Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of gene silencing on fHA-mediated IL-6 production in IVD cells . A ) siRNA-mediated knockdown of genes Toll like receptor ( TLR ) 2 , TLR4 , CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR ( n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C ) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( n = 4) ( B ) and lipopolysaccharide (LPS)- (25 ng/ml) ( n = 3). ( C ) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. ( D ) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.01.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of TLR2 inhibition on fHA-mediated IL-6 production in IVD cells . Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( A ) and hyaluronic acid fragment (fHA)-treated (20 μg/ml) ( B ) intervertebral disc (IVD) cells following antibody-mediated neutralization of Toll like receptor (TLR)2 activity ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.05 as compared to cells treated with non-specific IgG control antibody.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Inhibition, Neutralization, Activity Assay, Control

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Role of whole saliva in the efficacy of sublingual immunotherapy in seasonal allergic rhinitis.

doi: 10.1016/j.alit.2018.07.008

Figure Lengend Snippet: Fig. 4. Role of TLR2 and TLR4 signaling in whole saliva-induced IL-10 production by monocytes. (A) Effect of Pam3CSK4, Poly (I:C), LPS, and CpG on IL-10 production. (BeD) Effect of TL2.1 alone (B), PMB alone (C), and TL2.1 plus PMB (D) on IL-10 production. Bar represents the median. P-values were determined using the Dunn test (A; **P < 0.01) or the Wilcoxon signed-rank test (BeD).

Article Snippet: We purchased the following materials for the study: polymyxin B sulfate salt (PMB, SigmaeAldrich, St. Louis, MO), Pam3CSK4, Poly(I:C), LPS, CpG and mouse anti-human TLR2 mAb (TL2.1, Novus Biologicals, Littleton, CO), mouse IgG2a (R&D Systems, Minneapolis, MN); FITC-conjugated anti-human CD80 mAb (BD Biosciences, San Jose, CA), FITC-conjugated anti-human CD86 mAb (Agilent Technologies, Santa Clara, CA), PE-conjugated anti-human HLA-DR mAb (BD Biosciences), and FicollePaque (GE Healthcare BioSciences AB, Uppsala, Sweden).

Techniques:

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