DOI: 10.5061/dryad.d2547d8b5
title: RNA seq data from: A randomized, placebo-controlled trial shows that nicotinamide riboside reduces airway inflammation in COPD

description: Chronic obstructive pulmonary disease (COPD) is a progressive incurable
 disease associated with smoking and advanced age, ranking as the third
 leading cause of death worldwide. DNA damage and loss of the central
 metabolite nicotinamide adenine dinucleotide (NAD) may contribute to both
 aging and COPD, presenting a potential avenue for interventions. In this
 randomized, double-blinded, placebo-controlled clinical trial, we treated
 patients with stable COPD (n = 40) with the NAD precursor nicotinamide
 riboside (NR) for 6 weeks and followed up 12 weeks after. The primary
 outcome was change in sputum interleukin-8 (IL-8) from baseline to week 6.
 The estimated treatment difference between NR and placebo in IL-8 after 6
 weeks was -52.6% (95% confidence interval (CI), -75.7 to -7.6%; P =
 0.030). This effect persisted until the follow-up twelve weeks after the
 end of treatment (-63.7%: 95% CI, -85.7 to -7.8%; P = 0.034). For
 secondary outcomes, NR treatment increased NAD levels by more than
 two-fold in whole-blood while IL-6 levels in plasma remained unchanged. In
 exploratory analyses, treatment with NR showed indications of upregulated
 gene pathways related to genomic integrity in the airways and reduced
 epigenetic aging–possibly through a reduction in cellular senescence.
 These exploratory analyses need to be confirmed in future trials.
 ClinicalTrials.gov identifier NCT04990869.
description: <strong>Data
 collection</strong><em> </em>
 <em>Study participants</em> Forty
 patients with a diagnosis of COPD (post-bronchodilator
 FEV<sub>1</sub>/FVC &lt; 0.7) were enrolled in this study
 (Table 1). Inclusion criteria included age ≥ 60 years, BMI between
 18.5-40.0 kg·m<sup>-2</sup> and a weight ≥ 40 kg at enrolment,
 a smoking history of at least 10 pack years but currently ex-smokers
 within six months, no use of inhalation corticosteroids (ICS), reported
 experiencing worsening of symptoms in relation to respiratory infections
 and a blood eosinophil count of &lt; 0.3 x 10<sup>9</sup>
 cells/L. Exclusion criteria included having had an exacerbation of COPD or
 severe airway infection within the last two months, chronic use of
 supplements containing NR or vitamin B prior to and throughout the trial
 or a cancer diagnosis within 5 years. Furthermore, we enrolled a
 convenience sample of lung-healthy controls that were comparable with the
 patients with COPD in terms of age, sex, and body mass index (BMI) but who
 reported being never-smokers with no history of lung disease (Table 1).
 Two lung-healthy control participants were randomized to the placebo group
 but were later diagnosed with asthma and were therefore excluded from
 further analyses. <em>Study design,
 randomization, and intervention</em> This was a
 single-center, double blind, placebo-controlled clinical trial, with a
 6-week intervention phase and a 12-week follow-up period. The intervention
 consisted of ingesting 2 g NR or placebo for 6 weeks (four 250 mg capsules
 consumed with meals in the morning and evening). Participants attended a
 screening visit and were randomized only if all inclusion criteria and
 none of the exclusion criteria were fulfilled. All participants were
 allocated 1:1 to receive NR or placebo using permuted block randomization
 with a block size of four. Independent blocks were generated for COPD and
 controls, and patients with COPD were additionally randomized stratified
 by CAT score (0-15 and 16-40). Randomization was performed by a member of
 the study team not involved in the assessment of outcomes. The study
 participants and members of the study team involved in the collection and
 analysis of the outcomes were blinded to the treatment condition. The
 placebo capsules had the same size, color, smell and appearance as the
 active drug tablets. The study visits included pre- (week 1), post- (week
 6), and follow-up (week 18) assessments, for which participants arrived in
 the morning following an overnight fast. Participants took the last dose
 of NR/placebo 12-14 h prior to the post-intervention assessments. During
 each of the study visits, venous blood, sputum, and nasal brush samples
 were collected. Lung function was measured using spirometry according to
 standards set by the ATS/ERS<sup> 1</sup> Venous blood samples
 were collected into Vacutainer tubes. Leukocyte count was analyzed using
 standardized clinical assays at Bispebjerg Hospital. Blood for
 NAD<sup>+</sup> quantification was collected into sodium
 citrate tubes and immediately placed on wet ice. Aliquots of 0.1 mL blood
 were added to cryotubes containing 1 mL of 0.5 M perchloric acid, gently
 resuspended and stored at -80°C for later NAD<sup>+</sup>
 quantification. Adverse events were recorded during the post-intervention
 and follow-up assessments. <em>Nasal
 brushes</em> After participants cleaned their
 nose with isotonic saline, nasal brush samples were collected by inserting
 a brush (Gynobrush, Heinz Herenz Hamburg, Germany) ~4 cm into the left
 nostril towards concha media and rotating six times. The same procedure
 was repeated for the right nostril, and the brushes were put into a 15 mL
 falcon tube containing 4 mL PBS. The tube was vortexed for 20 s and
 centrifuged at 600 g for 10 min at 4°C after discarding the brushes. The
 supernatant was discarded, and the cell pellet was lysed in 350 µL RLT
 buffer (Qiagen, Hilden, Germany) containing dithiothreitol (20 µL 2 M DTT
 per 1 mL RLT). RNA was extracted using the QIAcube (Qiagen, Hilden,
 Germany) following the manufacturer’s instructions and samples were stored
 at -80°C. <em>RNA sequencing in nasal epithelial
 cells</em>. RNA sequencing was performed on 151 nasal epithelial
 cell samples collected by nasal brush as indicated above by BGI genomics
 using whole RNA sequencing analysis on samples with a RIN mean 8.0, range
 5.2-9.5. Paired-end reads were aligned to mm9 using bowtie2
 <sup>2</sup>.<sup>.</sup>Differential expression
 analysis was performed using Salmon, tximeta and, DESeq2
 <sup>3</sup>.<sup> </sup>Gene set enrichment
 analysis was performed on DESeq2 normalized counts and comparisons were
 made against baseline values and between COPD and controls. The gene set
 used was based on Gene Ontology (GO) terms. Terms were filtered for
 false-discovery rate (FDR) &lt; 0.05 and sorted by Normalized
 Enrichment Score. Gene sets with FDR&gt;0.05 that were differentially
 expressed following treatment with NR (pre to post) and did not overlap
 with placebo were visualized using EnrichmentMap in Cytoscape (v. 3.9.1).
 Similar gene sets were clustered together into functional groups using
 AutoAnnotate. References Miller, M.
 R. et al. Standardisation of spirometry. Eur Respir J 26, 319–338
 (2005). Langmead, B. & Salzberg, S. L. Fast
 gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359
 (2012). Patro, R., Duggal, G., Love, M. I., Irizarry,
 R. A. & Kingsford, C. Salmon provides fast and bias-aware
 quantification of transcript expression. Nat Methods 14, 417–419
 (2017).
description: # RNA seq data from: A randomized, placebo-controlled trial shows that
 nicotinamide riboside reduces airway inflammation in COPD
 [https://doi.org/10.5061/dryad.d2547d8b5](https://doi.org/10.5061/dryad.d2547d8b5) ## Description of the data and file structure The data includes DESeq2 normalized sequence counts for each participant in the ICON trial [https://clinicaltrials.gov/study/NCT04990869](https://clinicaltrials.gov/study/NCT04990869). RNA was extracted from nasal brushes. Each row in the data matrix is a gene as indicated in the first column, each subsequent column is an individual. The column header indicates the different groups in the study: Male, female, NR, placebo, COPD and/or healthy is indicated in the column header.    ### Files and variables #### File: DESeq2\_normalized\_count\_matrix2.tsv **Description:**  ##### Variables * Genes (first column) * COPD/Healthy (indicated in the column header) * NR/Placebo (indicated in the column header) * Male/Female (indicated in the column header) Here is an example of a column name: *01_baseline_COPD_Placebo_female* This is translated to Participant ID is: *01* The study time-point is: *baseline* Disease status is: *COPD* Treatment is: *placebo* Sex: *female*