Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 1 Coadministration of CD3/CD28 and CpG induce robust amounts of IL-10 expression in splenocytes when compared to either signal alone. (A) Splenocytes were treated with CpG, CD3/CD28, CD3/CD28/CpG, or left untreated for 72 hours and analyzed for IL0 production by ELISA. (B) Splenocytes were treated with CD3/CD28/CpG for 0, 48, or 72 hours, and IL-10 levels were measured by ELISA. (C) Supernatants of splenocytes treated with either CpG or control ODN (ctrl CpG) in the absence or presence of CD3/CD28 for 72 hours were analyzed for IL-10 production by ELISA. (D) Splenocytes were treated with CpG in the presence or absence of various T cell-activating antibodies (CD28, CD3, or CD3/CD28) for 72 hours, and IL-10 expression in the supernatants was measured via ELISA. (E) Supernatants from splenocytes treated as indicated for 72 hours were analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 2 Coordination between T cells and macrophages induces the highest expression of IL-10 in a cell-contact dependent manner. (A, B) Supernatants from wild type and nude (A, Balb/c background) or wild type and SCID (B, C3H background) splenocytes were treated with CD3/CD28/CpG for 72 hours and then analyzed for IL-10 production by ELISA. (C) Non-depleted splenocytes or the ones depleted for CD3, CD4, CD8, DC, and NK cells were treated with CD3/CD28/CpG for 72 hours, and the supernatants were analyzed for IL-10 expression via ELISA. (D) Purified splenic B cells, DC, CD4+ T cells, or peritoneal macrophages were coincubated in the presence or absence of purified CD4+ T cells, treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 expression in the supernatants by ELISA. (E) Peritoneal macrophages were coincubated in the presence or absence of purified NK, whole T (CD3+ T), CD4+ T, or a combination of CD4+ T and NK cells; treated with CD3/CD28/CpG for 72 hours; and analyzed for IL-10 expression in the supernatants via ELISA. (F) Peritoneal macrophages were coincubated with purified CD4+ cells in the presence or absence of a transwell barrier, treated with CD3/CD28/CpG for 72 hours, and analyzed for IL-10 expression in the supernatant via ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Purification

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 3 CD3/28/CpG induces IL-10 production in both macrophages and CD4+ T cells. (A) Diagram of FACS analysis. (B, C) Splenocytes left untreated or treated with CpG, CD3/CD28, CD3/CD28/CpG for 72 hours and number of CD4 (B) or F4/80 (C) were measured via FACS. (C, D) Median fluorescence intensity of IL10 in the CD4+ (D) or F4/80+ (E) gated cells. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 4 CD3/CD28/CpG regulates IL-10 via activation of NF-κB1,pERK, and STAT3. (A) Splenocytes were treated as indicated for 72 hours and IL-10 levels were measured by ELISA. (B) Supernatants from wild type or NF-κB1−/−splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were analyzed for IL-10 production by ELISA. (C) Peritoneal macrophages from wild type or NF-κB1−/−mice were coincubated with purified CD4+ T cells from either wild type or NF-κB1−/−mice, treated with CD3/CD28/CpG for 72 hours and the supernatants were measured for IL-10 expression by ELISA. (D) Splenocytes treated with CD3/CD28, CpG, or TX (CD3/CD28/CpG) for 72 hours were stained for CD4 and pERK or CD4 and pSTAT3. Histograms show pERK or pSTAT3 levels from gated T cells. (E) Quantification of median fluorescence intensity of pERK levels in CD4 cells (N = 3). (F) Splenocytes were treated with ERK inhibitor U0126 at various doses for 72 hours in presence of CD3/CD28/CpG, and IL-10 levels were measure by ELISA. (G) Splenocytes were treated with STAT3 inhibitor NSC 74859 at various doses for 72 hours in presence of CD3/ CD28/CpG, and IL-10 levels were measure by ELISA. (H) Wild type or NF-κB1−/−splenocytes in presence or absence of STAT3 inhibitor (STAT3i, 50 μM) were treated with CD3/CD28/CpG for 72 hours and analyzed for IL-10 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Staining, Fluorescence

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 5 Activation or inhibition of CD40/CD154 pathway in the presence of CD3/CD28/CpG acts as a switch in the expression of IL-10 or IL-30. (A) Supernatants from wildtype or CD40−/−splenocytes were treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (B) Supernatants from wild type or CD154−/−splenocytes treated with CD3/CD28/CpG for 72 hours were analyzed for IL-10 production by ELISA. (C, D) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 72 hours, and IL-10 (C) or IL-30 (D) expression was measured in the supernatants using ELISA. (E, F) Splenocytes from wild type mice were treated with the indicated treatments in the presence of control or anti-CD40 antibodies for 24, 48, or 72 hours, and IL-10 (E) or IL-30 (F) expression was measured in the supernatants using ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Communication and Signaling

Article Title: The cell-to-cell coordination between activated T cells and CpG-stimulated macrophages synergistically induce elevated levels of IL-10 via NF-κB1, STAT3, and CD40/CD154

doi: 10.1186/1478-811x-11-95

Figure Lengend Snippet: Figure 6 Regulation of IL-10 and IL-30 via activation of CD40 signaling. (A) Supernatants from splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), in the presence or absence of rIL-10 for 72 hours were analyzed for IL-30 production by ELISA. (B) Wild type or IL-10−/−splenocytes were treated as indicated for 72 hours, and IL-30 levels were measured by ELISA. (C) Splenocytes were treated with CD3/CD28/CpG in the presence of recombinant IL30 at 50 and 100 ng/ml for 72 hours, and IL-10 levels were measured by ELISA. (D) Wild type or IL-10−/−splenocytes were treated with CD3/CD28/CpG for 72 hours in the presence of control or anti-CD40 antibodies, and IL-30 levels were measured by ELISA. (E) Harvested pellets of splenocytes treated with anti-CD40 or control antibody for the indicated time points were probed for pSTAT3, p-p65, p50/105, and actin. (F) Splenocytes were treated with CD3/CD28/CpG in the presence or absence of anti-CD40 activating antibody and the levels of p-p65 and p-STAT3 in the CD4+ T cells or macrophages were measured via flow cytometry. (G) Supernatants from wild type or NF-κB1−/−splenocytes treated with CpG, CD3/CD28, TX (CD3/CD28/CpG), or CD3/CD28/CpG in the presence of activating anti-CD40 for 72 hours were analyzed for IL-30 production by ELISA. *, P <0.05. N = 3.

Article Snippet: Vendors for all reagents were as follows: thiol-modified CpG oligodeoxynucleotide (ODN) 1668 or control ODN (Sigma), anti-mouse CD3 (eBioscience), anti-mouse CD28 (Biolegend), activating anti-CD40 (Novus, NBP1-06657), recombinant mouse IL12, IFNγ, and IL-10 (eBioscience), LPS (Sigma), lipoteichoic acid (Invivogen), poly I:C (Invivogen), concanamycin A (Sigma), and rat IgG (eBioscience), QNZ (Cayman Chemicals), U0126 (Sigma Aldrich), and NSC 74859 (SelleckBio), pSTAT3, p-p65, STAT3 (Cell signaling), pERK (Santa Cruz), CD154, FOXP3 (eBioscience), recombinant IL27p28 (IL30, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Control, Flow Cytometry

Journal: Cardiovascular research

Article Title: Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via Toll-like receptor 2.

doi: 10.1016/j.cardiores.2006.11.004

Figure Lengend Snippet: Fig. 3. Resting HCAECs but not HUVECs express TLR2. RT-PCR was performed on RNA from unstimulated HUVECs and HCAECs using primers specific for β-actin, TLR1, 2, 4, or 6 or CD14. Numbers beneath images show abundance of each transcript relative to β-actin as determined by real-time PCR. Dash indicates not detected within 40 cycles. (A). TLR2 mRNA expression was also measured in HUVECs cultured in medium alone, 1 μg/ml E. coli LPS, 10 ng/ml IFN-γ or LPS and IFN-γ combined for 18 h (B). Western blot for TLR2, TLR4, TLR6 and loading control GAPDH was performed on 10 μg of whole cell lysate from the TLR-deficient cell line HEK-293, HEK-293 cells transfected with TLR2 or TLR4/MD2, HUVECs, HCAECs or the human monocytic cell-line THP-1 (C).

Article Snippet: Western blot was performed on 10 μg of whole cell lysates obtained from unchallenged HEK-293, HEK293 cells transfected with TLR2 or TLR4/MD2, HUVEC, HCAEC and THP-1 cells using antibodies to TLR2 (sc10739, Santa-Cruz, US), TLR4 (sc10741, Santa-Cruz), TLR6 (3653, Prosci, CA, US) or loading control GAPDH (sc25778, Santa-Cruz).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Western Blot, Control, Transfection

Journal: Cardiovascular research

Article Title: Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via Toll-like receptor 2.

doi: 10.1016/j.cardiores.2006.11.004

Figure Lengend Snippet: Fig. 4. NE-LPSs but not E. coli LPS stimulate TLR2-dependent signalling. TLR-deficient HEK-293 cells were transfected with CD14 and reporter construct alone (A), or with additional TLR4/MD2 (B) or TLR2 (C), then challenged with 0.1 to 1000 ng/ml of each type of LPS for 18 h. Data are expressed as fold induction of NF-κB dependent reporter expression normalised to internal transfection efficiency control vs cells cultured in medium alone, and are representative of at least four experiments. Ec = E. coli, Bf = B. fragilis, Pg = P. gingivalis, Pa = P. aeruginosa. (D) Phenol re-extraction removes all TLR2-stimulatory activity from a crude E. coli LPS preparation and neither TLR1 nor TLR6 is sufficient or required for responsiveness to NE-LPSs. HEK-293 cells were transfected with TLR1 and TLR6, TLR2 only, or the combination of TLRs 1, 2 and 6 together. Cells were then challenged with 1 μg/ml of crude E. coli LPS, phenol re-extracted E. coli LPS, or each of the NE-LPSs for 18 h. ⁎⁎Pb0.01 vs cells cultured in medium alone.

Article Snippet: Western blot was performed on 10 μg of whole cell lysates obtained from unchallenged HEK-293, HEK293 cells transfected with TLR2 or TLR4/MD2, HUVEC, HCAEC and THP-1 cells using antibodies to TLR2 (sc10739, Santa-Cruz, US), TLR4 (sc10741, Santa-Cruz), TLR6 (3653, Prosci, CA, US) or loading control GAPDH (sc25778, Santa-Cruz).

Techniques: Transfection, Construct, Expressing, Control, Cell Culture, Extraction, Activity Assay

Journal: Cardiovascular research

Article Title: Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via Toll-like receptor 2.

doi: 10.1016/j.cardiores.2006.11.004

Figure Lengend Snippet: Fig. 5. Whole non-enterobacterial organisms and lipid-As can stimulate TLR2-dependent signalling. TLR-deficient HEK-293 cells were transfected with CD14, or with additional TLR2 or TLR4/MD2 co-expression, as described in the legend of Fig. 4. Cells were then challenged with 107 whole heat-killed bacteria per ml (A), or 1 μg/ml of lipid-A (LA) of each organism, for 18 h (B). Responses to 10 ng/ml E. coli or P. gingivalis LPS are included as positive controls for TLR4 and TLR2 signalling respectively. Data are expressed as mean fold induction of NF-κB dependent reporter expression normalised to internal transfection efficiency control vs cells cultured in medium alone, and are representative of three experiments. Ec = E. coli, Bf = B. fragilis, Pg = P. gingivalis, Pa = P. aeruginosa. ⁎Pb0.05 vs medium alone.

Article Snippet: Western blot was performed on 10 μg of whole cell lysates obtained from unchallenged HEK-293, HEK293 cells transfected with TLR2 or TLR4/MD2, HUVEC, HCAEC and THP-1 cells using antibodies to TLR2 (sc10739, Santa-Cruz, US), TLR4 (sc10741, Santa-Cruz), TLR6 (3653, Prosci, CA, US) or loading control GAPDH (sc25778, Santa-Cruz).

Techniques: Transfection, Expressing, Bacteria, Control, Cell Culture

Journal: Cardiovascular research

Article Title: Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via Toll-like receptor 2.

doi: 10.1016/j.cardiores.2006.11.004

Figure Lengend Snippet: Fig. 6. TLR2-neutralizing antibody blunts HCAEC responses to NE — but not E. coli LPS. HCAECs were incubated with 10 μg/ml of a TLR2- neutralizing monoclonal antibody, TL2.5 (Anti-TLR2) or isotype matched antibody control, TLR3.7 (Irr. Ab) for 30 min prior to challenge with 1 μg/ ml of each LPS or 100 ng/ml Pam3CSK4 for 4 h (A). Alternatively, HCAECs were preincubated with 10 μg/ml of TLR6-neutralizing polyclonal antibody (Anti-TLR6) or irrelevant antibody before challenge with 1 μg/ml of each LPS or 10 ng/ml of the synthetic diacyl lipopeptide FSL-1 for 4 h (B). Cell- surface ELISA was then performed to measure relative expression of E- selectin. Results are representative of three experiments and are presented as mean+/−S.D. ⁎Pb0.05 by Student's t-test.

Article Snippet: Western blot was performed on 10 μg of whole cell lysates obtained from unchallenged HEK-293, HEK293 cells transfected with TLR2 or TLR4/MD2, HUVEC, HCAEC and THP-1 cells using antibodies to TLR2 (sc10739, Santa-Cruz, US), TLR4 (sc10741, Santa-Cruz), TLR6 (3653, Prosci, CA, US) or loading control GAPDH (sc25778, Santa-Cruz).

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Nocardia rubra cell wall skeleton-induced MARCO expression: implications for improved phagocytosis and cytokine secretion in tumor-associated macrophages

doi: 10.3389/fimmu.2026.1611476

Figure Lengend Snippet: Nr-CWS affects MARCO expression through TLR4. The expression of TLR4 significantly increased after Nr-CWS treatment [ (D-G) , scale bars = 100 μm], while the expression TLR2 was unchanged (A-C) . Subsequent inhibition of TLR4 expression resulted in decreased levels of MARCO [ (H-J) , scale bars = 100 μm]. n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following commercial antibodies (vendor, catalog number and dilution) utilized for western blot and immunohistochemical staining were employed in accordance with the manufacturers’ guidelines: mouse anti-CD68 antibody (Abcam, ab201340, 1:200), rabbit anti-MARCO antibody (Abcam, ab231046, 1:1000 for western blot and 1:100 for immunohistochemistry), rabbit anti-CD163 antibody (Abways Technology, CY6845, 1:500), mouse anti-CD86 antibody (Proteintech, 68674-2-Ig, 1:5000), mouse anti-TLR4 antibody (Santa Cruz Biotechnology, sc-293072, 1:1000 for western blot and 1:200 for immunohistochemistry), rabbit anti-TLR2 antibody(Abways Technology, CY5102, 1:2000), mouse anti-GAPDH antibody (Proteintech, 6004-1-Ig, 1:50000), HRP-goat anti-mouse recombinant secondary antibody (H+L)(Proteintech, RGAM001, 1:5000), goat anti-mouse IgG (H+L) Alexa Fluor 594 (Abways Technology, AB0152, 1:300), HRP-goat anti-rabbit recombinant secondary antibody (H+L) (Proteintech, RGAR001, 1:5000), goat anti-rabbit IgG (H+L) secondary antibody DyLightTM 594 (Report Biotech, S7002, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 488 (Report Biotech, S6001, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 594 (Abways Technology, AB0152, 1:500).

Techniques: Expressing, Inhibition

Journal: Journal of Inflammation Research

Article Title: Therapeutic Effect of C-C Chemokine Receptor Type 1 (CCR1) Antagonist BX471 on Allergic Rhinitis

doi: 10.2147/JIR.S254717

Figure Lengend Snippet: Relative quantification of NF-kB p65, TLR4, and TLR2 protein expression levels. ( A ) Western blot analysis for NF-kB p65, TLR4, and TLR2 relative expression from groups indicated. ( B ) summary graph for relative expression of NF-kB (n=8 per group). ( C ) summary graph for relative expression of TLR4 (n=8 per group). ( D ) summary graph for relative expression of TLR2 (n=8 per group). All values are represented as mean ± SEM. * p<0.05; ** p<0.01; and *** p<0.001 versus the vehicle control group.

Article Snippet: Samples were subsequently treated with anti β-actin antibody (BM3873, Boster Biological Technology, China), NF-kB p65 monoclonal antibody (A10609, Abclonal, USA.), TLR4 antibody (BA1717, Boster Biological Technology, China), or TLR2 antibody (BM4001, Boster Biological Technology, China), and were incubated overnight at 4°C.

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control

Journal: The Journal of Veterinary Medical Science

Article Title: Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves

doi: 10.1292/jvms.13-0534

Figure Lengend Snippet: Monoclonal antibodies used for flow cytometry

Article Snippet: CD282 , TLR2 , Monocytes , HCA151F , IgG , AbD-Serotec.

Techniques: Bioprocessing