Journal: BMB Reports

Article Title: Diesel exhaust particles disrupt blood–retina barrier integrity via TLR2 and TLR4 activation

doi: 10.5483/BMBRep.2025-0013

Figure Lengend Snippet: Expression levels of TLR2/TLR4 and TNF-α/IL-1β mRNA in U937 macrophages exposed to DEPs with TLR2 (C29) or TLR4 (TAK242) inhibitors. (A) Schematic representation of the experimental procedure. (B, C) TLR2 and TLR4 expression levels. Compared with those in the control, TLR2 (B) and TLR4 (C) levels in U937 macrophages were significantly elevated following DEP exposure. TLR2 and TLR4 expression levels were significantly restored following treatment with C29 and TAK242 before DEP exposure, respectively. (D, E) TNF-α and IL-1β mRNA expression. TNF-α (D) and IL-1β (E) mRNA levels were significantly lower in U937 macrophages treated with C29 or TAK242 before DEP exposure than in cells that received DEP exposure alone without TLR inhibitors. *P < 0.05 vs. control, † P < 0.05 vs. DEP.

Article Snippet: The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBSTw) for 1 h and were then incubated with the following primary antibodies: anti-mouse TLR2 (1:100, Santa Cruz Biotechnology, TX, USA), anti-mouse TLR4 (1:100, Santa Cruz Biotechnology), anti-mouse claudin-5 (1:500, Invitrogen), anti-rabbit ZO-1 (1:1000, Invitrogen) and anti-mouse actin (JLA20, 1:10, DSHB, IA, USA).

Techniques: Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression.

doi: 10.4049/jimmunol.174.9.5713

Figure Lengend Snippet: FIGURE 1. TLR2 expression in wild-type BALB/c mouse lung tissue. A, TLR2 mRNA expression was ex- amined by quantitative real-time RT-PCR and expressed as relative level using the comparative cycle of thresh- old method. Data expressed as medians (25–75% range). n 6–8 mice per group. B, TLR2 protein West- ern blot analysis. Raw 264.7 mouse macrophages in- fected with Mp at 50 CFU/cell serve as a positive con- trol. -Actin immunoblot onto the TLR2 Ab-stripped membrane was performed to confirm equal protein load- ing. C, TLR2 protein immunofluorescent staining. The green and blue colors indicate TLR2 and cell nuclei, respectively. On day 1, saline treated BALB/c mouse lung showing very weak TLR2 staining (a), while on day 1, Mp infected BALB/c mouse lung (b) showing strong TLR2 staining on bronchial epithelial cells (white arrows) and alveolar macrophages (red arrows). The specificity of TLR Ab staining was confirmed by no fluorescent staining in infected BALB/c mouse lung (day 1) incubated with an irrelevant goat IgG (c). On day 1 after Mp infection, wild-type (d), but not TLR2/ C57BL/6 mice (e), demonstrated airway epi- thelial TLR2 staining (white arrow). Original magnifi- cation, 200

Article Snippet: To localize TLR2 on the lung tissue, immunofluorescent staining of TLR2 protein was performed on deparaffinized lung tissue sections using a rabbit anti-mouse TLR2 Ab (1 g/ml; Santa Cruz Biotechnology) or an irrelevant rabbit IgG (1 g/ml) as a negative control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Saline, Infection, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression.

doi: 10.4049/jimmunol.174.9.5713

Figure Lengend Snippet: FIGURE 2. A, Mp infection increases recruitment of adaptor protein MyD88 to TLR2. Lung tissue lysates (400 g) from BALB/c mice after 24 h of Mp infection (Mp ) or saline inoculation (Mp ) were immu- noprecipitated with a goat anti-mouse TLR2 Ab or goat IgG control Ab. Coprecipitating MyD88 was detected using Western blot analysis. B, NF-B p65 activation levels were measured in BALB/c mouse lung tissue nuclear extracts by an ELISA-based NF-B p65 assay kit. OD value at 450 nm was used to represent the NF-B p65 activation levels. n 6–8 mice per group.

Article Snippet: To localize TLR2 on the lung tissue, immunofluorescent staining of TLR2 protein was performed on deparaffinized lung tissue sections using a rabbit anti-mouse TLR2 Ab (1 g/ml; Santa Cruz Biotechnology) or an irrelevant rabbit IgG (1 g/ml) as a negative control.

Techniques: Infection, Saline, Control, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression.

doi: 10.4049/jimmunol.174.9.5713

Figure Lengend Snippet: FIGURE 4. An anti-mouse TLR2-neutralizing Ab (TLR2Ab) signifi- cantly reduced Mp-induced lung tissue mucin MUC5AC mRNA expres- sion (A) and bronchoalveolar lavage fluid IL-6 protein levels (B) on day 3 after infection. Data are expressed as medians (25–75% range). n 5–6 mice per group. BP of TLR2-neutralizing Ab. , p 0.05, TLR2Ab Mp group vs IgG Mp and TLR2Ab BP Mp groups in A. , p 0.05, TLR2Ab Mp group vs IgG Mp group in B.

Article Snippet: To localize TLR2 on the lung tissue, immunofluorescent staining of TLR2 protein was performed on deparaffinized lung tissue sections using a rabbit anti-mouse TLR2 Ab (1 g/ml; Santa Cruz Biotechnology) or an irrelevant rabbit IgG (1 g/ml) as a negative control.

Techniques: Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression.

doi: 10.4049/jimmunol.174.9.5713

Figure Lengend Snippet: FIGURE 5. In wild-type C57BL/6 mice, Mp infection increased NF-B p65 activity and airway mucin protein levels on day 3 postinfection. How- ever, in TLR2/ mice, Mp infection failed to increase NF-B p65 activity and airway mucin protein levels on day 3 postinfection. n 5–6 mice per group. Sa, Saline.

Article Snippet: To localize TLR2 on the lung tissue, immunofluorescent staining of TLR2 protein was performed on deparaffinized lung tissue sections using a rabbit anti-mouse TLR2 Ab (1 g/ml; Santa Cruz Biotechnology) or an irrelevant rabbit IgG (1 g/ml) as a negative control.

Techniques: Infection, Activity Assay, Saline

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression.

doi: 10.4049/jimmunol.174.9.5713

Figure Lengend Snippet: FIGURE 7. Transient transfection of a human TLR2 dominant-negative mutant significantly reduced the NF-B luciferease activity (A) and mucin MUC5AC protein expression (B) by Mp-infected A549 lung epithelial cells. The results shown are from three different experiments.

Article Snippet: To localize TLR2 on the lung tissue, immunofluorescent staining of TLR2 protein was performed on deparaffinized lung tissue sections using a rabbit anti-mouse TLR2 Ab (1 g/ml; Santa Cruz Biotechnology) or an irrelevant rabbit IgG (1 g/ml) as a negative control.

Techniques: Transfection, Dominant Negative Mutation, Activity Assay, Expressing, Infection

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: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Primer names for PCR reactions.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Identified SNP variants within the coding sequence of bovine tlr2 in HF and BS breeds animals.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Residue

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Genotype frequency of Bos taurus and Bos indicus cattle breeds for TLR2 selected candidate SNP rs68343167 (H326Q).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Location of identified SNPs. Gene model for TLR2 from assembly UMD 3.1.1 with annotated SNPs. Missense SNPs are located on the first track below the gene model, with synonymous SNPS on the second track. Reading from left to right, exons and coding sequence (CDS; thick line) are shown. All mutations occur in the second exon, which codes for ligand binding side.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Ligand Binding Assay

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: TLR2-Ligand-Dependent NF-κB Activity and CXCL8 Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harboring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDuo-mcs. Cells were stimulated with either FSL-1 at 100 ng mL -1 (A, C) , Pam3CSK4 at 1 mg mL -1 (B, D) for 24 hrs. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm (A, B) . OD values were normalised against values for PMA stimulation at 200 ng mL -1 . Additionally, supernatants were assessed for CXCL8 concentration by ELISA (C, D) . Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: M. bovis BCG and M. tuberculosis -Dependent NF-κB Activity Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harbouring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDUO-mcs. Cells were stimulated with either live M. bovis BCG (MOI of 10) (A) or M. tuberculosis H37Rv (MOI of 5) (B) for 24 h. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm. OD values were normalised against values for PMA stimulation at 200 ng/ml. Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (**=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: CXCL8 production by bovine MØ in response to FSL-1, M. bovis BCG or MTB Ligand Stimulation. Bovine MØ of BS (heterozygous for TLR2 H326Q SNP) and HF MØ (homozygous for the wild-type TLR2 sequence) were stimulated with either FSL-1 at 100 ng mL -1 (A) , 19 kDa lipoprotein antigen (represents Rv3763 or Mb3789) (EMC microcollections, Germany) or infected with M. bovis BCG at MOI = 5 (B) for 24 hr. CXCL8 concentration in cell culture supernatant was assessed by ELISA. A total of n=8 animals per breed were stimulated with FSL-1 and n=6 animals per breed were infected with M. bovis BCG. Error bars represent standard deviation from the mean. Data was analysed with two-way ANOVA and Tukey’s post hoc comparison in GraphPad Prism V8 (GraphPad Inc., USA) (*=p<0.05, ***=p<0.001).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Infection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 2. Real-time RT-PCR analysis of TLR2 and TLR4 mRNA expression in colon extracts in TNBS-induced colitis and their down-regulation after VIP treatment. Colitis was induced by rectal instillation of TNBS in 50% ethanol, and 1 nmol VIP was injected i.p. on alternate days. Controls received PBS or 50% ethanol. mRNA was extracted from colons at days 1, 3, 5, and 7. TLR2 (A) and TLR4 (B) mRNA expressions were measured by real-time RT-PCR, values were rectified by mRNA expression of -actin for each sample, and arbitrary units were calculated with respect to control expression, being 1 (2– Ct). Each result is the mean

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Quantitative RT-PCR, Expressing, Injection, Control

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 3. Western blot analysis of TLR2 and TLR4 in colon extracts in TNBS-induced colitis down-regulation after VIP treatment. Colon extracts from control and ethanol-, TNBS-, and TNBS/VIP-treated animals were prepared and electrophoresed as described in Materials and Methods. Specific bands for TLR2 (A) or TLR4 (B) were quantified by densitometry and rectified with respect to -actin protein levels in each case. Each result is the mean SEM of at least four mice. , P 0.05; , P 0.01; , P 0.001, versus control animals, and *, P 0.05; **, P 0.01; ***, P 0.001, versus TNBS-treated animals. A representative experiment of three others is shown in the upper panels. O.D. optical density.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Western Blot, Control

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 4. Immunohistochemical analysis of TLR2 and -4 at day 5 in gut sections. Immunohistochemical study was performed to study TLR2 and TLR4 expressions in colon mucosa as described in Materials and Methods. Mono- nuclear cells positive to antibody against TLR2 within the lamina propria in control (untreated animals; A), TNBS (B), and TNBS VIP-treated animals (C; original 400). Gut crypts showing positive staining for TLR2 and TLR4 in control (untreated animals; D and G), TNBS (E and H), and TNBS VIP-treated animals (F and I). Original magnification, 200. Arrows indicate the positive staining. VIP reduced the TNBS-induced expression of TLR2 and TLR4 in epithelial cells from intestinal crypts and mononuclear cells.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Immunohistochemical staining, Control, Staining, Expressing

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 5. Flow cytometry analysis of TLR2 expres- sion in TNBS-induced colitis. Effect of VIP treat- ment. Colitis was induced as described in Materials and Methods, and mice were treated i.p. with 1 nmol VIP on alternate days over 7 days. Cell sus- pensions from mesenteric lymph node extracted from control and treated animals were collected at different time-points and processed for immunoflu- orescence staining by using a PE-conjugated TLR2 mAb. (A) Aliquots of control lymph node cells were incubated with isotype control antibodies or anti- bodies to TLR2 (IgG2b), as described. (B) Total cells subjected to single immunofluorescence stain- ing were analyzed by FACSCalibur. (C and D) Double immunofluorescence staining for TLR2 and a subpopulation marker (C, FITC-conjugated CD11c; D, FITC-conjugated CD11b). (E) Lympho- cytes were identified by a FSC versus SSC gate and subjected to TLR2 immunofluorescence staining (F). Each result is the mean SEM of two separated experiments (three mice/group/experiment). *, P 0.05; **, P 0.01; ***, P 0.001 (ANOVA), with respect to TNBS-treated mice. , P 0.01; , P 0.001 (ANOVA), with respect to con- trol animals.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Flow Cytometry, Control, Staining, Incubation, Marker

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 7. Representative example at day 5 of the TLR2 and -4 expressions in CD11c and CD11b cells and lymphocytes after TNBS-induced colitis and VIP effect. Flow cytometry analysis of CD11c and CD11b cells and lymphocytes from mesenteric lymph nodes of control and treated animals. Cell suspensions were processed for double immunofluorescence staining for TLR2 and TLR4 and a subpopulation marker (CD11c and CD11b). Lymphocytes were identified by a FSC versus SSC gate and subjected to TLR2 and TLR4 immunofluorescence staining. Cell suspensions were incubated with isotype control antibodies or antibodies to TLR2 (IgG2b) or TLR4 (IgG2a), as described. The values shown in right quadrants and histograms indicate the percentage of CD11c/CD11b or lymphocytes and TLR2/4-positive cells. The results presented are representative of two independent experiments using three mice per group.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Flow Cytometry, Control, Staining, Marker, Incubation

Journal: Journal of leukocyte biology

Article Title: Time-course expression of Toll-like receptors 2 and 4 in inflammatory bowel disease and homeostatic effect of VIP.

doi: 10.1189/jlb.1004564

Figure Lengend Snippet: Fig. 9. Hypothesis planned to explain the two possible VIP modes of action. Based on our observations, we hypothesized that TLR2 and -4 modulations, induced after VIP treatment, may be explained through two possible ways that are not mutually exclusive. The first is an indirect through the reduction of TLR2 and -4, caused by the VIP-mediated decrease of inflammatory mediators such as IL-1 and IFN-, which synergize with bacterial products contributing to the amplification of TLR (blue arrows). The second possible mechanism, which remains to be elucidated in this model, is a primary (black arrows) through the VIP-mediated decrease of NF- B, which would cause a down-regulation of TLR expression. IRAK, IL-1 receptor-activated kinase; TRAF6, TNF receptor-associated factor 6; IKK, I B kinase.

Article Snippet: Membranes were blocked for 2 h in Trisbuffered saline/Tween 20 (TBST; 2 mM Tris-HCl, pH 7.6, 13.7 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and then incubated at 4°C overnight with goat anti-mouse TLR2 or TLR4 polyclonal antibodies (1:250; Santa Cruz Biotechnology, CA) in TBST containing 3% notfat dry milk.

Techniques: Expressing

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: Mycobacteria increase alveolar TLR expression. TLR2 and TLR4 expression on alveolar epithelial cells. (A) Alveolar epithelial cells were infected with mycobacteria for 3 d and stained with monoclonal anti-TLR2 or anti-TLR4 Ab and expression was analyzed with flow cytometry (A,B) and confocal microscopy (C). Unstimulated alveolar epithelial cells have low basal level expression of both receptors, with TLR4 more abundant than the TLR2. The mean fluorescence intensity (MFI) was calculated as an increase from negative control cells. Mycobacterial infection significantly increased epithelial TLR2 expression as detected by confocal microscopy and quantified by flow cytometry, but TLR4 expression was less affected. Confocal images showing alveolar epithelial TLR2 or TLR4 expression after 3 d of mycobacterial infection (green). Cellular DNA was counter-stained with propidium iodide (red). Original magnification x 300. (D) Alveolar epithelial TLR2 and TLR4 expression upon mycobacterial infection for up to 5 d was analyzed by Western blot. Epithelial cells express continuous TLR2 that was further increased in response to mycobacterial infection, while TLR4 expression was not affected. Data are presented as mean ± SEM of four separate experiments; ** P < 0.01, *** P < 0.001, TLR2 expression compared to TLR4 expression.

Article Snippet: For the blocking experiments, monoclonal mouse anti-human TLR2 and/or monoclonal mouse anti-human TLR4 Abs (R&D Systems, Copenhagen, Denmark) 10 μg/ml were added to the Transwell system or to the individual cell groups 30 min before the addition of BCG.

Techniques: Expressing, Infection, Staining, Flow Cytometry, Confocal Microscopy, Fluorescence, Negative Control, Western Blot

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: TLR2 is important for neutrophil diapedesis. The impact of TLR2 and TLR4 on neutrophil diapedesis was investigated with the Transwell model. Alveolar epithelial cells were infected for 3 d with mycobacteria. After infection, epithelial cells were combined with endothelial cells and human neutrophils in the Transwell model and neutrophil diapedesis was recorded in the lower compartment after 3 h. Blocking with anti-TLR2 Abs or with both TLR2 and TLR4 Abs induced a significant decrease of mycobacteria-induced neutrophil diapedesis. Blocking of TLR4 did not have any impact. Data are presented as mean ± SEM of four separate experiments; ** P < 0.01, *** P < 0.001, compared with infected control.

Article Snippet: For the blocking experiments, monoclonal mouse anti-human TLR2 and/or monoclonal mouse anti-human TLR4 Abs (R&D Systems, Copenhagen, Denmark) 10 μg/ml were added to the Transwell system or to the individual cell groups 30 min before the addition of BCG.

Techniques: Infection, Blocking Assay, Control

Journal: Innate Immunity

Article Title: Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

doi: 10.1177/1753425911426591

Figure Lengend Snippet: TLR2 control cytokine secretion. Influence of TLR2 and TLR4 on mycobacteria induced cytokine secretion from neutrophils, monocytes and alveolar epithelial cells. The cells were treated with Abs against TLR2 and/or TLR4 before infection with live BCG. Secretion of CXCL8, IL-6, CCL2 and TNF-α was measured after 3 h for leukocytes and after 3 d for epithelial cells. Blocking of TLR2 significantly reduced the cytokine secretion of all the infected cells, while blocking of TLR4 had the highest inhibitory function on epithelial and monocyte cytokine secretion. Blocking of both receptors significantly reduced cytokine secretion of all the infected cells. Data are presented as mean ± SEM of four separate experiments; * P < 0.05, ** P < 0.01, *** P < 0.001, compared with infected control.

Article Snippet: For the blocking experiments, monoclonal mouse anti-human TLR2 and/or monoclonal mouse anti-human TLR4 Abs (R&D Systems, Copenhagen, Denmark) 10 μg/ml were added to the Transwell system or to the individual cell groups 30 min before the addition of BCG.

Techniques: Control, Infection, Blocking Assay