Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: Intratracheal administration of 0.5μg TNF (A and B) or 50μg poly(I:C) (C and D) to A20 AEC-KO or wild type littermates (A20 WT ). Absolute numbers of neutrophils or monocytes in bronchoalveolar lavages (BAL) as determined by flow cytometry at 6h and 24h post-treatment for TNF (A) or 24h post-treatment for poly(I:C) (C). IL-6, CXCL1 (KC), CCL2 (MCP-1) and TNF [only for poly(I:C)] protein levels in BAL fluid detected by Multiplex immunoassay (B and D). Data represent mean ± SEM of at least 4 mice per group (*p < 0.05; Student’s t -test).

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Flow Cytometry, Multiplex Assay

Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: (A) Absolute numbers of monocytes, neutrophils and alveolar macrophages in bronchoalveolar lavages (BAL) of A20 AEC-KO or A20 WT mice at 2, 5, 8 and 12 days post-infection (days p.i.) with 0.05 X LD 50 X-47. (B) Absolute numbers of resident CD11b - or recruited CD11b + macrophages in the lungs of A20 WT and A20 AEC-KO mice. (C) CCL2 (MCP-1) protein levels in BAL fluid measured by Multiplex immunoassay at indicated time points post-infection. (D) Weight loss of A20 AEC-KO and A20 WT mice infected with 0.05 X LD 50 X-47. At day 6 p.i. (indicated by an arrow) mice received intranasal treatment with 50 μg/kg recombinant CCL2 (rCCL2) or PBS. Data were analysed using Student’s t -test (A, B and C *p < 0.05) and 2-way ANOVA (D, *p < 0.05 for A20 AEC-KO PBS vs. A20 WT PBS and # p < 0.05 for A20 AEC-KO PBS vs A20 AEC-Cre rCCL2). Data represent mean ± SEM of at least 3 mice per group. Data are representative of at least 2 independent experiments.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Infection, Multiplex Assay, Recombinant

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 increases cell surface expression of P2X4R protein in microglial cells. P2X4R protein on the surface of primary cultured microglial cells was detected by cell surface biotinylation followed by Western blot analysis of P2X4R protein using its specific antibody. Total cellular P2X4R protein was detected in whole-cell lysates of microglial cells. Microglial cells in primary cultures were treated with either CCL2 (100 ng/mL), CX3CL1 (100 ng/mL), or IFN-γ (100 U/mL) for 30 min (a) with CCL2 (10, 30, and 100 ng/mL) for 30 min (b) and with CCL2 (100 ng/mL) for 30, 60, and 120 min (c). b, c histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCR2 mediates CCL2-induced surface P2X4R expression. a–c Microglial cells in primary cultures were pretreated with either CCL2 neutralizing antibodies (CCL2-Ab, 2.5 and 25 μg/mL) for 30 min (a), RS-504393 (RS, 1 and 10 μM) for 20 min (b) or forskolin (10 μM) for 10 min (c) before CCL2 (100 ng/mL) stimulation. d Microglial cells were stimulated by CCL12 (10 ng/mL) for 30 min. Surface proteins of microglial cells were biotinylated 30 min after CCL2 (a–c) and CCL12 (d) stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P < 0.01, ***P < 0.001 compared with unstimulated control microglia; #P < 0.05, ##P < 0.01 compared with CCL2-stimulated microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 is involved in fibronectin-induced surface P2X4R upregulation. a Biotinylation assay of surface P2X4R proteins. CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and normal goat IgG (IgG, 25 μg/mL) were pretreated for 30 min before fibronectin (FN, 10 μg/mL) stimulation. Surface proteins of microglial cells were biotinylated 24 h after fibronectin stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated five times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P <0.01 compared with unstimulated control microglia; #P < 0.05 compared with fibronectin-stimulated microglia. b CCL2 levels in supernatants of microglial cultures were measured by ELISA; primary cultured microglial cells were treated with or without fibronectin (10 μg/mL) for various time points of testing. The experiments were repeated six times, and the data are means ± SEM of the concentration of CCL2 (pg/mL). ***P < 0.001 compared with control at 1 h

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Surface Biotinylation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances movement of P2X4Rs. a Immunocytochemical analysis of P2X4Rs detected by its specific antibody. Primary cultured microglia were stimulated by CCL2 (100 ng/mL) for 30 min and were fixed by formaldehyde. Cells were pretreated with CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and forskolin (10 μM) for 30 and 10 min, respectively, before CCL2 stimulation. b Monitoring of P2X4R-GFP fluorescence particles in living microglial cells transduced with a lentiviral vector encoding P2X4R-GFP. Left Typical trajectories of P2X4R-GFP fluorescence particles in microglia treated with HBSS (control) or CCL2 (100 ng/mL). Right The total distance (μm, means ± SEM, n = 19) of the trajectories of P2X4R-GFP fluorescence particles from initial position. ***P < 0.001 compared with the control group by two-way ANOVA. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Culture, Fluorescence, Transduction, Plasmid Preparation, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: Dynamin inhibitor does not affect the effect of CCL2 on cell surface P2X4R expression. Microglial cells in primary cultures were pretreated with the dynamin inhibitor dynasore (80 μM) for 30 min before CCL2 (100 ng/mL) stimulation. Surface proteins of microglial cells were biotinylated 30 min after CCL2 stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, ***P < 0.001 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 causes lysosomal exocytosis in microglial cells. a Immunofluorescence for P2X4R (green) and LGP85 (red), a marker of lysosome, in primary cultured microglial cells treated with or without CCL2 (100 ng/mL) for 30 min. b The release of the lysosomal enzyme β-hexosaminidase from microglial cells was assayed by measuring its activity in the supernatants of microglial cultures collected at 30 min after CCL2 (100 ng/mL) or ionomycin (5 μM) stimulation. The experiments were repeated six times and the data are means ± SEM. *P < 0.05, **P < 0.01 compared with unstimulated control microglia. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Immunofluorescence, Marker, Cell Culture, Activity Assay, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances P2X4R-mediated Akt phosphorylation in microglial cells. Primary cultured microglia were treated with or without CCL2 (100 ng/mL) for 30 min. Western blot analysis of the levels of phosphorylated and total Akt protein in whole-cell lysate of CCL2-treated microglia 10 min after ATP (5 μM) stimulation. Cells were pretreated with TNP-ATP (100 μM) 5 min before ATP stimulation. A histogram of the relative band density ratio of Akt phosphorylation normalized to the levels of β-actin. The experiments were repeated five times, and the data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Phospho-proteomics, Cell Culture, Western Blot

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: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

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: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

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