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: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: PU.1 is upregulated in macrophages of advanced atherosclerotic lesions and promotes inflammation through IL-1β/NF-κB signaling. ( A ) Boxplot showing expression levels of PU.1 in early versus advanced atherosclerotic plaques based on the GSE43292 dataset. ( B ) UMAP plot of single-cell RNA sequencing data depicting major immune and stromal cell populations in atherosclerotic lesions. ( C ) UMAP feature plot showing SPI1 (encoding PU.1) expression predominantly enriched in macrophages. ( D ) Representative immunofluorescence staining of PU.1 (green) and CD68 (red) in aortic root sections from chow diet– and high fat diet–fed mice. Nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. ( E ) CUT&Tag analysis showing genome-wide binding of PU.1 in macrophages. Heatmap indicates PU.1 enrichment near transcription start sites (TSS). ( F ) Genomic distribution of PU.1 binding peaks identified by CUT&Tag. ( G – H ) Representative CUT&Tag tracks showing PU.1 binding at the promoters of pro-inflammatory cytokines. ( I ) Dual-luciferase reporter assay confirming the transcriptional activation of the IL-1β promoter by PU.1 overexpression (OE). ( J ) Western blot showing that PU.1 knockdown suppressed ox-LDL–induced IL-1β expression and NF-κB pathway activation (p-IκB and p-p65). ( K ) Western blot demonstrating that IL-1β knockdown reversed PU.1-induced NF-κB activation. ( L ) Western blot analysis showing that the PU.1 inhibitor DB1976 attenuated ox-LDL–induced IL-1β expression and NF-κB activation. ( M ) qRT-PCR analysis of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, MCP-1) in macrophages treated with ox-LDL with or without DB1976. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining, Genome Wide, Binding Assay, Luciferase, Reporter Assay, Activation Assay, Over Expression, Western Blot, Knockdown, Quantitative RT-PCR, Comparison, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: The anti-atherosclerotic effects of the D-FNVs. ( A - D ) BMDMs were co-treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs). The levels of IL-1β, IL-6, TNF-α, and MCP-1 in the supernatant were measured by ELISA. ( E , F ) Flow cytometry analysis and quantification of intracellular ROS levels in BMDM cells treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs), respectively, at 2 mM DB1976 for 24 h. ( G , H ) Flow cytometry analysis and quantification of apoptosis rates in BMDMs treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs) at 2 mM DB1976 for 24 h. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Comparison, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: D-FNVs ameliorated inflammation in an atherosclerotic mouse model. ( A , B ) Representative images of plaques within the aortic root subjected to immunofluorescent staining for the macrophage marker CD68. Scale bar: 100 μm. ( C – F ) Levels of IL-1β, IL-6, TNF-α, and MCP-1 in aortic tissues collected from atherosclerotic mice treated with various formulations (saline, DB1976, D-NVs, D-FNVs). ( G – J ) Levels of IL-1β, IL-6, TNF-α, and MCP-1 in blood serum collected from the same groups of atherosclerotic mice. The n values are all biological replicates. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, and *** p < 0.001. **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Staining, Marker, Saline, Comparison, Standard Deviation

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: Frontiers in Immunology

Article Title: CCR2 Signal Facilitates Thymic Egress by Priming Thymocyte Responses to Sphingosine-1-Phosphate

doi: 10.3389/fimmu.2018.01263

Figure Lengend Snippet: Restricted expression of CCR2 in CD69 − Qa-2 + mature CD4 single-positive (SP) thymocytes. (A,B) Genes encoding chemoattractant receptors were sorted out from the genes differentially expressed among the four subsets (SP1–SP4) of CD4 SP thymocytes. The expression patterns of these genes are depicted in heatmap (A) or line charts (B) . (C) Expression levels of Cxcr3, Cxcr6, S1pr4 , and Ccr2 were assessed in various subsets of CD4 SP thymocytes using qPCR. The experiments were repeated three times with triplicates for each sample and the data are presented as mean ± SEM. (D) CCR2 expression in CD69 + Qa-2 − (SP1 + SP2), CD69 − Qa-2 − (SP3), and CD69 − Qa-2 + (SP4) subsets of CD4 SP thymocytes as determined by flow cytometry. Representative dot plots are shown out of three independent experiments. (E) Purified CD69 − Qa-2 + SP4 thymocytes were tested for chemotaxis to CCL2 and S1P using transwell migration assay. Data from four independent experiments with duplicates are presented as mean ± SEM. Statistical differences between groups were determined by the Student’s t test. ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Purification, Chemotaxis Assay, Transwell Migration Assay

Journal: Frontiers in Immunology

Article Title: CCR2 Signal Facilitates Thymic Egress by Priming Thymocyte Responses to Sphingosine-1-Phosphate

doi: 10.3389/fimmu.2018.01263

Figure Lengend Snippet: CCR2-mediated chemokinesis and its impact on S1P-induced chemotaxis. (A) CCL2 concentration in the thymic homogenate and serum of wild-type (WT) mice as measured by ELISA. The experiments were repeated three times with duplicates. Data are presented as mean ± SEM. (B) Double-positive thymocytes, and Qa-2 − (SP1–3) and CD69 − Qa-2 + CD4 single-positive (SP) thymocytes were purified from wild-type (WT) and knockout (KO) mice and loaded onto thymic slices. Their migration speed were recorded using two-photon microscopy. The experiments were repeated three times. Each dot represents a single cell (WT SP1–3, n = 171; KO SP1–3, n = 120; WT SP4, n = 193; KO SP4, n = 198). Bars indicate the median. (C) Mean squared displacement (MSD) over a period of 20 min for SP4 thymocytes. Data from more than 100 cells at each time point are presented as mean ± SD. (D) CD69 − Qa-2 + CD4 SP thymocytes were plated in a two-dimensional migration assay. Cell movement under different conditions was tracked using time-lapse imaging. The trajectories of WT (red) and KO (green) CD4 SP4 cells are shown ( Upper ). The displacement of the WT SP4 cells from the original position is depicted at the Bottom . (E) Velocity of cell movement in the two-dimensional migration assay. The experiments were repeated three times. Each dot represents a single cell. Bars indicate the median. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance.

Article Snippet: Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Chemotaxis Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Purification, Knock-Out, Migration, Microscopy, Imaging

Journal: Frontiers in Immunology

Article Title: CCR2 Signal Facilitates Thymic Egress by Priming Thymocyte Responses to Sphingosine-1-Phosphate

doi: 10.3389/fimmu.2018.01263

Figure Lengend Snippet: Activation of the FoxO1–KLF2–S1P1 axis by CCR2 signaling through Stat3. (A) Surface expression of S1P1 in the CD69 + Qa-2 − (SP1 + SP2), CD69 − Qa-2 − (SP3), and CD69 − Qa-2 + (SP4) subsets of CD4 SP thymocytes from WT and KO mice. The experiments were repeated three times with six mice for each group. Representative dot plots are shown. (B) Percentage of S1P1 + cells are presented as mean ± SD. (C) Foxo1, Klf2, and S1pr1 mRNA expression in WT and KO CD4 SP thymocytes as determined by qPCR. The experiments were repeated three times with triplicates. Data are present as mean ± SEM. (D) CD69 − Qa-2 + SP4 thymocytes from WT and KO mice were stimulated with CCL2 in the presence or absence of STAT3 inhibitor Stattic. Foxo1, Klf2, and S1pr1 mRNA expression was detected by qPCR. The experiments were repeated three times with triplicates. Data are present as mean ± SEM. (E) Following stimulation with CCL2, WT SP4 thymocytes were examined for surface expression of S1P1. Representative histograms are shown out of three independent experiments. (F,G) Levels of phosphorylated Stat3 were examined by Western blotting (F) or intracellular staining (G) in SP4 thymocytes after stimulation with CCL2. Western blotting was performed at 30 min after stimulation. The experiments were repeated three times with similar results. (H–J) SP4 thymocytes were stimulated with CCL2 in the presence or absence of STAT3 inhibitor Stattic. Surface expression of S1P1 was assayed by flow cytometry (H) . Subcellular localization of FoxO1 was revealed by immunofluorescent staining (I) and nucleus Foxo1 was quantified by mean fluorescent intensity (MFI) (J) . Data are presented as mean ± SD. Two independent experiments were performed with similar results. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: ns, no significance; S1P1, sphingosine-1-phosphate receptor 1; SP, single-positive; WT, wild-type; KO, knockout; KLF2, Kruppel-like factor 2; FoxO1, forkhead box O1.

Article Snippet: Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Western Blot, Staining, Flow Cytometry, Knock-Out

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