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