Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs activate the NF-κB/AP-1 pathways in human reporter macrophages via TLR2 signaling. A dTHP1-XBlue cells were treated with Ef -EVs (100 − 50,000 EVs/cell). LPS (100 ng/mL) was used as a positive control. B dTHP1-XBlue cells were treated with EVs derived from clinical E. faecalis bloodstream isolates (1000-10,000 EVs/cell). LPS (100 ng/mL) and Pam 3 CSK 4 (100 ng/mL) were used as positive controls. C-E) dTHP1-XBlue cells were pretreated with increasing concentrations (from 0.1 to 5 µg/mL) of anti-hTLR2-IgA mAb (dark pink) or human IgA2 control mAb (light pink) for 1 h. Subsequently, cells were treated with either (C) EVs (7000 EVs/cell), (D) Pam 3 CSK 4 (TLR2 ligand, 1 ng/mL), or (E) LPS (TLR4 ligand, 1 ng/mL) in the presence of antibodies for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) in (C, blue), Pam 3 CSK 4 (1 ng/mL) in (D, dark blue), or LPS (1 ng/mL) in (E, red) were used as positive activating controls while cells treated with cell culture medium supplemented alone ( C - E , light green) were used as negative controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive controls. Data are shown as means ± SD of three independent experiments ( N = 3, n ≥ 3) and analyzed either by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test in (A and B) or by two-way analysis of variance (ANOVA) followed by Šídák multiple comparison post hoc test in ( C - E )

Article Snippet: SUVs were then functionalized with synthetic bacterial lipopeptide Pam 3 -Cys-Ser-Lys 4 (Pam 3 CSK 4 , cat. no. tlrl-pms, Invivogen) by post-insertion.

Techniques: Positive Control, Derivative Assay, Control, Cell Culture, Activation Assay, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 engagement by Pam 3 CSK 4 -SUVs triggers inflammatory activation but does not enhance SUV internalization. A - C ) dTHP1-XBlue cells were treated with Pam 3 CSK 4 -SUVs (6 µM) containing increasing amounts of Pam 3 CSK 4 (from 0 to 0.4 mol% total SUV lipid composition) for 18 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.4 mol% total SUV lipid composition) were used as positive activating controls. D - F ) dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) in the presence of antibodies for 18 h. Cells treated with cell culture medium containing Pam 3 CSK 4 -SUVs (6 µM, Pam 3 CSK 4 composition = 0.04 mol% total SUV lipid composition) were used as positive activating controls. For both experiments, cells incubated in cell culture medium alone were used as negative controls. Mean fluorescence intensity (B and E, upper panel) and SUV-positive cells (B and E, lower panel) were quantified after 18 h of treatment by measuring fluorescence intensity associated with SUVs on the APC channel. In C and F, NF-κB/AP-1 activation was measured after 18 h of SUV treatment as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are shown as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: SUVs were then functionalized with synthetic bacterial lipopeptide Pam 3 -Cys-Ser-Lys 4 (Pam 3 CSK 4 , cat. no. tlrl-pms, Invivogen) by post-insertion.

Techniques: Activation Assay, Incubation, Cell Culture, Control, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs modulate gene expression and metabolic activity in primary human macrophages. A and B ) HMDMs were incubated with Ef -EVs at different concentrations (1000-10,000 EVs/cell) for 24 (A) and 48 h (B). Data are presented as frequency distributions, median, and quartiles of gene expression results from three individual donors ( N = 3, n = 3) and normalized to medium-treated cells (0 EVs/cell) as control. C and D) HMDMs were treated with Ef -EVs (10,000 and 50,000 EVs/cell) or Pam 3 CSK 4 (10 ng/mL) for 24 h. Cells incubated in cell culture medium alone served as untreated control. C Normalized ECAR values were monitored after injections of glucose, oligomycin, and 2-DG, according to the glycolysis stress test. D Glycolytic activity is shown as the x-fold change of untreated control regarding the normalized ECAR values in glycolysis. Results C and D are shown as mean ± SEM of three individual donors ( N = 3, n = 6). Results were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: SUVs were then functionalized with synthetic bacterial lipopeptide Pam 3 -Cys-Ser-Lys 4 (Pam 3 CSK 4 , cat. no. tlrl-pms, Invivogen) by post-insertion.

Techniques: Gene Expression, Activity Assay, Incubation, Control, Cell Culture, Comparison