Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neurodegeneration Enhances the Development of Arthritis.

doi: 10.4049/jimmunol.1601472

Figure Lengend Snippet: FIGURE 2. Tau-related neurodegeneration induces immune activation and a proinflammatory state. Flow cytometric analyses of isolated spleen, blood, and brain cells from untreated (control) and arthritic (CIA) tau-tg mice and wt littermates at day 50 after induction of CIA. (A) Percentage of splenic CCR2-expressing leukocytes (CD45+CCR2+), inflammatory monocytes (CD45+Ly6ChiCD11b+), granulocytes (CD45+Ly6CintCD11b+), and plasmablasts (CD45+B220lowCD138+) and mean fluorescence intensity (MFI) of Ly6C-expressing inflammatory monocytes and granulocytes. (B) Representative dot plots for splenic inflammatory monocytes (black) and granulocytes (blue), (C) representative dot blots for plasmablasts. (D) Per- centage of blood CCR2-expressing leukocytes (CD45+CCR2+), inflammatory monocytes (CD45+Ly6ChiCD11b+), granulocytes (CD45+Ly6CintCD11b+), and plasmablasts (CD45+B220lowCD138+) and MFI of Ly6C-expressing inflammatory monocytes and granulocytes. (E) Representative dot plots for splenic inflammatory monocytes (black), granulocytes (blue), (F) representative dot plots for plasmablasts. (G) Percentage of brain CD45hi leukocytes (forward scatter, CD45hi) and CCR2-expressing leukocytes (CD45hiCCR2+) and MFI of Ly6C-expressing leukocytes (CD45+Ly6C+). (H) Representative dot plots for brain CD45hi leukocytes. Pooled data from three independent experiments with n = 5–11 per experimental group. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, Mann–Whitney U test.

Article Snippet: Flow cytometric analyses were performed using anti-mouse CD16/32 unconjugated clone 93 (rat) (dilution 1:200, cat. no. 101302, BioLegend), anti-mouse/human CD45R/B220 PE/Cy5 clone RA3-6B2 (rat) (dilution 1:2000, cat. no. 103210, BioLegend), anti-mouse CD45 Pacific Blue clone 30-F11 (rat) (dilution 1:1000, cat. no. 103126, BioLegend), anti-mouse CD138 PE/Cy7 clone 281-2 (rat) (dilution 1:1000, cat. no. 142514, BioLegend), anti-mouse Ly-6G/Ly-6C (Gr-1) PE clone RB6-8C5 (rat) (dilution 1:2000, cat. no. 108408, BioLegend), anti-mouse Ly-6C PE/Cy7 clone HK1.4 (rat) (dilution 1:3000, cat. no. 128017, BioLegend), antimouse CD45 allophycocyanin clone 30-F11 (rat) (dilution 1:500, cat. no. 17-0451-82, eBioscience), anti-mouse CD11b PerCP-Cyanine5.5 clone M1/70 (cat. no. 45-0112-82, eBioscience), and anti-mouse CCR2 DyLight 488 polyclonal (rabbit) (cat. no. NBP1-48338G, Novus).

Techniques: Activation Assay, Isolation, Control, Expressing, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Lactation-associated macrophages exist in murine mammary tissue and human milk

doi: 10.1038/s41590-023-01530-0

Figure Lengend Snippet: a , Representative flow cytometry histograms and graph (±s.d.) showing the percentage of tdTomato labeling in F4/80 lo macrophages (F4/80 lo Mac), F4/80 hi macrophages (F4/80 hi Mac), liMacs (pregated on CD45 + SiglecF – Ly6C – Ly6G – ) and Ly6C hi monocytes (Mo) (pregated on CD45 + SiglecF – Ly6G – cells) in mammary glands from lactating (day 10 pp) Ms4a3 Cre R26 Ai14 mice. Pooled data from three independent experiments, n = 9. b , Immunohistochemistry of lactating mammary glands from Ms4a3 Cre R26 Ai14 mice (day 10 pp). DAPI (blue), SMA (green), Dectin-1 (magenta), tdTomato (red). Right panels show DAPI and single stains for Dectin-1 and tdTomato, respectively, of the outlined region in the overview image on the left. Scale bar, 70 µm; n = 4. c , Representative flow cytometry histograms and graph (±s.d.) showing the percentage of tdTomato labeling in F4/80 lo Macs, F4/80 hi Macs, liMacs and Ly6C hi Mo as in a in the mammary glands of lactating (day 8–15 pp) Ccr2 CreER R26 Ai14 mice treated with tamoxifen on day 1 and day 3 pp. Pooled data of three experiments, n = 6 ( n = 4 from 8 days pp and n = 2 from 15 days pp). d , Representative immunofluorescence image of lactating mammary glands (day 15 pp) from Ccr2 CreER R26 Ai14 dams treated with tamoxifen on day 1 and day 3 pp. DAPI (blue), SMA (green), Iba1 (yellow) and tdTomato (red). Middle panels (1) and bottom panels right (2) show DAPI and single stains for Iba1 and tdTomato of the outlined region in the overview image on the left (1) or of the image on top (2), respectively. Scale bars, 70 µm for (1), 35 µm for (2); n = 6. e , Representative flow cytometry plots and violin plots show the frequency and total cell counts of monocytes, F480 hi Macs, F4/80 lo Macs and liMacs as in a in the mammary glands from lactating (days 13−15 pp) Ccr2 – / – and wild-type dams. Pooled data from five independent experiments; n = 8 (WT) and 7 ( Ccr2 −/− ). Two-tailed Mann–Whitney test was performed, ** P < 0.01; *** P < 0.001; NS, not significant. f , Representative flow cytometry plots and a graph (±s.e.m.) showing the percentage of EdU + F4/80 lo macrophages, F4/80 hi macrophages and liMacs in mammary glands from lactating (day 7 pp) wild-type mice 20 h post EdU injection. Pooled data from three independent experiments, n = 4. Kruskal–Wallis test followed by Dunnʼs multiple comparison test. * P < 0.05.

Article Snippet: Ccr2 CreER-mKate ( Ccr2 CreER ) mice were generated by Taconic Artemis .

Techniques: Flow Cytometry, Labeling, Immunohistochemistry, Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Injection, Comparison

Journal: JCI Insight

Article Title: A prometabolite strategy inhibits cardiometabolic disease in an ApoE –/– murine model of atherosclerosis

doi: 10.1172/jci.insight.191090

Figure Lengend Snippet: ( A ) Schema of female ApoE –/– mice on an HFD with 150 mM SerBut or NaBut on a butyrate basis, or water ad libitum for 6 weeks. ( B ) Oil Red O (ORO) staining of aortic root plaque. ( C ) CD68 + IHC staining of aortic root plaque. Original magnification, ×20. ( D ) Representative images of immunofluorescent staining of aortic root. ( E ) Quantification of ORO + aortic root valve area. ( F ) Histological Stary scoring of plaque severity, ( G ) quantified CD68 + area, and ( H ) necrotic core of CD68 + IHC staining. ( I ) Quantified DAPI + , ( J ) DAPI + per plaque area, ( K ) iNOS + , and ( L ) iNOS + cells per plaque area in immunofluorescence stain. ( M – O ) Flow cytometry of immune cells infiltrating the aorta: ( M ) CD45 + total leukocytes, ( N ) CD11b + monocytes, and ( O ) CCR2 + CD11b + monocytes. ( P ) Water consumed by cages for the first 19 days of experiment. Data represent mean water consumed between 2 cages (replicates) as weight change of bottle converted to water volume. Error bars represent SD. ( Q ) Plasma IL-6 and ( R ) IFN-γ. ( S ) Blood flow cytometry of Ly6G + CD11b + circulating neutrophils. n = 5/cage and n = 10 mice per group. Data points represent individual mice displayed with median ± SEM. Statistical analyses were performed using a 1-way ANOVA with Tukey’s, Welch’s (if SDs were significantly different by Bartlett and Brown-Forsyth tests), or Kruskal-Wallis (if data were not normally distributed determined by Shapiro-Wilk test) post hoc test. P values less than 0.10 are shown. Outliers were removed by ROUT testing at Q = 1% in I – L . Scoring in C was done blinded. Data in C and F – H represent 2 independent pooled experiments: n = 10 for water and SerBut groups and n = 10 for NaBut groups in a single experiment. “B6,NC” denotes age-matched C57BL/6 mice on normal diet as non-statistical comparison to visualize healthy examples. Scale bars: 200 μm.

Article Snippet: In this study, antibodies used included those against CD16/CD32 (BioLegend, clone 93, catalog 101302), CCR2 (PE; R&D Systems, catalog FAB5538P), CD11b (BV605; BioLegend, clone M1/70, catalog 101257), CD11c (PE-Cy7; BD Biosciences, clone HL3, catalog 558079, BD Biosciences), CD19 (APC; BioLegend, clone 1D3, catalog 152412), CD3e (BUV395; clone 145-2C11, catalog 563565), CD45 (BV786; BD Biosciences, clone 30-F11, catalog 564225), F4/80 (BUV496; BD Biosciences, clone T45-2342, catalog 750644), I-A/I-E (PerCP-Cy5.5; BioLegend, clone M5/114.15.2, catalog 107626), Ly-6C (APC-Cy7; BioLegend, clone HK1.4, catalog 128026), and Ly-6G (FITC; eBioscience, clone 1AB, catalog 11-9668-80).

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Flow Cytometry, Clinical Proteomics, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Proportion of CD62L + , CD64 + , CCR2 + , HLA,A,B,C + or HLA,DR,DP,DQ + cells among peripheral blood monocytes from donors with or without chronic toxoplasmosis. Plastic-adherent PBMCs were isolated from blood samples and were fluorescently labeled using FITC-conjugated anti-CD14 or antibodies directed against the indicated surface markers or isotype control antibodies and PE-conjugated secondary antibodies. Blood donors were serologically classified as chronically T. gondii -infected or non-infected using plasma from the blood samples. (A) CD14 + monocytes (R1) were back-gated and identified (R2) among FSC/SSC-analyzed total cells. R2-gated cells were then analyzed for expression of cell surface markers as indicated, and positive cells were identified after specific (anti-CCR2 in (A) ; see for the other surface markers) and isotype control labeling. (B–F) Percentages of monocytes from T. gondii seropositive or seronegative individuals positive for cell surface markers as indicated. Solid and dashed lines in the box-whisker plots indicate median and mean values, respectively; circles indicate individual data points. Data are from 5 T. gondii seropositive and 16 seronegative blood donors; outlyers were excluded. * p < 0.05 (Student's t -test).

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: Isolation, Labeling, Control, Infection, Clinical Proteomics, Expressing, Whisker Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Expression of cell surface receptors on monocytes from individuals with chronic toxoplasmosis and non-infected controls.

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Long-Term Impact of Toxoplasma gondii Infection on Human Monocytes

doi: 10.3389/fcimb.2019.00235

Figure Lengend Snippet: Impact of in vitro infection of monocyte-enriched PBMCs with T. gondii on expression of CD62L, CD64, CCR2, HLA-A,B,C, or HLA-DR,DP,DQ on monocytes from donors with or without chronic toxoplasmosis. Monocyte-enriched PBMCs were isolated from blood samples and were either directly FACS-analyzed (0 h) or were cultivated in vitro for 48 h and infected or not with T. gondii during the final 24 h as indicated and then FACS-analyzed. Expression of cell surface markers was determined for CD14-positive monocytes as outlined in . (A–E) Percentages of cells from T. gondii seropositive (gray bars) or seronegative (open bars) individuals with expression of surface markers above background staining. (F–J) Expression levels of surface markers as indicated on monocytes from T. gondii seropositive or seronegative blood donors. Data represent means ± S.E.M. from 5 T. gondii seropositive and from 13 out of 16 seronegative blood donors which had been randomly selected for in vitro infection assays; outlyers were excluded. Individual data points are also indicated. Significant differences between groups were identified by ANOVA [ *** p < 0.001; ** p < 0.01; * p < 0.05; a and b indicate dose-dependent increase or decrease, respectively, of HLA-DR,DP,DQ after parasite infection ( p < 0.01)].

Article Snippet: Alternatively, they were labeled with 2 μg/mL of mouse monoclonal antibodies directed against human CD62L (clone DREG-56), human CD64 (clone 10.01.13), human HLA-A,B,C (clone G46-2.6), human HLA-DR,DP,DQ (clone Tu39; all antibodies from BD Biosciences), or directed against human CCR2 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or they were incubated with appropriate isotype control antibodies (clones MOPC-21, 27-35 and G155-178; BD Biosciences) for 30 min at 4°C.

Techniques: In Vitro, Infection, Expressing, Isolation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2.

doi: 10.4049/jimmunol.1501908

Figure Lengend Snippet: FIGURE 2. CYTL1 induces CCR2B internalization. (A) HEK293-CCR2B were treated with CCL2 (20 nM) or CYTL1 (10 or 50 nM) for 1 h. CCR2B internalization was analyzed by FACS using mouse anti-CCR2 Abs as primary Abs. (B) The average CCR2B internalization extent from three independent experiments is shown, and significant differences were calculated compared with the control. *p , 0.05, **p , 0.01, ***p , 0.001. (C) HEK293 cells overexpressing CCR2B-EGFP were treated with CCL2 (20 nM) or CYTL1 (10 or 50 nM) for 1 h. Cells were fixed and observed by confocal microscopy. Scale bars, 25 mm. All of the results above were representative of at least three independent experiments. (D) Human monocytes were treated with CCL2 (20 nM) or CYTL1 (10, 50, or 500 nM) for 15 min. CCR2B internalization was analyzed by FACS using mouse anti-CCR2 Abs as primary Abs. (E) The average CCR2B internalization extent from three independent experiments is shown, and significant differences were calculated compared with the control. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Mouse anti-CCR2 Abs were obtained from R&D Systems (Minneapolis, MN).

Techniques: Control, Confocal Microscopy