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: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: Amelioration of LPS-Induced AKI in MBD2-KO Mice The MBD2-KO and -WT littermate mice were injected with 10 mg/kg LPS for days 0 to 7 or saline as control. (A) Representative of survival cure. (B and C) Blood samples were collected for the measurement of serum nitrogen (BUN) (B) and creatinine (C) concentration at 24 h. (D and E) Representative of TNF-α (Δ) and IL-1β (E). (F) The kidney sections were stained with hematoxylin and eosin (H&E). (G and H) Tubular damage scores of kidney cortex (G) and OSOM (H). Scale bar, 100 μM. For the survival, the animal number is 12; for other experiments, the data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2-WT with LPS group. Original magnification, ×200.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Injection, Saline, Control, Concentration Assay, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: MBD2-Mediated LPS Induced the Expression of TNF-α, IL-1β, and ICAM1 in BUMPT Cells BUMPT cells were transfected with MBD2 plasmid or MBD2 siRNA or primary macrophages from bone marrow of MBD2 and MBD2-KO mice, followed by treatment with 300 μg/mL LPS for 24 h. (A) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2. (B) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2. (C)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2. (D) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2 siRNA. (E) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2 siRNA . (F)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2 siRNA. (G) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary tubular cells with LPS treatment. (H) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary tubular cells with LPS treatment. (I) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary macrophage cells with LPS treatment. (J) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary macrophage cells with LPS treatment. Data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2 plasmid or MBD2 siRNA with LPS group; Δp > 0.05 versus the LPS group.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Saline