neutralizing anti-ccl2 monoclonal antibody  (Thermo Fisher)

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: Inflammatory ICAM1 + fibroblasts expand in a murine model of ligature-induced periodontitis. (A) Flow cytometry gating strategy for analysis of lineage-negative (CD45 - CD31 - EpCam - Ter119 - ) pericytes, gingival fibroblasts and ICAM1 + fibroblasts in non-ligated control (NL) and ligature induced periodontitis (LIP) group. (B) Quantification of percent fibroblasts (Lin - PDGFRA + ) and pericytes (Lin - CD146 + ) normalized by Lin - mesenchymal cell numbers in NL and LIP groups. Each dot represents one mouse as a split-mouth design. (C) Quantification of percent ICAM1 + fibroblasts (Lin - PDGFRA + ICAM1 + ) normalized by total fibroblast numbers. Each dot represents one mouse as a split-mouth design. (D) Representative immunofluorescent images of NL and LIP paraffin sections stained with antibodies specific against ICAM1 (red) and PDGFRA (green). Arrows point to ICAM1 + PDGFRA + cells. Scale bar, 100μm; inset scale bar, 10μm. (E) Quantification of ICAM1 fibroblast numbers normalized by lamina propria area (mm 2 ) comparing NL and LIP groups from the immunofluorescence experiments. (F) Left, representative immunocytochemistry images of primary gingival fibroblasts stained with ICAM1 antibody comparing control versus stimulated groups. Lipopolysaccharide from P. gingivalis (LPS, 1 ug/ml) and tumor necrosis factor alpha (TNF, 10 ng/ml) were used for stimulation. Scale bar, 20 μm. Right, quantification of ICAM1 + fibroblast numbers normalized by total fibroblast cells comparing control and LPS+TNF group. (G) Schematic diagram of qPCR for fibroblast-derived cytokines comparing FACS-sorted ICAM1 - and ICAM1 + fibroblasts in human and mouse models of periodontitis. (H, I) Quantification of relative mRNA expression by qPCR for CXCL13, CXCL1, CXCL2, CCL19, and CCL2 comparing sorted ICAM1 - and ICAM1 + fibroblasts. (H) Gingival tissue specimens from N=8 periodontitis patients; each dot represents individual patient. (G) Gingival tissues harvested from mice with LIP; each dot represents pooled samples from 2-3 mice for a total of 4 data points (N=10 mice). Data represent mean ± SEM. Welch’s t-test (B–H) and Mann-Whitney U test (I) comparing control vs. experimental group; *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Staining, Immunofluorescence, Immunocytochemistry, Derivative Assay, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: (A) Left, representative flow cytometry histogram of CCL2 signal in 7d ligated control and experimental mice. Middle and right, quantification of percent CCL2 + ICAM1 + fibroblasts (Lin - PDGFRA + ) and CCL2 + pericytes (Lin - CD146 + ) normalized by lineage-negative mesenchymal cell numbers. (B) Representative flow cytometry histogram of ICAM1 expression in fibroblasts pre-gated for CCL2 + signal in control or ICAM1 + oral fibroblast-enriched conditions (LPS + TNF) treated with or without BMS-345541 in vitro. (C) Left, quantification of ICAM1 + CCL2 + fibroblasts cell counts by flow cytometry analysis. Right, ELISA analysis of CCL2 concentration in the supernatant of cultured control or ICAM1 + enriched fibroblast conditions with or without BMS-345541. (D) Top, representative gating strategy for CCL2 + cell phenotyping by flow cytometry using tissues collected from CCL2 mCherry reporter mice that had ligature placed for 7 days. Bottom right, quantification of CCL2 + fibroblasts (CD45 - EpCAM - PDGFRA + ), leukocytes (CD45 + ), endothelial cells (CD31 + ), epithelial cells (EpCAM + ), and pericytes (CD31 - CD146 + ) normalized by total CCL2 + cells. Each dot represents one mouse (N=4). (E) Top, representative immunofluorescent images of non-ligated control (NL) and ligature induced periodontitis (LIP) from CCL2 mCherry reporter mice. Paraffin sections were stained with antibodies specific against PDGFRA (green) and red fluorescent protein (red), and immunopositivity in the lamina propria and periodontal ligament space (PDL) was examined. Scale bar, 20μm.Bottom, quantification of percent CCL2 + fibroblasts (CCL2 + PDFGRA + ) normalized by total nucleated cells in field of view. N=3, split mouth design. (F) Schematic diagram of in vitro phagocytosis assay using conditioned media from ICAM1 + enriched oral fibroblast culture and primary bone marrow-derived macrophages. (G) Top, flow cytometry gating strategy for identification of double positive F4/80 + fluorescence beads + from in vitro phagocytosis assay. Bottom, representative flow cytometry histogram of fluorescence beads signals showing three distinct peaks from control, conditioned media (CM), and CM + anti-CCL2 neutralization groups. (H) Quantification of fluorescence beads + F4/80 + macrophage numbers per 10 4 events. Left, number of beads + F4/80 + with a first peak (one bead) in the histogram; right, number of beads + F4/80 + with a second or third peak (two or three beads phagocytosed). N=3 each. All in vitro experiments were repeated independently twice. Data represents mean ± SEM. For (A) , one-way ANOVA test followed by pairwise t-test’s with Šidák’s correction was performed. For (C) Brown Forsythe ANOVA test with Dunnett’s T3 Multiple comparison test; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Staining, Phagocytosis Assay, Derivative Assay, Fluorescence, Neutralization, Comparison

Journal: Cellular & molecular immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2.

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Fig. 6 Desialylation of MDSCs downregulates MDSC functional markers and cytokines at the transcript level. Suppressive MDSC-like cells were generated in vitro by coculture with A549 or A549-sia cancer cell lines as described in Fig. 5. CD33+ cells were isolated on Day 7 and processed for single-cell RNA sequencing (scRNAseq). A Seurat analysis of the scRNAseq dataset projected in UMAP colored by cluster. n = 4 donors per treatment group. B The dataset was subdivided into individual groups showing MDSC-like cells generated by A549 (green, left) or A549-sia (pink, right) coculture. C Stacked bar plots showing the frequency of each cluster annotated in (A) subclustered in A549- and A549-sia- generated MDSC-like cells. D Gene Ontology (GO) enrichment analysis of the top 10 upregulated gene sets found in Cluster 2. Dot plot showing the mean normalized enrichment score (NES) of the GO gene sets. The color coding indicates the adjusted p values, and the dot size is proportional to the gene count found in the listed pathway. E Heatmap of selected genes per patient divided into A549- and A549-sia- generated MDSC-like cells. The genes were functionally categorized into 5 groups: (i) chemokines and chemotaxis genes; (ii) MDSC and macrophage marker genes; (iii) protumor function MDSC genes; (iv) other genes; and (v) adhesion, attachment and ECM-related genes. F Gene expression of selected markers (IL1B, S100A9, S100A8, MKi67 and CCL2) as a density plot. The expression density is shown as a scale from blue (low) to yellow (high). G Expression of CCL2 was normalized to that of the donor.The data are presented as the mean ± SD. A two- tailed paired t test (F, E) was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 μg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Functional Assay, Generated, In Vitro, Isolation, RNA Sequencing, Chemotaxis Assay, Marker, Gene Expression, Expressing, Two Tailed Test

Journal: Cellular & molecular immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2.

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Fig. 7 CCL2 is involved in T-cell suppression via the Siglec-sialoglycan axis in suppressive myeloid cells. A MCP-1/CCL2 found in the supernatant of murine MDSC:T-cell cocultures at the endpoint of the experiment from Fig. 4B–D. MDSCs were untreated, or were pretreated with sialidase or a Siglec-E blocking antibody. n = 3 donors per group. B Correlation of MCP-1/CCL2 levels measured in supernatants of murine MDSC:T-cell cocultures at the endpoint and percentage of proliferating CD8+ T cells from the same conditions from (A). n = 3 donors per group. C Experimental setup: Neutralization of CCL2 using a neutralization antibody in SigEΔLysM mice and SigEWT littermates bearing B16F10 tumors. Mice were injected with a CCL2 neutralization antibody up to 3 times a week (gray arrow) starting 1 day after subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored, and the suppressive capacity of MDSCs was analyzed in vitro. D Kaplan‒ Meier survival curves from pooled data from 2 independent experiments. n = 5–8 mice per group. E B16F10 tumors at the endpoint (C) were digested, cocultured with pervanadate and analyzed by phospho-flow cytometry for phosphorylated STAT3 (pSTAT3). The percentage of pSTAT3 gated on total MDSCs is shown. Representative histograms for each condition are shown on the right. n = 3 mice per group. F Suppressive capacity of MDSCs against naïve T cells. Percentage of proliferating CD8+ T cells cocultured without MDSCs, (G) with MDSCs from SigEWT mice or (H) MDSCs from SigEΔLysM mice with or without addition of CCL2 blocking antibody. N = 3–5 mice from N = 3 experiments. I Cytokine expression found in the supernatant of human primary CD33+:CD8+ cell cocultures at the endpoint of the experiment from Fig. 5H. CD33+ cells were left untreated or pretreated with sialidase. Z scores were calculated for each cytokine and are shown on a color scale from blue (low) to red (high). n = 3 donors per group. The data are presented as mean ± SD. Two-tailed paired t tests or unpaired t tests (E) were used. R shows the Pearson correlation coefficient. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 μg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Blocking Assay, Neutralization, Injection, In Vitro, Cytometry, Expressing, Two Tailed Test