Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus

Figure 2

Liposomes with nonbilayer phospholipid arrangements induce TLR-2/6 and TLR-4 signaling in TLR-expressing HEK cells. (a) HEK-TLR-4/MD2/CD14, HEK-TLR-2/TLR-6, HEK-TLR-5, and HEK-TLR-8 cells were stimulated for 24 h at 37°C with 10 μL of egg-yolk phosphatidylcholine/egg-yolk phosphatidic acid (2 : 1) liposomes (50 nmol phosphatidic acid in 50 μL TS buffer) alone or bearing nonbilayer phospholipid arrangements induced with 2 mM MnCl₂. As positive controls, cells were stimulated with their TLR agonist: 100 ng/mL LPS, 1 μg/mL FSL1, 1 μg/mL bacterial flagellin, or 2.5 μg/mL ssRNA40, respectively. As negative controls, cells were stimulated with liposomes alone or bearing nonbilayer phospholipid arrangements + 0.1 mM chloroquine (CQ). IL-8 levels and NF-κB activation were measured 24 h after stimulation. (b) HEK-TLR-4/MD2/CD14 and HEK-TLR-2/TLR-6 cells were stimulated with nonbilayer phospholipid arrangements induced with different MnCl₂ concentrations with (+CQ) or (−CQ) 0.1 mM chloroquine. IL-8 levels were measured 24 h after stimulation. Histograms and graphs represent the mean ± SD of three independent experiments. (c, d) TLR-2 and TLR-4 of bone marrow-derived macrophages (BMDM) were analyzed. (c) 4 × 10⁴ BMDM were stimulated with liposomes alone or bearing Mn²⁺-induced nonbilayer phospholipid arrangements (as in a). Where indicated, 10 μg/mL of anti-TLR-2 or isotype control (IgG1,κ) was added 2 h before liposomes addition. PGN (1 μg/mL) was the positive control. After 24 h at 37°C, TNF-α levels were determined. (d) 3 × 10³ BMDM were stimulated with 20 μL of liposomes (as in a). Where indicated, 20 μg/mL of anti-TLR-4 or isotype control (IgG2a,κ) was added 2 h before liposomes addition. LPS (10 ng/mL) was the positive control. TNF-α levels were determined as in (c). Histograms represent the mean ± SD of three independent experiments. Data were analyzed with one-way ANOVA and Tukey’s multiple comparisons test. , .

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