Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: A Experimental design of fibroblast and macrophage indirect co-culture assay. B Representative images of THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. C Flow cytometric analysis of CD68 in THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. D Immunofluorescence staining of CD206 (green) in experimental and control macrophages after 48 h of co-cultivation. Scale bars, 100 μm. E , F Western blot and RT-qPCR analysis of CD206 protein levels and relative mRNA levels expression in the indicated groups ( n = 3). GAPDH served as a loading control. G Expressions of CD68 and CD206 were determined by flow cytometry. H Quantification of the flow cytometry assay results (n = 3). Data are expressed as the means ± SD. * P < 0.05, ** P < 0.01, and *** P ≤ 0.001. THP-1, human acute monocytic leukemia cell line; PMA, phorbol 12-myrisate 13-acetate; APC, allophycocyanin; FITC, fluorescein-isothiocyanate; GMQ, 2-guanidine-4-methylquinazoline.

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Co-culture Assay, Derivative Assay, Immunofluorescence, Staining, Control, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: A Schematic diagram of establishing rabbit ear hypertrophic scar model and experimental plan. B Immunohistochemistry analysis of ASIC3 expression in rabbit ear tissue 21 days after wounding. Scale bars, 100 μm. C Immunofluorescence staining of rabbit ear tissue 21 days after wounding showing the total (CD68+, green) and M2 subtype (CD206+, red) macrophages after treatment by PBS, DMSO, or GMQ. Scale bars, 100 μm. D , E MFI quantification of CD68 and CD206 in (C) ( n = 3). F , G Western blot and RT-qPCR analysis of rabbit ear tissue 21 days after wounding. Total CD206 protein levels and relative mRNA levels expression in control, vehicle, and GMQ groups ( n = 3). H , I Relative mRNA expressions of M1 macrophage markers (iNOS, TNF-α) as evaluated by RT-qPCR ( n = 3). Data are expressed as the means ± SD. * P < 0.05, *** P ≤ 0.001. NS, not significant. iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor alpha.

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Immunohistochemistry, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Control

Journal: The Tohoku journal of experimental medicine

Article Title: C-reactive protein reduces the relative number of tumor-associated M2 macrophages and intratumoral angiogenesis in mice.

doi: 10.1620/tjem.233.249

Figure Lengend Snippet: Fig. 2. Macrophage identification. Photomicrographs of F4/80+ cells, which represent the total macrophage population in the CRP group (A) and the con- trol group (B). CD206+ cells represent the M2 macrophage population in the CRP group (C) and the control group (D).

Article Snippet: M2 macrophages were identified using a rat monoclonal anti-CD206 antibody (1:200 dilution, MCA2235; AbD Serotec).

Techniques: Control

Journal: The Tohoku journal of experimental medicine

Article Title: C-reactive protein reduces the relative number of tumor-associated M2 macrophages and intratumoral angiogenesis in mice.

doi: 10.1620/tjem.233.249

Figure Lengend Snippet: Fig. 3. Changes in the size of the macrophage fractions. Group data showing that tumors from CRP-treated mice contain significantly larger numbers of F4/80+ macrophages (A), whereas there is no significant difference in the CD206+ macrophage counts between CRP-treated and control mice (B). Consequently, tumors from CRP-treated mice showed significantly smaller M2 macrophage fractions (C). N.S., not sig- nificant.

Article Snippet: M2 macrophages were identified using a rat monoclonal anti-CD206 antibody (1:200 dilution, MCA2235; AbD Serotec).

Techniques: Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASC therapeutic effects on experimental periodontitis in rats. (A) The schematic diagram showed the induction of the experimental rat model of periodontitis for 21 days and ASC injection after the removal of ligatures. (B) Micro-CT imaging and bone parameters (CEJ-ABC distance, BV/TV, Tb. Th, and Tb. Sp) between healthy and experimental rats of periodontitis. Scale bar, 1 mm. (C) Micro-CT imaging displayed the alveolar bone of PBS-injected and ASC-injected rats on day 7, day 14, and day 21 Scale bar, 1 mm. (D) The CEJ-ABC distance and bone parameters (BV/TV, Tb. Th, and Tb. Sp) between the PBS-injected and ASC-injected rats. (E) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (F) The calculated ratio of iNOS + /CD206 + macrophages in PBS-injected and ASC-injected groups. CEJ-ABC, cementoenamel junction and alveolar bone crest. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Micro-CT, Imaging, Immunofluorescence, Staining

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASCs modulated macrophage polarization through NRF2. (A) Representative immunohistochemical staining of NRF2 in the PBS-injected and ASC-injected rats on day 7, day 14, and day 21. Scale bar, 50 μm. (B) Representative immunofluorescence staining of NRF2 (Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (C) Schematic diagram of knocking down NRF2 in macrophages and macrophages cocultured with ASCs and LPS stimulation. (D, E) RT-qPCR and western blotting were used to measure the NRF2 siRNA or siNC in macrophages. (F, G) The mRNA expression and protein levels of M1 markers were increased, while those of M2 markers were decreased after silencing NRF2 in the cocultured groups. R, root; PDL, periodontal ligament; AB, alveolar bone; siRNA, small interfering RNA; NC, negative control. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: Inhibition of IDO activity reduced the therapeutic potential of ASCs in experimental periodontitis and downregulated NRF2 expression. (A) Three-dimensional reconstruction of maxillary alveolar bone in ASC-injected and 1-MT pretreated ASC-injected rats on day 7, day 14, and day 21. Scale bar, 1 mm. (B) Analysis of bone parameters. (C) Immunohistochemical staining of IL-1β, OCN, and NRF2 in the ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 50 μm. (D) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 20 μm. (E) The mean IOD of IL-1β, OCN, and NRF2 and the calculated ratio of iNOS + /CD206 + . IOD, integrated optical density.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Expressing, Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Cells

Article Title: Statins Modulate Microenvironmental Cues Driving Macrophage Polarization in Simulated Periodontal Inflammation

doi: 10.3390/cells12151961

Figure Lengend Snippet: MyD88−dependent modulation of M2 polarization by simvastatin. ( A ) A schematic representation of simvastatin, MyD88 inhibitor treatment, and THP-1-macrophages (M0–M1–M2). ( B ) Cell surface antigen analysis of M2 macrophage markers CD68, CD163, and CD206 was conducted using flow cytometry. Expression levels were normalized using IgG isotype control, and values were expressed in percentage of positive cells (mean ± SD) from three independent preparations. ( C ) RT-PCR analysis and heat maps summarizing TLR and NFkB signaling from M0 (CCL2), M1 (LPS, IFNγ, TNFα), and M2 (IL4, IL10, TGFβ) macrophages. (n = 3) preparations per treatment group, and data found to be significantly different were plotted as heat maps. ( D ) Graphical summary of statin-mediated suppression of TLR and NFkB signaling.

Article Snippet: Phenotyping of macrophages was performed in 1% BSA and 3% human serum PBS according to standard methods using a panel of antibodies targeting CD68 (R and D Systems Cat# IC20401P, Minneapolis, MN, USA, RRID: http://scicrunch.org/resolver/AB_2074835 , accessed on 28 April 2023), CD163 (R and D Systems Cat# FAB1607P, RRID: http://scicrunch.org/resolver/AB_2074536 , accessed on 28 April 2023), and CD206 (R and D Systems Cat# FAB25342P, RRID: http://scicrunch.org/resolver/AB_10889015 , accessed on 28 April 2023); antibodies all from R and D Systems.

Techniques: Flow Cytometry, Expressing, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Nanobiotechnology

Article Title: Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration

doi: 10.1186/s12951-024-02507-3

Figure Lengend Snippet: A-PUAT membranes stimulate macrophage polarization to M2-phenotype. ( A ) SEM results revealing the morphology of RAW264.7 cells cultured on different membranes. ( B ) The viability of RAW264.7 cells seeded on each membrane at indicated time points. ( C ) Relative transcription levels of pro-inflammatory gene and anti-inflammatory gene in each group. ( D ) The contents of TNF-α and IL-10 secreted by RAW264.7 cells cocultured on different substrates. ( E ) Representative immunofluorescent images revealing the cytoskeleton of RAW264.7 cells (FITC, green) and M2-phenotype macrophages (CD206, red) after culturing on different membranes for 3 days. ( F ) Quantitative analysis of orientation of macrophages cultured on different membranes. ( G ) Quantitative analysis of CD206 positive cells in RAW264.7 cells seeded on different membranes. ( H ) Flow cytometry showing the expression of CD80 and CD206 in RAW264.7 cells cultured on different substrates.

Article Snippet: Then samples were cultured overnight at 4 ℃ with primary antibodies to detect CD206 (1:500, 24595, CST).

Techniques: Cell Culture, Membrane, Flow Cytometry, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration

doi: 10.1186/s12951-024-02507-3

Figure Lengend Snippet: Relevance between macrophages and SCs at 7 and 14 days after grafting. ( A ) Representative immunofluorescence images of the transverse sections of different NGCs showing the distribution of macrophages (M0, CD68, green) and SCs (S100β, red). ( B ) Quantitative analyses showed the number of infiltrated macrophages and SCs in each group. ( C ) Representative immunofluorescence images of M1-phenotype macrophages (iNOS, red), and M2-phenotype macrophages (CD206, red).

Article Snippet: Then samples were cultured overnight at 4 ℃ with primary antibodies to detect CD206 (1:500, 24595, CST).

Techniques: Immunofluorescence

Journal: Frontiers in Physiology

Article Title: Microglial reactivity in brainstem chemosensory nuclei in response to hypercapnia

doi: 10.3389/fphys.2024.1332355

Figure Lengend Snippet: Microglial expression of CD86 (a marker of inflammatory state of reactive microglia) and CD206 (a marker of regulatory state of reactive microglia) in control (Co., normocapnic normoxia) and stimulated (hypercapnic normoxia) CF-1 mice. (A) , confocal microscopy of double labeled immunofluorescence detecting Iba1 (green, first column) and CD86 (red, second column) in VRC, NTS, RN, Sp5, and hippocampus of CF-1 mice; Hoechst 33258 in blue, merge, and amplification of a selected field of the merge panel are shown in the third, fourth, and fifth columns, respectively. Note in the amplified merge panel that hypercapnia increases the number of microglia CD86 + in VRC, RN, and NTS. Arrow heads indicate Iba1+ CD86 + cells. Bars = 100 μm. (B) , confocal microscopy of double labeled immunofluorescence detecting Iba1 (green, first column) and CD206 (red, second column) in VRC, NTS, RN, Sp5, and hippocampus of CF-1 mice; Hoechst 33258 in blue, merge, and amplification of selected field of the merge panel are shown in the third, fourth, and fifth columns, respectively. Note in the amplified merge panel that hypercapnia did not elicit significant increase in the density of CD206+ microglia in any of the brainstem nuclei or hippocampus. Arrow heads indicate Iba1+ CD206+ cells. Bars = 100 μm. (C) , Analysis of the percentage of microglia expressing CD86 or CD206. Left and right graphs at the bottom, percentage of microglia expressing CD206 and CD86, respectively. Bars and vertical lines, mean ± SD. Control (open bars), exposed to hypercapnia (filled bars). Two-way mixed ANOVA show significant effects of hypercapnia upon the percentage of microglia expressing CD86 in VRC, RN, and NTS (df = 1, F (1,4) = 17.11, p = 0.0144); ** indicates p < 0.01 between hypercapnia-exposed mice and controls by the Tukey’s multiple comparison post hoc test. No significant effects of hypercapnia upon the percentage of microglia expressing CD 206; df = 1, F (1,4) = 0.02076, p = 0.893.

Article Snippet: Immunofluorescence microscopy was used for double labeling for Iba1 and CD86 (IgG monoclonal mouse antibody, sc-28347; Santa Cruz Biotechnology, United States) or for Iba1 and CD206 (IgG monoclonal mouse antibody, sc-58986, Santa Cruz Biotechnology, United States).

Techniques: Expressing, Marker, Control, Confocal Microscopy, Labeling, Immunofluorescence, Amplification, Comparison

Journal: Cell Reports Medicine

Article Title: Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer

doi: 10.1016/j.xcrm.2024.101397

Figure Lengend Snippet: T cell infiltration into tumors occurs independent of the gut and tumor microbiome (A) Study design for (B)–(K). (B) 16S rRNA levels in tumor and stool from mice orthotopically injected with cold (69) tumor cells (n = 10) or hot (2838c3) tumor cells and treated with (n = 15) and without (n = 20) antibiotics. (C and D) Quantification of CD3 + (C) and CD8 + and FOXP3 + (D) T cells from hot tumors of mice treated with (n = 19) or without (n = 13) antibiotics. (E) 16S rRNA levels in tumor from mice orthotopically injected with cold (69) tumor cells and treated with (n = 5) and without (n = 5) antibiotics. (F) Quantification of CD8 + and FOXP3 + T cells from cold tumors of mice treated with (n = 5) and without (n = 5) antibiotics. (G) Number of DEGs. (H) Bar graph displaying overrepresentation analysis of DEGs in indicated gene sets. (I and J) Tumor weights at day 20. (K) Quantification of intra-tumoral CD19 + cells. (L and M) Mean fluorescence intensity (MFI) of MHC class II (L) and CD206 (M) on CD11b + F4/80 + intra-tumoral macrophages. Data were pooled from two to three experiments (B–D and J–M) or are representative of two independent experiments (E–I). Statistical significance was calculated using one-way ANOVA with Dunnett’s test (B, L, and M) and a two-tailed Mann-Whitney test (C–F and I–K). Data are represented as scatterplots (mean ± SD). NS, not significant.

Article Snippet: Rat monoclonal anti-mouse CD206 (MR6F3) , eBioscience , 17-2061-82; RRID:AB_2637420.

Techniques: Injection, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer

doi: 10.1016/j.xcrm.2024.101397

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti-mouse CD206 (MR6F3) , eBioscience , 17-2061-82; RRID:AB_2637420.

Techniques: Recombinant, Sequencing, Software

Journal: British Journal of Pharmacology

Article Title: The suppression of bromodomain and extra‐terminal domain inhibits vascular inflammation by blocking NF‐κB and MAPK activation

doi: 10.1111/bph.13657

Figure Lengend Snippet: Effect of BET inhibition on TNF‐α‐induced NF‐κB activation in HUVECs. (A and B) Western blot analysis of phosphorylated IKK or phosphorylated and degradated IκBα in HUVECs treated with 10 ng·mL−1 TNF‐α for 15 min in the presence of DMSO or JQ1. Data represent relative quantities obtained by densitometry (lower panel) from five independent experiments (mean ± SEM; *P < 0.05 vs DMSO, # P < 0.05 vs TNF‐α treatment; one‐way ANOVA test). (C and D) Effect of JQ1 on nuclear translocation of p65. HUVECs were treated with TNF‐α (10 ng·mL−1) for 30 min in the presence or absence of JQ1. The representative images show immunofluorescence staining analysis of p65 localization (green). Nuclei were stained with DAPI. (D ) Mean intensity of nuclear p65 protein from 5 independent experiments (mean ± SEM; *P < 0.05 vs DMSO, # P < 0.05 vs TNF‐α alone; one‐way ANOVA test). (E and F) The expression of p65 protein, detected by western blot analysis, in nuclear and cytoplasmic fractions isolated from HUVECs. Results of semiquantitative densitometry of nuclear p65 protein expression are shown in (D), from five independent experiments (mean ± SEM; *P < 0.05 vs TNF‐α alone, Student's t‐test). (G) Western blot analysis of TNF‐α‐induced phosphorylation of IKKβ in HUVECs transfected with Brd2 shRNA or Brd4 shRNA or control shRNA in the presence or absence of TNF‐α stimulation. Results of semiquantitative densitometry of protein expression are shown in the right panel, from five independent experiments (mean ± SEM; *P < 0.05 vs control shRNA, # P < 0.05 vs TNF‐α treatment; one‐way ANOVA test).

Article Snippet: Equal amounts of the samples were then separated by SDS‐PAGE gel and transferred onto NC membranes and immunoblotted with the indicated antibodies: anti‐VCAM1 (Abcam), anti‐ICAM1 (Abcam), anti‐ E‐selectin (Abcam) anti‐JNK (Cell signalling), anti‐phospho‐JNK(Cell signalling), anti‐p38(Cell signalling), anti‐phospho‐p38(Cell signalling), anti‐ERK(Cell signalling), anti‐phospho‐ERK(Cell signalling), anti‐NF‐κB p65(Cell signalling), anti‐phospho‐IκBα (Cell signalling), anti‐IκBα (Cell signalling) and anti‐phospho‐IκB kinase (IKK) (Cell signalling).

Techniques: Inhibition, Activation Assay, Western Blot, Translocation Assay, Immunofluorescence, Staining, Expressing, Isolation, Transfection, shRNA