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: iScience

Article Title: Protective effect of Zymosan-A against radiation-induced premature ovarian insufficiency in a murine model

doi: 10.1016/j.isci.2026.115027

Figure Lengend Snippet: The TLR2-NF-κB signaling pathway plays a critical role in the radioprotective effect of Zymosan-A (A) Heatmap of differential gene expression between wild-type mice and Zymosan-A mice. (B) Scatterplot of differently expressed genes in ovary tissue after Zymosan-A treatment. Each dot stands for a gene. Red and green color dots indicate an increase or decrease, respectively. (C) Pathway enrichment analysis of KEGG pathways within the core network. (D) RNA level to verify the expression of DEGs, including TLR2, CCL3, CCL5, AKT1, MYD88, and IκBκB. (E) The expression of TLR2-NF-κB pathway-related proteins. (F) The serum E2 level, AMH level, FSH level, and LH level were measured at TLR2 KO + IR + NS group and TLR2 KO + IR + Zymosan-A group. (G) The cell viability in siTLR2 + NS + IR group and siTLR2 + Zymosan-A + IR group (error bars represent the mean ± SD of independent experiments; N.S., no statistical difference, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n = 3).

Article Snippet: The following antibodies were used: TLR2 (Cat no: 17236-1-AP, 1:1000, Proteintech), BCL-2 (Cat no: 60178-1-Ig, 1:1000, Proteintech), BAX (Cat no: 60267-1-Ig, 1:1000, Proteintech), PCNA (Cat no: 13110, 1:1000, CST), Cleaved Caspase-3 (Cat no: 9661, 1:1000, CST) and GAPDH (Cat no:5174, 1:1000, CST), p -IKKα/β (Cat no: 2697, 1:1000, CST), p-P65 (Cat no: 3033, 1:1000, CST), TLR2 (Cat no: 66645-1-Ig, 1:1000, Proteintech), Myd88 (Cat no: 50010, 1:1000, CST).

Techniques: Gene Expression, Expressing

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