Figure 4
miR-181b inhibition upregulates ALX/FPR2 expression and agonist-induced phagocytosis in CF-MΦs. (A) MΦs from healthy subjects were transfected with either a negative control (CTRL) or a miR-181b inhibitor for 24 h miR-181b expression was evaluated by real-time PCR. Bars represent mean ± SEM from 3 independent transfections. **p = 0.0013. (B) MΦs were treated as in (A) and total ALX/FPR2 expression was determined by flow cytometry. WRW4 (10 μM) was added to samples incubated with 0.1 nM LXA4. Bars are mean ± SEM from 3 independent transfections. *p = 0.019. The inset shows a representative histogram. (C) HS or CF-MΦs (5 × 105/well) transfected either with a negative control or with a miR-181b inhibitor, were exposed to the indicated concentrations of LXA4. Cells were incubated with FITC-labelled zymosan particles for 30 min at 37 °C and phagocytosis was assessed by measuring fluorescence with a Synergy H1 microplate reader. Data points are mean ± SEM from separate experiments with cells from 4 healthy donors and 4 CF patients. *p = 0.017 (HS vs CF, 0.01 nM LXA4); *p = 0.032 (HS vs CF, 0.1 nM LXA4); *p = 0.011 (HS vs CF, 1 nM LXA4); *p = 0.045 (HS vs CF, 10 nM LXA4); **p = 0.003 (CF vs CF + miR-181b inhibitor). (D) HS or CF-MΦs (2 × 105/well) were incubated for 1 h at 37 °C with PAO1-GFP at 1:200 cell:bacteria ratio. Phagocytosis of PA01-GFP was determined by measuring total fluorescence (Ex 485 nm/Abs 530 nm) using a plate reader (Synergy, BioTek). Data points are mean ± SEM from separate experiments with cells from 3 healthy donors and 3 CF patients. *p = 0.021 (CF vs CF + miR-181b inhibitor, 1 nM LXA4); *p = 0.012 (CF vs CF + miR-181b inhibitor, 10 nM LXA4); ** p = 0.009 (CF vs CF + miR-181b inhibitor, 0.1 nM LXA4); **p = 0.004 (HS vs CF, 1 and 10 LXA4); ***p = 0.0003 (CF vs CF + miR-181b inhibitor, 0.01 nM LXA4); ***p = 0.0007 (HS vs CF, 0.1 nM LXA4); ***p = 0.00062 (CF vs CF + WRW4, 1 nM LXA4); ****p = 0.000006 (HS vs CF, 0.01 nM LXA4); ****p = 0.000000006 (CF + miR-181b inhibitor vs CF + miR-181b inhibitor + WRW4, 1 nM LXA4); ****p = 0.00000009 HS vs HS + WRW4, 1 nM LXA4).
