Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: Microscale thermophoresis showing biophysical analysis of binding of human rMBL with human rTLR4/MD2. MST is based on the detection of a temperature-induced change in fluorescence of rTLR4/MD2 (target) as a function of the concentration of a non-fluorescent ligand (rMBL). By titrating MBL into the labeled TLR4 the K d (dissociation constant) was 9.07 × E −07 indicating strong binding.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Microscale Thermophoresis, Binding Assay, Fluorescence, Concentration Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: Effect of human rMBL or human rTLR4 on FD expression on differentiated 3T3-L1 cells at 48 h. (A) rMBL increased FD expression in a dose-dependent manner. (B) rMBL also effected the TLR4 expression. (C) LPS also increased FD expression dose-dependent manner. (D) LPS also decreased TLR4 expression with increasing doses. Data are shown as Mean ± SEM of three replicative experiments. * p < 0.05 considered significant.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: A hypothetical model in mouse showing how MBL or conjugates of MBL-MASP-1 or MBL-MASP-2 can regulate the transcription of FD to regulate the activation of the AP via TLR4 receptors. Circulating MBL alone or MBL-MASP-1 or MBL-MASP-2 complexes can directly interact with disguised TLR4 on adipocytes to activate the complement system via enhancing the expression of FD to regulate the AP pathway. (A) MBL or MBL-MASP-1 or MBL-MASP-2 conjugates under normal physiological conditions can bind to the TLR4 and regulate the expression of FD but MASP-2 might be dominant. (B) In contrast, under inflammatory conditions, MBL or MBL-MASP-1 or MBL-MASP-2 conjugates might be displaced by the LPS and modulate the transcription of FD through TLR4 receptors.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Activation Assay, Expressing

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Primary peritoneal macrophages from uPAR−/− mice were stimulated with suPAR with or without LPS for 15 min. Then, cells were fixed and stained for TLR4 (Alexa 488, green) and uPAR (Alexa 647, red). DAPI was used as nuclear stain. Scale bar 10 μm. B. Primary WT and uPAR−/− macrophages were stimulated with 100 ng/ml LPS and1μg/ml suPAR for 3 hrs C. Raw 264.7 cells were stimulated with 100 ng/ml LPS, fixed and stained as in A. Scale bar 12.5 μm. D. Raw 264.7 were stimulated with 1μg/ml biotin-LPS for 30 min, then cell lysis was performed. Protein complexes were precipitated using Streptavidin magnetic beads and analyzed by western blotting using anti-murine-uPAR antibody.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Staining, Lysis, Magnetic Beads, Western Blot

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Biotin-LPS binding was assessed in SiCo and uPARsi HK-2 cells as described. B. Duolink proximity ligation assay to assess uPAR/TLR4 and uPAR/CD36 interaction was performed on HK-2 cells stimulated with LPS for 15 min as described in Methods. C. Duolink images were quantified using Particles analysis tool of ImageJ. D. HK-2 cells were stimulated with LPS for 3 hrs after cell pre-treatment with CD36 inhibitor SSO. Expression of IL-6 and IL-8 was assessed by TaqMan RT-PCR.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Peritoneum of sham and LPS-injected WT mice was fixed and stained for uPAR and TLR4. DAPI used as nuclear stain­ scale bar 100μm. B. Expression of TNFα, MCP-1, IL-6 and IL-1 0 was assessed in mouse blood plasma before and 20 h after CLP surgery using Cytometric Beads Array. C: IL-6/IL-10 ratio in CLP mice 20 hrs after surgery.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Injection, Staining, Expressing, Clinical Proteomics

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: TLR4 mediates IL-8 expression in response to FnIII-1c and LPS in dermal fibroblasts. Monolayers of human dermal fibroblasts in 10% FBS/DMEM were treated for 24 h with ( A ) FnIII-1c or FnIII-13 (1-20 µg/mL), ( B ) LPS (1-100 ng/mL), ( C ) LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of the designated amounts of blocking antibody to TLR4 or TLR2. IgG served as control. ( D ) TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the TLR4 inhibitor, TAK-242. The wells without antibodies ( C ) or inhibitors ( D ) were set as 100%. IL-8 concentration in conditioned medium was determined by ELISA. The data represent the mean ± S.E. of triplicate assays from three separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Blocking Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: FnIII-1c-induced IL-8 expression requires membrane CD14. HEK cells expressing either TLR4/MD2 or TLR4/MD2/CD14 were incubated for 24 hours with the designated concentrations of LPS ( A , B ) or FnIII-1c ( C , D ) in either 10% FBS/DMEM ( A , C ) or 0.1% BSA/DMEM ( B , D ). ( E ) HEK-293 cells expressing TLR4-MD2 were treated with 1 µg/mL LPS or 20 µM FnIII-1c in 0.1% BSA/DMEM in the presence of the indicated concentration of exogenous soluble CD14 for 24 h. IL-8 concentration in the conditioned medium was measured by ELISA. The data represent the mean ± S.E. of triplicate assays from two ( A – D ) or three ( E ) separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Membrane, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay