Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: eNPM binds to TLR4 and the binding increases upon cytokines treatment in HaCaT and HFs. (A) Proximity ligation assay (PLA) of NPM and TLR4 interaction analysis in serum-starved HaCaT cells stimulated with a MIX of cytokines for 8 h (IL-17A, IL-22, TNF-α, and IFN-γ). Lower panels represent zoomed-in images of the original PLA images. Red dots indicate that even under physiological conditions, NPM interacts with TLR4. When a MIX of cytokines is administered, the rate of interaction increases. As a negative control, the IgG specific for each antibody (i.e., normal rabbit IgG used as the control of TLR4 antibody) and normal mouse IgG for NPM antibody (left panel) revealed the specificity of NPM and TLR4 interaction, since no red dots were present in the PLA. Scale bar 10 μm. (B) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 5; * p < 0.05). (C) PLA of NPM and TLR4 interaction analysis in serum-starved HFs derived from healthy subjects and stimulated with a MIX of cytokines for 8 h. Lower panels represent zoomed-in images of the original PLA images. Scale bar 10 μm. (D) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Binding Assay, Proximity Ligation Assay, Negative Control, Control, Derivative Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: rNPM promotes NF-κB translocation to the nucleus and induces inflammation in HaCaT and HFs via TLR4. (A) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NFκB translocated in the nucleus in HaCaT treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (B) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NF-κB translocated in the nucleus in HFs treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (C) HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05; ** p < 0.01). (D) HFs were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Translocation Assay, Immunofluorescence, Staining, Control, Negative Control, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: TRL4-based mechanism regulates NPM and miR-200c secretion in response to cytokine mix and miR-200c and NPM physically interact. HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with cytokine MIX (IL-17A, IL-22, TNF-α, and IFN-γ). (A) Media were collected and analyzed by ELISA assay for the presence of eNPM. eNPM increased levels by cytokine MIX were significantly decreased by TLR4i ( n = 5; * p < 0.05; ** p < 0.01). (B) qRT-PCR of miR-200c from the supernatant show that miR-200c increase elicited by cytokine MIX treatment is significantly reduced by TLR4i ( n = 6; ** p < 0.01; *** p < 0.001). (C) HaCaT were treated with cytokine MIX for 8 h then supernatants were collected and treated either with RNase or 200 μM NPM inhibitor (NPMi) or 2 μg/ml RNAse together with 200 μM NPMi for 15 min at 37°C ( n = 6, * p < 0.05; * * p < 0.01).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the myocardium of chronic heart failure ( CHF ) rats. ( A ) TLR 4 mRNA levels in infarct and remote myocardium of sham and CHF rats (n = 6/group). ( B ) Representative Western blot images and ( C ) quantification of TLR 4 proteins in infarct and remote myocardium of sham and CHF rats (n = 4/group). ( D ) Representative immunohistochemistry images of heart sections stained with TLR 4 (green) and CD 45 (red). The yellow box indicates the enlarged area shown on the right (data are means ± SD , * P < 0.05, ** P < 0.01 versus respective sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the surviving cardiomyocytes of chronic heart failure ( CHF ) rats. ( A ) Representative immunofluorescent images of TLR 4 in cardiomyocytes isolated from sham and CHF rats. ( B ) TLR 4 mRNA levels in cardiomyocytes isolated from sham and CHF rats. ( C ) Representative Western blot images and ( D ) quantification of TLR 4 proteins in cardiomyocytes isolated from sham and CHF rats (data are means ± SD , n = 6/group, ** P < 0.01 versus sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Isolation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4)‐sh RNA lentivirus reduced myocardial inflammation and improved heart function after myocardial infarction ( MI ). The rats received intra‐myocardial injection of normal saline ( NS ), control‐sh RNA lentivirus or TLR 4‐sh RNA lentivirus (1 × 10 9 TU /ml, 100 μl/heart) just after left anterior descending coronary artery ( LAD ) ligation or sham operation. All examinations were performed after 4 weeks of MI . ( A ) Expression of green fluorescent protein ( GFP ; green), the marker gene carried by TLR 4‐sh RNA lentivirus, in the myocardium. The nuclei were counter‐stained with Hoechst 33258 (blue). ( B ) Representative Western blot images and quantification of TLR 4 proteins in sham and chronic heart failure ( CHF ) myocardium. ( C ) tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 protein contents in infarct and remote myocardium. ( D ) Representative images of Masson's trichrome staining (upper panel) and quantification (lower panel) of post‐infarct failing hearts, showing that TLR 4‐sh RNA lentivirus reduced cardiac fibrosis. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with Masson's trichrome reagents. ( E ) Infarct size of post‐infarct failing hearts. ( F ) Fractional shortening (%) of the left ventricle (data are means ± SD , n = 4/group, a P < 0.05, A P < 0.01 versus respective sham‐ NS ; B P < 0.01 versus respective CHF ‐ NS ).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Injection, Saline, Control, Ligation, Expressing, Marker, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Enhanced binding activity of toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes to lipopolysaccharide ( LPS ) and heat shock protein 60 ( HSP 60). Isolated cardiomyocytes were cultured in a CO 2 incubator at 37°C for 24 hrs, then the binding assay was performed at 4°C for 30 min. To block TLR 4, cultured cardiomyocytes were incubated with TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) at 37°C for 15 min., and subsequently incubated with FITC ‐ LPS or OG ‐ HSP 60 at 4°C for 30 min. ( A ) Representative fluorescent images of isolated cardiomyocytes after the incubation with FITC ‐ LPS (green) or OG ‐ HSP 60 (green). ( B ) Binding curves of FITC ‐ LPS to cardiomyocytes. ( C ) Binding curves of OG ‐ HSP 60 to cardiomyocytes.

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Binding Assay, Activity Assay, Isolation, Cell Culture, Blocking Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased cytokine production mediated by toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes. Cultured cardiomocytes from sham and CHF rats were treated with lipopolysaccharide ( LPS ; 1 μg/ml) or heat shock protein 60 ( HSP 60; 1 μg/ml) for 6 hrs. TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) was added 15 min before LPS or HSP 60 treatment. ( A ) Tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 mRNA levels (n = 6/group). ( B ) The amount of TNF ‐α and IL ‐6 released into culture supernatant (n = 6/group). ( C ) Representative Western blot images and quantification of p65 in the nuclei of cardiomyocytes from three independent experiments (data are means ± SD , a P < 0.05, A P < 0.01 versus respective sham; b P < 0.05, B P < 0.01 versus sham‐blank; c P < 0.05, C P < 0.01 versus CHF ‐blank; d P < 0.05, D P < 0.01 versus respective LPS ; e P < 0.05, E P < 0.01 versus respective HSP60).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Cell Culture, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Primers for real‐time PCR

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

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. 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: International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience

Article Title: Ontogeny of white matter, toll-like receptor expression, and motor skills in the neonatal ferret

doi: 10.1016/j.ijdevneu.2018.05.006

Figure Lengend Snippet: TLR4 (A) and TLR3 (B) relative mRNA expression is present at low levels in the neonatal ferret brain, and significantly increases from P1 to P40. P12 ferrets exposed to 1 mg/kg LPS x 3 doses + hypoxia/hyperoxia have increased TLR4 immunopositive cells within meningeal vessels and expanding the meninges (C), relative to controls (saline followed by normoxia) (D). TLR4 positive cells are brown; hematoxylin counterstain; bars = 100 μm. Asterisks indicate p < 0.05.

Article Snippet: Immunohistochemistry Immunohistochemistry was performed on formalin fixed paraffin embedded sections using antibodies for TLR3 (monoclonal mouse anti ferret, 1:250 dilution, Clone 40C1285.6, LifeSpan BioSciences, LS-C545), TLR4 (polyclonal rabbit anti ferret, 1:2000 dilution, Novus Biological, NBP2-24538), MBP (monoclonal rat anti ferret, 1:500 dilution, Abcam, AB7349), Iba-1 (1:1500 dilution, WAKO Chemicals USA, catalog number 019-19741), and Olig2 (polyclonal rabbit anti ferret, 1:500 dilution, Millipore, Cat No. AB9610).

Techniques: Expressing, Saline