Lung Atelectasis Promotes Immune and Barrier Dysfunction as Revealed by Transcriptome Sequencing in Female Sheep

Abstract

Background Pulmonary atelectasis is frequent in clinical settings. Yet there is limited mechanistic understanding and substantial clinical and biologic controversy on its consequences. The authors hypothesize that atelectasis produces local transcriptomic changes related to immunity and alveolar–capillary barrier function conducive to lung injury and further exacerbated by systemic inflammation. Methods Female sheep underwent unilateral lung atelectasis using a left bronchial blocker and thoracotomy while the right lung was ventilated, with (n = 6) or without (n = 6) systemic lipopolysaccharide infusion. Computed tomography guided samples were harvested for NextGen RNA sequencing from atelectatic and aerated lung regions. The Wald test was used to detect differential gene expression as an absolute fold change greater than 1.5 and adjusted P value (Benjamini–Hochberg) less than 0.05. Functional analysis was performed by gene set enrichment analysis. Results Lipopolysaccharide-unexposed atelectatic versus aerated regions presented 2,363 differentially expressed genes. Lipopolysaccharide exposure induced 3,767 differentially expressed genes in atelectatic lungs but only 1,197 genes in aerated lungs relative to the corresponding lipopolysaccharide-unexposed tissues. Gene set enrichment for immune response in atelectasis versus aerated tissues yielded negative normalized enrichment scores without lipopolysaccharide (less than –1.23, adjusted P value less than 0.05) but positive scores with lipopolysaccharide (greater than 1.33, adjusted P value less than 0.05). Leukocyte-related processes (e.g., leukocyte migration, activation, and mediated immunity) were enhanced in lipopolysaccharide-exposed atelectasis partly through interferon-stimulated genes. Furthermore, atelectasis was associated with negatively enriched gene sets involving alveolar–capillary barrier function irrespective of lipopolysaccharide (normalized enrichment scores less than –1.35, adjusted P value less than 0.05). Yes-associated protein signaling was dysregulated with lower nuclear distribution in atelectatic versus aerated lung (lipopolysaccharide-unexposed: 10.0 ± 4.2 versus 13.4 ± 4.2 arbitrary units, lipopolysaccharide-exposed: 8.1 ± 2.0 versus 11.3 ± 2.4 arbitrary units, effect of lung aeration, P = 0.003). Conclusions Atelectasis dysregulates the local pulmonary transcriptome with negatively enriched immune response and alveolar–capillary barrier function. Systemic lipopolysaccharide converts the transcriptomic immune response into positive enrichment but does not affect local barrier function transcriptomics. Interferon-stimulated genes and Yes-associated protein might be novel candidate targets for atelectasis-associated injury. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

RNA Sequencing

“…8 This leaves anesthesiologists and intensivists alike caught in a veritable therapeutic dilemma. Although the study by Zeng et al 2 is unable to ultimately provide guidance to deal with this issue, it supports the concept that atelectasis, specifically in the context of systemic inflammation, may cause further harm, as evidenced at the molecular level.…”

“…In this issue of the Journal, Zeng et al 2 present their data exploring the molecular events related to atelectasis, a common clinical entity, by taking advantage of a translational large animal model with high fidelity to human disease. Atelectasis was induced with a bronchial blocker and thoracotomy on the left whole lung, while the right lung was ventilated in the presence or absence of systemic endotoxin infusion.…”

“…In addition, the authors found defective YESassociated protein signaling in atelectasis independent of endotoxin exposure. 2 YES-associated protein, a transcriptional coactivator of the transcriptional enhanced associate domain transcription factor family, which controls cell proliferation and stem cell functions, appears to be involved in the loss of pulmonary barrier function of the atelectatic lung. These are important discoveries and fit very well to our current understanding where interferon-stimulated genes have been linked to severe outcomes in acute respiratory distress syndrome (ARDS) patients because of their role of predisposing to additional lung injury.…”

“…The technology has been further developed by combining next generation sequencing with cell sorting (single-cell RNA sequencing) or, most recently, to measure spatially resolved gene expression, 10 opening the fascinating possibility of profiling the expression pattern of specific cell subtypes rather than RNA collected from inherently heterogeneous bulk tissue samples. It is unfortunate that Zeng et al 2 did not exploit this technology to its fullest by exploring the long noncoding RNAs, circular RNAs, and microRNAs, all of which are RNA transcripts not coding for proteins but important due to their chromatin and gene regulatory activities. In fact, the potential of RNAs as disease markers 11 and as powerful innovative therapeutics 12 has been widely recognized.…”

Isomer-Specific Effects of cis-9,trans-11- and trans-10,cis-12-CLA on Immune Regulation in Ruminal Epithelial Cells

“…The differential gene expression among the CON, LPS, c-9,t-11-CLA+LPS and t-10,c-12-CLA+LPS groups was analyzed using the DESeq2 R package (1.16.1), and the resulting p-values were adjusted (p adj ) using Benjamini and Hochberg's approach to control the false discovery rate. Genes with p adj < 0.05 and fold change (FC) > 1.5 were considered differentially expressed genes (DEGs) [30].…”