Effect of IL-13, IL-4 and/or LTD₄ on CysLT₁ expression by airway epithelial cells.

<p>Airway epithelial cells were treated with vehicle, 10 ng/ml IL-4, 10 ng/ml IL-13 and/or 100 nM LTD₄ for 6 hours. Incubations were stopped by removing the incubation media and cells were harvested, processed, and analyzed for CysLT₁ expression by flow cytometry as indicated in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. A. Representative histograms of CysLT₁ expression (control isotype, open bar; baseline expression, gray plot; IL-13-treated airway epithelial cells). B. Data represent the mean (± SEM) of three separate experiments. NS = not significant.</p

Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ <i>In Vitro</i> and <i>In Cellulo</i>

<div><p>LTB₄ is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results <i>in vitro</i> and <i>in vivo</i>, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC₄ synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB₄ biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB₄ biosynthesis in human neutrophils. At a concentration of 10 μM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB₄ biosynthesis, as assessed by the sum of LTB₄, 20-OH-LTB₄, and 20-COOH-LTB₄. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB₄ into 20-OH-LTB₄ by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB₄ levels. This was true for both endogenously biosynthesized and exogenously added LTB₄. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB₄ in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB₄ <i>in cellulo</i>, which might result in increased LTB₄ levels <i>in vivo</i>.</p></div

Effect of LTD₄ on eotaxin-3 release by airway epithelial cells pretreated with IL-13.

<p>After a 6 hour incubation of airway epithelial cells with 10 ng/ml IL-13, cells were further cultured for an additional 18 hours in the presence or absence of 100 nM LTD₄. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. The data represent the mean (± SEM) of three independent experiments.</p

Effect of LTD₄ on the IL-13-induced release of eotaxin-3 by airway epithelial cells.

<p><b>A</b>) Airway epithelial cells were incubated with 10 ng/ml IL-13 alone or in combination with 100 nM LTD₄ for up to 48 hours. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. Data are expressed as % of IL-13-treated cells for each incubation period. The data represent the mean (± SEM) of four independent experiments. NS = not significant. <b>B</b>) Airway epithelial cells were incubated with 50 ng LTD₄ for up to 48 hours. Incubations were stopped by adding one volume of cold incubation buffer. Samples were harvested then processed for the analysis of LTD₄ and LTE₄ by reverse-phase HPLC as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. Data are the mean (± SEM) of three independent experiments, each performed in duplicate.</p

Expression of CysLT₁ and CysLT₂ by airway epithelial cells.

<p>Total mRNA was extracted from resting airway epithelial cells (A549), human primary bronchial epithelial cells (HPBEC) and freshly isolated human eosinophils (Eo). The expression of CysLT₁ and CysLT₂ mRNA was then analyzed by RT-PCR as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. The data presented are a typical result of three independent experiments for eosinophils and airway epithelial cells, and four independent experiments for HPBEC.</p

Kinetic of eotaxin-3 release from airway epithelial cells.

<p>Airway epithelial cells were incubated with vehicle (open bars), 10 ng/ml IL-13 (closed bars), or 100 nM LTD₄ (gray bars) for different times. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043544#s4" target="_blank">Material and Methods</a></i>. The data represent the mean (± SEM) of four independent experiments. Insert represents a zoom of the 6 and 24 hours data points. * p = 0.002 vs 6 hours, # p = 0.003 vs 24 hours.</p

Removal of the inhibitory constraint exerted by CYP4F3A inhibitors on LTB₄ ω-oxidation in neutrophils.

<p>Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl₂) were incubated with <b>A,B)</b> 30 μM PF-4708671 or vehicle for 5 minutes, <b>C)</b> 30 μM PF-4708671 for 15 minutes, <b>D,E)</b> 30 μM 17-ODYA for 30 minutes, or <b>F)</b> 30 μM 17-ODYA for 15 minutes. Neutrophils were washed (or not) with autologous plasma or HBSS-CaCl₂ as described in methods. <b>A,D)</b> 100 nM thapsigargin or <b>B,C,E,F)</b> 1 μM LTB₄ were then added for 10 and 20 minutes, respectively. Samples then were processed and analyzed for 20-OH-LTB₄ and 20-COOH-LTB₄ as described in methods. Data are the mean (± S.D) of 4 independent experiments, each performed in duplicate.</p

Impact of the p70S6K1 inhibitors on LTB₄ biosynthesis and ω-oxidation in neutrophil suspensions.

<p><b>A-D)</b> Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl₂) were incubated with PF-4708671, LY2584702 or vehicle (DMSO) for 5 minutes, then stimulated with 100 nM thapsigargin for 10 minutes. <b>A,C,D</b>) Samples were processed and analyzed for LTB₄ biosynthesis as described in methods. Data are the mean (± S.D) of 5 independent experiments, each performed in duplicate. <b>A</b>) Leukotrienes represent the sum of LTB₄, 20-OH-LTB₄ and 20-COOH-LTB₄. <b>C,D</b>) ω-LTB₄ represents the sum of 20-OH-LTB₄ and 20-COOH-LTB₄. <b>B</b>) Samples were processed and analyzed for S6 and phospho-S6 content as described in methods. Data are from one experiment, representative of three.</p

Impact of p70S6K1 and CYP4F3A on the half-life of LTB₄ <i>in cellulo</i>.

<p>Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl₂) were incubated 5 minutes with vehicle (DMSO), <b>A)</b> PF-4708671 (30 μM), <b>B)</b> LY2584702 (10 μM), or 30 minutes with <b>C)</b> 17-ODYA (30 μM), then treated with 1 μM of LTB₄ for the indicated times. <b>D)</b> PF-4708671 (30 μM) or 17-ODYA (30 μM) were added to neutrophil suspensions for the indicated times before adding 1 μM LTB₄ for 20 minutes. <b>E)</b> LTB₄ (1 μM) was added for 20 minutes to either incubation medium, neutrophil supernatants (incubation medium incubated with neutrophils during 30 minutes) or neutrophil suspensions. <b>A-E)</b> Samples then were processed and analyzed for LTB₄ biosynthesis as described in methods. ω-LTB₄ represents the sum of 20-OH-LTB₄ and 20-COOH-LTB₄. Data are the mean (± S.D) of 4-5 independent experiments, each performed in duplicate.</p

Impact of PF-4708671 on human recombinant CYP4F3A activity.

<p><b>A)</b> Pre-warmed human recombinant CYP4F3A in phosphate buffer containing a NADPH regenerating system was incubated with increasing concentrations of PF-4708671 for 5 minutes, with LY2584702 (10^-5M) for 5 minutes or with 17-ODYA (10^-5M) for 30 minutes before the addition of 1 μM LTB₄ for 1 minute. <b>B)</b> Representative time curves of the ω-oxidation of LTB₄ par CYP4F3A. Initial rates were calculated by linear regession. <b>C)</b> Michaelis-Menten kinetics of the inhibition of CYP4F3A by PF-4708671. Human recombinant CYP4F3A was incubated with increasing concentrations of PF-4708671 before the addition of LTB₄ at different concentrations. LTB₄, and ω-LTB₄ (20-OH-LTB₄ and 20-COOH-LTB₄) were quantified by HPLC as described in methods and represent the addition of 20-OH-LTB₄ and 20-COOH-LTB₄. Data are shown as the mean (± S.D) of 8 independent experiments. <b>D)</b> Data from the Michaelis-Menten graph were transformed using the Lineweaver-Burk plot (double reciprocal plot). <b>E)</b> Legend for C and D.</p