale bars: 200 μm, 50 μm. qPCR data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting CD177: A Novel Therapeutic Strategy for NLRP3-Associated Autoinflammatory Diseases

doi: 10.3390/ijms27062841

Figure Lengend Snippet: Targeting CD177 suppresses the expression of IL-1β. ( A , B ) Flow cytometry detection of IL-1β protein expression levels in peripheral blood of mice treated with either IL-1β antibody or CD177 siRNA ( n = 6). ( C ) ELISA detection of serum IL-1β protein levels in mice treated with either IL-1β antibody or CD177 siRNA ( n = 3). ( D , E ) Flow cytometry detection of IL-6 protein expression levels in peripheral blood of mice treated with either anti-IL-1β antibody or CD177 siRNA ( n = 6). ( F ) ELISA detection of serum IL-6 protein levels in mice treated with either IL-1β antibody or CD177 siRNA ( n = 3). ( G – I ) qPCR detection of IL-6, IL-1β, and TNFα mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). ( J ) Schematic diagram shows the role of CD177 in the development of NLRP3 mutation-related autoinflammation. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as ** p < 0.01; *** p < 0.001.

Article Snippet: The levels of IL-1β and IL-6 in the serum were detected using mouse-specific IL-6 (CSB-E04639m, Cusabio, Wuhan, China) and IL-1β (CSB-E08054m, Cusabio, Wuhan, China) detection kits according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated macrophage pyroptosis and reduced IL-1β release into the myocardium and serum during I/R injury. A-C The RNA sequencing data were extracted from GSE 126772. The mRNA levels of Nlrp3 , Pycard ( ASC ) and Il-1b in macrophages isolated from heart after MI were shown as fold change compared to the Pre group (control group, n = 2,3). D Macrophages were identified and isolated from the heart by flow cytometry. E–F Immunoblotting was performed to assess the expression levels of NLRP3, GSDMD and IL-1β in macrophages after I/R injury with or without bufalin treatment. Each target band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Sham + PBS group (control group, n = 3). G Representative IL-1β expression and localization in murine hearts are shown by IHC analysis in longitudinal sections of whole hearts. For each mouse, 5 representative zones in the left ventricle were chosen randomly to determine the percentage of IL-1β-positive ratio by ImageJ ( n = 3). H IL-1β levels in the peripheral blood of mice that were subjected to I/R injury and were treated with or without bufalin were measured by ELISA ( n = 6). IHC (immunohistochemistry). Data are expressed as the mean ± SEM. P values were determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test ( A - C ) and two-way ANOVA with Tukey’s multiple comparisons test ( F and H )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: RNA Sequencing, Isolation, Control, Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated H/R-treated macrophage pyroptosis in vitro. Macrophages were subjected to 1 h of hypoxia and 24 h of reoxygenation treated with PBS or bufalin. A-B Proteins were extracted from cells and immunoblotting analysis was performed. The expressions of NLRP3, GSDMD, and IL-1β were normalized to that of β-actin and then compared with the Sham + PBS group (control group, n = 3). C PI staining was used to examine the plasma membrane integrity of macrophages. Positive cells were counted and normalized to the total number of cells ( n = 5). D Cell viability was assessed by measuring the quantity of ATP in macrophages subjected to different treatments ( n = 3, 6). E IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test ( B , C , and E ) or Šídák’s multiple comparisons test ( D )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: In Vitro, Western Blot, Control, Staining, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin restored autophagic flux by suppressing P62 pathway in H/R-induced macrophages. Macrophages were transfected with the p62-overexpression plasmid for 24 h and then subjected to H/R with or without bufalin. A - B The protein levels of P62 were measured by immunoblotting. The bands were normalized to the β-actin band, and the relative expressions were calculated as the fold changes compared to the control group ( n = 3). C The mRNA levels of p62 were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and then compared with the control group ( n = 3). D-E Proteins were extracted from H/R-treated macrophages that were infected with vector or OE-p62 plasmid in advance and treated with bufalin or PBS. Immunoblotting was performed to measure the expressions of NLRP3, GSDMD and IL-1β. Each band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Con + PBS group (control group, n = 3). F PI staining was used to assess pyroptosis in macrophages. Positive ratios were calculated as the percentage of positive cells compared with total number of cells ( n = 5). G IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Infection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Single-cell RNA sequencing reveals an IL1R2+Treg subset driving immunosuppressive microenvironment in HNSCC

doi: 10.1007/s00262-025-04015-1

Figure Lengend Snippet: IL-1β promoted tumor growth and immunosuppressive TME formation. A Schematic diagram showing the process for establishing the subcutaneous tumor models and treatments. B Images of isolated tumors from 3 groups ( n = 6). C Tumor growth after tumor cells injection and treatment. D Tumor weight after 24 days injection and treatment. E – H Flow Cytometry showing the average percentage of CD4+T cells ( E ), CD8+T cells ( F ), Tregs ( G ) and M2 macrophages (CD206, H ). I , J The proportion of IL1R2+Treg in Tregs ( I ) and the expression of IL1R2 in Treg ( J ). K Expression of CTLA4 and Tim3 in Tregs. L Expression and analysis of Granzyme B, Perforin and CTLA4 in CD8+T cells. i.t., intratumor injection. α-il1r1: anti-IL1R1. rmIL-1β: recombinant murine IL-1β. ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Tregs induced by naive CD4+T cells from the spleen of C57BL/6 mice, treated with IL-1β (50 ng/ml, TargetMol, #TMPY-02134).

Techniques: Isolation, Injection, Flow Cytometry, Expressing, Recombinant

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Single-cell RNA sequencing reveals an IL1R2+Treg subset driving immunosuppressive microenvironment in HNSCC

doi: 10.1007/s00262-025-04015-1

Figure Lengend Snippet: IL-1β promoted the expression of the CTLA4 by regulating NR4A1. A Representative mIHC staining of CD4 (green), FOXP3 (red), IL1R2 (violet), IL-1β (cyan) in human HNSCC tissues. Tregs (yellow) were identified using colocalization of CD4 and FOXP3. Intensity of IL1R2+Treg (violet) and IL-1β (cyan) was calculated using ImageJ according to the line on picture. Scale bar, left, 20 μm; right, 10 μm. B Flow Cytometry showing the expression of IL1R2 in Tregs after treating with recombinant murine IL-1β for 12 h. C Volcano Plot showing significant down-regulated and up-regulated nuclear protein after treating with rmIL-1β (50 ng/mL) measured by Pro DIA quantitative proteomics. D Venn diagram showed 16 genes both up-regulated significantly in rmIL-1β group and worked as transcription factor. E , F The expression of CTLA4 in Tregs after using DIM-C-pPhOH (NR4A1 inhibitor, 20 μM, 24 h) and 4μ8C (IRE1 inhibitor, 200 nM, 24 h) with (F) or without (E) treating with rmIL-1β. G The expression of NR4A1 in nucleus in Tregs after stimulating with rmIL-1β or blocking IL1R2. H The location and intensity of NR4A1 in Tregs with different treatments. Scale bar, left, 2 μm; right, 1 μm. Intensity of NR4A1 (red) and DAPI (blue) was calculated using ImageJ according to the line on picture. ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Tregs induced by naive CD4+T cells from the spleen of C57BL/6 mice, treated with IL-1β (50 ng/ml, TargetMol, #TMPY-02134).

Techniques: Expressing, Staining, Flow Cytometry, Recombinant, Quantitative Proteomics, Blocking Assay