Journal: Frontiers in cellular and infection microbiology

Article Title: Lipopolysaccharide Preparation Derived From Porphyromonas gingivalis Induces a Weaker Immuno-Inflammatory Response in BV-2 Microglial Cells Than Escherichia coli by Differentially Activating TLR2/4-Mediated NF-κB/STAT3 Signaling Pathways.

doi: 10.3389/fcimb.2021.606986

Figure Lengend Snippet: FIGURE 2 | Effects of P. gingivalis-LPS and E. coli-LPS on gene and protein expression of TLR2, TLR4, and CD14 in BV−2 microglial cells. (Ai, Bi) BV-2 microglial cells were treated with 1mg/mL P. gingivalis-LPS or E. coli-LPS for different times, and RT-PCR was performed; two-way ANOVA, **p < 0.01, and ***p < 0.001 compared to the 0hr group. (Aii, Bii) BV-2 microglial cells were treated with TAK-242 (1mM) or serum-free medium for 60min, followed by treatment with P. gingivalis-LPS, E. coli-LPS (1mg/mL) or serum-free medium for 24h and western blotting with anti-TLR4, anti-CD14, and anti-GAPDH antibodies; one-way ANOVA, *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group or the LPS groups. (Aiii, Biii) BV-2 microglial cells were treated with C29 (100mM) or serum-free medium for 60 min, followed by treatment with P. gingivalis-LPS, E. coli-LPS (1mg/mL) or serum-free medium for 24h and western blotting with anti-TLR2 and anti-GAPDH antibodies; one- way ANOVA, **p < 0.01 and ***p < 0.001 compared to the control group or the LPS groups. (C) BV-2 microglial cells were treated with 1mg/mL P. gingivalis-LPS, E. coli-LPS (1mg/mL) or serum-free medium for 24h and western blotting with anti-TLR2, anti-TLR4, anti-CD14, and anti-GAPDH antibodies; Student’s t-test, **p < 0.01 compared the P. gingivalis-LPS group with the E. coli-LPS group. Data from three independent experiments are presented as mean ± SD.

Article Snippet: We used a TLR4 inhibitor (TAK-242, 1mM, MedChemExpress, Monmouth Junction, NJ, USA) and a TLR2 inhibitor (C29, 100mM, MedChemExpress) in our experiments.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in cellular and infection microbiology

Article Title: Lipopolysaccharide Preparation Derived From Porphyromonas gingivalis Induces a Weaker Immuno-Inflammatory Response in BV-2 Microglial Cells Than Escherichia coli by Differentially Activating TLR2/4-Mediated NF-κB/STAT3 Signaling Pathways.

doi: 10.3389/fcimb.2021.606986

Figure Lengend Snippet: FIGURE 8 | Binding modes of lipid A from E. coli (A, B) and P. g (C–G) with TLR4-MD-2. (A) Top view of the symmetrical dimer of the TLR4-MD-2- LPS complex (PDB 3VQ2). (B) Hydrogen bond interactions of the 1-phosphate and 4’-phosphate residues of lipid A. Interaction distance in Ångstrom are indicated. (C, D) The predicted binding modes of 5P. g-lipid A, (E) 4aP. g-lipid A, (F) 4bP. g-lipid A, and (G) 3P. g-lipid A with the receptor. E. coli-lipid A is shown as magenta sticks, while the four types of P. g-lipid A are displayed as yellow, cyan, green, and lightblue sticks, respectively. Key residues are shown as sticks. Hydrogen bonds are depicted as yellow dashed lines. TLR4, TLR4*, MD-2 are colored green, cyan, and gray, respectively.

Article Snippet: We used a TLR4 inhibitor (TAK-242, 1mM, MedChemExpress, Monmouth Junction, NJ, USA) and a TLR2 inhibitor (C29, 100mM, MedChemExpress) in our experiments.

Techniques: Binding Assay

Journal: Cancers

Article Title: Bone Marrow Myeloid-Lymphatic Progenitors Expand Tumor Lymphatic Vasculature Through Cell Fusion.

doi: 10.3390/cancers17111804

Figure Lengend Snippet: Figure 2. TLR4 and Th2 pathways upregulate fusogenic proteins during differentiation of M-LECPs in a time-dependent manner. Expression of fusogenic markers Sirp-a (A), Trem-2 (B), and Stab1 (C) was determined by flow cytometry in freshly isolated BM cells (blue lines) and cells differentiated with LPS (red line), IL-4 (green line), or IL-10 (orange line). Secondary controls in representative histograms are indicated by black lines and gray shading. The numbers in histograms indicate the mean percentage of positive cells. The mean percentage of positive cells and mean fluorescent intensity (MFI) of fusogenic markers in naïve and differentiated cells are shown in (D,E), respectively. (F–H) panels show the percentage of positive cells for Th2 receptors, LECs, and fusogenic markers on differentiation days 0, 3, and 6, respectively. All analyses were performed in duplicate and reproduced twice. Significant differences in marker expression in naïve and differentiated cells were determined by Student’s t-test with p-values < 0.05, <0.01, and <0.001 indicated by *, **, and *** symbols, respectively.

Article Snippet: A > 99% pure monospecific TLR4 inhibitor TAK-242 (cat#S7455) was from Selleckchem (Houston, TX, USA).

Techniques: Expressing, Flow Cytometry, Isolation, Marker

Journal: Cancers

Article Title: Bone Marrow Myeloid-Lymphatic Progenitors Expand Tumor Lymphatic Vasculature Through Cell Fusion.

doi: 10.3390/cancers17111804

Figure Lengend Snippet: Figure 5. Primary M-LECPs and RAW264.7 macrophages fuse with LECs in vitro. (A,F) Representa- tive images of M-LECPs from BM of GFP+ mice (A) or GFP-expressing RAW264.7 cells (F) co-cultured with RLEC-mCherry (mCHR) for 5 days in 1% BSA medium. Hoechst-stained multiple nuclei are highlighted by white circles. Images were acquired at 400× magnification. (B,G) Flow cytometry analysis of M-LECP (B) and RAW264.7 (G) double-positive cells co-expressing GFP and mCherry on the fifth day of co-culture. Numbers in upper right quadrants indicate percentages of fused cells. (C,H) Daily quantification of fusion mediated by M-LECPs (C) or RAW264.7 cells (H) using flow cytometry. (D,I) Daily quantification of fusion mediated by M-LECPs (D) or RAW264.7 cells (I) using fluorescent microscopy. (E) Flow cytometry quantification of either fused ex vivo BM-GFP cells, post-CSF-1 treatment alone, or additionally differentiated with IL-4, IL-10, or LPS in the absence or presence of the TLR4 inhibitor TAK-242. (J). Flow cytometry quantification of fused RAW264.7 cells with LECs in the presence of anti-IL-4 IgG, anti-IL-10 antibody (5 µg/mL each), or TAK-242 (10 µM). Here, 1% BSA and 10% FBS media were used as a positive and a negative control, respectively. All experiments were performed in duplicate and reproduced at least three times. Statistical analyses determined by Student’s t-test yielded p-values < 0.05, <0.01, and <0.001 as indicated correspondingly by *, **, and *** symbols.

Article Snippet: A > 99% pure monospecific TLR4 inhibitor TAK-242 (cat#S7455) was from Selleckchem (Houston, TX, USA).

Techniques: In Vitro, Expressing, Cell Culture, Staining, Flow Cytometry, Co-Culture Assay, Microscopy, Ex Vivo, Negative Control

Journal: Cancers

Article Title: Bone Marrow Myeloid-Lymphatic Progenitors Expand Tumor Lymphatic Vasculature Through Cell Fusion.

doi: 10.3390/cancers17111804

Figure Lengend Snippet: Figure 9. Blocking TLR4 signaling reduces M-LECP fusion, lymphatic formation, and lymph node metastasis. Female CB-17 SCID mice were lethally irradiated and grafted with male BM from the same strain. After full reconstitution, mice were orthotopically implanted with MDA-MB-231 tumor cells. Mice were treated with 0.5% DMSO vehicle control or TAK-242 (5 mice per group). (A) Mean tumor volumes per group treated with vehicle or TAK-242. Asterisks indicate statistically significant but transient reduction in tumor growth rate in the TAK-242-treated versus control group, as determined by Student’s t-test (p < 0.05). (B) The density of fused lymphatic vessels was determined in 10 fields per section at 400× magnification and normalized per mm2. (C) The area-normalized density of tumor Lyve-1+ vessels in control and TAK-242-treated mice was determined as described for (B). (D) Metastatic burden was determined by measuring luciferase activity in tumor-adjacent lymph nodes. Data are presented as the mean relative luciferase units (RLU) normalized per mg of protein. Statistical significance of differences between control and TAK-242 groups was determined by Student’s t-test and is indicated by the p-values listed above the black bars.

Article Snippet: A > 99% pure monospecific TLR4 inhibitor TAK-242 (cat#S7455) was from Selleckchem (Houston, TX, USA).

Techniques: Blocking Assay, Irradiation, Control, Luciferase, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: α-Synuclein orchestrates Th17 responses as antigen and adjuvant in Parkinson’s disease

doi: 10.1186/s12974-025-03359-w

Figure Lengend Snippet: Functional alterations of α-synuclein-stimulated DCs. Monocyte-derived DCs (mDCs) generated from healthy subjects (Table 1) were used for the following assays. ( A – D ) mDCs were cultured in the presence or absence of the indicated concentrations of αS monomers or fibrils for 24 h. ( A , B ) The HLA-DR high CD86 + ratio in CD209 + mDCs was analyzed. Gating strategies are shown in Supplementary Fig. 7. A representative contour plot is shown in ( A ) and the HLA-DR high CD86 + ratio in CD209 + mDCs (n = 8) is shown in ( B ). ( C , D ) IL-1β, IL-6, and IL-23 concentrations in culture supernatants stimulated as indicated (n = 8) are shown in ( C ) and a comparison of IL-23 and IL-12 levels after high-dose αS fibril stimulation (n = 8) is shown in ( D ). ( E – G ) mDCs (n = 4) were cultured for 48 h in the presence of 1000-fold diluted αS seeds. Three αS seeds derived from PD patients (#1–#3) and one control product derived from a healthy donor (control) were used for stimulation. ( E , F ) CD86 expression in CD209 + mDCs was analyzed. A representative histogram is shown in ( E ) and the CD86 + ratio in CD209 + mDCs is shown in ( F ). ( G ) IL-1β, IL-6, and IL-23 concentrations in culture supernatants were measured. ( H , I ) mDCs were treated with a TLR4 inhibitor, TAK-242 (60 µM), or control vehicle for 2 h prior to 24 h stimulation with high-dose (50 µg/ml) αS fibrils (n = 6) or LPS (n = 4–5). The HLA-DR high CD86 + ratio in CD209 + mDCs is shown in ( H ), and IL-1β, IL-6, and IL-23 concentrations in culture supernatants are shown in ( I ). stimu, stimulation; mono, monomer; TAK, TAK-242. Each dot indicates the value of one individual. Data represent the mean ± SD ( B–D, F–I ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P-values were determined by the Friedman test ( B , C , F , G ) or Wilcoxon’s matched-pairs signed-rank test ( D, H, I )

Article Snippet: For inhibitor experiments, a TLR4 inhibitor (TAK-242), TLR1/2 inhibitor (TLR2-IN-C29), or XBP-1 s inhibitor (STF-083010) was purchased from Selleck Chem (Houston, TX, USA).

Techniques: Functional Assay, Derivative Assay, Generated, Cell Culture, Comparison, Control, Expressing

Journal: Journal of Neuroinflammation

Article Title: α-Synuclein orchestrates Th17 responses as antigen and adjuvant in Parkinson’s disease

doi: 10.1186/s12974-025-03359-w

Figure Lengend Snippet: XBP-1s expression in α-synuclein-stimulated DCs. ( A , B ) mDCs generated from healthy subjects (Table 1) were cultured in the presence or absence of high-dose (50 µg/ml) αS monomers or fibrils for 24 h. XBP-1s expression in CD209 + mDCs was analyzed. Gating strategies are shown in Supplementary Fig. 7. A representative histogram is shown in ( A ) and the XBP-1 s + ratio in CD209 + mDCs (n = 7) is shown in ( B ). ( C ) Cryopreserved PBMCs from PD patients (n = 8) and age-matched controls (n = 5) were subjected to flow cytometric analysis (Supplementary Table 4). Gating strategies are shown in Supplementary Fig. 12. The XBP-1 s + ratio in CD1c + type 2 conventional DCs (cDC2s) is shown. ( D – G ) mDCs generated from healthy subjects (Table 1, n = 6) were treated with a TLR4 inhibitor, TAK-242 (60 µM) ( D, E ), or an IRE1-XBP-1s inhibitor, STF-083010 (120 µM) ( F , G ), for 2 h prior to 24 h stimulation with high-dose (50 µg/ml) αS fibrils. Vehicle containing the same concentration of DMSO was used as a control. ( D – F ) XBP-1s expression in CD209 + mDCs was analyzed. A representative histogram is shown in ( D ), and the XBP-1s + ratio in CD209 + mDCs is shown in ( E , F ). ( G ) IL-6 and IL-23 concentrations in the culture supernatants were measured. mono, monomer; TAK, TAK-242; STF, STF-083010; XBP-1s, spliced X-box binding protein-1. Each dot indicates the value of one individual. Data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P-values were determined by the Friedman test ( B ), the Mann–Whitney U -test ( C ), or Wilcoxon’s matched-pairs signed-rank test ( E – G )

Article Snippet: For inhibitor experiments, a TLR4 inhibitor (TAK-242), TLR1/2 inhibitor (TLR2-IN-C29), or XBP-1 s inhibitor (STF-083010) was purchased from Selleck Chem (Houston, TX, USA).

Techniques: Expressing, Generated, Cell Culture, Concentration Assay, Control, Binding Assay, MANN-WHITNEY