Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A qPCR showed increased TLR4 expression in the 216911KO cell line. ** p < 0.01 by student T tests. B The diagram presented two pir-hsa-216911 binding sites in 3ʹUTR of TLR4. C The dual luciferase assay revealed that pir-has-216911 could bind both TLR4 binding sites TLR4S1 and TLR4S2. Plasmids carrying TLR4S1 and TLR4S2 generate a higher Firefly/Renilla Luciferase ratio in the 216911KO cell line, which pir-hsa-216911 was absent. **p < 0.01; ***p < 0.001 by student T tests. D TCGA-LIHC survival analysis found low expression of TLR4 to be unfavorable to the survival of HCC patients. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway. TLR4, p-p65, and NLRP3 levels were elevated in the 216911KO cell line, which pir-hsa-216911 was absent. F Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway through silencing TLR4. After silencing TLR4 with siRNA, pir-hsa-216911 absent could not elevate p-p65, and NLRP3 levels were elevated in the 216911KO cell line.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the xenograft modeling process. B Euthanized mouse to display the subcutaneous xenografted tumors. C 216911KO group resulted in much smaller tumors in size compared to the control group. D 216911KO group resulted in much smaller tumors in weight compared to the control group. The histogram represents the mean tumor weight of each group. ***p < 0.001 by student T tests. E Western blot analysis revealed pir-hsa-216911 modulating the TLR4/NFκB/NLRP3 signaling pathway in vivo. Both TLR4 and p-p65 levels were elevated in tumors formed in the 216911KO group compared to the control group.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Control, Western Blot, In Vivo

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: A The diagram presented the clinical HCC tumor tissue sampling process. B The immunohistochemistry demonstrates that TLR4 was downregulated in high pir-hsa-216911 tumor samples. The image shows typical IHC results. The box plot presents all IHC scores. *p < 0.01 by LSD test. C The immunohistochemistry demonstrates that c-GSDMD activation was suppressed in high pir-hsa-216911 tumor samples. The image shows typical IHC results. The box plot presents all IHC scores. * p < 0.01 by LSD test. D The tumor/adjacent tissue pir-hsa-216911 ratio was higher in HCC patients accompanied by steatosis. ** p < 0.01 by student T tests.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Sampling, Immunohistochemistry, Activation Assay

Journal: Cell Death Discovery

Article Title: pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

doi: 10.1038/s41420-024-02285-9

Figure Lengend Snippet: Correlation analysis between pir-hsa-216911 elevation, TLR4 expression, and GSDMD activation.

Article Snippet: The antibodies used in this study were Cleaved Caspase-1 Rabbit mAb (4199, Cell Signaling Technology, USA), Cleaved Gasdermin D Rabbit mAb (1:2000 dilute; 36425 Cell Signaling Technology, USA), anti-TLR4 rabbit polyclonal antibody (1:500 dilute; D121751, Sangon Biotech, China), anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (1:500 dilute; D155006, Sangon Biotech, China), anti-NLRP3 rabbit polyclonal antibody (1:250 dilute; D120143, Sangon Biotech, China), anti-ACTB rabbit polyclonal antibody (1:6000 dilute; D110001, Sangon Biotech, China), anti-GAPDH rabbit polyclonal antibody (1:4000 dilute; D110016, Sangon Biotech, China), and HRP-conjugated goat anti-rabbit IgG (1:6000 dilute; D110058, Sangon Biotech, China).

Techniques: Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Polarization-related signaling pathway of macrophages was activated after T.cp -MIF treatment. The macrophages binded with TLR4, and T.cp -MIF activated NF-κB signaling pathway 24 hours after induction. At this time, macrophages showed M1 type polarization, while PI3K/AKT/mTOR signaling pathway is activated at 48 hours, making macrophages show M2 type polarization.

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques:

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: Effect of T.cp -MIF on the expression of macrophage polarization-related proteins in different time. (A–D) The protein expression levers of TLR4, NF-κBp65, PI3K and AKT were analyzed by western blot and normalized against β-actin expression. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Dynamic changes in human THP-1-derived M1-to-M2 macrophage polarization during Thelazia callipaeda MIF induction

doi: 10.3389/fimmu.2022.1078880

Figure Lengend Snippet: The effect of TAK-242 on the expression of macrophage polarization-related proteins. (A–D) The cells were pretreated with TAK-242 (the TLR4 inhibitor) for 6 h, and the expression levels of TLR4, NF-κBp65, p-NF-κBp65, PI3K, p-PI3K, Akt and p-Akt were detected by western blot. * indicated P <0.05. Results are showed as the mean of three times of independent experiments ( x̄ ±SD, n=3).

Article Snippet: After blocking, the corresponding primary antibodies such as TLR4 (Wuhan Sanying Biotechnology Co., Ltd.), NF-κB (Wuhan Sanying Biotechnology Co., Ltd.), p-NF-κB (Cell signaling technology, USA), PI3K (Wuhan Sanying Biotechnology Co., Ltd.), p-PI3K (Abcam, UK), Akt (Wuhan Sanying Biotechnology Co. Ltd.), and β-actin (Hangzhou Huaan Biotechnology Co., Ltd.) was separately added to the box (antibody concentrations were diluted according to the instructions), incubated at 4°C for over 12 h. After incubation, the membrane was washed 3 times with TBST buffer for 10 min each time.

Techniques: Expressing, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes

doi: 10.1016/j.bbrc.2016.07.078

Figure Lengend Snippet: HMGB1 inhibits monocyte apoptosis induced by ABT-737 (A,C) or staurosporine (B,D), which is reversed when monocytes are pretreated with a blocking TLR4 antibody, as evaluated by Annexin V (A,B) and TMRE (C,D) stainings. (E) U0126, a specific MEK/ERK inhibitor, inhibits the effect of rHMGB1 on TMRE fluorescence in monocytes. (F) rHMGB1 (100 ng/ml) induces phosphorylation of ERK in monocytes, which does not occur when monocytes are pretreated with a blocking TLR4 antibody. Data are presented as mean ± SD for N≥4 and at least three separate experiments in all studies. * p<0.05, # p<0.05, ** p<0.01 (Student’s t test).

Article Snippet: When indicated, HMGB1 receptors were blocked on monocytes with anti-human RAGE polyclonal antibody (20 μg/ml, goat IgG), anti-human TLR2 monoclonal antibody (2 μg/ml, mouse IgG2b) or anti-human TLR4 polyclonal antibody (10 μg/ml, goat IgG) (R&D Systems, Wiesbaden, Germany).

Techniques: Blocking Assay, Fluorescence, Phospho-proteomics

Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 8. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for S100A8 and S100A9 in macrophages. We measured the mRNA expression levels of S100A8 and S100A9 to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) using a StepOnePlus Real-Time PCR System. (A) RT-PCR was carried out to detect S100A8 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. (B) RT-PCR was also carried out to detect S100A9 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statistical analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 9. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for inflammatory and anti-inflammatory cytokines in macrophages. We measured the mRNA expression levels of IL-1b, TNF-a, IL-6, IL- 10, and TGF-b to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone using a StepOnePlus Real-Time PCR System. (A–E) RT-PCR was carried out to detect these cytokine mRNAs in macrophages. Changes in the mRNA expression levels of IL-1b (Α), TNF-a (Β), IL-6 (C), IL-10 (D), and TGF-b (E), respectively, in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statisti- cal analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: The American Journal of Pathology

Article Title: Enhanced Susceptibility to Endotoxic Shock and Impaired STAT3 Signaling in CD31-Deficient Mice

doi: 10.1016/s0002-9440(10)62243-2

Figure Lengend Snippet: Figure 11. TLR4 expression of WT and CD31-deficient splenocytes and endothelial cells remains constant and does not change following LPS treat- ment. Representative FACS analyses of WT (A) and KO (B) splenocytes in the absence and presence of LPS; KO (C) and PECAM-1 reconstituted (D) lung endothelial cells and KO (E) and WT (F) brain endothelial cells illus- trating essentially no differences in TLR4 expression. This was confirmed by Western blot analyses (data not shown).

Article Snippet: Splenocytes were prepared as described, fixed in 2% PFA, then stained with 0.5 g of polyclonal anti-mouse TLR4 (Imgenex) in Hanks’ buffered salt solution plus 1% BSA for 1 hour on ice, then, following two washes, in donkey anti-rabbit IgG F(ab )2 PE conjugate (Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

doi: 10.1016/j.jbc.2022.102577

Figure Lengend Snippet: Figure 6. Potential roles of the DNA-mediated proteolytic processing of HMGB1 by neutrophil elastase in NETs. Due to the enhanced binding activities of the processed HMGB1 protein, this processing may promote (1) TLR4 signaling, (2) binding to biofilm DNA, and (3) DNA sensing by cGAS. Due to the loss of residues 177–215, the processing of HMGB1 may diminish (4) RAGE signaling and (5) nuclear localization. NET, neutrophil extracellular trap.

Article Snippet: Lyophilized TLR4 MD-2 complex was purchased from R&D Systems (catalog no.: #3146-TM-050).

Techniques: Binding Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 1 The FBG domains of tenascin-C, -R, and -W can induce NF-kB activation and cytokine synthesis, and bind to TLR4. a Tenascin-C, -R, -W, and -X each contain an assembly domain, a variable number of epidermal growth factor (EGF)-like repeats, a variable number of fibronectin type III-like repeats (these can be constitutively expressed (white rectangles) or alternatively spliced (gray rectangles) and a C-terminal fibrinogen-like globe (FBG) domain. The FBG domains exhibit a similar molecular weight, comprising between 229 and 240 amino acids each (FBG-C: 26.1 kDa, amino acids 1974–2201, FBG-R: 27.0 kDa, amino acids 1128–1359, FBG-W: 27.5 kDa, amino acids 1060–1300, FBG-X: 26.1 kDa, amino acids 4013–4243); protein accession numbers: tenascin-C (P24821), tenascin-R (Q92752), tenascin-W (Q9UQP3), tenascin-X (P22105). b THP1 NF-kB cells were stimulated with different concentrations of FBG-C, -R, -W, and -X, or were left unstimulated (−) for 24 h and NF-kB activation measured using QUANTI-Blue. Data are shown as mean ± SEM from three independent experiments. One-way ANOVA vs. non-stimulated, **p < 0.01, ***p < 0.001. c–e Primary human macrophages were stimulated with different concentrations of FBG-C,-R, -W, and -X, or were left unstimulated (−) for 24 h, and TNF (c), IL-6 (d), and IL-8 (e) levels measured by ELISA. Data are shown as mean ± SEM from three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001. f 96-well plates were coated with 1 µg ml−1 of FBG-C, -R, -W, or -X, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data in the graph are shown as mean ± SEM from four independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 2 Peptide mapping reveals specific regions in FBG-C involved in TLR4 activation and binding. a Nine peptides of ~30 amino acids long from FBG-C were synthesized; overlapping amino acid sequences are shown in bold. b THP1 NF-kB cells were stimulated with LPS (0.5 ng ml−1), FBG-C (0.5 µM), or 20, 50, or 100 µM of peptides 1–9 for 24 h and NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 4 independent experiments. One-way ANOVA vs. unstimulated cells. **p < 0.01, ***p < 0.001. c Increasing doses of TLR4 were pre-incubated with 200 µM of peptides before adding them to 96-well plates coated with 1 µg ml−1 of FBG-C. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM, n = 3. d THP1 NF-kB cells were left unstimulated (−) or pre-incubated with 100 µM peptides prior to stimulation with 0.5 µM of FBG-C for 24 h. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM, n = 3 independent experiments. Paired t-test vs. FBG- C only, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Binding Assay, Synthesized, Incubation

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 4 Pinpointing amino acids in loops 5, 7, and 10 of FBG-C that mediate TLR4 binding and activation. Upper panel: Sequences of wild-type FBG-C and mutants 1–7, highlighting wild-type amino acids in blue and mutations in red (loop 5 variants are shown in a, loop 10 in b, and loop 7 in c). Second panel: THP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 4 independent experiments. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Third panel: Primary human macrophages were left unstimulated (−) or stimulated for 24 h with LPS (1 ng ml−1), increasing doses (μM) of FBG-C or FBG-C mutants 1–7. Cytokines synthesis was measured by ELISA. Data shown as mean ± SEM. n = 4 independent donors. Paired t-test vs. FBG-C, *p < 0.05, **p < 0.01, ***p < 0.001. Bottom panel: 96-well plates were coated with 1 µg ml−1 of FBG-C or FBG-C mutants 1–7, and TLR4 was added in a dose-dependent manner. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data shown as mean ± SEM; n = 4

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 5 Mutations in FBG-X confer TLR4-activating ability. a FBG-X chimeric proteins were designed to introduce the amino acids found in FBG-C to activate and bind to TLR4 (red) into the FBG-X sequence (blue). b ThP1 NF-kB cells were left unstimulated (−) or stimulated for 24 h with 0.5 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. NF-kB activation was measured using QUANTI-Blue™. Data shown as mean ± SEM. n = 3 independent experiments. One-way ANOVA vs. FBG-C. c Primary human macrophages were left unstimulated (−) or stimulated for 24 h with 1 ng ml−1 of LPS or increasing doses (μM) of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4. Cytokine synthesis was measured by ELISA. Data shown as mean + SEM. n = 3 independent donors. One-way ANOVA vs. FBG-C. d 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FBG-X mutant 1, 2, 3, and 4, and TLR4 was added in a dose-dependent manner. Data shown as mean ± SEM. n = 4 independent experiments

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Introduce, Sequencing, Mutagenesis, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 6 A conserved cationic ridge in fibrinogen-related proteins (FRePs). a Simplified domain organization of human FRePs: each protein contains distinct N-terminal sequences but all possess a C-terminal FBG domain, including the four tenascin family members (shown in Fig. 1a), α, β, and γ chains of fibrinogen, the three angiopoietins, seven of the angiopoietin-like proteins (Angio-LPs), the three ficolins, fibroleukin, FIBCD-1, FGL1, and MFAP4. b The cationic loop 5 ridge present in FBG-C, -R, and -W, but absent in FBG-X, is conserved in a subset of FRePs, which possess a comparable structural epitope made up of residues from loops 5, 6, and 7. Homology models of the FBG domains of the three FRePs selected for further analysis are shown together with that of tenascin-C (FBG-C); these include two predicted TLR4 agonists; the fibrinogen γ chain (FIB-G) and ficolin-1 (FIC-1), and one FBG domain predicted to be incapable of activating TLR4; angiopoietin-like protein 4 (ALP-4). The region created by residues from loops 5, 6, and 7 on the surface of each FBG domain is shown in pale orange, within which positively charged residues are colored red

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques:

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 7 The FBG domains of FIB-G and FIC-1 exhibit pro-inflammatory effects in vitro and in vivo. a–c Primary human macrophages were stimulated with different concentrations of FBG-C, FIB-G, FIC-1, and ALP-4, or were left unstimulated (−) for 24 h. Cytokine levels were measured by ELISA. Data shown as mean ± SEM from at least three independent donors. One-way ANOVA vs. non-stimulated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. d Primary human macrophages were pre-incubated for 6 h with 3 µM TAK 242 prior to stimulation with FBG-C, FIB-G, FIC-1, and ALP-4 (1 µM), or no stimulation (−) for 24 h. Cytokine synthesis was measured by ELISA. Data shown as mean ± SEM from at least three independent donors. Paired t-test vs. non-treated, *p < 0.05, **p < 0.01, ***p < 0.001. e 96-well plates were coated with 1 µg ml−1 of FBG-C, FBG-X, FIB-G, FIC-1, and ALP-4, or PBS, and incubated with increasing doses of TLR4. Curves were fitted in GraphPad Prism using one-binding site hyperbola equation. Data are shown as mean ± SEM from three independent experiments. f, g Synovial inflammation was assessed 3 days post injection of each protein (1 µg) or PBS alone into the knees of DBA-1 mice. The histological score was calculated as the mean of seven sections from each knee joint per mouse. n = 5 mice per group except for FIC-1 (n = 4) (f). Mann–Whitney non-parametric test vs. PBS, *p < 0.05, **p < 0.01. Images show representative sections stained by haematoxylin and eosin (left panels) or safranin-O (right panels) (g). Mice injected with FBG-C, FIB-G, and FIC-1 exhibit cell infiltration into a thickened synovial lining layer, cellular invasion into the subchondral bone (arrows indicate bone erosion) and loss of articular cartilage proteoglycan (cp), pathological features not observed in mice injected with FBG-C mut or ALP-4.Scale bar left panels: 100 μM, right panels: 50 μM

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Injection, MANN-WHITNEY, Staining

Journal: Nature communications

Article Title: Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.

doi: 10.1038/s41467-017-01718-7

Figure Lengend Snippet: Fig. 8 A common danger domain revealed. Three distinct sites within the FBG domain of tenascin-C contribute to TLR4 activation (center panel); a cationic ridge made up of residues from loops 5–7 (pale orange with positive residues highlighted red), underneath which sits a triad of hydrophobic/polar residues from loop 7 (green, purple, and blue), plus a C-terminal cationic tail in loop 10 (positive residues highlighted red). The cationic ridge is the dominant inflammatory epitope; its deletion renders inflammatory stimuli inert and its ectopic expression can convert immunologically inactive proteins into TLR4 agonists. In addition to tenascin-C, in other proteins that contain FBG domains, possession of this inflammatory epitope also confers TLR4-activating capabilities, irrespective of protein family (*denotes validated domains). Together, these data reveal a common mechanism by which distinct inflammatory triggers, spanning a wide range of tissue locations, induced in response to a spectrum of different threats, can activate TLR4 to raise an immune response

Article Snippet: Recombinant human TLR4 was purchased from R&D systems.

Techniques: Activation Assay, Expressing