Journal: Cells

Article Title: Exercise-Induced Irisin Decreases Inflammation and Improves NAFLD by Competitive Binding with MD2

doi: 10.3390/cells10123306

Figure Lengend Snippet: Exercise blocks MD2-TLR4 pathway activation in mouse livers. ( A ) MD2-TLR4 complex formation levels in mouse liver tissues detected by co-immunoprecipitation. ( B ) Protein levels of MAPK pathway and NF-κB pathway components, including p-ERK, p-JNK, p-p38, p-p65, and IκB-α. The corresponding unphosphorylated proteins and tubulin were used as the loading controls. ( C ) Relative mRNA levels of several pro-inflammatory markers Il6, Il1b, Tnf, Ccl2, Icam1, and Vcam1 in mouse liver tissues. The data are presented as the mean ± SEM, n = 6 per group. # p < 0.05 vs. NCD group; * p < 0.05 vs. HFD group.

Article Snippet: Recombinant irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA) and recombinant human MD2 (rhMD2) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Immunoprecipitation

Journal: Cells

Article Title: Exercise-Induced Irisin Decreases Inflammation and Improves NAFLD by Competitive Binding with MD2

doi: 10.3390/cells10123306

Figure Lengend Snippet: Irisin blocks NF-κB and MAPK pathways, and reduces inflammatory factors in AML12 cells. ( A , B ) AML12 cells were pretreated with recombinant irisin (50 or 100 ng/mL) for 30 min followed by exposure to 200 μM PA for 2 h. ( A ) MD2-TLR4 complex formation levels in AML12 cells detected by immunoprecipitation. ( B ) Protein levels of MAPK pathway and NF-κB pathway components, including p-ERK, p-JNK, p-p38, p-p65, and IκB-α. The corresponding unphosphorylated proteins and tubulin were used as loading controls. ( C ) AML12 cells were pretreated with recombinant irisin (50 or 100 ng/mL) for 30 min followed by exposure to 200 μM PA for 12 h. Relative mRNA levels of Il6, Il1b, Tnf, Ccl2, Icam1, and Vcam1 were detected. The data are presented as the mean ± SEM. # p < 0.05 vs. CON group; * p < 0.05 vs. PA group.

Article Snippet: Recombinant irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA) and recombinant human MD2 (rhMD2) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Recombinant, Immunoprecipitation

Journal: Cells

Article Title: Exercise-Induced Irisin Decreases Inflammation and Improves NAFLD by Competitive Binding with MD2

doi: 10.3390/cells10123306

Figure Lengend Snippet: Irisin competitively binds to MD2 but not TLR4. ( A , B ) Immunoprecipitation analysis of the binding ability of recombinant irisin to MD2 ( A ) or TLR4 ( B ) in liver lysates. ( C ) ELISA analysis in the binding ability of recombinant irisin to MD2 or TLR4 in liver lysates. ( D ) Immunoprecipitation analysis in the binding ability of recombinant irisin to rhMD2. ( E ) ELISA analysis of the binding ability of recombinant irisin to rhMD2. ( F ) Surface plasmon resonance analysis between irisin with rhMD2. ( G ) ELISA analysis of the effect of recombinant irisin (0.1, 0.2, and 0.5 μg/mL) on the basal binding level of MD2-TLR4. ( H , I ) ELISA analysis of the competitive MD2 binding ability of recombinant irisin (0.1, 0.2, and 0.5 μg/mL) to PA or LPS. ( J ) Molecular docking of the dimeric irisin-MD2 complex. ( K ) ELISA analysis of irisin-MD2 binding levels in mouse liver tissue ( n = 6 per group). The data are presented as the mean ± SEM. # p < 0.05 vs. CON or NCD group; * p < 0.05 vs. rhMD2 or HFD group.

Article Snippet: Recombinant irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA) and recombinant human MD2 (rhMD2) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunoprecipitation, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: Frontiers in Medicine

Article Title: Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens

doi: 10.3389/fmed.2023.1105538

Figure Lengend Snippet: The role of TLR4 in HDM-induced moDC activation. (A) A HEK-293/TLR4 reporter cell line and the parental HEK-293 control cell line were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype control antibody. Subsequently, cultures were exposed for 24 h to 10 ng/mL LPS or 10 μg/mL HDM extract, after which the culture supernatants were collected and screened for by ELISA for IL-8, that is expressed upon TLR4 engagement. The measured data are shown relative to levels obtained from LPS stimulation. The data are presented as the mean ± SD ( n = 3–4 donors). (B) MoDCs were incubated for 1 h at 37°C with either TLR4-blocking antibody or the respective isotype controls and subsequently exposed for 24 h to 10 ng/mL LPS or 50 μg/mL HDM extract. Culture supernatants were collected and screened for the cytokines IL-6, IL-10, and IL-12p70 by ELISA. The measured data are shown relative to levels obtained from untreated (control group) cells stimulated with HDM extract. The data are presented as the mean ± SD ( n = 5 donors). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (RM-ANOVA analysis with Tukey’s multiple comparison test).

Article Snippet: After seeding the cultures into 96-well flat-bottom culture plates (Corning) and letting them adhere for 24 h, the cells were treated for 1 h with 30 μg/mL of either anti-human TLR4/MD-2 (clone 7E3, Hycult Biotech) or mouse IgG1 control antibody (Invivogen) prior to stimulation with 10 μg/mL HDM extract or LPS as a positive control.

Techniques: Activation Assay, Control, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Basal TLR4 expression by (A) primary adult baboon lung DCs and (B) KG-1-derived DCs under steady-state condition. Cell-surface expression of TLR4 was detected by flow cytometry after staining the cells with TLR4-specific antibody. The percent number and MFI values of cells stained with TLR4-specific antibody (TLR4) are compared with isotype control antibody-stained cells (I) in M1 region. (C): Western blot showing undetectable expression of TLR4 in 5 μg cell lysate protein of KG-1-derived DCs (KG1-DC). An equal amount of cell lysate protein of HEK293 cells stably transfected with TLR4 (HEK-TLR4) served as positive control.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Staining, Western Blot, Stable Transfection, Transfection, Positive Control

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Localization of exogenously-added recombinant TLR4-MD2 protein by confocal microscopy and flow-cytometry. Confocal microscopic images of KG-1-derived DCs pulsed with Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein for (A) 1h and (B) 4h. Vybrant DiO (green) dye stains the cytoplasm, and Hoechst 33342 (blue) dye stains the nucleus of the cell. The images were acquired using 63 X objective. (C) Flow-cytometric charts of KG-1-derived DCs pulsed with Alexa-fluor 495-conjugated recombinant TLR4-MD2 protein after 1h (dark line) and 4h (dotted line). The histogram chart of non-pulsed cells (negative control) is shown under the black area. Cells were gated in M region. Percent number of cells (and MFI values) positive for fluorescence are shown within the chart. Results are representative of two experiments.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Recombinant, Confocal Microscopy, Flow Cytometry, Derivative Assay, Negative Control, Fluorescence

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A, recombinant TLR4-MD2 protein and MD2 protein on phagocytic function of KG-1-derived DCs. (A): Confocal microscopic images of KG-1-derived DCs incubated with pHrodo-labeled E. coli bioparticles for 3h. Phagocytosed bioparticles fluoresce red. Cells without phagocytosed particles and extracellular bacteria do not fluoresce. Enlarged images of a cell (shown as circle) are also shown in the figure, at different z-stack slices. (B): The extracellular bacteria that are either settled at the bottom or lie towards the top do not emit any fluorescence. These images confirm that fluorescence is of phagocytosed bioparticles. Next, KG-1-derived DCs were incubated with (C): purified baboon lung SP-A (0.2 and 2 μM), (D): recombinant TLR4-MD2 protein (0.06–0.6 μM) and functional-grade anti-human TLR4 antibody (HTA125 clone, Imgenex, CA; control reaction), (E): recombinant MD2 protein (0.02–0.2 μM), and (F): purified baboon lung SP-A (2 μM) and TLR4-MD2 protein (0.6 μM), for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically at 550 nm excitation and 600 nm emission wavelengths. Results are mean (SEM) of three different experiments. * p<0.05 or ns: not significant as compared to basal phagocytosis.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Derivative Assay, Incubation, Labeling, Bacteria, Fluorescence, Functional Assay

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of simultaneous addition of purified SP-A and recombinant TLR4-MD2 protein on phagocytic function of primary (A) adult baboon lung DCs and (B) fetal baboon lung DC-precursor cells. The DCs were incubated with respective proteins for an hour prior to addition of pHrodo-labeled E. coli bioparticles. The phagocytic uptake of E. coli bioparticles was measured spectrofluorometrically. * p<0.05, ns: not significant or otherwise indicated. Results are mean (SEM) of three different experiments performed at different times.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling

Journal: Cellular immunology

Article Title: Surfactant protein-A and Toll-like receptor-4 Modulate Immune Functions of Preterm Baboon Lung Dendritic Cell Precursor Cells

doi: 10.1016/j.cellimm.2011.02.009

Figure Lengend Snippet: Effect of purified native SP-A and recombinant TLR4-MD2 proteins on TNF-α secretion by DCs against E. coli. (A) Primary adult baboon lung DCs or (B) fetal baboon lung DC-precursor cells were incubated with effector molecules for an hour prior to addition of pHrodo-labeled E. coli bioparticles. After 3h incubation at 37°C in 5% CO2 incubator, the cell-free supernatants were collected and subjected to ELISA for measurement of TNF-α. The results are representative of two experiments performed separately in triplicate. * p<0.05, ** p<0.001, ns: not significant.

Article Snippet: Cellular distribution of exogenously added TLR4-MD2 protein Since KG-1-derived-DCs, adult baboon lung DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein, the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN).

Techniques: Purification, Recombinant, Incubation, Labeling, Enzyme-linked Immunosorbent Assay