Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is upregulated in fibroblasts during mammary carcinogenesis. a Scheme of fibroblast isolation procedure. b Principal component analysis (PCA) of samples included in the microarray transcriptome analysis. Each focal point represents a cohort of mice. Normal n = 6 mice/cohort, Hyperplasia n = 6 mice/cohort, Carcinoma n = 4 mice/cohort, advanced carcinoma (A. Carcinoma) n = 3 mice/cohort. c Venn diagrams depicting overlap of differentially expressed genes in fibroblasts isolated from distinct stages of MMTV-PyMT mammary carcinoma. d Pro-inflammatory genes upregulated in CAFs isolated from MMTV-PyMT tumours. Inflammasome-related genes are highlighted. e Heat map of Nlrp3/Il1b pathway-related genes. f qRT-PCR analysis of Nlrp3/Il1b pathway related genes in fibroblasts isolated by FACS (PDGFRα + CD45 − EpCAM − ) from MMTV-PyMT mice or normal mammary glands. Data are presented as mean ± s.d of technical repeats; One-way analysis of variance followed by Tukey’s test. * p < 0.05, ** p < 0.005, *** p < 0.0005, n = pools of 3 mice/group. Results are representative of three independent biological experiments. g Representative IHC staining of NLRP3 in PyMT tumours or in normal mammary glands. Arrows indicate fibroblasts. Scale bar, 25 µm. h Quantification of staining shown in g . Multiple fields of 3 mice/group were analysed for the percentage of NLRP3 expressing fibroblasts out of total fibroblasts. Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Isolation, Microarray, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is up-regulated in CAFs in human breast tumours. a – d Expression levels of Nlrp3/Il1b pathway-related genes in tumour-associated stroma of breast cancer patients with infiltrating ductal carcinoma (IDC) staged 1/2 vs. 3 ( n = 53) or in normal breast stroma ( n = 6). Data was obtained from NCBI GEO (Dataset accession number GSE 9014) and is presented as mean ± s.e.m.; One-way analysis of variance test followed by Tukey’s multiple comparisons test. e – g Analysis of NLRP3 expression in fibroblasts in human IDC or in normal breast tissue sections. Images were obtained from the Human Protein Atlas. e Representative images, arrows in insets indicate fibroblasts. f Quantification of the percentage of NLRP3-expressing fibroblasts out of total fibroblasts for each sample. n = 2 samples for normal and 8 samples for IDC, error bars represent s.e.m; Welch’s t -test. g Quantification of staining intensity for NLRP3 in fibroblasts shown in e . h – j Staining for IL-1β in benign or malignant (DCIS/IDC) tissue sections from human breast cancer patients. h Representative images of stained benign and IDC breast tissue sections. Arrows indicate fibroblasts. Scale bar, 50 µm. i , j Quantification of the abundance i and staining intensity j of labelled fibroblasts, n = 73 (benign: n = 31, DCIS: n = 16, IDC: n = 26); Mann–Whitney test ( i ) or Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( j ). Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Fibroblasts function as DAMP sensors via NLRP3 inflammasome signalling. a – e Primary FVB/n NMFs were incubated for 24 h in control medium or in medium containing one of the following DAMPs: ATP 1 mM, H 2 O 2 300 μM, MSU 5 μg/mL, 5% necrotic fluid (extracted from late-stage PyMT tumours). Nlrp3 a and Il1b b expression in fibroblasts were analysed by qRT-PCR. Data are presented as mean ± s.d.; One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Representative of three independent experiments. c Caspase-1 processing was assessed by western blot of cell lysates with anti-Caspase-1. β-actin was utilised as a loading control. The samples shown are derived from the same experiment. Data are representative of three independent experiments. d ELISA quantification of IL-1β secretion. Data are presented as mean ± s.d. of biological replicates ( n = 4 per group); Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Representative of three independent experiments. e Primary FVB/n NMFs were incubated for 24 h with 5% necrotic fluid or control medium and expression of selected genes was analysed using qRT-PCR. Data are presented as mean ± s.d. of biological replicates ( n = 3 per group); Welch’s t -test. Representative of two independent experiments. f Schematic illustration of cutaneous wound assay. Cutaneous wounds were generated in 8 weeks old FVB/n mice using the dermal punch method. Wounded or control skin fibroblasts were isolated by FACS as PDGFRα + CD45 − EpCAM − cells. g qRT-PCR analysis of the expression of selected genes in wound-derived fibroblasts as compared to their expression in fibroblasts from normal skin. n = pool of 8 mice per group. Data are presented as mean ± s.d. of biological repeats; Welch’s t -test. Representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated, Isolation

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: CAF-derived NLRP3 inflammasome signalling facilitates tumour growth. a Scheme of experiments analysed in b – h . shNlrp3 AT3 cells were orthotopically co-injected with WT ( Nlrp3 +/+ ) or with Nlrp3 −/− NMFs (C57BL/6) into C57BL/6 mice. b Growth curves of AT3 tumours. n = 7 mice per group. c Tumour weights at termination of experiment. n = 6 and 7 mice per group (WT and Nlrp3 −/− , respectively). d Flow cytometry analysis of Gr1 + cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. e Representative images of staining with anti-Gr1 antibody in AT3 tumours. Scale bar, 100 μm. f Quantification of staining shown in d . Three fields of 3 mice/group were analysed; Data are presented as mean Gr1 + cells/field ± s.d; Mann–Whitney test. g , h Flow cytometry analysis of CD11b + Ly6C high Ly6G − and CD11b + Ly6G + Ly6C − cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. i Scheme of experiments analysed in j – r . Met-1 cells were orthotopically co-injected with NMFs depleted of Nlrp3 or Il1b expression (shNlrp3, and shIl1b), or with control NMFs (shScramble). j Growth curve and k weight at endpoint of Met-1 tumours. n = 5, 10, 20, 19 tumours (TCs only, shScramble, shNlrp3, and shIl1b, respectively). l – n Flow cytometry analysis of Gr1 + cell infiltration into Met-1 tumours. n = 7, 14, and 16 per group (shScramble, shNlrp3, and shIl1b, respectively). Data presented are percentage of CD45 + CD11b + cells, normalised to shScramble. o – r Met-1 tumours were analysed for the expression of selected genes using qRT-PCR. n = 6, 7, and 5–6 tumours per group (shScramble, shNlrp3, and shIl1b, respectively). Data are presented as fold change from shScramble. In b – d , g , h , j , k , data are presented as mean ± s.e.m; Welch’s t -test, * p < 0.05, ** p < 0.005. In j – r data are presented as mean ± s.e.m; One-way analysis of variance test followed by Tukey’s multiple comparisons test. Data are representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Injection, Flow Cytometry, Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Summary. Activation of the NLRP3 inflammasome in cancer-associated fibroblasts links tissue damage with tumour-promoting inflammation in breast cancer progression and metastasis. Figure preparation partially involved elements from the Servier Medical Art. Figure preparation involved using elements from the Servier Medical Art and somersault18:24 (somersault1824.com)

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Immunoprecipitation, Expressing, Control, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Gene Expression, Ubiquitin Proteomics, Protease Inhibitor

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Percentage of in vitro–generated Th17 cells obtained from CD4+ T cells of carrier and noncarrier patients (n = 8). (B and C) Representative flow zebra plots (B) and quantitative histogram (C) depicting the percentage of peripheral CD4+IL-17+ cells (n = 8). (D) IL-17 plasma levels of carrier and noncarrier patients (n = 10). (E) IL-17 levels (Luminex) measured in the supernatants of unstimulated 24-hour-cultured CD4+ T cells of carrier and noncarrier patients (n = 5). (F) Table summarizing the secretome profile (Luminex, n = 5) and primary phenotypic characteristics (flow cytometry, n = 4) of carrier and noncarrier polarized Th17 cells. (G and H) Normalized mRNA expression of Th2-related factors IL-4 (G) and GATA-3 (H) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 using silencing RNA (siRNA), before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (I–K) Normalized mRNA expression of the Th2-related factors IL-4 (I), IL-10 (J), and GATA-3 (K) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 (siRNA), upon BzATP exposure (n = 4). (L and M) Normalized mRNA expression of the Th2-related factors IL-4 (L) and GATA-3 (M) measured in carrier CD4+ T cells, in which NLRP3 was overexpressed, before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (N) Effects of various treatments (anti–IL-17 antibody, RMT1-10, cyclosporin A [CsA] and rapamycin [Rapa]) on in vitro–generated Th17 cells (n = 5). Experiments were run in triplicate (D, G, H, and N) or in duplicate (F and I–M). mRNA expression was normalized to ACTB. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 1-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: In Vitro, Generated, Clinical Proteomics, Luminex, Cell Culture, Flow Cytometry, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Transplantation Assay, Membrane, Activity Assay, Ubiquitin Proteomics

Journal: Antioxidants

Article Title: Indapamide Increases IRS1 Expression and Modifies Adiponectin/NLRP3/PPARγ Crosstalk in Type 2 Diabetic Rats

doi: 10.3390/antiox11040691

Figure Lengend Snippet: Impact of daily CANA and INDA treatment on epididymal adipose tissue NLRP3 expression: oral CANA and INDA treatments were initiated once daily for 4 weeks. Data represent the mean ± S.E.M. (6 rats/group). ANOVA followed by post hoc Tukey–Kramer test at ( p < 0.05) was used for statistical comparison; a against normal CTRL, b against diabetic CTRL, d against diabetic/CANA.

Article Snippet: Moreover, using Cusabio ELISA kits (USA), NLRP3 (Cat. no. CSB-EL015871MO) and Nrf2 (Cat. no. CSB-E16188m) were quantified in the supernatant of the adipose tissue and the soleus muscle supernatant was used for the determination of IRS1 (Cat. no. CSB-PA047827) according to the enclosed pamphlet from the manufacturer.

Techniques: Expressing, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Effects of Nlrp3 gene silencing on visfatin‐induced disruption of junction proteins in mouse vascular endothelial cells ( MVEC s). MVEC s were transfected scramble (Scr) or Nlrp3 sh RNA (Nlrp3sh) plasmids by Nucleofection and then stimulated with or without visfatin (Visf: 4 μg/ml) for 24 hrs. ( A ) Immunofluorescence stainings were performed with Alexa555‐conjugated antibodies against ZO ‐1, ZO ‐2, occludin or VE ‐Cadherin ( VE ‐Cad) for determination of the expression of these junction proteins. Representative images show the cell membrane of fluorescence of ZO ‐1, ZO ‐2, occludin or VE ‐Cadherin (red) are representative of at least three independent experiments. ( B – F ) Representative Western blot gel document and summarized data showing the protein expression of ZO ‐1, ZO ‐2, occludin, VE ‐Cadherin and β‐actin expression in the microsomes of MVEC s ( n = 3–4). * P < 0.05 versus Scr; # P < 0.05 versus Scr+Visf.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Transfection, Immunofluorescence, Expressing, Fluorescence, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: ( A ) Western blot documents and summarized data showing the effect of control or visfatin (0 or 4 μg/ml for 24 hrs) on the expression of high mobility group box protein 1 ( HMGB 1) or β‐actin in either cell culture medium (Medium) or homogenized cytoplasm (Homo) of mouse vascular endothelial cells ( MVEC s; n = 6). * P < 0.05 versus control. ( B ) MVEC s transfected with scramble sh RNA (Scr) or Nlrp3 sh RNA (Nlrp3sh) plasmids and then stimulated with or without visfatin (Visf: 4 μg/ml) for 24 hrs. Summarized data show the concentration of HMGB 1in culture medium of MVEC s as analysed by ELISA ( n = 6). * P < 0.05 versus Scr; # P < 0.05 versus Scr+Visf. ( C ) Cells were incubated with fresh culture medium before addition of recombinant HMGB 1 (15 μg/ml) for 24 hrs. Representative Western blot gel document and summarized data show the effect of recombinant HMGB 1 ( n = 3) on protein expression of ZO ‐1, ZO ‐2, occludin, VE ‐Cadherin and β‐actin in the microsomes of MVEC s. * P < 0.05 versus control.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Western Blot, Expressing, Cell Culture, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Nlrp3 gene deletion inhibits high‐fat diet ( HFD )‐induced disassembly of tight junction and adherens junction proteins in mouse coronary arterial endothelium. Wild‐type ( Nlrp3 +/+ ) or Nlrp3 knockout ( Nlrp3 −/− ) Mice were fed with either a normal diet ( ND ), or a high‐fat diet ( HFD ) for 6 weeks. ( A ) Frozen sections of mouse hearts were stained with Alexa555‐conjugated antibodies against ZO ‐1, ZO ‐2, Occludin, or VE ‐Cadherin; scale bar = 50 μm. ( B ) The summarized data show the fluorescence intensity of the endothelial layer ( n = 4–6). * P < 0.05 versus ND on Nlrp3 +/+ ; # P < 0.05 versus Nlrp3 +/+ with HFD .

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Staining, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Nlrp3 deficiency gene deletion inhibits high‐fat diet ( HFD )‐induced inflammasome activation in mouse coronary arterial endothelium. Wild‐type (Nlrp3 +/+ ) or Nlrp3 knockout (Nlrp3 −/− ) Mice were fed with normal diet ( ND ) and high‐fat diet ( HFD ) for 6 weeks. Frozen sections of mouse hearts were stained with FLICA , a green fluorescent probe specific for active caspase‐1, and Alexa555‐conjugated antibodies against an endothelium marker vWF in coronary arteries. ( A ) The merged images displayed yellow dots or patches indicating the colocalization of FLICA (green) with vWF (red). Enlarged images of area of interest ( AOI ) in merged images are shown; scale bar = 50 μm. ( B ) The summarized data show the colocalization coefficient of FLICA with vWF ( n = 5–7). * P < 0.05 versus Nlrp3 +/+ with ND ; # P < 0.05 versus Nlrp3 +/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Activation Assay, Knock-Out, Staining, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Nlrp3 deficiency inhibits high‐fat diet ( HFD )‐induced increases of high mobility group box protein 1 ( HMGB 1) release in coronary arterial endothelium. Wild‐type (Nlrp3 +/+ ) or Nlrp3 knockout (Nlrp3 −/− ) Mice were fed with normal diet ( ND ) and high‐fat diet ( HFD ) for 6 weeks. Frozen sections of mouse hearts were used for confocal immunofluorescent analysis. ( A ) Representative confocal fluorescence images of HMGB 1 with endothelium marker vWF in coronary arteries of mice. Enlarged images of area of interest ( AOI ) in merged images are shown; scale bar = 50 μm. ( B ) The summarized data show the colocalization coefficient ( PCC ) of HMGB 1 with vWF ( n = 4–8). * P < 0.05 versus Nlrp3 +/+ with ND ; # P < 0.05 versus Nlrp3 +/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Fluorescence, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter‐endothelial junction disruption: role of HMGB 1

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Nlrp3 deficiency blocks high‐fat diet ( HFD )‐induced T cell adhesion and infiltration in coronary arterial wall. Wild‐type ( Nlrp3 +/+ ) or Nlrp3 knockout ( Nlrp3 −/− ) Mice were fed with either a normal diet ( ND ), or a high‐fat diet ( HFD ) for 6 weeks. ( A ) Frozen sections of mouse hearts were stained with T cell marker ( CD 43). The fluorescence images displayed red dots indicating the CD 43‐positive cells infiltrated into the arterial wall. ( B ) Summarized data show the fluorescence intensity of the arterial wall area ( n = 4). * P < 0.05 versus Nlrp3 +/+ with ND ; # P < 0.05 versus Nlrp3 +/+ with HFD .

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Staining, Marker, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Induction of HNSCC in Nlrp3-deficient mice by 4NQO. a Haematoxylin and eosin (H&E) staining showed the pathological changes in mouse tongues after 4NQO exposure. The dotted line indicates the epithelial basement membrane. Bar, 200 μm. b Schematic of the 4NQO tumorigenesis protocol. c Representative pictures of oral lesions in control and Nlrp3−/− mice at 24 weeks. d Representative H&E histological sections of 4NQO induced HNSCC. Bar, 50 μm. e Tumor incidence in the Nlrp3−/− (n = 10) and control (n = 10) groups. ns, no significant difference. f The proportion of oral lesion types in the two groups at week 24. g Representative image of tongue SCC in the two groups of mice. The red dotted circle denotes the lesion area. h Quantitative analysis of tumor size and i number in the Nlrp3−/− (n = 7) and control groups (n = 8) at the experimental endpoint. *, P < 0.05. j The body weights of 4NQO-treated control and Nlrp3−/− mice from the beginning to the end of this experiment. *, P < 0.05; ***, P < 0.001. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Staining, Membrane, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 is overexpressed in the stromal cells of human HNSCC. a Representative immunofluorescence images of NLRP3 (red) and CK-14 (green) in human HNSCC tissue. Bar, 50 μm. b Flow cytometry to detect NLRP3 expression (mean ± SD) in HNSCC TILs, and cells were gated by CD14+. c, d Representative IHC images and corresponding quantification data of NLRP3 expression in oral mucosa (n = 20), dysplasia (n = 61) and HNSCC (n = 157) (***, P < 0.001). Bar, 50 μm. e Representative IHC staining of NLRP3 in HPV-negative (n = 142) and HPV-positive HNSCC tissue (n = 15) and f quantification analysis (**, P < 0.01). Bar, 50 μm. G Kaplan–Meier analysis using overall survival (OS) of NLRP3 expression in HNSCC patients. median cut-off, n = 148. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Immunofluorescence, Flow Cytometry, Expressing, Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Increased NLRP3 expression in TAMs predicts poor prognosis in HNSCC. a The ratio of M1 (CD64, CD68) and M2-like macrophage (MRC1, CD163) signatures in the low-expression (n = 259) and high-expression NLRP3 groups (n = 259) in the TCGA HNSCC database (***, P < 0.001). b Co-immunofluorescence staining of NLRP3 (red) and CD206 (green) in human HNSCC tissue. The white arrow shows co-expressed cell (yellow). T, tumor area; S, stromal area. Bar, 50 μm. c Flow cytometry to detect NLRP3 expression (mean ± SD) in CD206+ TAMs in human HNSCC PBMCs (n = 6) and TILs (n = 5). d Quantitative statistical analysis of NLRP3- and CD206-positive populations on gated cells (**, P < 0.01; ***, P < 0.001). e Correlation analysis of CD14+/CD206+/NLRP3+ and CD14+/CD206+ cell proportions in HNSCC patient PBMC and TIL, n = 11. f Representative IHC staining of NLRP3, CD206 and CD163 in human HNSCC using serial sections. Bar, 50 μm. g Correlation analysis between NLRP3, CD206 and CD163 protein expression in HNSCC tissue microarray using histoscore, n = 157. h Correlation analysis between NLRP3, MRC1 (CD206 encoding gene) and CD163 RNA expression in the TCGA HNSCC database. unit, expression (RSEM, Log2(Val + 1)), n = 520. i Hierarchical clustering plot of NLRP3, CD11b, CD206 and CD163 in HNSCC based on histoscore. n = 74. j Survival curves of high NLRP3 and CD206 expression vs. low NLRP3 and CD206 expression patients; high NLRP3 and CD163 expression vs. low NLRP3 and CD163 expression patients; low NLRP3 on high CD206 expression vs. high NLRP3 on CD206 high expression patients; and low NLRP3 on high CD163 expression vs. high NLRP3 on CD163 high expression patients. Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Immunohistochemistry, Microarray, RNA Expression

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 overexpression is positively correlated with TAMs in mouse HNSCC. a Representative IHC images of NLRP3, CD206 and F4/80 in Tgfbr1/Pten 2cKO and 4NQO-induced mouse HNSCC tumor tissues. Bar, 50 μm. b Correlation analysis between NLRP3, CD206 and F4/80 expression histoscores in mouse HNSCC tissues. n = 27. c Co-immunofluorescence staining of CD206 (green) and NLRP3 (red) in mouse HNSCC tissue. Bar, 50 μm. d, e Representative flow cytometry plots and quantification analysis showed the population change of NLRP3+ TAMs in the blood of normal wild-type and tumor-bearing individuals in two mouse models (n = 6 in 4NQO; n = 4 in 2cKO) (***, P < 0.001). Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Over Expression, Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: Knockdown of NLRP3 in THP-1 cells inhibits M2-like macrophage polarization. a THP-1 cells were treated with CAL27 cultured medium (CM) or IL-4 and IL-13 after treatment with PMA; NLRP3 and Caspase-1 expression were analyzed by western blot, and b IL-1β amounts in the supernatant were detected by ELISA (n = 3 per group; ***, P < 0.001). c Western blotting analysis of NLRP3 expression in NLRP3-knockout or control THP-1 cells. d, e Flow cytometry plots and quantification analysis of CD206 in NLRP3-deficient and control THP-1 macrophages after CAL27 CM culture (n = 3 per group; ***, P < 0.001). f ELISA results of IL-1β amounts in control and siNLRP3 THP-1 macrophage cultured medium (n = 3 per group; ***, P < 0.001). g, h NLRP3-deficient THP-1 macrophages were exposed to IL-1β (20 ng/ml) for 48 h after CAL27 CM treatment, and CD206 was analyzed by flow cytometry (n = 3 per group; ***, P < 0.001). i Growth curves of CAL27 cells in different groups as measured by a CCK-8 assay (n = 3 per group; ***, P < 0.001). Error bar, SD

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Knockdown, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Knock-Out, Control, Flow Cytometry, CCK-8 Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: NLRP3 in tumor-associated macrophages predicts a poor prognosis and promotes tumor growth in head and neck squamous cell carcinoma

doi: 10.1007/s00262-022-03357-4

Figure Lengend Snippet: NLRP3 deficiency reduced the proportion of TAMs and MDSCs. a Representative IHC images and quantification of IL-1β in tumor tissues from the two groups of mice. ***, P < 0.001, Bar, 50 μm. b ELISA results of IL-1β amounts in the blood of normal WT (n = 5), 4NQO-treated control (n = 8) and Nlrp3−/− (n = 7) tumor burden mice. **, P < 0.01; ***, P < 0.001; ns, no significant difference. c The Luminex liquid suspension chip analysis result. *, P < 0.05; **, P < 0.01, three mice in each group. d Flow cytometry to detect the percentage of CD11b+/F4/80+/CD206+ TAMs in the spleen of the two groups and e quantitative statistical analysis. *, P < 0.05. f Representative IHC images and quantification of CD206 in control and Nlrp3−/− mouse HNSCC tissues. **, P < 0.01, Bar, 50 μm. g Flow cytometry to detect the percentage of M-MDSCs and PMN-MDSCs in the spleen of the two groups and h quantitative statistical analysis. *, P < 0.05. i Representative IHC images and quantification of Gr-1 in control and Nlrp3−/− mouse HNSCC tissues. **, P < 0.01, Bar, 50 μm

Article Snippet: Flow Cytometry : PE anti-human/mouse NLRP3 (IC7578P) was obtained from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Luminex, Suspension, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Immunoprecipitation, Expressing, Control, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Gene Expression, Ubiquitin Proteomics, Protease Inhibitor

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Percentage of in vitro–generated Th17 cells obtained from CD4+ T cells of carrier and noncarrier patients (n = 8). (B and C) Representative flow zebra plots (B) and quantitative histogram (C) depicting the percentage of peripheral CD4+IL-17+ cells (n = 8). (D) IL-17 plasma levels of carrier and noncarrier patients (n = 10). (E) IL-17 levels (Luminex) measured in the supernatants of unstimulated 24-hour-cultured CD4+ T cells of carrier and noncarrier patients (n = 5). (F) Table summarizing the secretome profile (Luminex, n = 5) and primary phenotypic characteristics (flow cytometry, n = 4) of carrier and noncarrier polarized Th17 cells. (G and H) Normalized mRNA expression of Th2-related factors IL-4 (G) and GATA-3 (H) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 using silencing RNA (siRNA), before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (I–K) Normalized mRNA expression of the Th2-related factors IL-4 (I), IL-10 (J), and GATA-3 (K) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 (siRNA), upon BzATP exposure (n = 4). (L and M) Normalized mRNA expression of the Th2-related factors IL-4 (L) and GATA-3 (M) measured in carrier CD4+ T cells, in which NLRP3 was overexpressed, before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (N) Effects of various treatments (anti–IL-17 antibody, RMT1-10, cyclosporin A [CsA] and rapamycin [Rapa]) on in vitro–generated Th17 cells (n = 5). Experiments were run in triplicate (D, G, H, and N) or in duplicate (F and I–M). mRNA expression was normalized to ACTB. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 1-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: In Vitro, Generated, Clinical Proteomics, Luminex, Cell Culture, Flow Cytometry, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

Techniques: Mutagenesis, Transplantation Assay, Membrane, Activity Assay, Ubiquitin Proteomics