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. 2018 Jul 27;9:1688. doi: 10.3389/fimmu.2018.01688

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Copyright © 2018 Köffel, Wolfmeier, Larpin, Besançon, Schoenauer, Babiychuk, Drücker, Pabst, Mitchell, Babiychuk and Draeger.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Pneumolysin (PLY)-bearing liposomes induce phenotypic changes in human macrophages in vitro. PLY-bearing liposomes (PLY-Lipo) were produced by pre-incubation of 40 µg/ml cholesterol:sphingomyelin liposomes with 4 µg/ml PLY for 10 min at room-temperature. This protocol efficiently sequesters the total amount of added toxin and neither cytotoxicity nor binding of free PLY to cultured cells is observed (see Figure 2A). (A) PLY-liposomes induce a downregulation of MHCII and CD86 in macrophages. Day 7 M-CSF differentiated human macrophages were stimulated with PLY-liposomes (PLY-Lipo), liposomes without PLY (Lipo) or left untreated (CTRL) and analyzed by FACS gated for CD14⁺ macrophages after 48 h. PLY-Lipo stimulated macrophages showed a marked downregulation of MHCII and CD86 as compared to control cells (untreated or liposomes only; Lipo). A representative FACS blot of one of three independent experiments is shown. Mean fluorescence intensity of MHCII and CD86 of CD14⁺ gated cells of three independent experiments are shown (mean ± SD, n = 5; *p < 0.05; unpaired t-test; two-tailed). (B) PLY-liposomes induce IL-6 production in macrophages. Human macrophages were stimulated as in (A) and IL-6 production was determined after 48 h from culture supernatants performing Bio-Plex assays (BioRad). IL-6 production is shown to be significantly enhanced in PLY-Lipo stimulated macrophages (mean ± SD; n = 7; **p < 0.005; unpaired t-test; two-tailed). (C) PLY-liposomes induce TLR2 expression in macrophages. Day 7 M-CSF differentiated human macrophages were stimulated with PLY-Lipo or liposomes only (Lipo) and analyzed by FACS gated for CD14⁺ macrophages after 48 h. PLY-Lipo stimulated macrophages showed a significant increase in TLR2 as compared to control cells (liposomes only; Lipo). Percentages of TLR2- and TLR4-expressing CD14⁺ macrophages are shown (mean ± SD; n = 6; *p < 0.05; paired t-test; two-tailed). (D) PLY-liposomes induced macrophages express increased levels of NOD2. Day 7 M-CSF differentiated human macrophages were stimulated with PLY-Lipo or liposomes only (Lipo) for 48 h. RNA was isolated and RT-PCR was performed to analyze expression levels of NOD1 and NOD2 in these cells. PLY-liposome induced macrophages showed a significant increase in NOD2 expression as compared to control cells (liposomes only; Lipo). NOD1 levels remained unchanged (mean ± SD; n = 7; *p < 0.05; paired t-test; two-tailed).