Journal: JCI Insight

Article Title: Combined single-cell transcriptome and immune repertoire analysis reveals hepatic and renal immune injury by heat stroke

doi: 10.1172/jci.insight.189825

Figure Lengend Snippet: ( A ) Subclustering of myeloid cells identified 11 cell subclusters. ( B ) Dot plot illustrating the scaled expression of functional marker genes across each myeloid cell subcluster. ( C ) UMAP plot (left) and violin plot (right) displaying the expression of NLRP3 inflammasome feature scores in each myeloid cell subcluster. ( D ) UMAP plot (left) and violin plot (right) showing the expression of pyroptosis feature scores in each myeloid cell subcluster. ( E ) Bar plot illustrating the relative contribution of each of the 10 myeloid cell subclusters in different sample groups. P values were obtained using the 1-way Kruskal-Wallis test with post hoc Dunn’s test. ( F ) Dot plot showing the expression of inflammasome and pyroptosis features in myeloid cell subtypes derived from the HC, HE, and HS groups. ( G ) Ranking of significantly differentially expressed genes between HS-enriched and HC-enriched myeloid cells. ( H ) GSEA plot illustrating pathways enriched in HS-associated myeloid cell clusters compared with HC-associated clusters. NES, normalized enrichment score.

Article Snippet: The third and the fourth groups were intraperitoneally injected with NLRP3 inflammasome inhibitor (CY-09, Selleck) or TNF-α inhibitor QNZ (Selleck) for 7 days before the induction of the HS model, according to previous reports ( , ).

Techniques: Expressing, Functional Assay, Marker, Derivative Assay

Journal: JCI Insight

Article Title: Combined single-cell transcriptome and immune repertoire analysis reveals hepatic and renal immune injury by heat stroke

doi: 10.1172/jci.insight.189825

Figure Lengend Snippet: ( A ) Diagram of intervention procedures for HS induction in 4 groups. The third and fourth groups were intraperitoneally injected with the NLRP3 inhibitor CY-09 (3 mg/kg/day) or the TNF-α inhibitor QNZ (0.1 mg/kg/day) for 7 consecutive days before HS induction ( n = 10 per group). The other 2 groups received PBS as vehicle control. Mice were euthanized 2 hours after HS onset for sample collection. Serum samples were used for cytokine and biological function measurements. Hepatic and renal tissues were used for pathology staining and Western blotting. ( B ) Representative images of H&E-stained hepatic and renal tissues from the experimental groups. Original magnification, ×400. Scale bars: 50 μm. n = 3 repeats per group. ( C ) Clinical parameters measured include markers for hepatic function (ALT, AST), renal function (serum urea, creatinine), and rhabdomyolysis (creatine kinase); n = 9 repeats per group. Comparisons were conducted using the 1-way ANOVA followed by Tukey’s multiple-comparison test. ( D ) Key plasma cytokines (IL-6, IL-1β, IL-18, TNF-α) and chemokines (CCL2, CCL3, CCL4) quantified by ELISA; n = 9 repeats per group. Comparisons were conducted using 1-way ANOVA followed by Tukey’s multiple-comparison test. ( E and F ) Western blots and bar graphs displaying expression profiles of inflammasome- and pyroptosis-related proteins (NLRP3, TLR4, ASC, GSDMD, caspase 1, caspase 11) and inflammatory cytokines (IL-6, IL-1β, IL-18, TNF-α, MCP-1) in hepatic tissues, with β-actin as loading control; n = 3 repeats per group. Comparisons were conducted using 1-way ANOVA followed by Tukey’s multiple-comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The third and the fourth groups were intraperitoneally injected with NLRP3 inflammasome inhibitor (CY-09, Selleck) or TNF-α inhibitor QNZ (Selleck) for 7 days before the induction of the HS model, according to previous reports ( , ).

Techniques: Injection, Control, Staining, Western Blot, Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing

Journal: International journal of molecular sciences

Article Title: HERV-W Env Induces Neuron Pyroptosis via the NLRP3-CASP1-GSDMD Pathway in Recent-Onset Schizophrenia.

doi: 10.3390/ijms26020520

Figure Lengend Snippet: Figure 6. HERV-W env promoted NLRP3 inflammasome sensor. (A) Upregulation of NLRP3 mRNA in HERV-W env-transfected SH-SY5Y cells, as detected by RT-qPCR. (B) Upregulation of NLRP3 protein in HERV-W env-transfected SH-SY5Y cells, as detected by Western blot analysis. (C–E) HERV-W env did not affect the expression of NLRP2 (C), CARD8 (D), and NLRC4 (E) in SH-SY5Y cells, as detected by RT-qPCR. ns, not significant, ** p < 0.01, *** p < 0.001.

Article Snippet: To confirm whether HERV-W env initiates pyroptosis through the NLRP3 inflammasome sensor, we utilized NLRP3 specificity inhibitor CY-09 (MedChemExpress, HY-103666).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: International journal of molecular sciences

Article Title: HERV-W Env Induces Neuron Pyroptosis via the NLRP3-CASP1-GSDMD Pathway in Recent-Onset Schizophrenia.

doi: 10.3390/ijms26020520

Figure Lengend Snippet: Figure 7. HERV-W env induced pyroptosis via NLRP3. (A–D) CY-09 reduced the expression of NLRP3 (A), CASP1 (B), GSDMD (C), and IL1B (D) in HERV-W env-transfected SH-SY5Y cells, as detected by RT-qPCR. (E,F) CY-09 reduced the expression of NLRP3 (E) and the cleavage of Caspase- 1 and Gasdermin D (F) in HERV-W env-transfected SH-SY5Y cells, as detected by Western blot. (G) CY-09 reduced Caspase-1 activity in HERV-W env-transfected SH-SY5Y cells, measured using a Multiskan FC plate reader. (H) CY-09 reduced LDH release in HERV-W env-transfected SH-SY5Y cells, measured using the CytoTox 96 LDH Cytotoxicity Assay Kit according to the manufacturer’s protocol. (I) CY-09 reduced IL-1β release in the supernatant of HERV-W env-transfected SH-SY5Y cells, as measured by ELISA. Statistical analysis was performed by one-way analysis of variance (ANOVA). * p < 0.05; ** p < 0.01., *** p < 0.001.

Article Snippet: To confirm whether HERV-W env initiates pyroptosis through the NLRP3 inflammasome sensor, we utilized NLRP3 specificity inhibitor CY-09 (MedChemExpress, HY-103666).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: HERV-W Env Induces Neuron Pyroptosis via the NLRP3-CASP1-GSDMD Pathway in Recent-Onset Schizophrenia.

doi: 10.3390/ijms26020520

Figure Lengend Snippet: Figure 8. HERV-W env induced pyroptosis via NLRP3-CASP1-GSDMD pathway. HERV-W env promoted the expression of NLRP3, CASP1, GSDMD, and IL1B (as indicated by the red up arrows), along with the cleavage of Caspase-1 and Gasdermin D. This process leads to the release of LDH and IL-1β, which are markers of pyroptosis.

Article Snippet: To confirm whether HERV-W env initiates pyroptosis through the NLRP3 inflammasome sensor, we utilized NLRP3 specificity inhibitor CY-09 (MedChemExpress, HY-103666).

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: B.uniformis IM01-derived IAA alleviates asthma via AhR/NLRP3 pathways in mice

doi: 10.1007/s00018-025-05910-7

Figure Lengend Snippet: B.uniformis IM01 inhibits activation of NF-κB/NLRP3 pathway to improve lung barrier function in mice. ( A ) Measurement of cytokines IL-6 and IL-1β in the lung tissues of mice by ELISA ( n = 5). ( B ) Western blotting analysis of NLRP3, Caspase-1 p20, NF-κB p65 and p-NF-κB p65 expression in the lung of mice and quantification was done by using Image J software. ( C ) Experimental schema for D-J. Mice received daily oral gavage of either PBS or B.uniformis IM01 (1 × 10 9 CFU) for 24 days. Selected groups were sensitized with OVA on days 14 and 21, and challenged with OVA from days 28 to 30. Additionally, subgroups were administered the NLRP3 antagonist CY-09 (10 mg/kg) or vehicle from days 21–23. ( D ) Level of serum IgE detected by ELISA ( n = 5). ( E ) Measurement of cytokines IL-4, IL-5 and IL-13 in the lung tissues of mice by ELISA ( n = 5). ( F ) Measurement of cytokines IL-6, and IL-1β in the lung tissues of mice by ELISA ( n = 5). ( G ) mRNA expression of ZO-1 , Occludin and Claudin5 as detected by RT-qPCR ( n = 4). ( H ) Western blotting analysis of ZO-1, Occludin and Claudin5 expression in lung tissues of mice and quantification was done by using Image J software. ( I ) RT-qPCR analysis was conducted for β-catenin and E-cadherin mRNA expression in the lung homogenate of mice ( n = 4). ( J ) Western blotting analysis of β-catenin and E-cadherin expression in lung tissues of mice. Quantification was done by using Image J software. Data are presented as mean ± SD. P value was determined by one-way ANOVA with Dunnett’s post hoc test

Article Snippet: P value was determined by one-way ANOVA with Dunnett’s post hoc test For the treatment with NLRP3 inhibitor CY-09 (MedChemExpress, USA) and AhR antagonist CH223191 (MedChemExpress, USA), mice were treated with 10 mg/kg of CY-09 or CH223191 which were formulated in a vehicle containing 10% DMSO, 40% PEG300, 5% tween-80 and 45% saline by intraperitoneal injection 4 h before OVA challenge on days 21, 22 and 23, and the corresponding vehicle was used as a control.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Software, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: B.uniformis IM01-derived IAA alleviates asthma via AhR/NLRP3 pathways in mice

doi: 10.1007/s00018-025-05910-7

Figure Lengend Snippet: The AhR inhibitor CH223191 diminished the protection effect of B.uniformis IM01 in allergic asthma. ( A ) Experimental schema for B-H. Mice received daily oral gavage of either PBS or B.uniformis IM01 (1 × 10 9 CFU) for 24 days. Selected groups were sensitized with OVA on days 14 and 21, and challenged with OVA from days 28 to 30. Additionally, subgroups were administered the AhR antagonist CH223191 (10 mg/kg) or vehicle from days 21–23. ( B ) Level of serum IgE detected by ELISA ( n = 5). ( C ) Measurement of cytokines IL-4, IL-5 and IL-13 in the lung tissues of mice by ELISA ( n = 5) ( D - E ) Representative image of flow cytometric analysis of CD4 + CD25 + Foxp3 + Treg cells in the spleen. Quantification of CD4 + CD25 + Foxp3 + Treg cells was analyzed with FlowJo ( F ) Western blotting analysis of NLRP3, Caspase-1 p20, NF-κB p65 and p-NF-κB p65 expression in the lung of mice and quantification was done by using Image J software. ( G ) mRNA expression of ZO-1 and Occludin as detected by RT-qPCR ( n = 4). ( H ) Western blotting analysis of ZO-1 and Occludin expression in lung tissues of mice and quantification was done by using Image J software. Data are presented as mean ± SD. P value was determined by one-way ANOVA with Dunnett’s post hoc test Graphical abstract

Article Snippet: P value was determined by one-way ANOVA with Dunnett’s post hoc test For the treatment with NLRP3 inhibitor CY-09 (MedChemExpress, USA) and AhR antagonist CH223191 (MedChemExpress, USA), mice were treated with 10 mg/kg of CY-09 or CH223191 which were formulated in a vehicle containing 10% DMSO, 40% PEG300, 5% tween-80 and 45% saline by intraperitoneal injection 4 h before OVA challenge on days 21, 22 and 23, and the corresponding vehicle was used as a control.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Software, Quantitative RT-PCR

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: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

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: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

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: CD177 expression is upregulated in NLRP3 mutant mice. ( A ) Flow cytometry detection of CD177 protein expression in mouse peripheral blood ( n = 6). ( B ) qPCR detection of CD177 mRNA expression levels in mouse skin, liver, and spleen tissues ( n = 3). ( C – E ) Western blot analysis of CD177 protein expression levels in mouse skin, liver, and spleen tissues. qPCR Data are shown as mean ± s.d. Student’s t -test was used. Statistical significance was determined as *** p < 0.001; ns, not significant.

Article Snippet: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

Techniques: Expressing, Mutagenesis, Flow Cytometry, 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: 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: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

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: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

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: To prime the NLRP3 inflammasome pathway, cells were first stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, L5293, USA) for 2 h. Following priming, neutrophils were treated for 1 h with either the NLRP3 activator nigericin (10 μM; Selleck, Houston, TX, E4788, USA) alone or co-treated with nigericin and the NLRP3 inhibitor CY-09 (10 μM; Selleck, Houston, TX, S5774, USA).

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

Journal: JCI Insight

Article Title: Combined single-cell transcriptome and immune repertoire analysis reveals hepatic and renal immune injury by heat stroke

doi: 10.1172/jci.insight.189825

Figure Lengend Snippet: ( A ) Diagram of intervention procedures for HS induction in 4 groups. The third and fourth groups were intraperitoneally injected with the NLRP3 inhibitor CY-09 (3 mg/kg/day) or the TNF-α inhibitor QNZ (0.1 mg/kg/day) for 7 consecutive days before HS induction ( n = 10 per group). The other 2 groups received PBS as vehicle control. Mice were euthanized 2 hours after HS onset for sample collection. Serum samples were used for cytokine and biological function measurements. Hepatic and renal tissues were used for pathology staining and Western blotting. ( B ) Representative images of H&E-stained hepatic and renal tissues from the experimental groups. Original magnification, ×400. Scale bars: 50 μm. n = 3 repeats per group. ( C ) Clinical parameters measured include markers for hepatic function (ALT, AST), renal function (serum urea, creatinine), and rhabdomyolysis (creatine kinase); n = 9 repeats per group. Comparisons were conducted using the 1-way ANOVA followed by Tukey’s multiple-comparison test. ( D ) Key plasma cytokines (IL-6, IL-1β, IL-18, TNF-α) and chemokines (CCL2, CCL3, CCL4) quantified by ELISA; n = 9 repeats per group. Comparisons were conducted using 1-way ANOVA followed by Tukey’s multiple-comparison test. ( E and F ) Western blots and bar graphs displaying expression profiles of inflammasome- and pyroptosis-related proteins (NLRP3, TLR4, ASC, GSDMD, caspase 1, caspase 11) and inflammatory cytokines (IL-6, IL-1β, IL-18, TNF-α, MCP-1) in hepatic tissues, with β-actin as loading control; n = 3 repeats per group. Comparisons were conducted using 1-way ANOVA followed by Tukey’s multiple-comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The third and the fourth groups were intraperitoneally injected with NLRP3 inflammasome inhibitor (CY-09, Selleck) or TNF-α inhibitor QNZ (Selleck) for 7 days before the induction of the HS model, according to previous reports ( , ).

Techniques: Injection, Control, Staining, Western Blot, Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: The in vivo experiment schedule. To explore the role of the Nlrp3 inflammasome in the development of hemolytic uremic syndrome (HUS) induced by Stx2, two in vivo experiments were performed and each experiment included three groups of mice. There were six mice per group in experiment 1 and 10 mice per group in experiment 2 (A) ; To confirm whether the Nlrp3 inhibitors protect the host from HUS, two in vivo experiments were performed and each experiment included five groups of mice. There were six mice per group in experiment 1 and 10 mice per group in experiment 2 (B) .

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: In Vivo

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: The Stx2-induced IL-1β secretion requires the Nlrp3 inflammasome activation in vitro . The Stx2-triggered pro-inflammatory cytokine secretion and lactate dehydrogenase (LDH) leakage were compared among peritoneal macrophages (PMs) derived from wild-type (WT) C57BL/6J or deficient mice ( Nlrc 4 -/- , Aim2 -/- , Nlrp3 -/- ). PMs were primed with LPS for 4 h, followed by Stx2 or Stx2B treatment for 16 h. The supernatants were harvested and assayed for IL-1β (A) , TNF-α (B) , and LDH (C) . The culture supernatant and cell lysates were also harvested to examine the expression of IL-1β (p17), caspase-1 (p20), and their precursors (pro-IL-1β p31, pro-caspase-1 p45) using immunoblotting (D) . The data in panels (A) to (C) are the means ± standard deviations from three independent experiments. The data in panel (D) are obtained from one of two independent experiments. The univariate ANOVA test was used to compare the means of different groups and the Dunnett test was used for their multiple comparisons (A–C) . *p < 0.05, no significance (ns), p > = 0.05.

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: Activation Assay, In Vitro, Derivative Assay, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: Inhibitors reduce Stx2-Mediated IL-1β release in vitro . Primed peritoneal macrophages (PMs) were treated with Stx2 (2 μg/ml, 10 μl/well) for 16 h in the absence (PBS) or presence of the K + efflux blocker (KCl, 50 mM), ATP receptor inhibitor (oxidized ATP, oATP, 500 μM), cathepsin B inhibitor (CA-074Me, 10 μM), Nlrp3 inhibitors (MCC950, 20 μM; CY-09, 5 μM; Oridonin, 20 μM), and caspase-1 inhibitor (Z-YVAD-FMK, 10 μM). IL-1β in the supernatants was measured by ELISA (A) , and LDH was assayed using a Cytotox96 Kit (B) . The results are represented as the means ± standard deviations of three independent experiments. The univariate ANOVA test was used to compare the means of different groups and the Dunnett test was used for their multiple comparisons (A, B) . *p < 0.05; **p < 0.01.

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: Stx2-mediated IL-1β release requires Nlrp3 inflammasome activation in vivo . Three groups of eight-week-old mice (6 mice per group) were chosen to examine renal function changes after Stx2/LPS treatment. Group 1 (WT-PBS group) C57BL/6J WT mice were injected i.p. with PBS as the negative control. Group 2 (WT-Stx2/LPS group) C57BL/6J WT mice were injected i.p. with Stx2 plus lipopolysaccharide (LPS) according to the hemolytic uremic syndrome (HUS) inducement procedure as the positive control, Group 3 ( Nlrp3 -/– Stx2/LPS group) Nlrp3 -/- mice were treated as group 2 was. Sera were harvested on day 4 after injection for detecting serum CRE (A) , BUN (B) , and serum IL-1β via ELISA kit (C) . The other similar three groups of mice (treated the same way as above with 10 mice per group) were monitored survival every day up to 6 days after injection (D) . The data in panels (A–C) are the mean ± standard deviation from three independent experiments. The data in panels (D) are obtained from one of two independent experiments. The univariate ANOVA test was used to compare the means of different groups and the Bonferroni test was used for their multiple comparison (A–C) . The survivals of different groups of mice were plotted with the Kaplan–Meier method, and their multiple comparisons were performed using the log-rank method (pairwise comparison over strata) (D) . *p < 0.05, **p < 0.01.

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: Activation Assay, In Vivo, Injection, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: Renal histopathological and glomerular ultrastructural findings in mice 4 days after the challenge. Renal histopathological examination (H-E staining, 100×) are shown in panel (A–D) , six mice per group. (A) Wild-type (WT)-PBS group (WT mice treated with PBS only). No abnormal findings were observed. (B) The WT-Stx2/lipopolysaccharide (LPS) group (WT mice treated by Stx2/LPS). Renal multifocal inflammatory cell (mainly neutrophils) infiltration (black arrow), a small abscess, pyonephrosis (yellow arrow). (C) Nlrp3 -/- -Stx2/LPS group ( Nlrp3 -/- mice treated by Stx2/LPS). Most of the mice showed almost normal renal histopathological findings, and only two mice had mild inflammatory cell infiltration. (D) Renal histopathological scores of the above three groups of mice were also presented. Panels (E–G) are the representative transmission electron micrographs of glomeruli from the above three groups of mice. (E) The WT-PBS group, normal. (F) The WT-Stx2/LPS group, neutrophil infiltration (red arrow), endothelial cell swelling, capillary lumen stenosis (blue arrow). (G) The Nlrp3 -/- -Stx2/LPS group, erythrocyte deposition in a capillary lumen in individual mice (green arrow). E, endothelial cell; P, podocyte; R, Red blood cell. Bar = 5 µm, Magnification of ×6,000 in panel (A) ; Bars = 2 µm, Magnifications of ×8,000 in panel (B) through (F) . The univariate ANOVA test was used to compare the means of different groups and the Bonferroni test was used for their multiple comparisons (D) . *p < 0.05, **p < 0.01.

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: Staining, Transmission Assay

Journal: Frontiers in Immunology

Article Title: Activation of the Nlrp3 Inflammasome Contributes to Shiga Toxin-Induced Hemolytic Uremic Syndrome in a Mouse Model

doi: 10.3389/fimmu.2020.619096

Figure Lengend Snippet: Nlrp3 inhibitors reduce serum IL-1β level and protect renal function in mouse hemolytic uremic syndrome (HUS) models. Eight-week-old C57BL/6J wild-type (WT) mice were randomly divided into five groups, six mice per group. Group 1 (mock group as a negative control) mice were injected i.p. with PBS. Group 2 (Stx2/LPS group as a positive control, namely HUS model group) mice were injected i.p. with Stx2 plus LPS according to the HUS inducement procedure. Group 3 (the MCC950+Stx2/LPS group), group 4 (the CY-09+Stx2/LPS group), and group 5 (Oridonin+ Stx2/LPS group) mice were injected i.p. with three kinds of Nlrp3 inhibitors (MCC950, CY-09, and Oridonin, respectively) before Stx2/LPS treatment. Sera were harvested on day 4 after Stx2/LPS injection for detecting serum CRE (A) , BUN (B) , and serum IL-1β (C) . The other five groups of mice (treated as above but 10 mice per group) were adopted to monitor their survival every day up to 6 days after Stx2/LPS injection (D) . The data in panel (A–C) are the means ± standard deviation from three independent experiments. The data in panel (D) are obtained from one of two independent experiments. The univariate ANOVA test was used to compare the means of different groups and the Dunnett test was used for their multiple comparison (A) . The Kruskal-Wallis test was done to compare the means of different groups with the pairwise comparisons performed (B, C) . The survivals of different groups of mice were plotted with the Kaplan–Meier method, and their multiple comparisons were performed using the log-rank method (pairwise comparison over strata) (D) . *p < 0.05, **p < 0.01.

Article Snippet: In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K + efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro .

Techniques: Negative Control, Injection, Positive Control, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

doi: 10.3390/ijms222212413

Figure Lengend Snippet: CXCL2 secreted by macrophages is critical for liver fibrosis. RNA sequencing analysis of DEGs between CCL 4 -induced liver tissues from control and MyD88 Lyz-KO mice. ( A ) Volcano diagram of DEGs, the threshold is padj < 0.05. ( B ) Heatmap view of the most significant differential expressed genes. ( C ) KEGG enriched signal pathway analysis with up-regulated genes. ( D ) Analysis of fold change of DEGs, * p < 0.05. ( E ) The relative RNA expression of CXCL2 in LPS treated BMDMs were measured by q-PCR, * p < 0.05. ( F ) The secretory protein levels of CXCL2 in LPS treated BMDMs were measured by ELISA, ** p < 0.01. ( G ) Double immunofluorescence staining and statistical analysis for F4/80 and CXCL2 in liver tissues. Scale bar, 50 μm. * p < 0.05. ( H ) ELISA verification for serum CXCL2 levels in MyD88 Lyz-KO or control mice treated with CCL 4 . * p < 0.05.

Article Snippet: LX-2 cells were exposed to inhibitor of NLRP3 CY-09 (10 μM) (MedChemExpress, Princeton, NJ, USA) for 2 h. After incubation, the cells were challenged with 200 ng/mL CXCL2 recombinant protein (Sino Biological, Beijing, China) for 24 h for further analysis.

Techniques: RNA Sequencing Assay, Control, RNA Expression, Enzyme-linked Immunosorbent Assay, Double Immunofluorescence Staining

Journal: International Journal of Molecular Sciences

Article Title: MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

doi: 10.3390/ijms222212413

Figure Lengend Snippet: CXCL2 secreted by macrophages promotes the activation of HSCs. ( A – C ) LX-2 cells were cultured with control and MyD88 Lyz-KO mice BMDM CM for 24 h. ( A ) Immunofluorescence staining for α-SMA in LX-2 cells. Scale bar, 50 μm. ( B ) The mRNA levels of the activation-related genes α-SMA and collagen I in LX-2 cells were measured using qPCR analysis. ** p < 0.01. ( C ) The protein levels of α-SMA in LX-2 cells were detected by Western blot. The densities of proteins were quantified by densitometry. α-SMA were normalized to GAPDH. * p < 0.05. ( D – F ) LX-2 cells were treated with CXCL2 for 24 h. ( D ) Immunofluorescence staining for α-SMA in LX-2 cells. Scale bar, 50 μm. ( E ) The mRNA levels of α-SMA and collagen I in LX-2 cells were measured using qPCR analysis. ** p < 0.01. ( F ) The protein levels of α-SMA in LX-2 cells were detected by Western blot. The densities of proteins were quantified by densitometry. α-SMA were normalized to GAPDH. * p < 0.05. ( G , H ) LX-2 cells treated with exogenous CXCL2 co-cultured with BMDMs isolated from MyD88 Lyz-KO mice for 24 h. ( G ) The protein levels of α-SMA in LX-2 cells were detected by Western blot. The densities of proteins were quantified by densitometry. α-SMA were normalized to GAPDH. ** p < 0.01. ( H ) The mRNA levels of α-SMA and collagen I in LX-2 cells were measured using qPCR analysis. * p < 0.05 and ** p < 0.01.

Article Snippet: LX-2 cells were exposed to inhibitor of NLRP3 CY-09 (10 μM) (MedChemExpress, Princeton, NJ, USA) for 2 h. After incubation, the cells were challenged with 200 ng/mL CXCL2 recombinant protein (Sino Biological, Beijing, China) for 24 h for further analysis.

Techniques: Activation Assay, Cell Culture, Control, Immunofluorescence, Staining, Western Blot, Isolation

Journal: International Journal of Molecular Sciences

Article Title: MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

doi: 10.3390/ijms222212413

Figure Lengend Snippet: CXCL2 activates NLRP3 inflammasome in HSCs. ( A ) Groups of control and MyD88 Lyz-KO mice ( n = 5 per group) were subjected to CCl 4 -induced liver fibrosis. Double immunofluorescence staining and statistical analysis for NLRP3 and α-SMA in liver tissues. Scale bar, 50 μm. ** p < 0.01. ( B ) Control and MyD88 Lyz-KO mice BMDM CM-treated LX-2 cells for 24 h; the expression levels of NLRP3 inflammasome-associated protein were detected by Western blot analysis. The densities of proteins were quantified by densitometry. NLRP3, Cleaved-Caspase 1 and ASC were normalized to GAPDH. ** p < 0.01. ( C ) After pretreatment of LX-2 cells with or without CY-09 (NLRP3 inhibitor) for 2 h, 200 ng/mL CXCL2 recombinant protein was incubated for 24 h, and the expression of NLRP3 inflammasome-associated protein was detected by Western blot analysis. The densities of proteins were quantified by densitometry. NLRP3, Cleaved-Caspase 1 and ASC were normalized to GAPDH. ** p < 0.01. ( D ) Representative electron microscopy of LX-2 cells treated with or without 300 ng/mL CXCL2 for 24 h. Scale bar, 1 μm (left). Scale bar, 0.5 μm (right). ( E ) Immunofluorescence staining for NLRP3 in LX-2 cells. Scale bar, 50 μm. ( F ) cells were stimulated with 200 ng/mL CXCL2 recombinant protein for 24 h, and CXCR2 protein level was determined by Western blot analysis. ( G ) LX-2 cells were pretreated with or without SB225002 (CXCR2 inhibitor) for 30 min, and then treated with CXCL2 for 24 h, and the expression levels of NLRP3 inflammasome-related proteins were detected by Western blot analysis. The densities of proteins were quantified by densitometry. NLRP3, Cleaved-Caspase 1 and ASC were normalized to GAPDH. The data are representative of at least three independent experiments. * p < 0.05 and ** p < 0.01.

Article Snippet: LX-2 cells were exposed to inhibitor of NLRP3 CY-09 (10 μM) (MedChemExpress, Princeton, NJ, USA) for 2 h. After incubation, the cells were challenged with 200 ng/mL CXCL2 recombinant protein (Sino Biological, Beijing, China) for 24 h for further analysis.

Techniques: Control, Double Immunofluorescence Staining, Expressing, Western Blot, Recombinant, Incubation, Electron Microscopy, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MyD88 in Macrophages Enhances Liver Fibrosis by Activation of NLRP3 Inflammasome in HSCs

doi: 10.3390/ijms222212413

Figure Lengend Snippet: Schematic of MyD88 signaling in macrophages in CCL 4 -induced liver fibrosis model. Deficiency of MyD88 in macrophages reduces the secretion of CXCL2, thus inhibiting the activation of HSC and fibrosis, which is characterized by the activation of NLRP3 inflammasomes.

Article Snippet: LX-2 cells were exposed to inhibitor of NLRP3 CY-09 (10 μM) (MedChemExpress, Princeton, NJ, USA) for 2 h. After incubation, the cells were challenged with 200 ng/mL CXCL2 recombinant protein (Sino Biological, Beijing, China) for 24 h for further analysis.

Techniques: Activation Assay