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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand.

doi: 10.4049/jimmunol.181.10.6702

Figure Lengend Snippet: FIGURE 2. FimH elicits an innate antiviral response in cells expressing TLR4. A, Peritoneal macrophages from B6 or TLR4/ mice were treated with LPS, FimH, or poly(I:C) or left un- treated and supernatants were assayed for TNF- levels. B, Human lung fibro- blasts (HEL; TLR4 negative), human foreskin fibroblasts (BJ; TLR4 positive) and murine B6 fibroblasts (B6 MEFs; TLR4 positive) were left untreated (control) or treated with poly(I:C), LPS, or FimH as indicated. Twenty-four hours posttreatment, cells were left un- infected (negative (ve) control) or in- fected with VSV-GFP. GFP fluores- cence was visualized 24 h postinfection.

Article Snippet: To determine whether FimH can directly bind TLR4, we performed an in vitro protein binding assay (Fig. 4D) using recombinant TLR4 fused to the Fc fragment of human IgG1 (TLR4-Fc; R&D Systems).

Techniques: Expressing, Control, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand.

doi: 10.4049/jimmunol.181.10.6702

Figure Lengend Snippet: FIGURE 3. FimH-induced innate immunity is independent of LPS. The FimH protein purification protocol was performed using bacteria containing the FimH expression plasmid (FimH prep) or containing an empty vector (FimH prep). LPS contamination levels were determined by Limulus amebocyte lysate assay. A, TLR4 BJ fibroblasts were treated with either the FimH or FimH preparation or left untreated (control). Twenty-four hours posttreatment, cells were left uninfected (–ve control) or infected with VSV-GFP. B, Peritoneal macrophages from B6 or TLR4/ mice were treated with either the FimH or FimH prep- aration and assayed for TNF-. C, Intact, trypsin-digested, or heat-inactivated FimH was used to treat peritoneal macrophages from B6 or TLR4/ mice and the levels of TNF- were measured.

Article Snippet: To determine whether FimH can directly bind TLR4, we performed an in vitro protein binding assay (Fig. 4D) using recombinant TLR4 fused to the Fc fragment of human IgG1 (TLR4-Fc; R&D Systems).

Techniques: Protein Purification, Bacteria, Expressing, Plasmid Preparation, Control, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand.

doi: 10.4049/jimmunol.181.10.6702

Figure Lengend Snippet: FIGURE 4. FimH signals in cells unresponsive to LPS and directly interacts with TLR4. A, Total RNA was isolated from 293, 293-hTLR4, and 293-hTLR4- CD14-Md2 cells for RT-PCR (RT) analysis. B, 293, 293-hTLR4, and 293-hTLR4-CD14-Md2 cells were cotransfected with pCMV-gal and pNFB-luc. Sixteen hours later, cells were left untreated (Mock) or treated with FimH (2 g/ml) or LPS (10 ng/ml). Six hours later, cells lysates were harvested and luciferase assays were performed. Luciferase levels were standardized to internal -galactosidase levels as a transfection control and reported as fold increase relative to mock-treated controls for each cell line. The luciferase experiments were performed in triplicate three times. C, 293 and 293-hTLR4 cells were left untreated (mock) or treated with FimH (2 g/ml) or LPS (10 ng/ml). Twenty-four hours posttreatment the supernatants were collected to measure the production of IL-8. D, FimH binding to TLR4 was assessed in an ELISA-like protein binding assay using a TLR4-Fc fusion protein. ELISA plates were coated with different concentrations (0.0625–8 g/ml) of FimH or LPS. Immobilized FimH or LPS was then incubated with 2 g/ml TLR4-Fc fusion protein. Specific TLR4 binding was measured through the Fc domain of the fusion protein using a HRP-conjugated anti-human IgG. Binding of immobilized FimH or LPS to IL-15R-Fc was assayed similarly. Data shown are representative of three independent experiments.

Article Snippet: To determine whether FimH can directly bind TLR4, we performed an in vitro protein binding assay (Fig. 4D) using recombinant TLR4 fused to the Fc fragment of human IgG1 (TLR4-Fc; R&D Systems).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Protein Binding, Incubation

Journal: Science advances

Article Title: A neuroimmune pathway drives bacterial infection.

doi: 10.1126/sciadv.adr2226

Figure Lengend Snippet: Fig. 4. The TLR4-MYD88-MAPK3 pathway mediates CREB1 phosphorylation and function. (A) Dose-dependent impact of PKA inhibitor H-89 on ACOD1 expression post- LPS stimulation (100 ng/ml, 6 hours). (B) ACOD1 expression following LPS exposure and cotreatment with dopamine (0.5 mM) and increasing doses of PKA activator forskolin (10, 50, 100, and 200 μM). (C) Effects of cAMP analog 8-Br-cAMP on ACOD1 expression in dopamine-cotreated cells after LPS stimulation. (D) CREB1 phosphorylation and ACOD1 expression in TLR4-deficient monocytes challenged with LPS for varying durations (1 and 6 hours). (E and F) IP analyses revealing interactions within the TLR4 signal- ing complex in response to LPS and dopamine in native and DRD2-deficient monocytes. (G) Phosphorylation heatmap illustrating kinase activity shifts over time (1, 3, and 6 hours) post-LPS and dopamine treatment. (H) Effects of MAPK1/3 inhibition (VX-11e, pluripotin, ulixertinib, all 10 μM) on CREB1 and ACOD1 regulation following LPS stimu- lation. (I and J) Influence of MAPK3 knockdown or overexpression on CREB1 phosphorylation and ACOD1 expression post-LPS challenge. (K) MAPK3 activation dynamics in TLR4-knockdown THP1 cells after LPS exposure. (L) Protein expression profiling in DRD2-deficient monocytes under LPS stimulation for 1 hour. All the semiquantitative data are presented as means ± SD; n = 3 biologically independent samples. Statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Surface plasmon resonance assay Surface plasmon resonance assay was used to investigate the interactions between recombinant DRD2 (rDRD2) and recombinant TLR4 (rTLR4) proteins (OriGene) using a Biacore system.

Techniques: Phospho-proteomics, Expressing, Activity Assay, Inhibition, Knockdown, Over Expression, Activation Assay