Journal: Cell reports

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation

doi: 10.1016/j.celrep.2024.114193

Figure Lengend Snippet: (A) Conditioned medium (CM) was collected from primary WT and Slc4a4 KO astrocytes and subjected to LC-MS/MS-based unbiased proteomics and cytokine/chemokine array. (B) Angiogenic factors detected from LC-MS/MS-based unbiased proteomics. (C) Cytokine/chemokines changed in the CM from WT and Slc4a4 KO astrocytes. Pooled CM from three independent cultures per genotype were used (D) CCL2 level was measured by ELISA in CM collected under either normal or oxygen-glucose-deprivation (OGD) condition cortices from uninjured and stroked (1 dpi) brains from WT and Slc4a4-icKO mice. Each dot represents CM or cortices collected from an individual animal. n = 3–5 per genotype. * p < 0.05, ** p < 0.01 by Student’s t test. In vivo astrocytic CCL2 expression after stroke at 4 dpi was quantified from double immunostaining. n = 17–21 cells collected from 3–5 mice per genotype. * p < 0.05 by Student’s t test. (E) Representative images of astrocytic CCL2 expression at 4 dpi by double immunostaining of CCL2/GFAP. (F and G) Endothelial CCR2 mRNA expression was visualized by RNA scope and CD31 staining and quantified by counts of CCR2+ puncta. Each dot represents an individual blood vessel. n = 49 vessels collected from 4 mice per genotype. **** p < 0.0001 by Student’s t test. (H and I) Representative images and quantification of endothelial CCR2 expression from double immunostaining. Each dot represents an individual animal. n = 4–5 per genotype. * p < 0.05 by Student’s t test. (J) Proposed model for the Slc4a4-CCL2-CCR2 axis regulating astrocyte-endothelial interaction. All data are presented as mean ± SEM.

Article Snippet: CCR2 antagonist , Tocris , 2517/10.

Techniques: Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vivo, Expressing, Double Immunostaining, RNAscope, Staining

Journal: Cell reports

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation

doi: 10.1016/j.celrep.2024.114193

Figure Lengend Snippet:

Article Snippet: CCR2 antagonist , Tocris , 2517/10.

Techniques: Purification, Virus, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Mutagenesis, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCR2/CCL2 axis promoted pro-inflammatory macrophages through the phosphorylation of p65. (A) Relative mRNA expression of Nos2 in untreated, CCL2 treated and LPS treated macrophages. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (B) Representative immunofluorescence staining of iNOS in untreated, CCL2 treated and LPS treated macrophages. (C) Relative mRNA expression of inflammatory factors in macrophages treated with LPS or IL-4, and RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (D) Relative mRNA expression of Ccr2 in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. (E, F) Relative mRNA expression of pro-inflammatory (E) and anti-inflammatory factors (F) in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (G) Representative immunofluorescence staining of p65 in macrophages in untreated, treated with CCL2, and CCL2+RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (H) Relative mRNA expression of inflammatory factors Il6 , Il1β and nos2 treated with CCL2 or p65 inhibitor. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05, ns, no significance.

Article Snippet: For RS504393 treatment, 5mg/kg RS504393 (HY-15418; MCE) or vehicle was intraperitoneal injected in mice daily after orthodontic devices application ( ).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCL2 treatment promoted the phosphorylation of p65 in macrophages. (A) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 for 0, 30, 60 and 360 minutes. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (B) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 and RS504393. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. ns, no significance. (C, D) Western bolt analysis of p-p65 and p65 expression in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (E, F) The distance of OTM in TM and TM+CCR2i group and representative 3D micro-CT reconstruction. White arrow: direction of force and tooth movement. Values are mean ± SD. n = 5. ***P < 0.001. (G) Graphic abstract of this study.

Article Snippet: For RS504393 treatment, 5mg/kg RS504393 (HY-15418; MCE) or vehicle was intraperitoneal injected in mice daily after orthodontic devices application ( ).

Techniques: Western Blot, Expressing, Quantitation Assay, Micro-CT

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. Panel 1 , the effect of 10 μM concentration of CCR2 RS504393 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. CCR2 RS504393 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Notably, levels of specific mRNAs from non-stimulated cells were not significantly different than those obtained from cultures that proceeded with the CCR2 inhibitor alone. Panel 2 , the effect of 1 μM concentration of MMP-1 inhibitor 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was diluted in incomplete RPMI. The results presented are from six independent experiments showing the mean and standard deviations from the mean. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Of note, levels of specific mRNAs from non-stimulated cells were not significantly different from those obtained from cultures that proceeded with the 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide inhibitor alone. The inhibitors’ concentrations were selected from dose response-experiments.

Article Snippet: THP-1 cells (1×10 6 cells/ml) were stimulated with the indicated amounts of H37Rv M. tuberculosis lysate obtained after sonication (sonicated H37Rv M. tuberculosis ) as described previously (Ganachari et al. 2010), or the indicated amounts of CCR2 RS504393 (Tocris), MMP-1 4-Aminobenzoyl-Gly-ProD-Leu-D-Ala hydroxamic peptide (SIGMA), PAR-1 SCH79797 (Tocris) inhibitors, and human purified MMP-1 (hMMP-1) activated with p-aminophenylmercuric acetate (EMD Chemicals).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Sonication, Control

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We used three-color FACS analysis for these experiments. Exposure of quiescent THP-1 cells to sonicated H37Rv M. tuberculosis for 24 hr induced the differentiation of these cells into CD14-positive/CD16-negative cells. In Panels 1 and 2, Section A shows a low proportion of CD14-positive/CD16-negative cells in quiescent THP-1 cells; Section B shows an increment in the proportion of CD14-positive/CD16-negative THP-1 cells in response to sonicated H37RV M. tuberculosis exposure.; Section C shows minimal (non-significant) variation in this response to sonicated H37Rv M. tuberculosis exposure in the presence of the MMP-1 inhibitor (Panel 1) or CCR2 inhibitor (Panel 2). In Sections D, E, and F of Panel 1, we show that the presence of MMP-1 inhibitor 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide prevents the down-regulation of PAR-1 expression by THP-1 cells exposed to sonicated H37Rv M. tuberculosis exposure. In Sections D, E, and F of Panel 2, we show that the presence of CCR2 inhibitor RS504393 also prevents the down-regulation of PAR-1 expression by THP-1 cells exposed to sonicated H37Rv M. tuberculosis exposure. We acquired 100,000 events for experiments in Panel 1, while we acquired 10,000 events for experiments in Panel 2, according to the number of live cells gathered in each experiment. Of note, the MMP-1 inhibitor was dissolved in incomplete RPMI while the CCR2 inhibitor was dissolved in DMSO to obtain a DMSO final culture concentration of 0.01%. Three experiments were done for the assessment of each inhibitor.

Article Snippet: THP-1 cells (1×10 6 cells/ml) were stimulated with the indicated amounts of H37Rv M. tuberculosis lysate obtained after sonication (sonicated H37Rv M. tuberculosis ) as described previously (Ganachari et al. 2010), or the indicated amounts of CCR2 RS504393 (Tocris), MMP-1 4-Aminobenzoyl-Gly-ProD-Leu-D-Ala hydroxamic peptide (SIGMA), PAR-1 SCH79797 (Tocris) inhibitors, and human purified MMP-1 (hMMP-1) activated with p-aminophenylmercuric acetate (EMD Chemicals).

Techniques: Sonication, Expressing, Concentration Assay

Journal: Respiratory Research

Article Title: Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells

doi: 10.1186/s12931-019-0973-6

Figure Lengend Snippet: Suppressive effect of a CCR2 antagonist on the expression of S100a8 , S100a9 , Slc26a4 , and Il5 in Adgrf5 −/− lungs. Two-week-old Adgrf5 −/− mice were administered RS504393 (2 mg/kg body weight) or vehicle once daily via subcutaneous injection for 8 days. The mRNA expression of Ccl2 , S100a8 , S100a9 , Saa3 , Slc26a4 , Clca1 , Tgfb1 , Il5 , and Il13 was analyzed by qPCR using total RNA isolated from post-lavage lungs of injected mice and non-injected WT mice at 3 weeks of age. The data were normalized to Gapdh levels and are expressed as values relative to those from corresponding vehicle-treated Adgrf5 −/− mice. Values are presented as the mean ± SEM ( n = 3–4). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: RS504393 (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in dimethylformamide at 10 mg/ml and then diluted to 0.4 mg/ml with a 0.9% sterile NaCl solution.

Techniques: Expressing, Injection, Isolation

Journal: Chemokines

Article Title: Selective and Dual Targeting of CCR2 and CCR5 Receptors: A Current Overview

doi: 10.1007/7355_2014_40

Figure Lengend Snippet: CCR2 antagonists with spiropiperidine structure, inhibitory effects on CCL2 binding to human CCR2 receptor

Article Snippet: A prominent example of the spiropiperidine class of CCR2 inhibitors, RS504393 ( 12 ), was reported by Roche/Iconix.

Techniques: Binding Assay