t2 5 monoclonal anti tlr2 ab  (Hycult Biotech)

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: Expression of TLRs in MSCs and effect of TLR2 stimulation on PTGS2/COX2-PGE2 pathway. A. Experimental scheme. Human BM MSCs were treated with either Pam2CSK4 or anti-TLR2 Ab for 24 h. Alternatively, MSCs were cocultured in a Transwell system with PMA-differentiated THP-1 macrophages (THP-1 macro) for 18 h or with GM-CSF-stimulated murine BM monocytes (BM mono) for 5 d. Then, MSCs were assessed for expression of TLRs and PTGS2 and production of PGE2. B-D. Representative and quantitative flow cytometry results for TLR2, TLR3 and TLR4 in MSCs. FMO (fluorescence minus one) control for each Ab was used as gating control. MSCs were treated with Pam2CSK4 (100 ng/mL) or anti-TLR2 Ab (10 μg/mL). E, F. qRT-PCR quantification for TLR2 , TLR3 and TLR4 in MSCs cocultured with THP-1 macrophages or BM monocytes. The mRNA levels are presented as the fold changes relative to MSCs cultured alone. G. qRT-PCR for PTGS2 and ELISA for PGE2 production in MSCs treated with Pam2CSK4 (0-1000 ng/mL). Shown are the mRNA levels relative to untreated MSCs. Data represent means ± SD from 2-3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant, as analyzed by one-way ANOVA and Tukey's test ( C, G ) or by Student's t -test ( D-F ).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Expressing, Flow Cytometry, Fluorescence, Control, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: TLR2 signaling in MSC is crucial to induction of immunosuppressive monocytes/macrophages. A. Experimental scheme. THP-1 macrophages were cocultured with human MSCs in a Transwell system. Prior to coculturing, MSCs were subjected to pretreatment with Pam2CSK4 (100 ng/mL) or anti-TLR2 Ab (10 μg/mL) or transfected with TLR2 siRNA or SCR siRNA for 24 h. After 18 h of coculturing, THP-1 macrophages were analyzed for PTGS2 mRNA levels and the secreted levels of PGE2, IL-10 and AREG. B. qRT-PCR for PTGS2 in THP-1 macrophages . The mRNA levels are fold changes relative to THP-1 macrophages cultured alone. C. ELISA for PGE2, IL-10 and AREG in supernatants of THP-1 macrophages with or without MSC coculturing. D. Experimental scheme. Murine BM cells were cocultured with human MSCs under GM-CSF stimulation for 5 d. Prior to coculturing, MSCs were pretreated with Pam2CSK4 (100 ng/mL) or anti-TLR2 Ab (10 μg/mL) or transfected with TLR2 siRNA or SCR siRNA for 24 h. After 5 d of coculturing, BM cells were assayed. E. Representative and quantitative flow cytometry results for CD11b hi Ly6C hi Ly6G lo and CD11b mid Ly6C mid Ly6G lo cells in BM cells. F. qRT-PCR for Arg1 , Nos2 and Ptgs2 in BM cells. Shown are the mRNA levels relative to BM cells cultured alone without GM-CSF. G. ELISA for PGE2, IL-10 and active TGF-β1 levels in cell-free supernatants of BM cells with or without MSC coculturing. Mouse-specific ELISA kits were used for measurement of IL-10 and active TGF-β1. The PGE2 assay kit recognizes both human and murine PGE2. H. Experimental scheme. MSCs were isolated from WT C57BL/6 or TLR2 KO mice. BM cells from WT C57BL/6 mice were cocultured with WT or TLR2 KO MSCs under GM-CSF for 5 d, and then subjected to assays. I, J. Representative flow cytometry results for CD11b, Ly6C, Ly6G and MHC class II in BM cells. K. Quantitative flow cytometric analysis for CD11b hi Ly6C hi Ly6G lo cells, CD11b mid Ly6C mid Ly6G lo cells and MHC class II hi Ly6C hi Ly6G lo cells in BM cells. L. ELISA for murine TNF-α and IL-1β in supernatants of cell cultures. Data are means ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as analyzed by one-way ANOVA and Tukey's test, Kruskal-Wallis test and Dunn's multiple-comparison test (CD11b hi Ly6C hi Ly6G lo and MHC class II hi Ly6C hi Ly6G lo cells in K) or Student's t -test (L).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Comparison

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: TLR2 signaling in MSCs is essential for suppression of inflammatory angiogenesis in the cornea following sterile injury. A. Experimental protocol. Immediately after corneal suturing injuries, MSCs, Pam2CSK4-pretreated MSCs, TLR2 siRNA-transfected MSCs, SCR siRNA-transfected MSCs or HBSS (vehicle) were administered via tail vein injection in BALB/c mice. Seven days later, the corneas were subjected to assays. B, C. Representative corneal photographs and microphotographs of CD31/LYVE1-stained corneal whole-mounts. Scale bar: 500 μm for the first and second rows and 200 μm for the third row (magnified images of yellow-outlined insets from the first row). D, E . Clinical scoring of corneal NV and quantification of CD31-stained area in corneal whole-mounts. F, G. qRT-PCR for pro-angiogenic factors and pro-inflammatory cytokines in cornea. The mRNA levels are presented relative to those in BALB/c control corneas that had not received injury or treatment. H. Experimental protocol. Corneal sutures were applied to TLR2 KO mice, and either MSCs or HBSS were intravenously injected. After 7 d, the corneas were subjected to assays. I, J. Representative corneal photographs and microphotographs after CD31/LYVE1 immunostaining. Scale bar: 500 μm for the upper row and 200 μm for the lower row (magnified images of yellow-outlined insets from the upper row). K, L. Clinical scoring of corneal NV and measurement of CD31- and LYVE1-stained areas. Data represent means ± SD, where a circle indicates the data from an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as analyzed by one-way ANOVA and Tukey's test or by Student's t -test (Injury + MSC vs. Injury + MSC/Pam2CSK4 in F, G).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Sterility, Transfection, Injection, Staining, Quantitative RT-PCR, Control, Immunostaining

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: Single-cell transcriptional profiling of Pam2CSK4-treated and untreated MSCs. A. Experimental scheme. MSCs treated with Pam2CSK4 100 ng/mL for 24 h and untreated MSCs were subjected to scRNA-seq on the 10x Genomics Chromium platform. B. Combined UMAP and tSNE plots of Pam2CSK4-treated MSCs (blue) and untreated MSCs (orange) showing overlapping. C. tSNE plots of Pam2CSK4-treated MSCs and untreated MSCs depicting 7 clusters (C0-C6) in both cell libraries. D, E. Feature and dot plots displaying positive and negative markers for MSCs. F. Heatmap of top 10 DEGs in Pam2CSK4-treated MSCs vs. untreated MSCs as determined by RNA-Seq analysis. AKR1C1 was identified as the top DEG upregulated in Pam2CSK4-treated MSCs relative to untreated MSCs. G. ELISA for secreted levels of AKR1C1 in cell-free supernatants of MSC cultures treated with Pam2CSK4 (0-100 ng/mL). H. qRT-PCR and ELISA for mRNA and protein levels of AKR1C1 in MSCs transfected with TLR2 siRNA or control SCR siRNA. Means ± SD are presented. ** p < 0.01 as analyzed by Kruskal-Wallis test and Dunn's multiple-comparison test (G) or Student's t -test (H).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, Control, Comparison

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: (A and B) Western blot shows TLR1 and TLR6 in NK cells. (C and D) Immunoprecipitations were performed with anti-TLR2 in non-stimulated (lane a) and LPG-stimulated NK cells after 1 h of co-incubation (lane b). (C) Immunoprecipitates were subjected to Western blotting and probed with anti-TLR1 and (D) anti-TLR6 antibodies, respectively. A representative immunoblot from four different experiments is shown. (E) NK cell lysates in non-stimulated and LPG-stimulated NK cells were immunoprecipitated with anti-TLR2, anti-MyD88, anti-IRAK-1 and anti-TRAF-6 antibodies and Western blotted with anti-MyD88, anti-IRAK-1, anti-TRAF-6 and anti-IKK-γ antibodies. (F) Nuclear translocation of p50 and p65 NF-κB isoforms were analyzed in non-stimulated and LPG-stimulated NK cells. (G) Phosphorilation of pIKK-α/β and pIκB-α were analyzed in non-stimulated NK cells (Non-stim) or in cells stimulated with PMA (15 min) or LPG (15, 30, 45 and 60 min). A representative immunoblot from two different experiments is show.

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Western Blot, Incubation, Immunoprecipitation, Translocation Assay

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: (A) Representative flow CD56 + /CD3 - expression in membranes of NK cells; right image: NK cells were gated and analyzed for TLR2 (TLR2 + /CD56 + ) expression. (B) Representative histograms showing the MFI levels of TLR2 expression on NK cells from control and patients (LCL and DCL). (C) Quantitation of TLR2 expression on NK cells. □ Non-stimulated cells, ▪ LPG-stimulated cells. Data are representative of healthy controls (n = 21), LCL patients (n = 28) and DCL patients (n = 6). These results are the mean ±SEM. *p≤0.05 was considered significant.

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: (A) Analysis according to disease form. (B) TLR2, TLR1 and TLR6 expression according to age (≤25 or ≥26 years) in LCL patients. □ Non-stimulated NK cells, ▪ LPG-stimulated NK cells. Cell surface expression is indicated by MFI. These results are the mean ±SEM. *Significant differences were observed between LCL and DCL patients for all TLRs in LPG-stimulated and non-stimulated cells.

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Expressing

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: (A) Double immunohistochemistry staining of TLR expression (TLR1, TLR2 and TLR6) on NK cells (CD57). (B) Number of NK cells expressing TLRs per mm 2 . Results are the mean ±SEM. Scale bar = 50 µm. Black arrows show double positive cells.

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: (A) Representative flow cytometry dot plot (TLR2 vs. CD56) and histogram of TLR2 expression in NK cells. (B) This gate was subsequently analyzed for TLR2 expression on NK-cell subsets. Dot plot (CD16 + /CD56 + ) and histogram for CD56 bright (blue box) and CD56 dim (green box) NK cells. (C) TLR1 expression. (D) TLR2 expression. (E) TLR6 expression. □ Non-stimulated NK cells; ▪ LPG-stimulated NK cells. Lane 1: CD56 dim ; lane 2: CD56 bright NK cells. Cell surface expression is indicated by MFI. Results are the mean ±SEM. *Significant differences were observed between NK cell subsets of LCL and DCL patients for all 3 TLRs in LPG-stimulated and non-stimulated cells. LCL patients (n = 9), DCL patients (n = 4) and controls (n = 4).

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

doi: 10.1371/journal.pone.0112410

Figure Lengend Snippet: Transcript expression by quantitative real time PCR in non-stimulated (NS) and LPG-stimulated NK cells (S).

Article Snippet: Afterwards the cells were washed and stained with 2 µL of Ab goat anti-human TLR2, TLR1 or TLR6 (sc- 8680, sc-8687, sc-30001) and FITC conjugated rabbit anti-goat IgG (Zymed 81-1611) or isotype controls.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: The TLR-NF-kB axis contributes to the monocytic inflammatory response against a virulent strain of Lichtheimia corymbifera , a causative agent of invasive mucormycosis

doi: 10.3389/fimmu.2022.882921

Figure Lengend Snippet: TLR4 contributes to the activation of the inflammatory response by primary human monocytes against L corymbifera and M. circinelloides . Neutralizing TLR2-antibody and TLR4 inhibitor were incubated for three hours before infection with the Mucorales. (A) Pro-inflammatory cytokines concentrations after inhibition of TLR2 or (B) TLR4. PAM3CSKA was used as a specific ligand for TLR2 activation and LPS forTLR4 activation. Dots and lines represent paired values of every single donor. Data were obtained from three independent experiments, each with two or three different donors, and differences among groups were assessed by paired t-test. * p < 0,05; ** p <0,01. ns indicates not significant. L corymbifera (L. cor), R. delemar (R. del), M. circinelloides (M. cir).

Article Snippet: The isolated cells were incubated in a 24-well plate at 37 °C and 5% CO 2 , and stimulated for three hours with specific inhibitors reported in previous studies: 5 µM Interleukin-1 Receptor-Associated Kinase (IRAK) inhibitor (CAS 509093-47-4, Merck), 5 µM Spleen tyrosine kinase (Syk) inhibitor (ER 27319 maleate, R&D systems), 10 µM NF-KB inhibitor (Bay11-7082, In vivo gen), 10 µg/mL human TLR2 Neutralizing antibody (Ab) (PAb-hTLR2, In vivo gen), and 5 µM TLR4 inhibitor (CLI-095, In vivo gen) ( – ).

Techniques: Activation Assay, Incubation, Infection, Inhibition

Journal: Frontiers in Immunology

Article Title: The TLR-NF-kB axis contributes to the monocytic inflammatory response against a virulent strain of Lichtheimia corymbifera , a causative agent of invasive mucormycosis

doi: 10.3389/fimmu.2022.882921

Figure Lengend Snippet: TLR4 contributes to the activation of the inflammatory response by primary human monocytes against L corymbifera and M. circinelloides . Neutralizing TLR2-antibody and TLR4 inhibitor were incubated for three hours before infection with the Mucorales. (A) Pro-inflammatory cytokines concentrations after inhibition of TLR2 or (B) TLR4. PAM3CSKA was used as a specific ligand for TLR2 activation and LPS forTLR4 activation. Dots and lines represent paired values of every single donor. Data were obtained from three independent experiments, each with two or three different donors, and differences among groups were assessed by paired t-test. * p < 0,05; ** p <0,01. ns indicates not significant. L corymbifera (L. cor), R. delemar (R. del), M. circinelloides (M. cir).

Article Snippet: The isolated cells were incubated in a 24-well plate at 37 °C and 5% CO 2 , and stimulated for three hours with specific inhibitors reported in previous studies: 5 µM Interleukin-1 Receptor-Associated Kinase (IRAK) inhibitor (CAS 509093-47-4, Merck), 5 µM Spleen tyrosine kinase (Syk) inhibitor (ER 27319 maleate, R&D systems), 10 µM NF-KB inhibitor (Bay11-7082, In vivo gen), 10 µg/mL human TLR2 Neutralizing antibody (Ab) (PAb-hTLR2, In vivo gen), and 5 µM TLR4 inhibitor (CLI-095, In vivo gen) ( – ).

Techniques: Activation Assay, Incubation, Infection, Inhibition