The lower airway microbiota and inflammation in chronic obstructive pulmonary disease (COPD) : Thoughts on where to measure it, how to interpret it, and why it might matter

Bakgrunn: Kronisk obstruktiv lungesjukdom (KOLS) er ein kompleks, inflammatorisk sjukdom som forårsakar millionar av dødsfall årleg. Bakteriane i dei nedre luftvegane (mikrobiotaet) og immunresponsar kan spele ei viktig rolle i KOLS. Målingar av dei begge kan tenkast å vera påverka av prøvetakingsmetode. Måla med denne oppgåva var å studere KOLS-kohortar med tanke på 1) høve for vekselbruk av indusert og spontant sputum for målingar av inflammasjonsmarkørar og mikrobiota, 2) endringar i inflammasjonsmarkørar og mikrobiota under sjukdomsforverringar samanlikna med stabil sjukdomsfase og 3) forskjellar i mikrobiota i bronkialskyllevæske (BAL) frå pasientar med KOLS samanlikna med friske kontrollar. Metode: Sputum data kjem frå studiane BergenKOLS og tilhøyrande eksaserbasjonsstudie med 433 pasientar med KOLS inkludert, og 356 som vart følgt med tanke på forverringar. BAL data kjem frå studien MikroKOLS med 130 pasientar med KOLS og 103 friske kontrollar inkludert. Inflammasjonsmarkørar i sputum vart målt med bead based multiplex immunoassay og antimikrobielle peptid med enzyme linked immunosorbent assay. DNA sekvensar vart reinska ved hjelp av både enzym og mekanisk lysering. PCR-amplifisering av 16S rRNA og paired-end sequencing med Illumina MiSeq System vart utført. Data vart analysert i QIIME 1&2, Stata og R. Resultat: Inflammasjonsmarkørar og mikrobiota var signifikant forskjellige i indusert og spontant sputum, og i stabil fase av KOLS samanlikna med under pågåande forverring. I høve til sjukdomsfase var ulikskapane heterogene når ein såg på kvart individ. Mikrobiota i BAL var meir ujamn og rikare på Firmicutes hos pasientar med KOLS samanlikna med friske. Kjønn, alder, røyking, sjukdomsgrad og bruk av inhalasjons-kortikosteroider var ikkje tydeleg assosiert til mikrobiotaet i dei nedre luftvegane. Konklusjon: Sputumprøvetaking påverkar målingar av inflammasjonsmarkørar og mikrobiota. KOLS forverringar og KOLS i seg sjølv er begge assosiert med endringar i luftvegsmikrobiotaet. Det tyder på at mikrobiotaet spelar ei rolle i KOLS.Background: Chronic obstructive pulmonary disease (COPD) is a complex inflammatory disease causing the death of millions annually. The lower airway bacterial community (microbiota) and immune responses could be important for the pathogenesis of COPD. The aims for this thesis were to study COPD cohorts considering if measures of inflammatory markers and microbiota 1) are affected by sputum sampling techniques, 2) differ with COPD state, and 3) if the microbiota differ in bronchoalveolar lavage (BAL) comparing patients with COPD with controls. Methods: Sputum data originated from the Bergen COPD Cohort and exacerbations studies in which 433 patients with COPD were enrolled and 356 followed for exacerbations. BAL data originated from the MicroCOPD study in which 130 patients with COPD and 103 controls were enrolled. Inflammatory markers in sputum were measured by a bead based multiplex immunoassay and antimicrobial peptides by enzyme linked immunosorbent assay. DNA sequences were obtained by enzymatic and mechanical lysis extraction methods, PCR-amplification of the 16S rRNA gene and paired-end sequencing using the Illumina MiSeq System. Data were analysed in QIIME 1&2, Stata, and R. Results: Inflammatory markers and microbiota differed significantly between induced and spontaneous sputum, and between stable state COPD and exacerbations. Differences related to disease state showed great heterogeneity looking at individual participants. The microbiota in BAL sampled in the COPD cohort had lower evenness and higher abundances of Firmicutes compared with controls. Sex, age, smoking, disease severity and use of inhaled corticosteroids were not clearly associated with the lower airway microbiota. Conclusion: Sputum sampling methods influences on measurements of inflammation and microbiota. Exacerbations in COPD and the presence of disease are both associated with microbiota dysbiosis which indicate importance of the lower airway microbiota in the pathogenesis in COPD.Doktorgradsavhandlin

The lower airways microbiome and antimicrobial peptides in idiopathic pulmonary fibrosis differ from chronic obstructive pulmonary disease

Background The lower airways microbiome and host immune response in chronic pulmonary diseases are incompletely understood. We aimed to investigate possible microbiome characteristics and key antimicrobial peptides and proteins in idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Methods 12 IPF patients, 12 COPD patients and 12 healthy controls were sampled with oral wash (OW), protected bronchoalveolar lavage (PBAL) and right lung protected sterile brushings (rPSB). The antimicrobial peptides and proteins (AMPs), secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 & hBD-2), were measured in PBAL by enzyme linked immunosorbent assay (ELISA). The V3V4 region of the bacterial 16S rDNA gene was sequenced. Bioinformatic analyses were performed with QIIME 2. Results hBD-1 levels in PBAL for IPF were lower compared with COPD. The predominant phyla in IPF were Firmicutes, Bacteroides and Actinobacteria; Proteobacteria were among top three in COPD. Differential abundance analysis at genus level showed significant differences between study groups for less abundant, mostly oropharyngeal, microbes. Alpha diversity was lower in IPF in PBAL compared to COPD (p = 0.03) and controls (p = 0.01), as well as in rPSB compared to COPD (p = 0.02) and controls (p = 0.04). Phylogenetic beta diversity showed significantly more similarity for IPF compared with COPD and controls. There were no significant correlations between alpha diversity and AMPs. Conclusions IPF differed in microbial diversity from COPD and controls, accompanied by differences in antimicrobial peptides. Beta diversity similarity between OW and PBAL in IPF may indicate that microaspiration contributes to changes in its microbiome.publishedVersio

Sputum microbiota and inflammation at stable state and during exacerbations in a cohort of chronic obstructive pulmonary disease (COPD) patients

Background: Exacerbations of chronic obstructive pulmonary disease (COPD) are debilitating events and spur disease progression. Infectious causes are frequent; however, it is unknown to what extent exacerbations are caused by larger shifts in the airways’ microbiota. The aim of the current study was to analyse the changes in microbial composition between stable state and during exacerbations, and the corresponding immune response. Methods: The study sample included 36 COPD patients examined at stable state and exacerbation from the Bergen COPD Cohort and Exacerbations studies, and one patient who delivered sputum on 13 different occasions during the three-year study period. A physician examined the patients at all time points, and sputum induction was performed by stringent protocol. Only induced sputum samples were used in the current study, not spontaneously expectorated sputum. Sputum inflammatory markers (IL-6, IL-8, IL-18, IP-10, MIG, TNF-α) and antimicrobial peptides (AMPs, i.e. LL-37/hCAP-18, SLPI) were measured in supernatants, whereas target gene sequencing (16S rRNA) was performed on corresponding cell pellets. The microbiome bioinformatics platform QIIME2TM and the statistics environment R were applied for bioinformatics analyses. Results: Levels of IP-10, MIG, TNF-α and AMPs were significantly different between the two disease states. Of 36 sample pairs, 24 had significant differences in the 12 most abundant genera between disease states. The diversity was significantly different in several individuals, but not when data was analysed on a group level. The one patient case study showed longitudinal dynamics in microbiota unrelated to disease state. Conclusion: Changes in the sputum microbiota with changing COPD disease states are common, and are accompanied by changes in inflammatory markers. However, the changes are highly individual and heterogeneous events.publishedVersio

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.publishedVersio

COPDindspon_mapping_DRYAD

Metadatafile for Comparing microbiota profiles in induced and spontaneous sputum samples in COPD patients, allowing rerun of analysis

COPDindspon_commands_DRYAD

Command file for correct sample selection and creation of sub populations as in article: Comparing microbiota profiles in induced and spontaneous sputum samples in COPD patients

uc_fast_params

Parameter file necessary for OTU-picking

The gut microbiota in chronic obstructive pulmonary disease varies by CT-verified emphysema status

Background and aim The association of the gut microbiota to chronic obstructive pulmonary disease (COPD) phenotypes is underexplored. We aimed to compare stool samples from COPD patients and healthy controls and relate findings to common COPD phenotypes. Methods Single-centre case-control study with 62 current and former smoking COPD patients and 49 controls. DNA was extracted from stool samples, and the V3V4-region of the bacterial 16S-rRNA gene was sequenced. Emphysema was defined based on thoracic computed tomography (CT thorax) low attenuating areas &ge;/</span

Data from: Comparing microbiota profiles in induced and spontaneous sputum samples in COPD patients

Background: Induced and spontaneous sputum are used to evaluate the airways microbiota. Whether the sputum types can be used interchangeably in microbiota research is unknown. Our aim was to compare microbiota in induced and spontaneous sputum from COPD patients sampled during the same consultation. Methods: COPD patients from Bergen, Norway, were followed between 2006/2010, examined during the stable state and exacerbations. 30 patients delivered 36 sample pairs. DNA was extracted by enzymatic and mechanical lysis methods. The V3-V4 region of the 16S rRNA gene was PCR-amplified and prepared for paired-end sequencing. Illumina Miseq System was used for sequencing, and Quantitative Insights Into Microbial Ecology (QIIME) and Stata were used for bioinformatics and statistical analyses. Results: Approximately 4 million sequences were sorted into 1004 different OTUs and further assigned to 106 different taxa. Pair-wise comparison of both taxonomic composition and beta-diversity revealed significant differences in one or both parameters in 1/3 of sample pairs. Alpha-diversity did not differ. Comparing abundances for each taxa identified, showed statistically significant differences between the mean abundances in induced versus spontaneous samples for 15 taxa when disease state was considered. This included potential pathogens like Haemophilus and Moraxella. Conclusion: When studying microbiota in sputum samples one should take into consideration how samples are collected and avoid the usage of both induced and spontaneous sputum in the same study

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients

Background: Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD). Methods: 433 COPD patients aged 40¿76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-?) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman¿s rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant. Results: Agreement between the two measurements was generally low for all six markers. TNF-? was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured. Conclusion: We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum