Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy

doi: 10.3389/fcell.2023.1286011

Figure Lengend Snippet: Comparative Analysis between Macrophages Cultured in Hypoxic Medium in the Presence and Absence of the TGF-β Neutralizing Antibody 1D11. (A) Macrophages were cultured in hypoxic medium and treated with TGF-β in the presence and absence of the TGF-β neutralizing antibody 1D11 and Western blotting was performed. (B–D) scRNA-Seq datasets from macrophages cultured in hypoxic MSC conditioned medium were integrated with macrophages cultured with the addition of the TGF-β neutralizing antibody 1D11 and comparative analyses were performed. (B) UMAP representation. The fraction (C) and (D) cells from each sample within each cluster are shown.

Article Snippet: To block effects of TGF-β isoforms, MSC conditioned media were incubated with the TGF-β isoform neutralizing antibody 1D11 (30 μg/mL, Thermo Fisher, #MA523795) for 2 h prior to use.

Techniques: Cell Culture, Western Blot

Journal: Military Medical Research

Article Title: Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

doi: 10.1186/s40779-023-00494-4

Figure Lengend Snippet: GPR65 promotes HSCs activation indirectly by paracrine secretion of TGF-β from macrophage. The CM from control, GPR65-overexpressed, pH 6.6-treated or GPR65 agonist-treated macrophage cells, with 20 μg/ml TGF-β neutralization antibody 1D11 or mouse IgG, were used to treat primary HSCs for 24 h. qRT-PCR was used to assess the mRNA level of Gpr65 , Acta2 , Col1α1 , Col1α2 , Timp1 , Mmp2 and Tgfβ1 ( n = 3) ( a-c ); Western blotting was used to determine the protein level of COL1α1, α-SMA, MMP2 and TIMP1 ( d ); the expression and location of α-SMA and COL1α1 was assessed by confocal microscopy ( e ). Scale bar = 20 μm. qRT-PCR was used to assess the expression of Tgfβ1 and Tgfβ3 in GPR65-KO ( f ), Gpr65 -overexpressed ( g ), or GPR65 agonist/inhibitor ( h ) treated HMs ( n = 3). i TGF-β1 level in the supernatant of HMs transfected with pcDNA3.1 or pcDNA3.1-GPR65 was detected by ELISA ( n = 4). HMs isolated from WT and GPR65-KO mice were treated with or without GPR65 agonist ( j ) or pH 6.6 ( k ) for 24 h, TGF-β1 level in the supernatant was detected by ELISA ( n = 3). * P < 0.05 vs. pcDNA3.1, pH 7.4, DMSO, WT, or WT + DMSO/pH 7.4; # P < 0.05 vs. pcDNA3.1-GPR65, pH 6.6, GPR65 agonist, or WT + GPR65 agonist/pH 6.6. CM conditioned medium, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, HSC hepatic stellate cell, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, COL1α1 collagen type I alpha 1, α-SMA α-smooth muscle actin, MMP2 matrix metalloproteinase 2, TIMP1 tissue inhibitor of metalloproteinases 1, TGF-β1 transforming growth factor-β1

Article Snippet: The CM with 20 μg/ml transforming growth factor-β (TGF-β) neutralization antibody 1D11 (Thermo Fisher Scientific, USA, Cat# 16-9243-85; RRID: AB_2573124) or mouse IgG, was used to treat primary HSCs.

Techniques: Activation Assay, Neutralization, Quantitative RT-PCR, Western Blot, Expressing, Confocal Microscopy, Transfection, Enzyme-linked Immunosorbent Assay, Isolation, Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: Military Medical Research

Article Title: Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

doi: 10.1186/s40779-023-00494-4

Figure Lengend Snippet: Targeting GPR65 alleviates hepatic macrophage inflammation and fibrosis by suppressing the JNK/NF-κB pathways. a Western blotting was used to determine the level of p-IKK, p-IĸBα, p-NF-κB p65, IKK, IĸBα, NF-κB p65, p-MLK3, p-MKK4, p-MKK7, p-JNK, JNK, p-p44/42, p44/42, p-p38, p38, p-GSK-3β, p-Akt, Akt, p-PDK1, p-PTEN and p–c-Raf in liver tissues from WT, WT + BDL, GPR65-KO and GPR65-KO + BDL mice. b Western blotting was used to determine the level of p-IKK, IKK, p-IĸBα, IĸBα, p-p65, p65, p-MLK3, p-MKK7, p-JNK and JNK in GPR65-KO, GPR65-overexpressed, various pH-treated and GPR65 agonist/inhibitor-treated HMs. The specific inhibitors of JNK, SP600125 (10 μmol/L) and JNK-IN-8 (5 μmol/L), as well as the specific inhibitors of NF-κB, BAY 11–7082 (5 μmol/L) and APDC (20 μmol/L), were used to treat GPR65-overexpressed HMs for 24 h, and qRT-PCR was used to assess the expression of Tnfα , Il6 and Tgfβ3 ( n = 3) ( c ); TGF-β1 and IL-6 level in the supernatant were detected by ELISA ( n = 3) ( d ); the expression and location of TNF-α was assessed by confocal microscopy ( e ). Scale bar = 20 μm. * P < 0.05 vs. DMSO + pcDNA3.1; # P < 0.05 vs. DMSO + pcDNA3.1-GPR65. CCl 4 carbon tetrachloride, BDL bile duct ligation, ELISA enzyme-linked immunosorbent assay, HM hepatic macrophage, KO knockout, qRT-PCR quantitative real-time reverse transcription-polymerase chain reaction, IKK inhibitor of ĸB kinase, IĸBα inhibitor of ĸB α, NF-κB p65 nuclear factor κB p65 subunit, MLK3 mixed lineage kinase 3, MKK4 mitogen-activated protein kinase kinase 4, MKK7 mitogen-activated protein kinase kinase 7, JNK c-Jun N-terminal kinase, GSK-3β glycogen synthase kinase 3β, Akt protein kinase B, PDK1 pyruvate dehydrogenase kinase 1, PTEN phosphatase and tensin homolog, c-Raf c-rapidly accelerated fibrosarcoma, TGF-β1 transforming growth factor-β1, IL-6 interleukin-6, TNF-α tumor necrosis factor-α

Article Snippet: The CM with 20 μg/ml transforming growth factor-β (TGF-β) neutralization antibody 1D11 (Thermo Fisher Scientific, USA, Cat# 16-9243-85; RRID: AB_2573124) or mouse IgG, was used to treat primary HSCs.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Ligation, Knock-Out, Reverse Transcription Polymerase Chain Reaction

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: Elevated active transforming growth factor‐beta (TGF‐ β ) levels in human tendinopathy. A) HE staining of normal tendon (left) and tendinopathic tendon (middle and right) and B) quantification of nuclei counts per bone marrow area (mm 2 ). Black arrows, tenocytes; black arrowheads, chondrocyte‐like cells; open arrowheads, blood vessels. Second row of A) shows high magnification of boxed area in first row. C) Immunostaining of type‐2 collagen (Col2) in both normal and tendinopathic tendons. D,F) Immunostaining and E,G) quantification and D,E) CD68 + cells and F,G) MMP13 + staining area per tissue area (mm 2 ). Black arrowheads, CD68 + macrophages. H,I) Quantification of H) active and I) total TGF‐ β 1 in tendons. J) Immunostaining and K) quantification of pSmad2 + cells per tissue area (mm 2 ). Scale bars: 200 µm. All data are shown as the mean ± standard deviation ( n = 8 per group). * p < 0.05 compared with normal tendon as determined by unpaired, 2‐tailed Student's t ‐test.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Staining, Immunostaining, Standard Deviation

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: Elevated active TGF‐ β levels are associated with tendinopathy in mice. A) A schematic diagram of the DI model. DI devices are applied to the right feet of mice to maintain dorsiflexion for 12 h each day for 1, 4, and 8 weeks as noted in results. Non‐immobilized littermates without devices are used as controls and allowed to move freely in the cages. B) Illustrations of observed area. The mid‐portion of Achilles tendon (Ac) is used as an area of interest (boxed area) from 2 to 6 mm proximal to the calcaneus (Ca). Ga, Gastrocnemius. (C, G, H, J, N, and P) Staining of sham‐processed tendon (first column) or tendons 1 week (second column), 4 weeks (third column), and 8 weeks (fifth column) after tendinopathy induction by DI. The fourth column is the boxed area of graphs of either 4 or 8 weeks after DI in high magnification. C) HE staining of normal tendon and tendinopathic tendons. Black arrowheads, chondrocyte‐like cells with rounded nuclei; open arrowheads, normal tenocytes with spindle‐like nuclei. Scale bar: 200 µm. D) Quantification of nuclei after sham or DI processing. E) The orientation of collagen fibers is analyzed using an OrientationJ plugin of ImageJ by evaluating every patch of the image (Yellow segments). F) Frequency distribution of orientation degree. (G) SOFG staining of normal tendon and tendinopathic tendons. Proteoglycan (red) and collagenous matrix (green). Black arrowheads, chondrocyte‐like cells; open arrowheads, normal tenocytes. Scale bar: 200 µm. H) Immunohistochemical staining and I) quantitative analysis of Col2 + chondrocytes per tissue area (mm 2 ) in Achilles tendons. Black arrowheads, chondrocytes. Scale bar: 100 µm. J,N,P) Immunohistochemical staining and K,O,Q) quantitative analysis of J,K) CD68 + macrophages, N,O) pSmad2 + cells, and P,Q) MMP13 + area per tissue area (mm 2 ) in Achilles tendons. Black arrowheads, MMP13 + cells. Scale bar: 50 µm. L,M) The concentration of L) active and M) total TGF‐ β 1 in Achilles tendon in sham and DI mice. All data are shown as the mean ± standard deviation ( n = 8 mice per group). * p < 0.05 compared with sham operated mice as determined by one‐way analysis of variance to determine significance between groups.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Staining, Immunohistochemical staining, Concentration Assay, Standard Deviation

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: CED mice show an Achilles tendon tendinopathy phenotype. A) Concentration of active TGF‐ β 1 in Achilles tendons of CED and WT mice. B) Message RNA expression of Scx in Achilles tendons of CED and WT mice. C) TUNEL staining and D) quantitative analysis of apoptotic cells in tendons. Scale bar: 50 µm. E) SOFG staining of normal tendon and CED mouse tendons. Proteoglycan (red) and collagenous matrix (green). Scale bar: 200 µm. F) Immunohistochemical staining and G) quantitative analysis of Col2 + chondrocytes per tissue area (mm 2 ) in Achilles tendons. Scale bar: 100 µm. H) Immunofluorescent staining of CD31 + cells and I) quantification of the fold change of blood vessels in CED mice normalized to that of WT mice in the Achilles tendons. Scale bar: 100 µm. J) Quantitative analysis of maximum tensile force and stiffness of Achilles tendons from WT and CED mice. K) Immunostaining of Col2 + cells (red) and yellow fluorescent protein (YFP) – positive cells (green) in Scx‐creERT2::EYFP mice 8 weeks after DI. Scale bar: 50 µm. L) Ratio of YFP + and YFP − cells in Col2 + cells in tendinopathic tendon 8 weeks after DI in Scx‐creERT2::EYFP mice. All data are shown as the mean ± standard deviation ( n = 8 per group). * p < 0.05 compared with WT littermates as determined by unpaired, 2‐tailed Student's t ‐test.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Concentration Assay, RNA Expression, TUNEL Assay, Staining, Immunohistochemical staining, Immunostaining, Standard Deviation

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: Inhibition of TGF‐ β signaling attenuates tendinopathy. A–K) TGF‐ β neutralizing antibody attenuates tendinopathy. Mice are treated with 5 mg kg −1 body weight of the TGF‐ β neutralizing antibody 1D11 or control antibody 13C4 once per week for 8 weeks. A) Immunohistochemical staining and B) quantitative analysis of pSmad2 + cells per tissue area (mm 2 ) in Achilles tendons. Scale bar: 50 µm. C) SOFG staining of tendons in 13C4‐ or 1D11‐treated mice. Proteoglycan (red) and collagenous matrix (green). Scale bar: 50 µm. D) Frequency distribution of Achilles tendon orientation degree in 13C4‐ or 1D11‐treated mice. E) Immunohistochemical staining and F) quantitative analysis of Col2 + chondrocytes per tissue area (mm 2 ) in Achilles tendons. Scale bar: 100 µm. G) TUNEL staining and H) quantitative analysis of apoptotic cells in tendons. Scale bar: 100 µm. I) Immunofluorescent staining of CD31 + cells and J) quantification of the fold change of blood vessels in 1D11‐treated mice normalized to that of 13C4‐treated mice in the Achilles tendons. Scale bar: 100 µm. K) Quantitative analysis of maximum tensile force and stiffness of Achilles tendons measured 8 weeks after DI operation with 13C4 or 1D11 treatment. All data are shown as the mean ± standard deviation ( n = 8 mice per group). * p < 0.05 compared with 13C4 treated mice as determined by unpaired, 2‐tailed Student's t ‐test. L–V) Inducible knockout of Tgfbr2 in Scx + cells attenuates tendinopathy in mice. L) Immunohistochemical staining and M) quantitative analysis of pSmad2 + cells per tissue area (mm 2 ) in Achilles tendons. Scale bar: 50 µm. N) SOFG staining of tendons in Scx‐creERT2::Tgfbr2 f/f mice or its Tgfbr2 f/f littermates 8 weeks after DI. Proteoglycan (red) and collagenous matrix (green). Scale bar: 50 µm. O) Frequency distribution of Achilles tendon orientation degree in Scx‐creERT2::Tgfbr2 f/f mice or its wildtype controls. P) Immunohistochemical staining and Q) quantitative analysis of Col2 + chondrocytes per tissue area (mm 2 ) in Achilles tendons. Scale bar: 100 µm. R) TUNEL staining and S) quantitative analysis of apoptotic cells in Achilles tendons. Scale bar: 100 µm. T) Immunofluorescent staining of CD31 + cells and V) quantification of the fold change of blood vessels in Scx‐creERT2::Tgfbr2 f/f mice normalized to that of Tgfbr2 f/f mice in the Achilles tendons. Scale bar: 100 µm. T) Quantitative analysis of maximum tensile force and stiffness of Achilles tendons of Scx‐creERT2::Tgfbr2 f/f mice and their Tgfbr2 f/f littermates 8 weeks after DI. All data are shown as the mean ± standard deviation ( n = 8 mice per group). * p < 0.05 compared with Tgfbr2 f/f mice as determined by unpaired, 2‐tailed Student's t ‐test.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Inhibition, Immunohistochemical staining, Staining, TUNEL Assay, Standard Deviation, Knock-Out

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: Integrin α v β 6 active latent TGF‐ β in response to mechanical stress drives the progression of tendinopathy. A) Representative images and B) quantification of immunostaining of tendon sections with antibodies against α v β 6 at 1, 4, and 8 weeks after DI or sham processing. Scale bar: 50 µm. All data are shown as the mean ± standard deviation ( n = 8 mice per group). * p < 0.05 compared with sham group as determined by one‐way analysis of variance. C) A schematic diagram of the ex vivo tensile model. D) Frequency distribution of tendon fiber orientation. E,G) Representative images and F,H) quantification of E,F) pSmad2 + cells and (G, H) TUNEL + cells. Scale bars: 50 µm. I) Western blot of pSmad2 and Smad2 levels in tendon lysates. J) Quantification of pSmad2 relative density relative to physiological loading group (20 g) in tendon lysates after above‐mentioned treatment. K) Quantitative analysis of maximum tensile force and stiffness of tendons. All data from B, F, H, J, and K are shown as the mean ± standard deviation ( n = 8 per group). * p < 0.05 compared with 200 g loaded group as determined by one‐way analysis of variance.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Immunostaining, Standard Deviation, Ex Vivo, TUNEL Assay, Western Blot

Journal: Advanced Science

Article Title: Inhibition of Integrin α v β 6 Activation of TGF‐ β Attenuates Tendinopathy

doi: 10.1002/advs.202104469

Figure Lengend Snippet: Inducible knockout of αv in Scx + cells attenuates tendinopathy. A) Active TGF‐ β 1 concentration of tendon sections from Scx‐creERT2:: αv f/f and their WT αv f/f littermates upon tamoxifen injection. B) Immunostaining and C) quantification of pSmad2 + cells in tendons. Scale bar: 50 µm. D) SOFG staining of tendons in WT or Scx‐creERT2:: αv f/f mice. Proteoglycan (red) and collagenous matrix (green). Scale bar: 50 µm. E) Frequency distribution of tendon fiber orientation. F) Immunohistochemical staining and G) quantitative analysis of Col2 + chondrocytes per tissue area (mm 2 ) in Achilles tendons. Scale bar: 100 µm. H) TUNEL staining and I) quantitative analysis of apoptotic cells in tendons. Scale bar: 100 µm. J) Immunofluorescent staining of CD31 + cells and K) quantification of the fold change of blood vessels in Scx‐creERT2:: αv f/f mice normalized to that of WT mice in the Achilles tendons. Scale bar: 100 µm. Quantitative analysis of L) maximum tensile force and stiffness of tendons. All data are shown as the mean ± standard deviation ( n = 8 mice per group). * p < 0.05 compared to αv f/f mice as determined by unpaired, 2‐tailed Student's t ‐test.

Article Snippet: TGF‐ β neutralizing antibody 1D11 (5.0 µg mL −1 , R&D Systems), RGD peptide (2 µmol L −1 , 3498, Tocris Bioscience, Bristol, UK), integrin α v β 6 antibody (100 µg mL −1 , ab77906, Abcam), or phosphate‐buffered saline (as a control) was added to Dulbecco's modified Eagle's medium (D5030, Sigma‐Aldrich) supplemented with 1% penicillin‐streptomycin (MT30001CI, Fisher Scientific, Pittsburgh, PA).

Techniques: Knock-Out, Concentration Assay, Injection, Immunostaining, Staining, Immunohistochemical staining, TUNEL Assay, Standard Deviation