Figures
- FIGURE 1
Decreased interferon (IFN)-γ-mediated responses in monocytes isolated from individuals with cystic fibrosis following initiation of ivacaftor therapy. a) Comparison of gene expression 3 h after stimulation ex vivo with IFN-γ (100 ng·mL−1) in monocytes isolated at day D0 and D7 of ivacaftor treatment; HPRT was used as a housekeeping gene. n=10, p-values were calculated using Wilcoxon signed rank testing. Peripheral blood mononuclear cells were recovered from blood by density centrifugation and monocytes were isolated by CD14-positive selection magnetic bead separation (Miltenyi, Auburn, CA, USA). Monocytes were plated with or without recombinant human IFN-γ (Peprotech, Rocky Hill, NJ, USA) in multiwell plates. Cells were lysed with RLT buffer (Qiagen, Valencia, CA, USA) for RNA analysis after 3 h. RNA was isolated using the RNeasy Plus Kit (Qiagen) per manufacturer's instructions. Real-time reverse transcription PCR was performed on total RNA using TaqMan Gene Expression Assays primer/probe sets for target genes. b) Ivacaftor dampens monocyte responses to IFN-γ within 2 days of starting therapy; n=6. *: p<0.05; **: p≤0.005 by ANOVA. c–f) Monocytes isolated from subjects at specified time points after initiation of ivacaftor were exposed to IFN-γ for 1 h then lysed with Laemmli buffer (Alfa Aesar, Haverhill, MA, USA). Immunoblots were performed to determine abundance of STAT1 and phosphorylated STAT1 (pSTAT1); β-actin was used as a housekeeping control protein. c) Representative immunoblots from two subjects showing results from D0 and D7 lysates. d) Densitometry was performed to quantitate changes in pSTAT abundance; n=8, p-value calculated using Wilcoxon signed rank testing. e) Representative immunoblots from subject 3 showing results from D0, D2 and D7. f) Densitometry comparing abundance of pSTAT at day 2 versus day 0; n=4, p-value calculated using Student's t-test. g) Ivacaftor did not cause changes in plasma IFN-γ concentrations during the first week of treatment, as measured by Meso Scale Discovery biomarker assay. Statistical comparisons noted above were performed using Excel (Student's t-test) or GraphPad Prism 7 (Wilcoxon signed rank testing and ANOVA); p<0.05 was considered significant.
