Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Experimental periodontitis induced by oral challenge with P. gingivalis . Groups of mice were administered P. gingivalis in CMC vs. CMC alone by repeated oral gavage. Six weeks later, the maxillae were harvested and alveolar bone volume was measured by μCT from the cemento-enamel junction to a reference line. The residual bone volume of P. gingivalis infected mice was compared to vehicle-treated mice ( n = 8–10 per group). (A) WT, Tlr2 −/− , Myd88 −/− , and Tlr2/Myd88 double knock-out mice (DKO) infected with P. gingivalis ATCC 381. (B) An independent experiment examined bone loss in WT vs. Myd88 −/− mice infected with P. gingivalis ATCC 381 vs. P. gingivalis ATCC 53977. Ns, non-significant. ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Infection, Knock-Out

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Detection of P. gingivalis in mouse tissue following the third oral challenge.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: IFN-γ priming enables TLR2-dependent, MYD88-independent signaling in macrophages in response to P. gingivalis challenge. (A) Naïve Myd88 −/− BMM vs. BMM primed with IFN-γ (100 ng/ml) for 2 h were challenged with P. gingivalis (MOI 100) vs. Pam3CSK4 (10 μg/ml). In (B) Myd88 −/− BMM primed with IFN-γ were challenged with increasing MOI of P. gingivalis . (A,B) Supernatants were collected after overnight stimulation and tested for TNF by ELISA. ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: Kinase involvement in the Myd88 −/− response to P. gingivalis . (A) Ly6C+ BM neutrophils or (B–D) BMM were prepared from Myd88 −/− mice and primed with IFN-γ (100 ng/ml for 2 h). (A,B) Inhibitors for PI3K (LY 2940002 100 μM), p38 MAPK (SB 202190 50 μM), mTORC1 (RaPamycin 60 nM) and RAC1 inhibitor (NSC 23766, 50 μM) were added 30 min before challenge with P. gingivalis (MOI 100). DMSO was used as a control at the highest concentration used in the inhibitor wells. Supernatants were collected after overnight stimulation and the percent inhibition of TNF production is shown. (C) Myd88 −/− BMM were similarly primed and Ly294 was added at increasing concentrations 30 min prior to challenge with P. gingivalis . (D) Myd88 −/− BMM were primed with IFN-γ and antibodies (20 μg/ml anti-TLR2 or TLR4 vs. isotype control, I.C.) or LY294 were added prior to challenge with P. gingivalis . * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Concentration Assay, Inhibition

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K plays a non-redundant role in the murine and human macrophage response to P. gingivalis . WT murine BMM (A) , RAW264.7 macrophages (B) , and PMA-differentiated human THP-1 cells (C) were primed with IFN-γ. TLR2 and TLR4 were inhibited with blocking antibodies vs. isotype control (I.C.) for 1 h and PI3K was blocked with LY294 prior to challenge with P. gingivalis (MOI 10). Supernatants were collected after overnight incubation and TNF was measured by ELISA. Background (BG) represents IFN-γ primed cells not challenged with P. gingivalis . Cells challenged with P. gingivalis without any blocker are referred to in the graphs as (–). Representative graphs of >3 repeats are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K signaling suppresses phagocytosis and enhances intracellular survival. (A) RAW264.7 or (B) PMA-differentiated THP-1 cells were treated with blocking antibodies or PI3K inhibitor and then challenged with FITC-labeled P. gingivalis at MOI 10 for 1 h. Cells were then washed, extracellular fluorescence was quenched with trypan blue, and phagocytosis was determined using a fluorescence plate reader (RFU, relative fluorescence units). (C,D) RAW 264.7 cells were treated with TLR blocking antibodies or the PI3K inhibitor prior to challenge with P. gingivalis at MOI 10 for 1 h. Cells were then washed and extracellular bacteria were killed by incubating the cells with Metronidazole and Gentamycin for 1 h. Cells were allowed to recover in fresh media for an additional hour after which they were lysed by DDW for 20 min and lysates were plated on blood agar plates in serial dilution. CFU were enumerated after 7 days of anaerobic growth. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Labeling, Fluorescence, Serial Dilution

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

doi: 10.3389/fcimb.2017.00359

Figure Lengend Snippet: TLR2-PI3K signaling enhances intracellular survival by blocking phago-lysosomal maturation. RAW 264.7 cells were seeded at 3 × 104 cells/ well in Ibidi 8 well m-slides. The cells were untreated (A) or treated with anti-TLR2 (B) , anti-TLR4 (C) , or with the PI3K inhibitor LY 294002 (D) for an hour. Cells were then infected with FITC-labeled P. gingivalis at MOI 10 for 1 h. LysoTracker red was added at 50 nM for the last 10 min of infection. Cells were washed and fixed with 2% formaldehyde and mounted with mounting media. Images were captured using a NIKON confocal microscope at 60X magnification. Yellow color indicates co-localization of P. gingivalis (green) with lysosomes (red). In each field (A–D) the cell in the box is further magnified and shown in the upper right corner. (E) The percent of co-localization was determined by counting cells that demonstrate co-localization as a percentage of all FITC positive cells. (F) Schematic representation of the pathway used by P. gingivalis to evade bactericidal activity without preventing inflammation. *** P ≤ 0.005.

Article Snippet: T2.5 monoclonal antibody (mAb) against mouse and human TLR2 was from Hycult Biotech (Uden, Netherlands), mAb 1A6 was a gift from Greg Elson (NovImmune, Geneva, Switzerland), and isotype control mAbs were from BioLegend (San Diego, USA).

Techniques: Blocking Assay, Infection, Labeling, Microscopy, Activity Assay

Journal: PLoS Pathogens

Article Title: Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

doi: 10.1371/journal.ppat.1006914

Figure Lengend Snippet: A) Exogenous expression of human SR-A6 in L-929 cells (low CAR expression) promotes binding of HAdV-C5 to the cells. L-929 cells were transfected with a plasmid directing the synthesis of human SR-A6 from the cytomegalovirus major immediate early promoter and transfected cells were identified by immunostaining with anti-human SR-A6 antibody PLK1. Alexa-Fluor488-labeled HAdV-C5 particles were added to transfected L-929 cells at 4°C for 60 min. Fixed samples were imaged by confocal microscopy and virus particles associated with PLK1-positive and PLK1-negative cells were scored from maximum projections of confocal stacks. The plot shows number of virus particles per cell, one dot representing one cell. Horizontal bars represent mean values. Number of cells analyzed is indicated. The difference between PLK1-positive and -negative cells was statistically highly significant (P<0.0001, Kolmogorov-Smirnov test). The right-hand panel shows a representative image as a maximum projection of confocal stacks. In the overlay panel virus particles are shown in green, SR-A6-positive cells in red and nuclei (DAPI) in blue. Scale bar = 10 μm. B) Exogenous expression of human SR-A6 in HDF-TERT cells boosts HAdV-C5-mediated gene transduction. SR-A6 was expressed in the cells from a plasmid that directed synthesis of the protein from a bi-cistronic SR-A6-IRES-Tomato mRNA. Transfected HDF-TERT cells were infected with two different amounts of HAdV-C5_dE1_GFP, and nuclear GFP signals were scored at 30 h post infection (pi) by microscopy. Non-transfected cells or cells transfected with an empty vector were used as controls. The mean nuclear GFP intensities of Tomato-positive and Tomato-negative cells are shown as Tukey box plots. The difference between Tomato-positive and Tomato-negative cells in the SR-A6 transfection was statistically highly significant with a P-value <0.0001 (Kolmogorov-Smirnov test), whereas the difference in empty vector transfections did not reach P<0.0001 significance levels. Over 300 Tomato-positive cells and more than 5000 Tomato-negative cells were scored for each sample. C) Mouse SR-A6 expression in HDF-TERT cells leads to higher HAdV-C5_dE1_GFP infection efficiency than human SR-A6 expression. Both mouse (mSR-A6) and human (hSR-A6) proteins were expressed from bi-cistronic SR-A6-IRES-Tomato mRNAs. The mean nuclear GFP intensities of Tomato-positive and Tomato-negative cells are shown as Tukey box plots. Over 500 Tomato-positive cells and more than 4500 Tomato-negative cells were scored for each sample.

Article Snippet: Transfected cells were identified by staining for surface SR-A6 using anti-human SR-A6 antibody PLK1 (HM2208, Hycult Biotech) and Alexa-Fluor 594-conjugated secondary anti-mouse antibodies as described [ ].

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Immunostaining, Labeling, Confocal Microscopy, Transduction, Infection, Microscopy