Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF and FGF up-regulate PD-L1 and skew macrophage polarization . (a, b) Expression of PD-L1 on the surface of fibroblast-like cells detected by FCM. (c, d, f, g) Expression of CD86 and CD206 in WT-MEF or PD-L1 KO MEF detected by FCM. Macrophages were co-cultured with untreated, or FGF-2 and TGF-β1 pretreated fibroblasts were washed with PBS, blocked in 5% FBS and two color-stained to identify M1 (CD86) and M2 (CD206) macrophage subpopulations.(e, h) Expression of CD16, CD86 and CD206 in macrophages co-cultured with pretreated fibroblast-like cells detected by immunofluorescence staining.(i) Pretreating WT or PD-L1-/- fibroblasts (fixed with Paraformaldehyde or not) affect activated macrophages TNF-α, IL-6 and IL-10 production in a contact-dependent manner. *p < 0.05, **p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF-β1 together with FGF-2 significantly upregulated fibroblast-like cells PD-L1 at translational levels. (a, b) Polysome profiles of MEF cells treated 24 h with FGF-2 and TGF-β1. One representative profile from three independent experiments is shown. Percentage of transcripts in each polysomal fraction obtained by sucrose-gradient ultracentrifugation was quantified by qRT-PCR (n = 3). (c) Western blot analysis of the indicated proteins in MEF when stimulated by FGF-2 and TGF-β1. Representative blots from two independent experiments are shown. (d, e) PD-L1 is visualized by flow cytometry in MEF cells stimulated by FGF-2 and TGF-β1 and eIF4E inhibitor. One representative experiment of two is shown. *p < 0.05,**p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: PD-L1 positive fibroblast-like cells pretreated by TGF-β1 and FGF-2 promote chronic refractory wound healing in vivo. (a) Representative photographs showing macroscopic excisional wound closure treated with PD-L1 positive and negative MEF in PD-L1-/- C57 BL6 mice. The wild-type and PD-L1-/- mice fibroblast-like cells were isolated and cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 units/mL streptomycin (Sigma). Followed by pretreated with TGF-β1 and FGF-2, approximately 1x 10 5 fibroblast-like cells were further suspended in saline and sprayed over the wound area of the mice. (b) Planimetric analysis of wound photographs reveals significantly delayed at 3-5 days after wounding when PD-L1-/- MEF were used. (c) The schematic diagram of the mechanism of PD-L1 regulating wound healing.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: In Vivo, Isolation, Cell Culture, Modification, Saline

Journal: Scientific Reports

Article Title: Toll-like Receptor-4 Activation Boosts the Immunosuppressive Properties of Tumor Cells-derived Exosomes

doi: 10.1038/s41598-019-44949-y

Figure Lengend Snippet: Expression of TGFβ on the surface of exosomes and its effect on regulatory T cells expansion. The expression of TGFβ1 ( A ) and TGFβ2 ( B ) on the surface of exosomes were evaluated using the TGF-β Magnetic Luminex Performance Assay on exosomes released by unstimulated (CTRL, white column) o LPS-activated tumor cells (LPS, black column). Data are shown as mean (n = 8) ± SD. *Difference with exosomes released by unstimulated tumor cells, P < 0.05. ( B ) Exosomes were coupled to ExoFlow beads, stained with monoclonal antibodies against TGFβ1 and analyzed by FACS. Representative plots of the FACS analyses for staining of control (black line) and of LPS-derived exosomes (red line) are shown. ( D–F ) CD4+ T cells, isolated from PBMCs by negative selection, were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence of exosomes released by unstimulated (CTRL, white column) o LPS-activated tumor cells (LPS, black column). The percentage of effector (CD4+/CD25−/FoxP3−) or regulatory T cells (CD4+/CD25+/FoxP3+) was determined by fluorescence activated flow cytometry (FACS) on day 4. Representative plots for CD25 and Foxp3 staining are shown and histograms represent the ratio between the percentage of effector and regulatory T cells (T eff /T reg ). In a separate set of experiments, exosomes were pre-treated with neutralizing anti-TGFβ1 antibody. Data are shown as mean (n = 6) ± SD. *difference with the untreated cells, P < 0.05 ; **difference with untreated cells, P < 0.005 , $ difference with exosomes treated cells released by LPS-activated cells.

Article Snippet: In a separate set of experiments, SW480-, SW620- and U87-MG-derived exosomes were pre-incubated for 15 min at 37 °C with 0.5 µg/ml of anti-TGFβ1 neutralizing antibody (R&D system) before adding to cells.

Techniques: Expressing, Luminex, Staining, Derivative Assay, Isolation, Selection, Fluorescence, Flow Cytometry

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: Mφ from S. japonicum-infected mice are sufficient to induce Tregs and exhibit a tolerogenic phenotype characterized by expressing high levels of TGF-β and IL-10. (a, b) Purified CD4+ T cells from normal mice were co-cultured with Mφ from normal or S. japonicum-infected mice for 3 days. CD4+CD25+Foxp3+ Tregs were analyzed by FCM and cells were gated on CD4+ T cells. (c, d) At 13 weeks post-infection, single-cell suspensions of splenocytes or liver cells from S. japonicum-infected mice were stimulated with phorbol myristate acetate and ionomycin in the presence of Golgistop for 6 h. Cells were surface stained with anti-CD11b and anti-F4/80 then intracellularly stained with antibodies against TNF-α, IL-4, IL-6, IL-10, TGF-β1, or isotype antibody and analyzed by FCM. Cells were gated on CD11b+F4/80+ Mφ. FCM plots are representative of three independent experiments. Data are means ± s.d. of triplicate cultures or 5 mice and representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Infection, Expressing, Purification, Cell Culture, Staining

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-treated Mφ display a phenotype resembling that of tolerogenic Mφ characterized by expressing high levels of TGF-β and IL-10. (a) Purified Mφ from normal mice were cultured for 2 days in medium alone or in the presence of 0.1 μg mL−1 SjHSP60 or 1 μg mL−1 LPS. The mRNA expression of cytokines in Mφ were determined by qPCR and expressed as fold increases over control. (b) ELISA quantification of cytokines in supernatants of Mφ cultures. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Expressing, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: Production of TGF-β by SjHSP60-Mφ is necessary for conversion of CD4+CD25− T cells into Tregs. (a) At 0, 3, 5, 8 and 13 weeks post-infection, serum TGF-β levels were measured by ELISA. (b) ELISA measurements of TGF-β in serum samples of mice injected with SjHSP60 or PBS. (c) ELISA measurements of cytokines in supernatants of purified CD4+CD25− T cells cultured for 3 days in medium alone or with Mφ that had been unstimulated or exposed to SjHSP60 or LPS for 2 days. (d) Mφ were prestimulated with 0.1 μg mL−1 SjHSP60 or medium for 2 days. Purified CD4+CD25− T cells were cultured for 3 days with medium, SjHSP60, or prestimulated Mφ above. Co-cultures were set up with or without SB-431542 (20 μmol L−1) or neutralizing TGF-β1 antibody (5 μg mL−1). Frequency of CD4+CD25+Foxp3+ Tregs was determined by FCM. FCM plots are representative of three independent experiments with similar results and cells were gated on CD4+ T cells. (e) The bar graph indicates average percentages ± s.d. of Tregs. Data are expressed as means ± s.d. of 6 mice or triplicate cultures and representative of two or three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Injection, Purification, Cell Culture

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production in Mφ is TLR4-dependent. (a, b) Purified Mφ from TLR2−/−, TLR4−/− mice or control WT littermates were stimulated with or without 0.1 μg mL−1 SjHSP60 for 2 days. In some cultures, purified Mφ from control littermates were pretreated with medium alone, or with 20 μg mL−1 of anti-TLR2, anti-TLR4 blocking or isotype-matched antibodies for 1 h at 4°C. After stimulation, TGF-β production was detected by ELISA. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Purification, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production and Treg conversion are MyD88-independent. (a, b) MyD88+/+ or MyD88−/− Mφ were stimulated with 0.1 μg mL−1 SjHSP60 for 2 days. TGF-β mRNA expression was detected by qPCR and expressed as fold increases over unstimulated MyD88+/+ Mφ (a). Culture supernatants were collected for determining TGF-β production by ELISA (b). (c, d) Purified CD4+CD25− T cells were co-cultured with MyD88+/+ or MyD88−/− Mφ prestimulated with 0.1 μg mL−1 SjHSP60 or medium. After 3 days, percentages of CD4+CD25+Foxp3+ Tregs were analyzed by FCM. FCM plots are representative of three independent experiments with consistent results and cells were gated on CD4+ T cells. The bar graph indicates average percentages ± s.d. of Tregs. Data are means ± s.d. of triplicate cultures and representative of two or three independent experiments. **P < 0.01.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: Immunology and cell biology

Article Title: SjHSP60 induces CD4 + CD25 + Foxp3 + Tregs via TLR4-Mal-drived production of TGF-β in macrophages

doi: 10.1111/imcb.12160

Figure Lengend Snippet: SjHSP60-triggered TGF-β production and Treg conversion are Mal-dependent. (a) Purified Mφ from MyD88−/− mice or control WT littermates were cultured with or without 0.1 μg mL−1 SjHSP60 for 2 days. Mal mRNA levels were determined by qPCR and expressed as fold increases over unstimulated MyD88+/+ Mφ. (b, c) Effect of specific siRNA on endogenous Mal expression in RAW264.7 cells was analyzed by qPCR (b) and Western blot (c). (d, e) RAW264.7 cells transfected with Mal siRNA or control siRNA were stimulated with 0.1 μg mL−1 of SjHSP60. After 2 days, TGF-β levels in supernatants were detected by ELISA (e), while cells were washed and co-cultured with purified CD4+CD25− T cells for three additional days. Percentages of CD4+CD25+Foxp3+ Tregs in co-cultures were measured by FCM and cells were gated on CD4+ T cells (e). (f) The bar graph indicates average percentages ± s.d. of Tregs. Western blots and FCM plots are representative of three independent experiments with consistent results. Data are means ± s.d. of triplicate cultures and representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In some co-cultures, a neutralizing antibody to TGFβ1 (5 μg mL −1 ; R&D Systems, Inc. Minneapolis, MN) or an inhibitor of TGF-βRI signaling (20 μmol L −1 ; SB-431542; Sigma-Aldrich) was added.

Techniques: Purification, Cell Culture, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay