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Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis

Katherine B. Hisert, Timothy P. Birkland, Kelly Q. Schoenfelt, Matthew E. Long, Brenda Grogan, Suzanne Carter, W. Conrad Liles, Edward F. McKone, Lev Becker, Anne M. Manicone
ERJ Open Research 2020 6: 00318-2019; DOI: 10.1183/23120541.00318-2019
Katherine B. Hisert
1Dept of Medicine, University of Washington, Seattle, WA, USA
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  • ORCID record for Katherine B. Hisert
  • For correspondence: hisertk@njhealth.org
Timothy P. Birkland
1Dept of Medicine, University of Washington, Seattle, WA, USA
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Kelly Q. Schoenfelt
2Ben May Dept for Cancer Research, University of Chicago, Chicago, IL, USA
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Matthew E. Long
1Dept of Medicine, University of Washington, Seattle, WA, USA
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Brenda Grogan
3Dept of Medicine, St Vincent's University Hospital, Dublin, Ireland
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Suzanne Carter
3Dept of Medicine, St Vincent's University Hospital, Dublin, Ireland
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W. Conrad Liles
1Dept of Medicine, University of Washington, Seattle, WA, USA
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Edward F. McKone
3Dept of Medicine, St Vincent's University Hospital, Dublin, Ireland
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Lev Becker
2Ben May Dept for Cancer Research, University of Chicago, Chicago, IL, USA
4These authors contributed equally
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Anne M. Manicone
1Dept of Medicine, University of Washington, Seattle, WA, USA
4These authors contributed equally
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    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.

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Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis
Katherine B. Hisert, Timothy P. Birkland, Kelly Q. Schoenfelt, Matthew E. Long, Brenda Grogan, Suzanne Carter, W. Conrad Liles, Edward F. McKone, Lev Becker, Anne M. Manicone
ERJ Open Research Apr 2020, 6 (2) 00318-2019; DOI: 10.1183/23120541.00318-2019

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Ivacaftor decreases monocyte sensitivity to interferon-γ in people with cystic fibrosis
Katherine B. Hisert, Timothy P. Birkland, Kelly Q. Schoenfelt, Matthew E. Long, Brenda Grogan, Suzanne Carter, W. Conrad Liles, Edward F. McKone, Lev Becker, Anne M. Manicone
ERJ Open Research Apr 2020, 6 (2) 00318-2019; DOI: 10.1183/23120541.00318-2019
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