nod1 agonist tri dap  (InvivoGen)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: TLR8 agonists remodel the tumor immune microenvironment through PF4-dependent T cell recruitment and ancillary mechanisms

doi: 10.1007/s00262-026-04329-8

Figure Lengend Snippet: Dynamics of immune cell subsets in response to agonist stimulation. A Schematic of the experimental workflow. Immune profiling by flow cytometry was performed 48 h after a single intratumoral injection of PRR agonists. B Percentage of CD45 + leukocytes among live cells following intratumoral administration of different agonists: TLR3 agonist poly IC (25 µg/tumor), TLR7 agonist imiquimod hydrochloride (25 µg/tumor), TLR8 agonist TL8-506 (10 µg/tumor), TLR9 agonist CpG ODN 2395 (50 µg/tumor), STING agonist ADU-S100 ammonium salt (25 µg/tumor), NOD1 agonist Tri-DAP (10 µg/tumor), and NOD2 agonist murabutide (5 µg/tumor). C Proportion of cDCs within the CD45 + population. D Percentage of ZsGreen + cells among DCs. E Proportion of macrophages within the CD45 + population. F Percentage of ZsGreen + cells among macrophages. G Proportion of CD8 + T cells within the CD45 + compartment. H Proportion of cCD4 + T cells within the CD45 + compartment. I Proportion of Tregs within the CD45 + compartment, pooled from two independent experiments. J Ratio of cCD4 + T cells to Tregs. (K) Ratio of CD8 + T cells to Tregs. Data represent the mean ± SEM ( n = 8 mice per group). Statistical comparisons were performed using one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: When tumors reached approximately 200 mm 3 , PRR agonists were administered via intratumoral injection at the following doses: TLR3 agonist poly(I:C) (Sigma-Aldrich, #42,424–50-0; 25 μg/tumor); TLR7 agonist imiquimod hydrochloride (MedChemExpress, #HY-B0180A; 25 μg/tumor); TLR8 agonist TL8-506 (InvivoGen, #tlrl-tl8506; 10 μg/tumor); TLR8 agonist motolimod (MedChemExpress, #HY-13773; 50 μg/tumor); TLR9 agonist CpG ODN 2395 (Class C) (InvivoGen, #tlrl-2395–1; 50 μg/tumor); STING agonist ADU-S100 ammonium salt (MedChemExpress, #HY-12885B; 25 μg/tumor); NOD1 agonist Tri-DAP (InvivoGen, #tlrl-tdap; 10 μg/tumor); and NOD2 agonist murabutide (InvivoGen, #tlrl-mbt; 5 μg/tumor).

Techniques: Flow Cytometry, Injection

Journal: Inflammation

Article Title: CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

doi: 10.1007/s10753-021-01497-w

Figure Lengend Snippet: Impact of CTRP-3 on NOD1 mediated pro-inflammatory cytokine release from pre-adipocytes and mature adipocytes. 3T3-L1 fibroblasts/pre-adipocytes as well as mature adipocytes were pre-incubated with 10 μg/ml recombinant CTRP-3 (30 min) prior to overnight (18 h) treatment with 10 ng/ml Tri-DAP. MCP-1 and RANTES protein concentrations in cell supernatants were quantified by ELISA. CTRP-3, C1q/TNF-related protein-3; TD, Tri-DAP. A CTRP-3 inhibits Tri-DAP-induced MCP-1 release from 3T3-L1 fibroblasts/pre-adipocytes (n = 6). B CTRP-3 inhibits Tri-DAP-induced RANTES release from 3T3-L1 fibroblasts/pre-adipocytes (n = 6). C CTRP-3 inhibits Tri-DAP-induced MCP-1 release from mature 3T3-L1 adipocytes (n = 10–11). D CTRP-3 inhibits Tri-DAP-induced RANTES release from mature 3T3-L1 adipocytes (n = 24).

Article Snippet: The synthetic NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) [ ] (10 ng/ml; Invivogen, San Diego, CA, USA) and recombinant CTRP-3 (expressed in insect cells, see below) were used for co-stimulation experiments (incubation time 18 h).

Techniques: Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

doi: 10.1007/s10753-021-01497-w

Figure Lengend Snippet: Inhibition of NOD1 mediated pro-inflammatory cytokine release from THP-1 cells. Monocyte-like THP-1 cells were pre-incubated with 10 μg/ml recombinant CTRP-3 (30 min) prior to overnight (18 h) treatment with 10 ng/ml Tri-DAP. MCP-1 and RANTES protein concentrations in cell supernatants were quantified via ELISA. CTRP-3, C1q/TNF-related protein-3; TNFα, tumor necrosis factor α; TD, Tri-DAP. A CTRP-3 inhibits Tri-DAP-induced MCP-1 release from THP-1 macrophages (n = 6). B Tri-DAP induced TNFα release from THP-1 monocyte-like cells pre-incubated with CTRP-3 (n = 6).

Article Snippet: The synthetic NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) [ ] (10 ng/ml; Invivogen, San Diego, CA, USA) and recombinant CTRP-3 (expressed in insect cells, see below) were used for co-stimulation experiments (incubation time 18 h).

Techniques: Inhibition, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

doi: 10.1007/s10753-021-01497-w

Figure Lengend Snippet: CTRP-3 attenuates LPS-induced NOD1 gene expression in vivo in murine adipose tissue. C57BL/6 mice were injected i.p. with recombinant CTRP-3 (10 μg/animal) 30 min prior to i.p. injection of LPS (1 μg/animal). After 2 h, intra-abdominal adipose tissue was obtained post-mortem. NOD1 gene expression levels were quantified by RT-PCR and normalized to GAPDH gene expression. AT, adipose tissue; CTRP-3, C1q/TNF-related protein-3; i.p., intraperitoneal; ia, intra-abdominal; LPS, lipopolysaccharide; sc, subcutaneous; TD, Tri-DAP. A LPS induces NOD1 gene expression in intra-abdominal adipose tissue in vivo in mice. B CTRP-3 attenuates LPS-induced NOD1 gene expression in intra-abdominal adipose tissue in mice. C and D LPS and CTRP-3 do not affect NOD1 gene expression in subcutaneous adipose tissue in mice.

Article Snippet: The synthetic NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) [ ] (10 ng/ml; Invivogen, San Diego, CA, USA) and recombinant CTRP-3 (expressed in insect cells, see below) were used for co-stimulation experiments (incubation time 18 h).

Techniques: Gene Expression, In Vivo, Injection, Recombinant, Reverse Transcription Polymerase Chain Reaction

Journal: Inflammation

Article Title: CTRP-3 Regulates NOD1-mediated Inflammation and NOD1 Expression in Adipocytes and Adipose Tissue

doi: 10.1007/s10753-021-01497-w

Figure Lengend Snippet: Impact of adipocyte CTRP-3 deficiency on NOD1 gene expression in adipose tissue and primary adipocytes. Intra-abdominal adipose tissue was obtained from control mice and mice with an adipocyte-specific CTRP-3 knockout. Primary pre-adipocytes were derived from intra-abdominal adipose tissue and were differentiated to mature adipocytes ex vivo . NOD1 gene expression levels were quantified via RT-PCR and normalized to GAPDH gene expression. Ctrl., control mice; CTRP-3, C1q/TNF-related protein-3; KO, knockout mice; TD, Tri-DAP. A NOD1 gene expression levels are elevated in intra-abdominal adipose tissue from female CTRP-3 KO mice (n = 17) when compared to littermate controls. B NOD1 gene expression levels are not significantly altered in primary intra-abdominal adipocytes from female CTRP-3 KO mice (n = 6).

Article Snippet: The synthetic NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) [ ] (10 ng/ml; Invivogen, San Diego, CA, USA) and recombinant CTRP-3 (expressed in insect cells, see below) were used for co-stimulation experiments (incubation time 18 h).

Techniques: Gene Expression, Control, Knock-Out, Derivative Assay, Ex Vivo, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent

doi: 10.1038/s41598-021-85347-7

Figure Lengend Snippet: List of bacterial strains and ligands used in this study.

Article Snippet: Figure 2 L. salivarius UCC118 stimulated NF-κB activity and cytokine responses were MyD88 dependent in THP-1 monocytes. ( a ) NF-κB transcriptional activity and ( b ) TNF-α cytokine secretion was measured in WT and MyD88 −/− THP1 cell lines that were non treated (NT) or treated with NOD1 agonist (TriDAP), TLR2 agonist (HKLM- heat-killed Listeria monocytogenes ), TLR1/2 agonist (Pam3csk4), TLR2/6 agonist (FSL-1), TLR4 agonist (LPS) or co-cultured with L. salivarius UCC118 at MOI of 10 for 20 h. NF-κB transcriptional activity was measured indirectly by quantifying the activity of an NF-κB regulated reporter protein: secreted embryonic alkaline phosphatase, by using QUANTI-Blue (Invivogen) whose colour changes from blue to pink at 630 nm (A 630 ) in THP1-XBlue and THP1-XBlue-defMyD cell lines upon addition of substrate to the cells.

Techniques:

Journal: Scientific Reports

Article Title: Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent

doi: 10.1038/s41598-021-85347-7

Figure Lengend Snippet: L. salivarius UCC118 stimulated NF-κB activity and cytokine responses were MyD88 dependent in THP-1 monocytes. ( a ) NF-κB transcriptional activity and ( b ) TNF-α cytokine secretion was measured in WT and MyD88 −/− THP1 cell lines that were non treated (NT) or treated with NOD1 agonist (TriDAP), TLR2 agonist (HKLM- heat-killed Listeria monocytogenes ), TLR1/2 agonist (Pam3csk4), TLR2/6 agonist (FSL-1), TLR4 agonist (LPS) or co-cultured with L. salivarius UCC118 at MOI of 10 for 20 h. NF-κB transcriptional activity was measured indirectly by quantifying the activity of an NF-κB regulated reporter protein: secreted embryonic alkaline phosphatase, by using QUANTI-Blue (Invivogen) whose colour changes from blue to pink at 630 nm (A 630 ) in THP1-XBlue and THP1-XBlue-defMyD cell lines upon addition of substrate to the cells. TNF-α cytokine secretion in THP1-XBlue and THP1-XBlue-defMyD cell lines were quantified by ELISA. Data shown are the average of triplicate wells of three independent experiments (n = 3). Statistical analysis was performed with two-tailed t-test in GraphPad Prism. p < 0.05 (denoted by *) is considered statistically significant. ns nonsignificant.

Article Snippet: Figure 2 L. salivarius UCC118 stimulated NF-κB activity and cytokine responses were MyD88 dependent in THP-1 monocytes. ( a ) NF-κB transcriptional activity and ( b ) TNF-α cytokine secretion was measured in WT and MyD88 −/− THP1 cell lines that were non treated (NT) or treated with NOD1 agonist (TriDAP), TLR2 agonist (HKLM- heat-killed Listeria monocytogenes ), TLR1/2 agonist (Pam3csk4), TLR2/6 agonist (FSL-1), TLR4 agonist (LPS) or co-cultured with L. salivarius UCC118 at MOI of 10 for 20 h. NF-κB transcriptional activity was measured indirectly by quantifying the activity of an NF-κB regulated reporter protein: secreted embryonic alkaline phosphatase, by using QUANTI-Blue (Invivogen) whose colour changes from blue to pink at 630 nm (A 630 ) in THP1-XBlue and THP1-XBlue-defMyD cell lines upon addition of substrate to the cells.

Techniques: Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

doi: 10.3389/fimmu.2018.03082

Figure Lengend Snippet: The augmented inflammatory response is dependent on the intact of both TLR and NOD signaling. Peritoneal macrophages and BMMs isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 12 h. Macrophages incubated with PBS were used as the control. TNF-α concentrations in the supernatants were assessed by cytometric bead array. Data are expressed as mean ± SD from five to six independent experiments in duplicate. * p < 0.05, ** p < 0.01 vs. macrophages incubated with PBS; ≠ p < 0.05, ≠≠ p < 0.01 vs. macrophages stimulated with LPS, BLP, Tri-DAP, or MDP alone.

Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

Techniques: Isolation, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

doi: 10.3389/fimmu.2018.03082

Figure Lengend Snippet: Stimulation of macrophages with the combined TLR and NOD agonists amplifies downstream NF-κB activation and augments NF-κB p65 binding to TNF-α and IL-6 promoters. Isolated BMMs were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations either for 30 min (A) or for the indicated time periods (B,C) . Cytoplasmic proteins were extracted and subjected to immunoblotting for detection of either TLR4, NOD1, NOD2, MyD88, IRAK1, RIP2, and CARD9 (A) or total and phosphorylated p65 (P-p65), total and phosphorylated IκBα (P- IκBα), and total and phosphorylated p38 (P-p38) (B,C) . Results shown represent one experiment from a total of three to four separate experiments. The intensity of P-65, total IκBα, P-IκBα, and P-p38 signal in each band was normalized by GAPDH (D,E) . Data are expressed as mean ± SD from three to four separate experiments. * p < 0.05, ** p < 0.01 vs. macrophages stimulated with LPS, Tri-DAP, or MDP alone. Isolated BMMs were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (F) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (G) for 1 h. The binding of NF-κB p65 to TNF-α and IL-6 promoters was assessed by ChIP analysis and expressed as percentage of input. Data are expressed as mean ± SD from four independent experiments in duplicate. * p < 0.05, ** p < 0.01 vs. macrophages incubated with PBS; ≠ p < 0.05, ≠≠ p < 0.01 vs. macrophages stimulated with LPS, BLP, Tri-DAP, or MDP alone.

Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

Techniques: Activation Assay, Binding Assay, Isolation, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection

doi: 10.3389/fimmu.2018.03082

Figure Lengend Snippet: TLR and NOD signaling are both required for an efficient macrophage-mediated intracellular bacterial killing. Peritoneal macrophages isolated from wild-type, TLR4- and TLR2-deficient, and NOD1- and NOD2-deficient mice were stimulated with LPS (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (A) or BLP (10 ng/ml), Tri-DAP (5 μg/ml), MDP (5 μg/ml), and their combinations (B) for 6 h, and further incubated with live S. typhimurium ( S. typhi ) (A) or live S. aureus (B) for 60 min to assess intracellular bacterial killing. Data are expressed as mean ± SD from four to five independent experiments in triplicate. * p < 0.05, ** p < 0.01 vs. macrophages stimulated with LPS, BLP, Tri-DAP, or MDP alone.

Article Snippet: The NOD1 agonist L-Ala-γ-D-Glu-mDAP (Tri-DAP) and NOD2 agonist MDP were obtained from InvivoGen.

Techniques: Isolation, Incubation