Journal: The Journal of Clinical Investigation

Article Title: The β 3-adrenergic receptor agonist mirabegron improves glucose homeostasis in obese humans

doi: 10.1172/JCI134892

Figure Lengend Snippet: To characterize macrophage polarization, adipose tissue sections were doubly stained for (A) CD86 and CD68 (M1) and (B) CD163 and CD68 (M2). (C) UCP1 was present in CD163+ cells in SC WAT. Yellow arrows point to UCP1+CD163+DAPI+ cells (scale bar: 50 μm). (D) UCP1+CD163+ cells were quantified in SC WAT before and after mirabegron treatment. Data indicate the mean ± SEM (n = 13). *P < 0.05 and ***P < 0.001, by paired, 2-tailed Student’s t test.

Article Snippet: The number of CD163 macrophages that costained with UCP1 was determined using mouse anti-CD163 (HM2157, Hycult Biotech) and rabbit anti-UCP1 (custom antibody J2648, ECM Biosciences).

Techniques: Staining

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 1. Changes of cytokine concentration in THP-1 macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF- α (D), MCP-1 (E), and IL-6 (F) protein concentration in THP-1 macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. *P < 0.05, vs. LPS, #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 2. Changes of cytokine concentration in primary human macrophages challenged with a single LPS stimulation and a CD163 antibody. Quantification for IL-10 (A), TGF-β (B), IL-1 β (C), TNF-α (D), MCP-1 (E), and IL-6 (F) protein concentration in primary human macrophages with a single LPS stimulation and incubated with the anti-CD163 antibody (RM3/1) or its isotype control (Iso) for 24 h (72 h after the addition of LPS). N = 6–10 per group. #P < 0.05, vs. isotype control by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Concentration Assay, Protein Concentration, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 3. CD163 mRNA induction in THP-1 macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in LPS-stimulated THP-1 macrophages at 24, 48, 72 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point. N = 5–6 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 4. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Microscopic images of nuclear staining using DAPI (blue), CD163 protein (red) and mannose receptor (CD206, green) in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative average fluorescence intensity of CD163 (B) or mannose receptor (CD206, C) in LPS-stimulated THP-1 macrophages from 48 to 96 h after transfection. The quantification of the relative average fluorescence intensity was normalized to the respective levels in the Man-PEI group, which was assigned a value equal to 1. N = 3 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Staining, Transfection, Plasmid Preparation

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 5. CD163 protein induction in THP-1 macrophages challenged with a single LPS stimulation. Representative Western blot images of CD163 and Beta-Actin proteins in LPS-stimulated THP-1 macrophages transfected with Man-PEI complexed with an empty vector (pEmp or pEmpty), a plasmid encoding for CD163 gene (pCD163) or the Man-PEI nanoparticle alone (A). Quantification of the relative expression (band density) of CD163 in LPS-stimulated THP-1 macrophages from 48 h after transfection (B). The quantification of the relative density was normalized to the respective levels in the pEmpty group, which was assigned a value equal to 1. N = 10 for pCD163 or pEmpty group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 6. Changes in cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 24, 48, 72 and 96 h after transfection. The concentration of each cytokine/chemokine was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 4–9 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 7. CD163 mRNA induction in THP-1 macrophages challenged with a double LPS stimulation. Quantification for CD163 mRNA using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in THP-1 macrophages with a double LPS stimulation at 4 and 24 h after the second stimulus (+4 and +24, respectively). The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group and then calculated as fold change against the expression of the control group (Man-PEI) at each time point, which was assigned a value equal to 1. N = 4–6 per group. *P < 0.05 against the pEmpty transfected group by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Expressing, Control, Transfection

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 8. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in THP-1 macrophages with a double LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 4 and 24 h after the second stimulus (+4 and +24, respectively). The concentration of each molecule was normalized to the control group (Man-PEI), which was assigned a value equal to 1. N = 5–13 per group. *P < 0.05, vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 9. Changes of cytokine expression in CD163-overexpressing THP-1 macrophages challenged with a double LPS stimulation and a CD163 antibody. Quantification for IL-10 (A) and IL- 1ra (B) protein concentration in THP-1 macrophages transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) and incubated with either anti- CD163 antibody (RM3/1) or its isotype control antibody at 4 (for IL-10) and 24 (for IL-1ra) h after a second LPS stimulus. The protein concentration of each group was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–12 per group. *P < 0.05 using Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Incubation, Control

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 10. CD163 mRNA induction in primary human macrophages challenged with a single LPS stimulation. Quantification for CD163 mRNA induction using Man-PEI complexed with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) in primary human macrophages with a single stimulation of LPS at 48 and 96 h after transfection. The expression of CD163 mRNA was normalized to the respective levels of β-actin expression in each group, and then calculated as fold change against the expression of the pEmpty group at each time point, which was assigned a value equal to 1. N = 6–9 per group. *P < 0.05 against the pEmpty group by Student's t- test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Plasmid Preparation, Transfection, Expressing

Journal: Immunobiology

Article Title: Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

doi: 10.1016/j.imbio.2017.05.011

Figure Lengend Snippet: Fig. 11. Changes of cytokine expression in CD163-overexpressing primary human macrophages challenged with a single LPS stimulation. Quantification for IL-10 (A), IL-1ra (B), TGF-β (C), TNF-α (D), MCP-1 (E), IL-1β (F), and IL-6 (G) protein concentration in primary human macrophages with a single LPS stimulation and transfected with either a plasmid encoding for CD163 gene (pCD163) or the empty vector (pEmpty) at 48 and 96 h after transfection. The concentration of each molecule was normalized to the control group (pEmpty), which was assigned a value equal to 1. N = 5–8 per group. *P < 0.05 vs. pEmpty by Student's t-test.

Article Snippet: Cells were then incubated overnight at 4 °C with a mouse antibody against human CD163 (Serotec, Raleigh, NC, USA, 1:150) and a rabbit antibody against human CD206 (Abcam, Cambridge, MA, USA, 1:250).

Techniques: Expressing, Protein Concentration, Transfection, Plasmid Preparation, Concentration Assay, Control

Journal: Immunity, Inflammation and Disease

Article Title: Monocyte‐derived macrophages: The supplements of hepatic macrophage in Echinococcus multilocularis infected mice

doi: 10.1002/iid3.699

Figure Lengend Snippet: Immunohistochemical positive cells area of liver tissue in mice infected with Echinococcus multilocularis . * p < .05, ** p < .01. (A) CX3CL1; (B) CX3CR1; (C) CD11b; (D) iNOS; (E) CD163.

Article Snippet: The sections were blocked in goat serum for 30 min and incubated overnight at 4°C with the following primary antibodies: rabbit anti‐CX3CL1 (1:500; Bioss), rabbit anti‐CX3CR1 (1:500; Bioss), rabbit anti‐iNOS (1:400; Bioss), rabbit anti‐CD163 (1:500; Bioss), and rabbit anti‐CD11b (1:4000; Abcam).

Techniques: Immunohistochemical staining, Infection

Journal: Journal of Leukocyte Biology

Article Title: Pleiotropic effects of extended blockade of CSF1R signaling in adult mice

doi: 10.1189/jlb.2A0114-006R

Figure Lengend Snippet: Mice were injected with 200 μg rat IgG control or M279, 3× weekly for 6 weeks. Serum was collected, as well as tissue, which was fixed and processed as described in Materials and Methods. Graphs show the mean ± sem . Significance is indicated by * P < 0.05, using unpaired t -tests. (A) Testis and seminal vesicle weight. (B) Representative sections show macrophages within the testicular interstitium, stained with a macrophage marker, Mac-2 or CD163. (C) Serum levels of testosterone and LH.

Article Snippet: MP-7401 (rabbit CD163), 7402 (mouse CD68), and 7404 (rat Mac-2); Vector Laboratories, Burlingame, CA, USA] for 1 h. Samples were washed in TBS, and 3,3′-diaminobenzidine detection (ImmPACT peroxidase substrates, Cat. No. SK-4105; Vector Laboratories) was used to resolve sites of immunolocalization, whereas hematoxylin was used as a counterstain.

Techniques: Injection, Staining, Marker

Journal: Nature Communications

Article Title: The neonatal Fc receptor (FcRn) is a pan-arterivirus receptor

doi: 10.1038/s41467-024-51142-x

Figure Lengend Snippet: A Growth of SHFV and PRRSV-2 in wild-type MA-104 cells, inoculated at MOI = 0.01 ( n = 2 biological replicates). Data shows representative results from one of two independent experiments. B Cytopathic effect (CPE) of arterivirus infection of MA-104 cells at 4 days post-inoculation, taken at ×100 magnification. Data shows representative results from one of two independent experiments. C Frequency of sgRNAs, as determined via deep sequencing of genomic DNA from MA-104 cells, prior to and after infection with arteriviruses. D Robust rank aggregation scores (RRA) scores for gene knockouts enriched in surviving MA-104 cells compared to pre-infection; top 10 hits for each screen are shown as colored datapoints. E RRA scores of SHFV vs. PRRSV-2, with CD163 , FCGRT (FcRn), and B2M circled and labeled. F Infection of cells transduced with single-guide RNA targeting grivet (for MA-104) or human (for ACHN) FCGRT (ΔFcRn), or empty vector control. Viruses tested on these cells are arranged in terms of relatedness to arteriviruses from left to right, with taxonomic relationships shown along the top. Data are presented as mean values ± SEM. Infections were performed with n = 3 biological replicates at an MOI of 0.01. Supernatant was titrated via plaque assay or focus-forming assay at the indicated time points; asterisks represent p-values (**p ≤ 0.01; *** p ≤ 0.001) derived from a two-tailed unpaired t-test with Welch correction and multiple comparisons testing. G Brightfield photographs of arterivirus infections from F taken at ×100 magnification at 3 days post-inoculation. Data shows representative results from one of two independent experiments. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies, including CD163 (goat polyclonal anti-CD163 antibody [R&D Systems, #AF1607]), FcRn (rabbit polyclonal anti-FcRn [Abcam, #ab193148]), and beta-actin (mouse monoclonal 8H10D10 [Cell Signaling Technology, #3700 S]) were diluted in TBST + 5% nonfat dried milk and incubated either overnight at 4 °C (CD163 and FcRn) or for 1 h at ambient temperature (beta-actin).

Techniques: Infection, Sequencing, Labeling, Transduction, Plasmid Preparation, Control, Plaque Assay, Focus Forming Assay, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The neonatal Fc receptor (FcRn) is a pan-arterivirus receptor

doi: 10.1038/s41467-024-51142-x

Figure Lengend Snippet: A Western blot showing gCD163 and gFcRn expression in wild-type (WT) MA-104 cells and MA-104 cells with ectopic expression of gCD163, gFcRn, or both. B Cells from A were infected with SHFV (MOI = 30) and assayed for intracellular SHFV RNA 4 h later using single molecule RNA fluorescence in situ hybridization (smRNA FISH). Confocal microscopy (×600 magnification) was used to capture 16–18 fields of view taken at random (one representative shown). Blue = nuclei; red = smFISH detecting viral (v)RNA, Scale bar = 25 μm. C Quantification of microscopy performed in B . From each field of view, the percent vRNA positive cells, average number of puncta per infected cell, and average puncta FISH intensity per infected cell were determined and graphed as independent points. Error bars represent the mean ± SEM. Data shows results from one experiment. D Time course of SHFV production from wild-type (WT) MA-104 cells (red) compared to dual-overexpressing gCD163/gFcRn cells (black) at a low (0.03, left) and high (3, right) MOI, n = 3 biological replicates. E Cytopathic effect of SHFV infection of wild-type (WT) MA-104 cells (red) compared to dual-overexpressing gCD163/gFcRn cells (black) at a low MOI (0.03) 24 hrs after inoculation. Data shows results from one representative experiment in D . F Photographs of plaque assay wells (6-well plate) fixed and stained with crystal violet 2 days after inoculation. Note the larger and more well-circumscribed plaques in the CD163/FcRn-dual-expressing cells. G Quantification of SHFV via plaque assay using MA-104 cells (WT, +gCD163, +gFcRn, or +gCD163 & +gFcRn) as substrate for infection with data presented as mean values ± SEM, n = 3 biological replicates, normalized to WT cells and analyzed using one-way ANOVA with multiple comparisons relative to WT cells: ns, not significant; ***p ≤ 0.001; ****p ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies, including CD163 (goat polyclonal anti-CD163 antibody [R&D Systems, #AF1607]), FcRn (rabbit polyclonal anti-FcRn [Abcam, #ab193148]), and beta-actin (mouse monoclonal 8H10D10 [Cell Signaling Technology, #3700 S]) were diluted in TBST + 5% nonfat dried milk and incubated either overnight at 4 °C (CD163 and FcRn) or for 1 h at ambient temperature (beta-actin).

Techniques: Western Blot, Expressing, Infection, Fluorescence, In Situ Hybridization, Confocal Microscopy, Microscopy, Plaque Assay, Staining

Journal: Nature Communications

Article Title: The neonatal Fc receptor (FcRn) is a pan-arterivirus receptor

doi: 10.1038/s41467-024-51142-x

Figure Lengend Snippet: A FcRn orthologs from natural and potential arterivirus hosts—grivet (g), human (hu), mouse (m), pig (p), and horse (ho)–were introduced into MA-104ΔFcRn cells and inoculated with SHFV (red), PRRSV-2 (green), or EAV (blue) at an MOI of 0.01, n = 2 biological replicates. FcRn orthologs derived from the natural host of an arterivirus (i.e., positive control) are denoted by a black outline. Productive infection was then assessed via plaque assay of supernatant at 2 days post inoculation. B Similar experimental setup to A , except with CD163 orthologs in MA-104ΔCD163 cells. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies, including CD163 (goat polyclonal anti-CD163 antibody [R&D Systems, #AF1607]), FcRn (rabbit polyclonal anti-FcRn [Abcam, #ab193148]), and beta-actin (mouse monoclonal 8H10D10 [Cell Signaling Technology, #3700 S]) were diluted in TBST + 5% nonfat dried milk and incubated either overnight at 4 °C (CD163 and FcRn) or for 1 h at ambient temperature (beta-actin).

Techniques: Derivative Assay, Positive Control, Infection, Plaque Assay