The Microbiome in Bronchial Biopsies from Smokers and Ex-Smokers with Stable COPD - A Metatranscriptomic Approach

Abstract

Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n = 5) and ex-smokers (n = 6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways.

Trial registration::

• ClinicalTrials.gov Identifiers: NCT00158847

Keywords:

• COPD
• metatranscriptomics
• microbiome
• bronchial biopsy

Acknowledgements

The author would like to thank Arnau Vich Vila (University of Groningen, Groningen, The Netherlands) for his scientific input regarding the development of the RNA-seq analysis pipeline in this study.

Author contributions

B.D., J.B., V.G., MvdB., A.F. contributed to the study concept and design. P.H., W.T., MvdB., H.K., coordinated patient inclusion and data collection for GLUCOLD. MvdB., C.B., M.N., M.G., G.T., organized and performed the RNA sequencing. B.D., J.B., V.G., MvdB., A.F., N.C., J.R. analyzed and interpreted data. B.D drafted the manuscript which was critically read and revised by the other coauthors.

Funding

The submitted work is cofinanced by the Ministry of Economic Affairs and Climate Policy by means of the PPP. AF was supported by a junior longfond grant (4.2.16.132JO). The study was funded by unrestricted grants from the Stichting Astma Bestrijding, the Netherlands Asthma Foundation, Netherlands Organization for Scientific Research (ZonMw), GlaxoSmithKline, the Royal Dutch Academy of Sciences and Arts, the University Medical Center Groningen and Leiden University Medical Center and the NIH R01 HL095388 (Spira/Lenburg). RNA-sequencing of the bronchial biopsies was funded by Genentech (San Francisco, CA, USA).

Availability of data and materials

The data that support the findings of this study are available from Genentech but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Genentech.

Ethics approval and consent to participate

The ethical committee of the University Medical Center Groningen (Medisch Ethische Toetsingscommisie) approved the study and all included subjects gave their written informed consent

Consent for publication

Not applicable.

Compete of interest

HK reports unrestricted grants, from Boehringer Ingelheim, Novartis and GlaxoSmithKline. He also reports fees advisory board participation for AstraZeneca, Boehringer Ingelheim, Novartis and GlaxoSmithKline. All above paid to his institution. GWT reports and I am a full time employee of Genentech, Inc and hold stock and options in the Roche. PH reports grants from Boerhinger Ingelheim, grants from Galapagos, outside the submitted work; MAG reports I am full time employee of Genentech, Inc and hold stock and options in the Roche Group. WT reports personal fees from Roche Diagnostics/Ventana, personal fees from Merck Sharp Dohme, personal fees from Bristol-Myers-Squibb, personal fees from AbbVie, outside the submitted work. JR reports personal fees from IDbyDNA Inc., from null, from null, outside the submitted work; and Dr. Rossen is currently an employee of IDbyDNA. IDbyDNA did not have any influence on the interpretation of reviewed data and conclusions drawn, nor on the drafting of the manuscript, and did not (financial) support the study. MN reports I am a full time employee of Genentech, Inc and hold stock and options in the Roche Group. BD, JB, NC, CAB, VG, AF and MvdB have nothing to disclose.