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Modulation, microbiota and inflammation in the adult CF gut: A prospective study

Open AccessPublished:June 25, 2022DOI:https://doi.org/10.1016/j.jcf.2022.06.002

      Highlights

      • Ivacaftor does not produce significant change in gut microbiota in an adult cohort.
      • Ivacaftor treatment does not significantly alter gut inflammation in adults with CF.
      • In an adult cohort ivacaftor does not produce a significant change in faecal elastase levels.

      Abstract

      Background

      Cystic Fibrosis (CF) has prominent gastrointestinal and pancreatic manifestations. The aim of this study was to determine the effect of Cystic fibrosis transmembrane conductance regulator (CFTR) modulation on, gastrointestinal inflammation, pancreatic function and gut microbiota composition in people with cystic fibrosis (CF) and the G551D-CFTR mutation.

      Methods

      Fourteen adult patients with the G551D-CFTR mutation were assessed clinically at baseline and for up to 1 year after treatment with ivacaftor. The change in gut inflammatory markers (calprotectin and lactoferrin), exocrine pancreatic status and gut microbiota composition and structure were assessed in stool samples.

      Results

      There was no significant change in faecal calprotectin nor lactoferrin in patients with treatment while all patients remained severely pancreatic insufficient. There was no significant change in gut microbiota diversity and richness following treatment.

      Conclusion

      There was no significant change in gut inflammation after partial restoration of CFTR function with ivacaftor, suggesting that excess gut inflammation in CF is multi-factorial in aetiology. In this adult cohort, exocrine pancreatic function was irreversibly lost. Longer term follow-up may reveal more dynamic changes in the gut microbiota and possible restoration of CFTR function.

      Keywords

      1. Introduction

      Cystic Fibrosis (CF) is a multisystem disorder with prominent respiratory involvement but also gastrointestinal and pancreatic manifestations. Ivacaftor is the first medication which potentiates CFTR protein function in the setting of class III (gating) and class IV (conductance) CFTR mutations [
      • Barry PJ
      • Ronan N
      • Plant BJ.
      Cystic fibrosis transmembrane conductance regulator modulators: the end of the beginning.
      ]. Ivacaftor produces significant clinical benefit in people with CF (PWCF) who carry the G551D-CFTR mutation, with significant improvement in lung function (FEV1), BMI and respiratory symptoms, as well as reduction in sweat chloride and pulmonary exacerbations reported [
      • Ramsey BW
      • Davies J
      • McElvaney NG
      • Tullis E
      • Bell SC
      • Dřevínek P
      • et al.
      A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.
      ,
      • Davies JC
      • Wainwright CE
      • Canny GJ
      • Chilvers MA
      • Howenstine MS
      • Munck A
      • et al.
      Efficacy and safety of ivacaftor in patients aged 6 to 11 years with cystic fibrosis with a G551D mutation.
      ]. Currently, the G551D mutation, is the most prevalent of the CFTR gating mutations with a worldwide prevalence of 4-5% and is amenable to treatment with ivacaftor monotherapy [
      • Bobadilla JL
      • Macek Jr, M
      • Fine JP
      • Farrell PM
      Cystic fibrosis: a worldwide analysis of CFTR mutations–correlation with incidence data and application to screening.
      ]. However, there is significant regional variation with a greater prevalence in the Cork Adult Cystic Fibrosis Centre of 23% [
      • De Boeck K
      • Zolin A
      • Cuppens H
      • Olesen H
      • Viviani L.
      The relative frequency of CFTR mutation classes in European patients with cystic fibrosis.
      ,
      • Ronan NJ
      • Einarsson GG
      • Twomey M
      • Mooney D
      • Mullane D
      • NiChroinin M
      • et al.
      CORK study in cystic fibrosis: sustained improvements in ultra-low-dose chest CT scores After CFTR modulation with Ivacaftor.
      ].
      Studies applying the PillCam SB imaging technology (Given Imaging, Yokneam, Israel) have demonstrated increased inflammation in the CF gut [
      • Werlin SL
      • Benuri-Silbiger I
      • Kerem E
      • Adler SN
      • Goldin E
      • Zimmerman J
      • et al.
      Evidence of intestinal inflammation in patients with cystic fibrosis.
      ]. Analysis of gut lavage samples from children with CF demonstrated increased levels of inflammatory markers including neutrophil elastase, IL-1β, IL-8 and IgG. An increased mononuclear cell influx into the duodenal mucosa with consequent increased expression of IL-2, ICAM-1 and IFN-γ has also been reported in children with CF [
      • Raia V
      • Maiuri L
      • de Ritis G
      • de Vizia B
      • Vacca L
      • Conte R
      • et al.
      Evidence of chronic inflammation in morphologically normal small intestine of cystic fibrosis patients.
      ]. Faecal calprotectin (FCP) is present in high levels in individuals with intestinal inflammation and has been shown to be elevated in PWCF compared to non-CF controls [
      • Werlin SL
      • Benuri-Silbiger I
      • Kerem E
      • Adler SN
      • Goldin E
      • Zimmerman J
      • et al.
      Evidence of intestinal inflammation in patients with cystic fibrosis.
      ,
      • Summerton CB
      • Longlands MG
      • Wiener K
      • Shreeve DR.
      Faecal calprotectin: a marker of inflammation throughout the intestinal tract.
      ]. Probiotic treatment has been reported to reduce faecal calprotecin levels in patients with CF [
      • Bruzzese E
      • Raia V
      • Gaudiello G
      • Polito G
      • Buccigrossi V
      • Formicola V
      • et al.
      Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration.
      ]. Furthermore, faecal lactoferrin (FLF) is also regarded as a marker of gut inflammation and has been detected in the gut of PWCF [
      • Kane SV
      • Sandborn WJ
      • Rufo PA
      • Zholudev A
      • Boone J
      • Lyerly D
      • et al.
      Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation.
      ].
      The KIWI study demonstrated improvements in exocrine pancreatic function, as reflected by faecal elastase (FE), in young children with CF aged 2-5 years treated with ivacaftor. Moreover, a further case report highlighted dynamic changes in faecal elastase in an older child treated with ivacaftor [
      • Davies JC
      • Cunningham S
      • Harris WT
      • Lapey A
      • Regelmann WE
      • Sawicki GS
      • et al.
      Safety, pharmacokinetics, and pharmacodynamics of ivacaftor in patients aged 2-5 years with cystic fibrosis and a CFTR gating mutation (KIWI): an open-label, single-arm study.
      ,
      • Howlett C
      • Ronan NJ
      • NiChroinin M
      • Mullane D
      • Plant BJ
      Partial restoration of pancreatic function in a child with cystic fibrosis.
      ]. Similarly, the Arrival study in children aged 12 to <24 months with gating mutations commenced on ivacaftor demonstrated an absolute increase in faecal elastase of 164.7 μg/g after 24 weeks of treatment [
      • Rosenfeld M
      • Wainwright CE
      • Higgins M
      • Wang LT
      • McKee C
      • Campbell D
      • et al.
      Ivacaftor treatment of cystic fibrosis in children aged 12 to <24 months and with a CFTR gating mutation (ARRIVAL): a phase 3 single-arm study.
      ]. Six of nine children who were pancreatic insufficient before commencing ivacaftor were pancreatic sufficient after 24 weeks of treatment, with increases in faecal elastase being noted after 2 weeks of treatment and sustained to 24 weeks [
      • Rosenfeld M
      • Wainwright CE
      • Higgins M
      • Wang LT
      • McKee C
      • Campbell D
      • et al.
      Ivacaftor treatment of cystic fibrosis in children aged 12 to <24 months and with a CFTR gating mutation (ARRIVAL): a phase 3 single-arm study.
      ]. Immunoreactive trypsinogen – a marker of pancreatic injury – were also noted to improve from week 2 of treatment and had improved by 56% after 24 weeks of treatment [
      • Rosenfeld M
      • Wainwright CE
      • Higgins M
      • Wang LT
      • McKee C
      • Campbell D
      • et al.
      Ivacaftor treatment of cystic fibrosis in children aged 12 to <24 months and with a CFTR gating mutation (ARRIVAL): a phase 3 single-arm study.
      ]. Additionally, improvements in serum lipase and amylase were reported from 4 days after commencing treatment and maintained to 24 weeks. In infants aged 4 to <12 months with gating mutations ivacaftor resulted in a 166  μg/g increase in faecal elastase after 24 weeks [
      • Davies JC
      • Wainwright CE
      • Sawicki GS
      • Higgins MN
      • Campbell D
      • Harris C
      • et al.
      Ivacaftor in Infants Aged 4 to <12 Months with Cystic Fibrosis and a Gating Mutation. Results of a Two-Part Phase 3 Clinical Trial.
      ]. Thus, results from paediatric cohorts aged under 5 years commencing ivacaftor have reported improvement in exocrine pancreatic function. We aimed to evaluate response in an adult cohort.
      Emerging evidence, although limited by sample size, reveals a unique gut microbiota in paediatric PWCF with lower species richness, evenness and diversity being reported compared to healthy paediatric controls [
      • Bruzzese E
      • Callegari ML
      • Raia V
      • Viscovo S
      • Scotto R
      • Ferrari S
      • et al.
      Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial.
      ,
      • Nielsen S
      • Needham B
      • Leach ST
      • Day AS
      • Jaffe A
      • Thomas T
      • et al.
      Disrupted progression of the intestinal microbiota with age in children with cystic fibrosis.
      ,
      • Coffey MJ
      • Nielsen S
      • Wemheuer B
      • Kaakoush NO
      • Garg M
      • Needham B
      • et al.
      Gut microbiota in children with cystic fibrosis: a taxonomic and functional dysbiosis.
      ]. While recurrent courses of antibiotics certainly contribute to this, disturbances in gut microbiota have been reported in young children with CF [
      • Bruzzese E
      • Callegari ML
      • Raia V
      • Viscovo S
      • Scotto R
      • Ferrari S
      • et al.
      Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial.
      ]. The aetiology of this is likely multifactorial with CFTR dysfunction and its resultant changes in intestinal environment and gut transit, pancreatic enzyme replacement therapy, a traditional CF high fat diet and gut inflammation all contributing [
      • Coffey MJ
      • Nielsen S
      • Wemheuer B
      • Kaakoush NO
      • Garg M
      • Needham B
      • et al.
      Gut microbiota in children with cystic fibrosis: a taxonomic and functional dysbiosis.
      ,
      • Sutherland R
      • Katz T
      • Liu V
      • Quintano J
      • Brunner R
      • Tong CW
      • et al.
      Dietary intake of energy-dense, nutrient-poor and nutrient-dense food sources in children with cystic fibrosis.
      ,
      • Debyser G
      • Mesuere B
      • Clement L
      • Van de Weygaert J
      • Van Hecke P
      • Duytschaever G
      • et al.
      Faecal proteomics: a tool to investigate dysbiosis and inflammation in patients with cystic fibrosis.
      ,
      • Vernocchi P
      • Del Chierico F
      • Russo A
      • Majo F
      • Rossitto M
      • Valerio M
      • et al.
      Gut microbiota signatures in cystic fibrosis: loss of host CFTR function drives the microbiota enterophenotype.
      ]. In this study, we hypothesised that treatment with ivacaftor reduces inflammation within the gut of PWCF and changes gut microbiota composition and structure. Given that previous studies have demonstrated an increase in lung microbiota diversity with ivacaftor treatment, we further hypothesised that it may also lead to increased diversity in the gut microbiome [
      • Ronan NJ
      • Einarsson GG
      • Twomey M
      • Mooney D
      • Mullane D
      • NiChroinin M
      • et al.
      CORK study in cystic fibrosis: sustained improvements in ultra-low-dose chest CT scores After CFTR modulation with Ivacaftor.
      ,
      • Rowe SM
      • Heltshe SL
      • Gonska T
      • Donaldson SH
      • Borowitz D
      • Gelfond D
      • et al.
      Clinical mechanism of the cystic fibrosis transmembrane conductance regulator potentiator Ivacaftor in G551D-mediated cystic fibrosis.
      ].

      2. Materials and methods

      2.1 Patient assessment

      Fourteen PWCF (age ≥16 years) with at least one copy of the G551D mutation provided written informed consent to participate in the study. This cohort represents a subset of patients from our previously published study [
      • Ronan NJ
      • Einarsson GG
      • Twomey M
      • Mooney D
      • Mullane D
      • NiChroinin M
      • et al.
      CORK study in cystic fibrosis: sustained improvements in ultra-low-dose chest CT scores After CFTR modulation with Ivacaftor.
      ]. Patients were assessed between March 2013 and March 2014 and followed for a median of 12 months after ivacaftor treatment. Participants attended the Cystic Fibrosis Day Centre when clinically stable and free from pulmonary exacerbation before commencing ivacaftor and routinely on a three-monthly basis thereafter for the first year of ivacaftor in keeping with our routine requirements for monitoring patients commenced on ivacaftor at the time. Patients performed spirometry in keeping with ATS/ERS guidelines, a sweat test and had their BMI recorded at each visit. They also completed the Cystic Fibrosis Questionnaire Revised (CFQR) at baseline [
      • Quittner AL
      • Buu A
      • Messer MA
      • Modi AC
      • Watrous M.
      Development and validation of the cystic fibrosis questionnaire in the United States: a health-related quality-of-life measure for cystic fibrosis.
      ]. The number of intravenous (IV) antibiotic courses for treatment of pulmonary exacerbations was documented prospectively for 6 - 12 months after commencement of ivacaftor and for the same matched period in the previous year depending on the period of patient follow-up. The patients consented to donate a stool sample before commencing ivacaftor and at routine three monthly follow-up, when possible. Stool samples were collected in sterile universal containers and frozen at -80⁰C for later DNA extraction, and FCP, FLF and FE-1 measurement. Ethical approval was obtained from the Clinical Research Ethics Committee of the Cork Teaching Hospitals (ECM 4 [j] February, 7, 2013).

      2.2 Intestinal inflammation

      Levels of FCP and FLF were evaluated in 14 baseline samples each with at least one follow-up time-point after commencement of ivacaftor. FCP was measured using the BÜHLMANN Calprotectin ELISA for faecal samples (BÜHLMANN, Switzerland) in keeping with manufacturers recommendations with further details provided in the online supplement. FLF was measured using IBD-Scan Techlab ELISA in keeping with manufacturers guidelines

      2.3 Gut microbiota sequencing and analysis

      Stool samples were stored at -80ºC for up to 9 months prior to DNA extraction. In brief, DNA was extracted using the Repeated Bead Beating (RBB) DNA extraction method previously described by Yu and Morrison (2004), followed by sequencing of the V4 region of the 16S rRNA marker-gene on the Illumina MiSeq platform. Further details regarding sample handling, processing and library construction are provided in the online supplement.

      2.4 Statistical analysis

      Data was analysed using SPSS version 22.0 and STATA. Baseline pre ivacaftor stool sample was compared to a paired stool sample from the same patient post ivacaftor. The paired Wilcoxon signed rank test was used to analyse changes in clinical parameters, inflammatory markers (FCP and FLF) and FE-1 levels. Change in categorical variables were analysed using the Chi squared test.
      Changes between before and after ivacaftor treatment in specific phyla and families were based on centred-log-ratio (clr) transformed data. Further details on this can be found in the online supplement.

      3. Results

      Fourteen PWCF participated, with a median duration of ivacaftor treatment of 12 months (range 3-12 months) at the time of post ivacaftor stool analysis. Table 1 summarises baseline characteristics. All patients had a class I-III mutation as their second mutation.
      Table 1Baseline characteristics.
      Median Age (range)29 years (18-39)
      Gender
      Male % (n)64.3% (9)
      Genotype % (n)
      G551D/F508del78.58% (11)
      G551D/G551D7.14% (1)
      G551D/R533X7.14% (1)
      G551D/3028delA

      Total Number of IV antibiotic courses pre ivacaftor

      Total Number of IV antibiotic courses post ivacaftor
      7.14% (1)

      12

      4

      3.1 Clinical response

      Significant increases in FEV1 (p < 0.05) and BMI (p < 0.05) and a decrease in sweat chloride (p < 0.05) were observed with ivacaftor therapy (Table 2). A significant improvement in CFQ-R weight domain was also observed (p = 0.02). There was no statistically significant change in the CFQ-R eating domain (p = 0.60), body image domain (p = 0.20) or digestive domain (p = 0.72). A significant 75% reduction in intravenous antibiotic prescribing was observed after ivacaftor treatment (p = 0.047).
      Table 2Clinical parameters before and after ivacaftor (median, range).
      BeforeAfterp-value
      FEV1 % predicted65.5 (31.0-91.0)72.0 (44.5-106.5)2.07*10−3
      Sweat Chloride (mmol/l)98.0 (81.0-155.0)41.5 (24.0-93.0)1.22*10-4
      BMI (kg/m2)22.0 (18.2-29.5)23.0 (19.7 – 30.6)1.43*10−2
      CFQ-R
      CFQ-R, Cystic Fibrosis Questionnaire revised (ranging between 0 and 100, with a higher score indicates a higher patient-reported quality of life with regard to respiratory and emotional status).
      Eating100 (77.8-100)100 (77.8-100)1.00
      Body Image77.8 (11.1-100)83.35 (11.1-100)0.20
      Digestive88.9 (44.4-100)88.9(66.7-100)0.90
      Weight66.7 (0-100)100 (33.3-100)0.02
      CFQ-R, Cystic Fibrosis Questionnaire revised (ranging between 0 and 100, with a higher score indicates a higher patient-reported quality of life with regard to respiratory and emotional status).

      3.2 Faecal elastase & gut inflammatory markers

      No change in levels of biomarkers associated with inflammatory processes were observed between samples collected before and after ivacaftor treatment with levels of FE-1 (p = 0.614), FCP (p = 0.22) and FLF (p = 0.27) similar (Fig. 1). All patients had an FE-1 level less than 100 μl/l before and after treatment, consistent with severe pancreatic insufficiency. There was no significant change in FCP c (p = 0.12) or FLF category (p = 1.00) following treatment.
      Fig 1
      Fig. 1Inflammatory biomarker levels prior to commencing ivacaftor treatment (PRE) and at ivacaftor follow-up (POST) visit. p < 0.05 denotes statistical significance (n = 14 for figure A, B&C).

      3.3 Changes in the Gut microbiota with ivacaftor treatment

      Following treatment with ivacaftor, no difference was observed in number of taxa present in stool samples (p > 0.05) (Fig. 2). At the phylum level, communities were predominately composed of Bacteroidetes and Firmicutes, with lower level of Proteobacteria detected in a number of samples (Fig. 2A). Overall, we observed a shift in community composition for the main phyla from communities with a higher relative abundance of Firmicutes to communities with a higher relative abundance of Bacteroidetes following treatment (Fig. 2A and B). While a reduction in Firmicutes (p = 0.04) and an increase in Bacteroidetes observed (p = 0.05); following adjustment for multiple testing, these changes were not statistically significant.
      Fig 2
      Fig. 2(A) Relative abundance (%) for the main phyla PRE- and POST-ivacaftor treatment, (B) Firmicutes/Bacteroidetes ratio (log2-fold change) between PRE- and POST-ivacaftor treatment, (C) Relative abundance (%) of families between PRE- and POST-ivacaftor treatment for each individual. Values shown depict percentage relative abundance >1% of the total bacteria detected.
      In total, we detected 50 different bacterial families at the combined visit time-points (total PRE: 48 [range 21-32]; total POST: 46 [range 20-31] (Fig. 2C). Amongst taxa that accounted for >1% of total read number at the family level, 13/14 were shared between PRE- and POST-ivacaftor treatment visits (Supplemental Table 1). Only two bacterial families, Bacteroidaceae and Porphyromonadaceae, were observed to be significantly different between the PRE- and POST-ivacaftor visits (Supplementary Table 1). However, these differences were not statistically significantly following adjustment for multiple testing (p > 0.05). Furthermore, following treatment with ivacaftor, there was no significant change in the main ecological community indices such as taxonomic richness [S], Shannon-Wiener diversity [H’], evenness [eH/S] or dominance [D] (Fig. 3).
      Fig 3
      Fig. 3Changes in ecological indices following treatment with ivacaftor. Comparison between PRE- and POST-ivacaftor treatment visits; (A) taxonomic richness [S], (B) community diversity (Shannon Wiener Index [H’], (C) community evenness [eH/S] and taxonomic dominance [D]. p < 0.05 denotes statistical significance.

      4. Microbial variance explained by clinical and other covariates

      Principle coordinate analysis (PCoA) of clr-transformed OTU counts showed that there were no clear clusters of samples based on sample groups (PRE- vs. POST-ivacaftor) (Supplementary Figure 1A). Moreover, we employed Bray-Curtis dissimilarity measurements between PRE- and POST-ivacaftor treatment time-points (beta-diversity) in order to identify and compare the underlying bacterial community structures. Dispersion using the permuted betadisper test, as implemented within the vegan package [ver. 2.5-4] [

      Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O'Hara RB, et al. Vegan: community ecology package. R package vegan, vers. 2.2-1 2015.

      ] was not significant between PRE- and POST-ivacaftor visits (p = 0.26) (Supplemental Figure 2A). Furthermore, the adonis (Permutational Multivariate Analysis of Variance Using Distance Matrices) function as implemented within vegan package (ver. 2.5-4) [

      Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O'Hara RB, et al. Vegan: community ecology package. R package vegan, vers. 2.2-1 2015.

      ] in R was used to test for location shift of the spatial medians based on sample time-points, and showed non-significant variation in community structure between PRE- and POST-ivacaftor visits (adonis test, F = 1.16, R2 = 0.04, p = 0.21) (Supplementary Figure 2B).

      5. Discussion

      In a clinically responsive cohort, we did not observe a significant change in markers of gut inflammation following treatment with ivacaftor. Given that partial restoration of CFTR function did not result in a reduction in gut inflammation, this would suggest that additional mechanisms give rise to this process in CF. Previous work in children with CF demonstrated a reduction in faecal calprotectin levels after treatment with probiotic in the form of Lactobacillus rhamnosus [
      • Bruzzese E
      • Callegari ML
      • Raia V
      • Viscovo S
      • Scotto R
      • Ferrari S
      • et al.
      Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial.
      ]. It has been suggested that there may be a relationship between intestinal microbiota composition and inflammation. The lack of a significant change in the gut microbiota in this study would support this hypothesis in CF. A study in ten patients treated with ivacaftor demonstrated an improvement in gastrointestinal pH but no change in gastro-intestinal motility with enhancement of CFTR function [
      • Gelfond D
      • Heltshe S
      • Ma C
      • Rowe SM
      • Frederick C
      • Uluer A
      • et al.
      Impact of CFTR Modulation on Intestinal pH, Motility, and Clinical Outcomes in Patients With Cystic Fibrosis and the G551D Mutation.
      ]. However, despite the ability of ivacaftor to improve gut pH no effect was noted on inflammation. In contrast to reports in young children with CF, there was no significant change in FE-1 in our adult cohort after treatment with ivacaftor [
      • Davies JC
      • Cunningham S
      • Harris WT
      • Lapey A
      • Regelmann WE
      • Sawicki GS
      • et al.
      Safety, pharmacokinetics, and pharmacodynamics of ivacaftor in patients aged 2-5 years with cystic fibrosis and a CFTR gating mutation (KIWI): an open-label, single-arm study.
      ,
      • Howlett C
      • Ronan NJ
      • NiChroinin M
      • Mullane D
      • Plant BJ
      Partial restoration of pancreatic function in a child with cystic fibrosis.
      ]. This suggests that exocrine pancreatic function may be irreversibly damaged in adults. Notwithstanding the lack of enhancement of exocrine pancreatic function, a significant weight gain was observed in our cohort. Thus, it appears that other mechanisms underlie this weight gain.
      We did not demonstrate a significant change in the gut microbiota with ivacaftor treatment. Studies assessing the effect of ivacaftor on the lung microbiota have demonstrated subtle changes in composition, a reduction in the relative abundance of Pseudomonas aeruginosa, a reduction in likelihood of culturing Pseudomonas aeruginosa and an increase in diversity [
      • Ronan NJ
      • Einarsson GG
      • Twomey M
      • Mooney D
      • Mullane D
      • NiChroinin M
      • et al.
      CORK study in cystic fibrosis: sustained improvements in ultra-low-dose chest CT scores After CFTR modulation with Ivacaftor.
      ,
      • Rowe SM
      • Heltshe SL
      • Gonska T
      • Donaldson SH
      • Borowitz D
      • Gelfond D
      • et al.
      Clinical mechanism of the cystic fibrosis transmembrane conductance regulator potentiator Ivacaftor in G551D-mediated cystic fibrosis.
      ,
      • Harris JK
      • Wagner BD
      • Zemanick ET
      • Robertson CE
      • Stevens MJ
      • Heltshe SL
      • et al.
      Changes in airway microbiome and inflammation with Ivacaftor treatment in patients with cystic fibrosis and the G551D mutation.
      ]. Studies in non-CF cohorts have demonstrated that antibiotic treatment results in a reduction in gut microbial diversity and changes in relative abundances of bacterial species, with some of these changes persisting for protracted periods of time after discontinuation of treatment [
      • Jakobsson HE
      • Jernberg C
      • Andersson AF
      • Sjölund-Karlsson M
      • Jansson JK
      • Engstrand L
      Short-term antibiotic treatment has differing long-term impacts on the human throat and Gut microbiome.
      ,
      • Dethlefsen L
      • Relman DA.
      Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation.
      ]. Despite the fact that our cohort experienced a 75% reduction in IV antibiotic therapy with ivacaftor treatment, they have had recurrent courses of oral and IV antibiotics over a lifetime of CF.
      In contrast to our results, a recent study by Ooi et al., evaluating changes in the gut microbiota in 16 patients with CF (8 adults and 8 children) after treatment with ivacaftor, demonstrated a significant reduction in faecal calprotectin [
      • Ooi CY
      • Syed SA
      • Rossi L
      • Garg M
      • Needham B
      • Avolio J
      • et al.
      Impact of CFTR modulation with Ivacaftor on gut microbiota and intestinal inflammation.
      ]. It is possible that age might contribute to differences between the two studies; the median age of the cohort reported by Ooi et al. was 17.1 years, while the median age of our cohort was over a decade older at 29 years. Gender is another factor which may have contributed to differences as females accounted for 62.5% of the cohort in the Ooi study compared to 35.7% in our group. Additionally, our patient cohort had more severe disease. The median baseline FEV1 was 76% (range 56.3-96%) in the Ooi cohort compared to a median FEV1 65.5% (range 31-91%) in our patient group. In keeping with the findings of Ooi et al., we did not find a significant change in alpha or beta diversity [
      • Ooi CY
      • Syed SA
      • Rossi L
      • Garg M
      • Needham B
      • Avolio J
      • et al.
      Impact of CFTR modulation with Ivacaftor on gut microbiota and intestinal inflammation.
      ].
      Kirstensen et al. demonstrated a significant increase in gut alpha diversity after ivacaftor in a cohort of 16 patients with CF with the S1251N mutation [
      • Kristensen MI
      • de Winter-de Groot KM
      • Berkers G
      • Chu M
      • Arp K
      • Ghijsen S
      • et al.
      Individual and group response of treatment with Ivacaftor on airway and gut microbiota in people with CF and a S1251N mutation.
      ]. However, the mean age of their cohort (22.5 years) was younger than our current cohort and mean baseline FEV1 was higher at 76% predicted [
      • Kristensen MI
      • de Winter-de Groot KM
      • Berkers G
      • Chu M
      • Arp K
      • Ghijsen S
      • et al.
      Individual and group response of treatment with Ivacaftor on airway and gut microbiota in people with CF and a S1251N mutation.
      ]. Similarly, in vitro work demonstrated greater potentiation of the S1251N by ivacaftor than the G551D mutation [
      • Yu H
      • Burton B
      • Huang C-J
      • Worley J
      • Cao D
      • Johnson JP
      • et al.
      Ivacaftor potentiation of multiple CFTR channels with gating mutations.
      ].
      Interestingly Pope et al. assessed faecal elastase levels in 12 pancreatic sufficient adult patients with the R117H mutation on ivacaftor and 8 with the F508del mutation treated with lumacaftor-ivacaftor and did not demonstrate a significant change in faecal elastase levels in either cohort [
      • Pope CE
      • Vo AT
      • Hayden HS
      • Weiss EJ
      • Durfey S
      • McNamara S
      • et al.
      Changes in fecal microbiota with CFTR modulator therapy: a pilot study.
      ]. Similarly they did not demonstrate a significant overall change in gut microbiota;, however, they did note subtle changes in individual taxa with a non-statistically significant decrease in Bacteroidetes and an increase in Firmicutes in the F508del group after lumacaftor-ivacaftor in contrast to our cohort [
      • Pope CE
      • Vo AT
      • Hayden HS
      • Weiss EJ
      • Durfey S
      • McNamara S
      • et al.
      Changes in fecal microbiota with CFTR modulator therapy: a pilot study.
      ].
      A limitation of this study is the small sample size (n = 14) thus the study may have been underpowered to detect a statistically difference. The lack of a control cohort is also a limitation. However, given that the global prevalence of the G551D-CFTR mutation is 4-5% this represents a relatively large sample size, from a single centre. An additional limitation is that it is predominantly an adult cohort. It is possible that ivacaftor treatment in younger children, with a shorter duration of pancreatic insufficiency, may result in a greater improvement of exocrine pancreatic function.

      6. Conclusion

      There was no significant change in exocrine pancreatic function in an older cohort of patients with CF after commencement of ivacaftor after short term follow up suggesting that it does not enhance exocrine pancreatic function in the short term. This may reflect established long standing pancreatic destruction which is irreversible, however, a longer period of follow up may be required to delineate longer term changes. There was no significant change in gut inflammation nor gut microbiota composition and structure after treatment. This is likely due to either a longer duration of treatment may be required to observe significant change or additional factors contributing to inflammation and microbiota changes in the CF gut.

      Funding

      We would like to acknowledge funding from CFMATTERS which is funded under the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n°603038.

      CRediT authorship contribution statement

      NJ Ronan: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing. GG Einarsson: Conceptualization, Formal analysis, Data curation, Writing – original draft, Writing – review & editing. J Deane: Investigation, Methodology, Writing – review & editing. F Fouhy: Investigation, Methodology, Writing – review & editing. M Rea: Investigation, Methodology, Writing – review & editing. C Hill: Investigation, Methodology, Writing – review & editing. F Shanahan: Investigation, Methodology, Writing – review & editing. JS Elborn: Investigation, Conceptualization, Methodology, Writing – original draft, Writing – review & editing. RP Ross: Resources, Supervision, Methodology, Writing – review & editing. M McCarthy: Investigation, Methodology, Writing – review & editing. DM Murphy: Investigation, Methodology, Writing – review & editing. JA Eustace: Investigation, Methodology, Data curation, Writing – review & editing. Tunney MM: Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing. C Stanton: Investigation, Resources, Writing – review & editing. BJ Plant: Conceptualization, Methodology, Investigation, Resources, Writing – original draft, Writing – review & editing, Funding acquisition, Project administration, Supervision.

      Appendix. Supplementary materials

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