Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 1 | DcR3.Fc- or HBD.Fc-treated M-Mφ and GM-Mφ secreted low amount of interleukin-1β (IL-1β) under particle stimulation. Six-day-cultured M-Mφ (A,C) and GM-Mφ (B) were derived from wild-type (WT) B6 and cocultured with hIgG (3 µg/ml), DcR3.Fc (3 µg/ml), or HBD.Fc (3 µg/ml). Cells were primed with lipopolysaccharides (LPS; 100 ng/ml) for 4 h and treated with monosodium urate (MSU) crystal, alum, or silica (150 or 300 µg/ml) for 6 h or ATP (3 mM) for 1 h. The concentration of IL-1β was measured by ELISA. All data shown were mean ± SD from three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 were obtained by comparing the DcR3.Fc- or HBD.Fc-treated group to the hIgG-group. “NS” means no statistical significance.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 2 | DcR3 suppressed MSU-induced caspase-1 activation but did not affect the expression of NLRP3 or pro-IL-1β in LPS-treated macrophages. M-Mφ (A,C) and GM-Mφ (B,C) were treated with hIgG, DcR3.Fc, or HBD.Fc (3 µg/ml for each) for 6 days during differentiation stage in M-Mφ and GM-Mφ. Cells were treated with LPS (100 ng/ml) for 4 h, and total RNA was extracted. The mRNA levels of pro-IL-1β and NLRP3 were measured by real-time PCR (A,B). In some experiments, after LPS priming, cells were treated with MSU (300 µg/ml) for 3 h. The supernatants were harvested for caspase 1 p10 ELISA analysis (C). Data indicated mean ± SD of three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05 and ***p < 0.001 were obtained by comparing DcR3.Fc- or HBD.Fc-treated group to hIgG-group.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Hepatocyte nuclear receptor SHP suppresses inflammation and fibrosis in a mouse model of nonalcoholic steatohepatitis

doi: 10.1074/jbc.ra117.001653

Figure Lengend Snippet: FIGURE 3. SHP level is decreased in the livers of mice NASH. A, qPCR analysis of relative mRNA levels of genes related to lipid metabolism, inflammation, and fibrosis in the livers of mice fed chow or HFCF for 1 and 5 moths. N=5 mice/Group. *p < 0.05 HFCF vs. Chow. B, Serum level of CCL2 was measured by enzyme-linked immunosorbent assay (ELISA). N=5 mice/Group. *p < 0.05 HFCF vs. Chow. C, qPCR analysis of Shp mRNA level in the livers of mice fed chow or HFCF. N=5 mice/Group. *p < 0.05 HFCF vs. Chow. D, Left: Western blotting analysis of SHP protein in the livers of mice fed chow or HFCF for 5 months. SHP (H-160) is a rabbit polyclonal antibody and SHP (H-5) is a mouse monoclonal antibody. Both antibodies recognize the epitope corresponding to amino acids 1-160 mapping at the N-terminus of SHP protein. Right: Band intensities were calculated using Image J software. The level of SHP was normalized to the expression of loading control β-actin and fold changes relative to that of the controls are plotted. N=5 mice/Group. *p < 0.05 HFCF vs. Chow. E, Representative images of liver sections stained with hematoxylin-eosin (H&E) in mice fed chow or methionine-choline deficient (MCD) diet for 1 month. Original magnification, x40. F, qPCR analysis of gene expression in the livers of mice fed chow or MCD diet for 1 month. N=5 mice/Group. Data are presented as mean ± SD. *p < 0.05 versus respective controls.

Article Snippet: Recombinant mouse CCL2 protein (479-JE-010) and anti-mouse CCL2 antibody (AF-479-SP) for neutralization were obtained from RD system.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Software, Expressing, Control, Staining, Gene Expression

Journal: Oncotarget

Article Title: Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse.

doi: 10.18632/oncotarget.4470

Figure Lengend Snippet: Figure 2: Tumor expression of chemokine decoy receptors (CDR) and host genotype of CDR jointly affect breast cancer relapse. A. Effect of tumor phenotype of CDR on relapse-free survival (RFS). P for log rank = 7.5 × 10−6. The RFS curve was derived from the Kaplan-Meier estimate, and the survival differences between groups were compared by log-rank test. B. Effect of host genotype of CDR on RFS. P for log rank = 0.002 C. Joint effect of tumor phenotype and host genotype of CDR on RFS. P-values of the differences between high expression/minor genotype group and high expression/major genotype group, high expression/major genotype group and low expression/ minor genotype group, and low expression/minor genotype group and low expression/major genotype group are 0.007, 0.354, and 0.047, respectively. High expression indicates co-expression of DARC and D6, otherwise low expression. Minor genotype indicates patients with at- least-one protective minor allele, otherwise major genotype. D. Chemokine levels in the supernatant of cells detected by ELISA after 24-hour incubation. For transient transfection, 1 μg pDARC-42G or -42A, 1 μg pD6-373S or -373Y, or the combination of 1 μg variant-type DARC- 42A and 1 μg variant-type pD6-373Y were transfected. An empty expression vector was also used as a control. 72 hours after transfection, the levels of human CCL2 and CCL5 in cell supernatants were determined with a sandwich ELISA. Columns represent the mean of three independent experiments; bars, standard error; *, P < 0.05. E. ROC curves assessing the discriminatory performance of the CDR phenotype/ genotype model and the CDR phenotype model for the prediction of disease relapse. P = 0.02 for AUC comparison.

Article Snippet: After 72 hours of transfection, the levels of human CCL2 and CCL5 in cell supernatants were determined with a sandwich ELISA (R&D systems, USA).

Techniques: Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Variant Assay, Plasmid Preparation, Control, Sandwich ELISA, Comparison

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 1. Exposure to high altitude results in PH and increased secretion of inflammatory classical monocyte ligands from the lungs. (A) Schematic showing hypoxia exposure time course in wildtype mice. Duration of hypoxia exposure is directly proportional to (B) RVSP and RV hypertrophy as measured by Fulton Index (N=6-13/group). At 3 days of hypoxia, increased protein expression of classical monocyte ligands (C) CCL2 (N=6-11/group) and (D) CCL12 (N=6- 11/group), whereas significantly lower levels of nonclassical monocyte ligand (E) CX3CL1 (N=6/group) in the lungs. (F) Higher CCL2 gradient in lungs and in the (G) peripheral blood of wildtype mice following 3 days of hypoxia exposure (N=5/group). Data in all panels were obtained from female mice. Statistical analysis was conducted using ANOVA, followed by Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. N=number of animals, mean±SD, CI=confidence interval.

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 4: Genetic and pharmacologic blockade of CCR2-CCL2 axis protects from hypoxic PH. (A) Schematic showing the BM reconstitution of Ccr2-/- and WT BM into lethally irradiated wildtype mice. Wildtype mice reconstituted with Ccr2-/- BM were protected from hypoxic PH by attenuated (B) RVSP (N=7-11/group) and (C) RV hypertrophy (N=7-11/group) as measured by Fulton Index, compared to wildtype mice that were reconstituted with wildtype BM. (D) Schematic showing pharmacological blockade of CCR2 ligands CCL2 or CCL7 using anti-CCL2 or anti-CCL7 neutralizing antibody treatment. Hypoxia exposed wildtype mice treated with CCL2 NAb but not CCL7 NAb showed lower (E) RVSP (N=6/group) and (F) RV hypertrophy (N=6/group). TSP-1 levels in (G) lungs (N=6/group) and (H) blood (N=6/group); and TGF-β1 levels in (I) lungs (N=6/group) and (J) blood (N=6/group) compared to wildtype mice treated with isotype control antibody. Data in all panels followed a normal distribution. ANOVA with the Tukey test was performed for multiple comparisons. Data were obtained from the female mice. mean ± SD

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Irradiation, Control

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 5: Resident IMs are a major source of CCL2 and recruited IMs are a major source of pathologic TSP-1 in hypoxic PH. (A) Flow cytometry analysis using Ccl2RFP-flox reporter mice showed a higher number of CCL2+ IMs (N=14/group; N=14/group, 9F and 5M in Nx; 8F and 6M in Hx), and (B) FOLR2+ IMs are a major source of CCL2 (N=14/group). (C) Hypoxia exposed wildtype mice following intracellular CCL2 staining by flow cytometry also showed a higher number of CCL2+ IMs (N=7/group, female mice). (D). IM subpopulation analysis using flow

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry, Staining

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 8: DEX prophylaxis blunts CCL2 production by resident IMs and blocks the recruitment of TSP-1 producing CCR2+ IMs in hypoxia. (A) DEX prophylactically-treated, hypoxia-exposed Ccl2RFP-flox reporter mice exhibited a significant reduction in CCL2+ IMs, particularly in (B) CCL2RFP+ resident IMs (N=7/group). Additionally, (C) intracellular CCL2 flow cytometry analysis in DEX prophylactically-treated hypoxia-exposed wildtype mice revealed a

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry

Journal: Biomedicines

Article Title: Regulation of Cancer Stem Cells and Epithelial-Mesenchymal Transition by CTNNAL1 in Lung Cancer and Glioblastoma.

doi: 10.3390/biomedicines11051462

Figure Lengend Snippet: Figure 4. Effects of CCL2 regulated by CTNNAL1 on lung cancer cells. (A) Cytokine analysis using cytokine array. A549 cells were transfected with siRNA to knock down the CTNNAL1 gene. (B) Determination of CCL2 expression at the gene level. The expression of CTNNAL1 was knocked down using siRNA in A549 cells. (C) After treatment with CCL2 neutralizing antibody, the expression of CTNNAL1 was confirmed. (D) After treatment with CCL2 neutralizing antibodies, the expression of CSC marker proteins CD44, ALDH1A1, and ALDH1A3 was confirmed by WB. (E) Comparative analysis of EMT marker proteins E-cadherin, N-cadherin, and Vimentin after treatment with CCL2 neutralizing antibody.

Article Snippet: Antibody CCL2 (2 μg/mL; cat. no. MAB679; R&D SYSTEMS, Minneapolis, MN, USA) and normal mouse IgG1 antibody (1 μg/mL; cat. no. sc-3877; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used in the neutralization assay.

Techniques: Transfection, Knockdown, Expressing, Marker