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: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: ( A ) Correlation of MD1 with RP105 levels and MD2 with TLR4 levels from publicaly available SSc transcriptome data sets (GSE32413), representing skin biopsies from 22 patients with dcSSc and 9 healthy controls. ( B ) Correlation of RP105 with ASMA mRNA levels using SSc ( n = 16) and healthy controls fibroblasts ( n = 6). Pearson’s correlation coefficient. ( C ) Skin fibroblasts from SSc ( n = 10) and healthy control ( n = 5) were immunolabeled using antibodies against RP105 and ASMA (left panel). Each dot represents a single biopsy (right panel) and represent the mean fluorescent intensity of both RP105 and ASMA was measured in the same cell from 3 different fields containing at least 3–4 cells/hpf. Representative images are shown. Scale bar: 10 μm (left panel). Pearson’s rank correlation (right panel). ( D and E ) Results from PLA assays using explanted SSc and healthy control skin fibroblasts. ( D ) RP105-TLR4 and ( E ) TLR4-TLR4 PLA signals ( F and G , left panels) and PLA quantification ( D and E , right panels). Blue, DAPI; green, PLA signal. Mann-Whitney U test. Representative images are shown. Scale bar: 20 μm. ( F ) Skin biopsies from SSc ( n = 5) and healthy control ( n = 4) were immunolabeled using antibodies against MD1. Representative images are shown (left panel). Quantitative data using SSc and healthy control skin biopsies were immunolabeled using antibodies against MD1. Average intensity from 3 cells/hpf from 4 different areas per skin biopsy. Mann-Whitney U test. Scale bar: 10 μm; 20 μm (inset). ( G ) Skin fibroblasts from SSc ( n = 4) and healthy control ( n = 4) were immunolabeled using antibodies against MD1 and ASMA (left panel). Each dot represents a single biopsy (right panel) and represent mean fluorescent intensity of MD1 from 3 different fields containing at least 3–4 cells/hpf from the indicated number of participants. Mann-Whitney U test. Representative images are shown. Scale bar: 10 μm; 20 μm (SSc). Each dot represents a single biopsy.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Immunolabeling, MANN-WHITNEY

Journal: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: Human foreskin fibroblasts were transfected with RP105/MD1 duo plasmid and control plasmid (100 or 250 ng) followed by incubation with tenascin-C (2 μg/ml) for 72 hours. ( A ) Real-time quantitative PCR expressed as fold change compared with vector control. Results, normalized with GAPDH, are shown as the mean ± SD of triplicate determinations from 2 separate experiments. ( B ) Whole-cell lysates examined by Western analysis. Representative immunoblots are shown. Type I collagen levels, normalized for tubulin, are shown below. ( C ) Immunofluorescence confocal microscopy. Representative images are shown. Scale bar: 50 μm.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Transfection, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: ( A ) Bone marrow–derived macrophages and ( B ) skin fibroblasts were isolated from 8-week-old WT and RP105-KO mice. Cultures were incubated in media with or without LPS at indicated concentrations, and after 24 hours, IL-6 concentration in blood and secreted in media and cellular RNA were examined by ( A and B , left, and C ) ELISA or ( B , right) qPCR analysis. Results are shown as the mean ± SD of triplicate determinations from 3 mice/group. The quantitative concentrations of IL-6 are plotted and compared with a standard curve by ELISA from secreted media from macrophages and fibroblasts and from the whole blood ex vivo assay. The control and LPS-treated WT and KO IL-6 levels were plotted. For qPCR, results, normalized with GAPDH, are shown as the mean ± SD of triplicate determinations expressed as fold change (control and LPS-treated WT and control and KO-treated group fold-change values were compared). P values were determined by unpaired 2-tailed Student’s t test. ( C ) Whole blood was collected from RP105-KO and C57/BL6 mice and incubated with LPS for indicated periods. Sera were examined by ELISA. Results are shown as the mean ± SD of triplicate determinations from 3 mice/group.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Derivative Assay, Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: ( A and B ) Skin fibroblasts explanted from RP105-KO and C57/BL6 mice were incubated in the presence or absence of LPS. ( A ) Real-time quantitative PCR. Results, normalized with GAPDH, are shown as the mean ± SD of triplicate determinations. One-way ANOVA followed by Šidák’s multiple comparisons test. ( B ) In vitro wound healing assays. Results are shown as the mean ± SD of triplicate determinations in 4 randomly selected fields. Mann-Whitney U test. ( C and D ) Skin fibroblasts were incubated with tenascin-C in presence of TAK242 for 72 hours. ( C ) Real-time quantitative PCR. Results, normalized with GAPDH, are shown as the mean ± SD of triplicate determinations. One-way ANOVA followed by Šidák’s multiple comparisons test. ( D ) Immunostaining using antibodies against CgnI and ASMA. Nuclei were identified by DAPI (blue). Representative confocal microscopic images. Scale bar: 10 μm (red); 5 μm (green). Quantification of immunofluorescence (inset). The results shown the mean ± SD from 3 randomly selected hpf/slide.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Incubation, Real-time Polymerase Chain Reaction, In Vitro, MANN-WHITNEY, Immunostaining, Immunofluorescence

Journal: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: Eight- to 12-week-old female RP105-KO and WT mice received daily s.c. injections of bleomycin or PBS for 2 weeks (5 days per week) and were sacrificed on day 22, when the skin was harvested for analysis. ( A ) Trichrome stain (yellow arrows indicate dermal thickness). Representative images are shown. Scale bar: 100 μm. Dermal thickness (mean ± SD of 8 determinations/hpf; middle). Intradermal adipose thickness (mean ± SD of 8 determinations/hpf; right). One-way ANOVA followed by Šidák’s multiple comparisons test. ( B ) Second-harmonic generation microscopy images of dermal collagen fibrils. Representative images are shown ( n = 12 mice, 3 for each group). For scale bars, the original scan dimensions were 387.65 microns square. ( C ) Collagen content. Dots represent the mean ± SD from duplicate determination from the indicated number of mice. One-way ANOVA followed by Šidák’s multiple comparisons test. ( D ) Immunolabeling with antibodies against F4/80 and CD3 and F4/80. Representative images are shown. Scale bar: 50 μm. ( E ) Immunofluorescence using antibodies against phospho-p65 (left). Scale bar: 50 μm. Quantitation of immunopositive cells (number/hpf from 4 different areas from each skin section, n = 4 mice). One-way ANOVA followed by Šidák’s multiple comparisons test.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Staining, Microscopy, Immunolabeling, Immunofluorescence, Quantitation Assay

Journal: JCI Insight

Article Title: Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis

doi: 10.1172/jci.insight.160684

Figure Lengend Snippet: Twelve-week-old RP105-KO; Tsk1/+ and Tsk1/+ transgenic mice were sacrificed, and skin was harvested for analysis. ( A ) Trichrome stain (left). Representative images are shown (dotted lines indicate hypodermis and yellow arrows show hypodermal thickness). Hypodermal thickness (right) (mean ± SD of 8 determinations/hpf). Scale bar: 50 μm. Student’s t test. ( B ) Collagen content of the skin. Dots represent the mean ± SD from duplicate determinations from the indicated number of mice. One-way ANOVA followed by Šidák’s multiple comparisons test.

Article Snippet: Subconfluent fibroblast cultures were transfected with pDUO-MD1/RP105 (InvivoGen) using Lipofectamine reagent (Thermo Fisher Scientific), followed by tenascin-C treatment for 72 hours.

Techniques: Transgenic Assay, Staining

Journal: Molecular oral microbiology

Article Title: Mechanism and implications of CXCR4-mediated integrin activation by Porphyromonas gingivalis.

doi: 10.1111/omi.12021

Figure Lengend Snippet: Figure 6 Exploitation of CXCR4 by Porphyromonas gingivalis. In macrophages, P. gingivalis interacts with CD14 and the Toll-like receptor 2 (TLR2)/TLR1 signaling complex resulting in inside-out signaling for activating and binding CR3, which leads to a relatively ‘safe’ uptake of these organisms by macrophages (Hajishengallis et al., 2006, 2007; Wang et al., 2007). The signaling pathway that activates the high-affinity state of CR3 is mediated by Rac1, PI3K and cytohesin 1 (Cyt1) (Harokopakis & Hajishengallis, 2005; Harokopakis et al., 2006; Hajishengallis et al., 2009). The P. gingivalis-activated TLR2/TLR1 also induces a MyD88-dependent pathway that can potentially promote the killing of this bacterium (Hajishengallis et al., 2008, 2009). However, by means of its fimbriae, P. gingivalis instigates a crosstalk between CXCR4 and TLR2 which interferes with this antimicrobial mechanism (Hajishengallis et al., 2008). In this study, P. gingivalis was shown to also use CXCR4 to induce phosphatidylinositol-3 kinase (PI3K) -dependent activation of CR3, independently of TLR2, which further contributes to its capacity to evade killing. CXCR4 exploitation requires fully functional fimbriae, i.e. containing both the FimA and FimCDE components, which can directly bind CXCR4 (Pierce et al., 2009).

Article Snippet: Briefly, CHO-CR3 cells were transfected with human TLR2 and CD14, using a single plasmid (pDUO-hCD14/TLR2; Invivogen), with or without co-transfection with human CXCR4 (pORF-hCXCR4; Invivogen).

Techniques: Binding Assay, Activation Assay, Functional Assay

Journal: Microbial Cell Factories

Article Title: Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

doi: 10.1186/1475-2859-11-161

Figure Lengend Snippet: TLR2 mediated signalling using HEK293T cells. HEK293T cells were transfected with pUNO-hTLR2 (Invivogen) alone (TLR2 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the corresponding plasmids as described in Material & Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml) and LTA (10 μg/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. * p-value < 0.05 compared to the negative control (TLR2(−)).

Article Snippet: These cells were then transfected with either pNiFty-SEAP alone or in combination with pUNO1-hTLR2 or pDUO-hTLR6/TLR2 (Invivogen Inc., San Diego, USA) using branched polyethylenimine (MW ~ 25.000).

Techniques: Transfection, Plasmid Preparation, Incubation, Activity Assay, Negative Control

Journal: Microbial Cell Factories

Article Title: Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

doi: 10.1186/1475-2859-11-161

Figure Lengend Snippet: TLR2/6 mediated signalling using HEK293T cells. HEK293T cells were transfected with pDUO-hTLR6/TLR2 (Invivogen) alone (TLR2/6 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the respective plasmids as described in Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml), LTA at 0.1, 1 and 10 μg/ml, mLTA1 (10 μg/ml), mLTA2 (10 μg/ml), LGG wild-type or dltD mutant cells (10 8 CFU/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. * p-value < 0.05 compared to the negative control (TLR2/6(−)).

Article Snippet: These cells were then transfected with either pNiFty-SEAP alone or in combination with pUNO1-hTLR2 or pDUO-hTLR6/TLR2 (Invivogen Inc., San Diego, USA) using branched polyethylenimine (MW ~ 25.000).

Techniques: Transfection, Plasmid Preparation, Incubation, Mutagenesis, Activity Assay, Negative Control