RNA Sequencing the Twenty-First Century Stethoscope in Pulmonary Medicine

Editorial

RNA Sequencing The Twenty-first Century Stethoscope in Pulmonary Medicine

Eliana Lucchinetti, Ph.D., Michael Zaugg, M.D., M.B.A., F.R.C.P.C.

UNG atelectasis was first were found to be overrepresented Ldescribed by the French phy- in atelectatic tissue. Systemic endo-

sician René Théophile Hyacinthe toxin was capable of upregulating Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/133/5/976/513318/20201100.0-00012.pdf by guest on 23 September 2021 Laënnec in his iconic book enti- the immune response in atelec- tled, De L’Auscultation Médiate; ou tatic tissue, while genes linked to Traité du Diagnostic des Maladies des alveolar-capillary barrier integrity Poumons et du Cœur published in and function remained depressed. 1819 in Paris,1 where he compiled Results from transcription factor his observations of lung and heart prediction analysis were even more sounds derived from the use of his intriguing. The data suggested the newly invented stethoscope, a sim- involvement of the interferon reg- ple wooden tube carved by him- ulatory factor family of transcrip- self. Europe was recovering from tion factors in endotoxin-exposed the French Revolutionary and the atelectasis. These transcription Napoleonic Wars and was battling factors, which play pivotal roles with poverty, famine, and tuber- in many aspects of the immune culosis, a mysterious, untreatable “Since the discovery of atelec- response, including immune cell pulmonary disease, reminiscent of development and differentiation, today’s SARS-CoV-2. tasis…we have learned quite a are likely involved in the enhanced In this issue of the Journal, bit, but maybe not enough to response of the atelectatic tissue to Zeng et al.2 present their data have a significant impact on systemic inflammation. In addition, exploring the molecular events the authors found defective YES- related to atelectasis, a common patient outcome.” associated protein signaling in atel- clinical entity, by taking advantage ectasis independent of endotoxin of a translational large animal model with high fidelity to exposure.2 YES-associated protein, a transcriptional coact- human disease. Atelectasis was induced with a bronchial ivator of the transcriptional enhanced associate domain blocker and thoracotomy on the left whole lung, while transcription factor family, which controls cell proliferation the right lung was ventilated in the presence or absence of and stem cell functions, appears to be involved in the loss systemic endotoxin infusion. Physiologic parameters were of pulmonary barrier function of the atelectatic lung. These recorded, and imaging techniques were used to visualize are important discoveries and fit very well to our current regional aeration. After 8 h, lung tissue was collected from understanding where interferon-stimulated genes have aerated and atelectatic lung regions and used for next gener- been linked to severe outcomes in acute respiratory distress ation RNA sequencing. The authors2 used gene set enrich- syndrome (ARDS) patients because of their role of predis- ment analysis, a pattern recognition tool assessing whether posing to additional lung injury.3 Likewise, YES-associated functionally related genes are coordinately and consistently protein signaling is known to play a major role in respira- regulated, and transcription factor prediction analysis, a tool tory epithelial cell regeneration and regulation of the cyto- pinpointing the transcription factors that drive the tran- skeleton,4 and it is conceivable that lack of cyclic stretch in scriptomic differences among groups, to reveal a wealth of atelectatic tissue downregulates this pathway. novel findings. Downregulated genes associated with host Does this study have translational implications for the defense, barrier integrity, and tissue regeneration or repair practicing clinician? Since the discovery of atelectasis, a

Image: J. P. Rathmell. This editorial accompanies the article on p. 1060. Accepted for publication July 29, 2020. Published online first on August 17, 2020. From the Department of Anesthesiology and Pain Medicine and Cardiovascular Research Centre (E.L., M.Z.), and the Department of Pharmacology (M.Z.), University of Alberta, Edmonton, Canada. Copyright © 2020, the American Society of Anesthesiologists, Inc. All Rights Reserved. Anesthesiology 2020; 133:976–8. DOI: 10.1097/ALN.0000000000003524

frequent finding in patients with infectious pulmonary dis- RNA sequencing is quantitative and can be used to deter- eases, in patients undergoing general anesthesia as well as in mine RNA expression levels more accurately than microar- patients with ARDS, we have learned quite a bit, but maybe rays, allowing for a robust comparison between diseased and not enough to have a significant impact on patient outcome. healthy tissues. The technology has been further developed Much of our current understanding is based on physiologic by combining next generation sequencing with cell sorting studies and not on the fundamental underlying molecular (single-cell RNA sequencing) or, most recently, to measure events. Atelectasis in patients undergoing general anesthesia spatially resolved gene expression,10 opening the fascinat- results from a reduction of functional residual capacity due to ing possibility of profiling the expression pattern of specific loss of respiratory muscle tone, positioning, denitrogenation, cell subtypes rather than RNA collected from inherently and surfactant impairment, and is believed to be a key cul- heterogeneous bulk tissue samples. It is unfortunate that

prit in the pathogenesis of postoperative pulmonary compli- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/133/5/976/513318/20201100.0-00012.pdf by guest on 23 September 2021 Zeng et al.2 did not exploit this technology to its fullest by cations.5 In ARDS, atelectasis is accompanied by significant leukocyte infiltration and lung aeration is even more hetero- exploring the long noncoding RNAs, circular RNAs, and geneously distributed (e.g., “baby lung” so named because of microRNAs, all of which are RNA transcripts not cod- the occurrence of atelectasis in the dependent lung, causing ing for proteins but important due to their chromatin and baby-size aerated lungs). It is typically not amenable to rapid gene regulatory activities. In fact, the potential of RNAs as reversal. Previously applied “open lung ventilation strategies” disease markers11 and as powerful innovative therapeutics12 used high sustained airway pressures followed by high posi- has been widely recognized. Noncoding RNAs have been tive end-expiratory pressure to achieve alveolar patency, but linked to human disease such as heart failure,13 and it would this aggressive approach has been abandoned in favor of a have been exciting to see whether the expression profile of cautious approach to avoid mechanical lung injury in already noncoding RNAs as revealed by RNA sequencing could jeopardized lungs. Some experts even promote “permis- discriminate between the atelectatic and aerated lung. Such sive hypercapnia,” the therapeutic use of hypercapnia, and discoveries could indeed open new therapeutic approaches 6 more recently even “permissive atelectasis.” However, in in pulmonary medicine as, for example, short-term induc- this context, it is eye-opening to remember that atelectasis, tion of members of the microRNA family miR 130/301 when combined with higher tidal volumes, causes injury not activates YES-associated protein and could potentially ame- only to the atelectatic airways but also to the nonatelectatic liorate lung injury.14 Irrespectively, the authors should be airways and alveoli, resulting in a generalized trauma to all congratulated for their contribution toward a fundamen- distal airways in the entire ventilated lung.7 On the other tal understanding of the underlying molecular events trig- hand, prolonged hypercapnia reduces host defense and repair gered by atelectasis and inflammation. However, significant mechanisms. For example, prolonged hypercapnia increases the bacterial load in pneumonia, increasing the severity of challenges remain, such as the mechanistic decoding of the lung injury.8 This leaves anesthesiologists and intensivists new discoveries as well as the development of a therapeutic alike caught in a veritable therapeutic dilemma. Although the approach, potentially RNA-based, to mitigate lung injury in study by Zeng et al.2 is unable to ultimately provide guidance patients with atelectasis and inflammation. As we all know, to deal with this issue, it supports the concept that atelecta- the demonstration of a robust lung protective effect in a het- sis, specifically in the context of systemic inflammation, may erogeneous patient population requires a powerful treatment cause further harm, as evidenced at the molecular level. effect first and foremost. What makes transcriptomics with next generation RNA sequencing to such a powerful means comparable Research Support to Laënnec’s wooden tube 200 yr ago? Next generation This work was supported by grants from the Heart and sequencing has fundamentally changed genome research in the biomedical sciences. In contrast to the classic sequencing Stroke Foundation of Canada (Ottawa, Canada) and the technology (i.e., Sanger sequencing), which delivers “one Swiss National Science Foundation (Berne, Switzerland, target per reaction,” next generation sequencing is charac- SINERGIA grant No. 177225). terized by the ability to perform and capture data from mil- lions of sequencing reactions simultaneously (massive parallel Competing Interests sequencing). This technology can be applied to transcrip- The authors are not supported by, nor maintain any finan- tomics studies by converting all RNA species into comple- cial interest in, any commercial activity that may be associ- mentary DNA fragments (a complementary DNA library). ated with the topic of this article. Next generation RNA sequencing is considered superior to microarray hybridization, the more traditional technology Correspondence used in transcriptomics,9 because owing to its single base resolution, it has the ability to distinguish between different Address correspondence to Dr. Zaugg: michael.zaugg@ isoforms and to explore allele-specific expression. Moreover, ualberta.ca

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