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: 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

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: 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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder

doi: 10.1073/pnas.2020858118

Figure Lengend Snippet: Flow cytometry analysis of PBMCs from HCs and iRBD patients. PBMCs in blood from iRBD patients and HCs were analyzed based on TLR2 and CD56 expression. NK cells were identified as TLR2−/CD56+ and divided into (A) CD56 dim mature NK cells and (B) CD56 bright precursor NK cells. TLR2 was used as a pan marker for Mo and DCs and subtyped based on CD14, CD16, and HLA-DR expression. Mo&DCs are shown with respect to the percentage expressing the following surface markers: (C) CD11b (D) CCR2, and (E) CD163; and their MFI of (F) TLR4 and (G) HLA-DR. (H) MFI of CCR2 on DCs. Percentage of the subpopulations in Mo&DCs population: (I) classical monocytes (CD14++/CD16−), (J) intermediate monocytes (CD14++/CD16+), (K) nonclassical monocytes (CD14low/CD16++), and (L) DCs (CD14low/CD16−). The MFI of HLA-DR shown for the Mo&DCs subpopulations: (M) classical Mo, (N) intermediate Mo, (O) nonclassical Mo, and (P) DCs. Lines show means. Mann–Whitney test for B and C, and unpaired t test for A and D–P. *P < 0.05, **P < 0.01, ***P < 0.001. Bonferroni correction (P value set to 0.0125) was applied for I–L and M–P.

Article Snippet: Cells were stained using: 3 μL mouse IgG2b K anti-human CD14-BV421 Mϕp9 (BD Horizon), 2.5 μL Mouse IgG2a K anti-human HLA-DR-BV650 L243 (BioLegend), 1.25 μL mouse IgG1 K anti-human CD11b-SuperBright780 ICRF44 (eBioscienceTM Thermo Fischer Scientific), 5 μL mouse IgG2a K anti-human CD192 (CCR2)-FITC K036C2 (BioLegend), 10 μL mouse IgG2a anti-human TLR4-PerCP 610015 (Novus, R&D systems), 1 µL Mouse IgG1 anti-human CD163-R-PE MAC2-158 (IQProducts), 2 μL Mouse IgG1 anti-human CD56-ECD N901 (Beckman Coulter), 2.5 μL Mouse IgG1 anti-human CD16-PC7 3G8 (Beckman Coulter), and 2 μL recombinant human IgG1 anti-TLR2 (CD282)-APC REA109 (MACS Miltenyi Biotec) with 50 μL Brilliant Stain Buffer (BD Horizon) in a total volume of 100 μL DPBS with 1% Albumin fraction V, from bovine serum (Merck).

Techniques: Flow Cytometry, Expressing, Marker, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder

doi: 10.1073/pnas.2020858118

Figure Lengend Snippet: Surface expression of monocytic markers and PET data or UPSIT score in iRBD patients. Each patients’ PBMC parameter of choice (x axis) was paired with its PET or UPSIT score (y axis), all points plotted (n = 15 in all, except A and B, n = 13 [two outliers by Grubb’s test]) and analyzed for correlation. (A–D) Graphs show individual patients paired values and regression lines with 95 confidence interval (discontinued) lines for the correlation between surface markers in monocytes and BPND of 11C-PK11195 in the SN and influx constant (Ki) of 18F-DOPA in putamen. Due to lateralization for each ligand, left (black) and right (blue) hemisphere PET data were correlated separately to the patients’ immune marker: (A) %CD163+ of live cells versus BPND of 11C- PK11195 in the SN. (B) %CD163+ of live cells versus Ki of 18F-DOPA in the putamen. (C) MFI of TLR4 on classical Mo versus BPND of 11C-PK11195 in the SN. MFI of TLR4 on (D) classical Mo and (E) nonclassical Mo versus Ki of 18F-DOPA in the putamen. (F) Graphs show individual patients paired values and regression lines with 95 confidence interval lines for the correlation of TLR2 MFI on Mo&DCs with UPSIT scores (n = 15). Pearson correlation analysis P and R values are shown. Associations were tested for covariates by multiple regression analysis (SI Appendix, Tables S1–S3), An asterisk (*) indicates significance remained after correction *P < 0.05, otherwise corrected P value is indicated in brackets (P).

Article Snippet: Cells were stained using: 3 μL mouse IgG2b K anti-human CD14-BV421 Mϕp9 (BD Horizon), 2.5 μL Mouse IgG2a K anti-human HLA-DR-BV650 L243 (BioLegend), 1.25 μL mouse IgG1 K anti-human CD11b-SuperBright780 ICRF44 (eBioscienceTM Thermo Fischer Scientific), 5 μL mouse IgG2a K anti-human CD192 (CCR2)-FITC K036C2 (BioLegend), 10 μL mouse IgG2a anti-human TLR4-PerCP 610015 (Novus, R&D systems), 1 µL Mouse IgG1 anti-human CD163-R-PE MAC2-158 (IQProducts), 2 μL Mouse IgG1 anti-human CD56-ECD N901 (Beckman Coulter), 2.5 μL Mouse IgG1 anti-human CD16-PC7 3G8 (Beckman Coulter), and 2 μL recombinant human IgG1 anti-TLR2 (CD282)-APC REA109 (MACS Miltenyi Biotec) with 50 μL Brilliant Stain Buffer (BD Horizon) in a total volume of 100 μL DPBS with 1% Albumin fraction V, from bovine serum (Merck).

Techniques: Expressing, Marker