Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Activation of TLR2 is dependent on PPAD expression and activity. U251 MG cells overexpressing the TLR2 receptor were infected (MOI=100) or treated for 4 h with (A) ATCC 33277 strain or Pam3CSK4 (1 μg/ml), n=5-7; (B) various P. gingivalis strains and ATCC-derived isogenic mutants of catalytically inactive PPAD (C351A PPAD), n=3-5; (C) ATCC 33277 wild-type and PPAD mutant strains and sonicates (cells were lysed by sonication (sonic.)), n=4; or (D) outer membrane vesicles (OMVs) isolated from ATCC 33277 strain and its isogenic PPAD mutants (2 µg/ml), n=4. Results in (A) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Results in (B–D) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Data are normalized to those from cells stimulated/infected with the same factor and transfected with an empty vector. Mean + SD; ****p < 0.0001; ***p < 0.001; ns, no statistical significance; 1-way ANOVA both followed by Tukey’s multiple comparisons test. ATCC WT- ATCC 33277.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Expressing, Activity Assay, Infection, Derivative Assay, Mutagenesis, Sonication, Membrane, Isolation, Luciferase, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Fimbriae purified from the wild-type ATCC 33277 strain activate the TLR2 receptor. U251 MG cells overexpressing TLR2 were: (A) infected for 4 h with WT ATCC 33277 and its isogenic major fimbriae (delFimA) mutant strain (MOI=100), n=4-5 (B) infected for 4 h with various ATCC 33277-derived PPAD and FimA mutants as well as the WT W83 strain at different MOI (all strains MOI=20-100 with an additional MOI=5 for WT ATCC 33277), n=3 or (C) infected for 2, 4 or 6 h with WT ATCC 33277 or the PPAD mutant strains (MOI=100), n=3; and (D) treated for 4 h with purified fimbriae (10 µg/ml) from WT ATCC 332771 (FimA WT) or the PPAD mutant (FimA delPPAD) strains; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (E) HEK Blue cells overexpressing TLR2 were treated with purified fimbriae (10 µg/ml) isolated from the WT ATCC 33277 strain (FimA WT) or the PPAD mutant (FimA delPPAD), n=3. Results are presented as the mean ± SD fold induction compared to control (unstimulated) cells. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. In (B) each condition was compared to control uninfected cells and in (C) comparisons were performed for each timepoint separately and significant differences compared to WT ATCC 33277 are depicted in the graph.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Purification, Infection, Mutagenesis, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Isolation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Both active PPAD and fimbriae are crucial for activation of TLR2. U251 MG cells overexpressing the TLR2 receptor were infected for 4 h (MOI=100) with various ATCC 33277-derived isogenic mutants expressing different forms of PPAD (the T1 form, the T2-hyperactive form, and the catalytically inactive C351A mutant form) or FimA; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. ****p < 0.0001; ns, no statistical significance; 1-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Infection, Derivative Assay, Expressing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: The ability of clinical strains to activate TLR2 is much weaker than that of ATCC 33277. (A) Cells were infected for 4 h (MOI=100) with various clinical strains (k1-k10), ATCC 33277 (ATCC WT), or W83, n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (B) Western blot analysis of laboratory and clinical strain cultures (adjusted to OD 600 ) to detect FimA. (C) PPAD activity in whole laboratory and clinical strain cultures (adjusted to OD 600 ), n=6. Results were compared with those from the ATCC 332777 strain. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) The FimA type determined by sequencing of the fimA gene in clinical strains.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Sequencing

Journal: Frontiers in Pharmacology

Article Title: A Series of Genes for Predicting Responses to Anti-Tumor Necrosis Factor α Therapy in Crohn’s Disease

doi: 10.3389/fphar.2022.870796

Figure Lengend Snippet: Experimental validation of core predictors. (A) Coculture system of THP1 and Caco2 cells. (B) Validated Western blotting results for TLR2 overexpression and knockdown. (C) CCK8 results for infliximab and LPS in Caco2 cells. (D) RT-qPCR results for cocultured Caco2 cells after infliximab treatment in the context of LPS-induced inflammation (one-way ANOVA, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were further performed to validate the overexpression and knockdown of TLR2 in THP1 cells (Proteintech, United States).

Techniques: Biomarker Discovery, Western Blot, Over Expression, Knockdown, Quantitative RT-PCR

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 5. Modified alkane polymers induce activation of TLR-1 and TLR-2 signaling pathways. a) Luciferase activity expressed by human TLR1/2, TLR2, TLR3 and TLR4 stable HEK 3T3 transfectant (pNF-kB-LUC Stratagene). Cells were left untreated or treated for a different time period with 50 mg/ml of unPE, mPE or pre and post implant PE and respective positive controls; PGN(10 mg/ml) for TLR1/2 and TLR2, Poly (I:C)(1 mg/ml) for TLR3 and LPS (10 mg/ml) for TLR4. doi:10.1371/journal.pone.0002438.g005

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Modification, Activation Assay, Protein-Protein interactions, Luciferase, Activity Assay, Transfection

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 6. Direct binding of oxidized alkane polymers to soluble TLR-2 molecules. a) Left panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with two different concentrations (exponential and plateau) of each analyzed polymer. Central panels; normalized fluorescence data (DF) for each concentration point as a function of the free ligand concentration. Right panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with each analyzed polymer at two different concentrations (exponential and plateau) in presence of an anti TLR2 mAb known to block the TLR2 binding groove (stoichiometric ratio 2:1 Ab to soluble TLR2 receptor). b) Comparison of the fluorescence emission scans collected for soluble TLR2, mPE and unPE. doi:10.1371/journal.pone.0002438.g006

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Binding Assay, Fluorescence, Polymer, Concentration Assay, Blocking Assay, Comparison