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.
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.
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).
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.
Pulmonary atelectasis is common during and after major surgery with general anesthesia and may be associated with adverse outcome
The role of inflammatory mediators in response to atelectasis is controversial, and most data are limited to small animal preparations
The authors postulated that local transcriptomic changes activating inflammatory processes may occur in response to atelectasis, and such changes would be exacerbated by systemic inflammation induced by lipopolysaccharide infusion
A large animal ovine model of one lung collapse mimicking human conditions was used, and a variety of imaging techniques were used to guide tissue sampling for next-generation sequencing and transcriptome analysis
Atelectasis alone dysregulated the local pulmonary transcriptome with negatively enriched immune response and alveolar–capillary barrier function
With associated systemic inflammation, the local immune response was positively enhanced while barrier function response remained negatively enriched
Interferon-simulated genes and Yes-associated protein appear to have important regulatory roles and may be novel candidate targets for therapy of atelectasis-associated injury
ATELECTASIS, a condition characterized by complete or partial collapse of lung regions with loss of aeration, is widely pervasive in clinical settings. Approximately 90% of 230 million patients undergoing major surgery with general anesthesia and mechanical ventilation develop atelectasis.1–3 It is also found in bedridden patients and in virtually all patients with the acute respiratory distress syndrome, which affects 10 to 86 patients per 100,000 each year in the United States, accounting for 10% of intensive care unit admissions.4 Considered an injurious process,5,6 atelectasis is usually reduced by open lung strategies, which are thought to be beneficial for patient outcomes.7 However, recent clinical trials have suggested “permissive atelectasis” as a favorable management strategy in conditions such as abdominal surgery8,9 and extracorporeal membrane oxygenation.10
Biologic and molecular information on mechanisms underlying atelectasis that might be conducive to lung injury are limited and contradictory. A few reports have shown increased inflammatory activity with neutrophil infiltration and potentiation of the neutrophil oxidative response in the atelectatic lung.11,12 In contrast, others found no increase in neutrophil recruitment and inflammatory cytokine production13,14 or even an inflammatory dysfunction for alveolar macrophages.15 In vitro studies suggest that pulmonary endothelial cell immobility, as present in atelectatic tissue, impaired pulmonary barrier recovery ability and increased lung permeability in response to thrombin.16 However, such findings, derived mostly from a small number of rodent studies, are limited in describing the structural and functional heterogeneity of the human lung, and could have been affected by the stress response, hypoxia, and mechanical injury beyond atelectasis. In vitro pulmonary cell studies cannot provide the in vivo understanding of atelectatic lung tissue exposed not only to mechanical stretch but also to systemic and local chemical stimuli. Furthermore, no study identified the molecular basis for the observed changes in cellular function accompanying atelectasis. Therefore, there is a remarkable lack of knowledge on the early molecular processes underlying atelectatic lung tissue. This gap markedly limits not only the mechanistical understanding of this common potential morbid entity but also the advance of therapeutic interventions.
We hypothesized that atelectasis would produce local transcriptomic changes in lung tissue conducive to lung injury and that these changes would be exacerbated by systemic inflammation. To investigate this hypothesis, we constructed a clinically relevant “pure” atelectasis large animal (sheep) model of one-lung collapse and mechanical ventilation with global and regional physiologic properties comparable to that of humans. In vivo regional lung endophenotypes were assessed by tracer kinetics modeling of positron emission tomography showing metabolic and inflammatory activity, and barrier dysfunction, and by computed tomography images showing lung expansion. We used these imaging techniques to guide tissue sampling for deep next-generation sequencing and transcriptome analysis.
Materials and Methods
Protocols were approved by the Subcommittee on Research Animal Care and the Institutional Animal Care and Use Committee of the Massachusetts General Hospital (Boston, Massachusetts), and followed National Institutes of Health (Bethesda, Maryland) and Animal Research: Reporting of In Vivo Experiments guidelines.
The experimental approach to imaging and tissue analysis is summarized in figure 1 and detailed in the Supplemental Digital Content 1 (http://links.lww.com/ALN/C457). Twelve female sheep (18.2 ± 2.3 kg) were anesthetized, paralyzed, and intubated. One-lung atelectasis (left lung) was achieved using a bronchial blocker (Arndt, Cook Medical, USA) and lateral thoracotomy, while the right lung was ventilated (tidal volume = 10 ml/kg, positive end-expiratory pressure = 2 cm H2O) for 8 h. Animals were divided into lipopolysaccharide (LPS)-unexposed (LPS[–], n = 6, 18.1 ± 2.4 kg) or LPS-exposed (LPS[+], n = 6, 18.3 ± 2.4 kg, 5 to 10 ng · kg-1 · min-1; Escherichia coli O55: B5, List Biologic Laboratories Inc., USA) group. Physiologic data and samples were acquired at baseline, the beginning of lung collapse (Atelectasis-0h) and the end of the study (Atelectasis-8h).
Image Acquisition and Analysis
Computed tomography images were acquired for aeration and strain analysis (end-inspiration and end-expiration) and at mean lung volume for positron emission tomography attenuation correction and delineation of regions of interest.17 Voxel gas fraction was quantified as voxel Hounsfield units/–1,000 with air = –1,000 Hounsfield units and tissue = 0 Hounsfield units. Tidal strain at the voxel level was computed by image registration of the end-inspiratory to the end-expiratory computed tomography images (voxel volume change/end-expiratory volume including lung tissue and air).17 Dynamic positron emission tomography images of 2-deoxy-2-[18F] fluoro-D-glucose were acquired to measure parameters of tissue metabolism representative of local inflammation: phosphorylation rate, tissue-normalized net uptake rate, and distribution volumes immediately or not immediately available for 2-deoxy-2-[18F] fluoro-D-glucose phosphorylation.18,19 Mean aeration, median strain, and 2-deoxy-2-[18F] fluoro-D-glucose kinetics were quantified in two regions of interest: the atelectatic left lung and a homogeneously normally aerated region of the ventilated right lung.
Lung tissue samples were collected from atelectatic and aerated lung regions based on computed tomography images within 15 min after euthanasia, and from the atelectatic lung at the time of thoracotomy (control). We assessed lung water content by wet/dry ratios, Yes-associated protein distribution by immunofluorescent staining, histological lung injury by hematoxylin and eosin stain, myeloperoxidase by immunohistochemistry, protein levels by immunoblotting, and other cytokines by enzyme-linked immunosorbent assay.
RNA-sequencing Analysis and Validation
Total RNA from lung tissues was extracted using PAXgene tissue RNA kit (Qiagen, Germany). Gene expression was quantified by RNA-sequencing (Illumina HiSeq2500). Differentially expressed genes were defined by an absolute fold change greater than 1.5 and an adjusted P value (Benjamini–Hochberg) less than 0.05, assessed with DESeq2 Bioconductor package.20 Functional analysis of enriched biologic processes was performed with gene set enrichment analysis (adjusted P value <0.05) in R, based on templates from the bcbioRNASeq package.21 Transcription factor prediction analysis was performed in iRegulon (version 1.3).22 Plots were visualized in Cytoscape (http://www.cytoscape.org; accessed May 15, 2018), GraphPad Prism software v.7.0 (GraphPad Software, USA), or Rstudio (R version 3.4.4, https://www.r-project.org; accessed June 4, 2018). Gene expression was validated using real-time polymerase chain reaction.
Our primary outcomes were the differential regional (atelectatic vs. aerated) lung transcriptomic signatures (differential gene expression, functional analysis) as assessed by RNA-sequencing. Secondary outcomes were the imaging measures of regional aeration, strain, and 2-deoxy-2-[18F] fluoro-D-glucose kinetics. Exploratory outcomes and associated confirmatory variables consisted of the immunofluorescence assessment of tissue Yes-associated protein, lung injury score and its components, tissue myeloperoxidase, and protein levels.
No statistical power calculation was conducted before the study, and sample size was determined based on a previous study indicating six biologic replicates as a general guideline for RNA- sequencing experiments.23 Data are presented as mean ± SD if normally distributed and median and interquartile interval (25%, 75%) otherwise. Two-way ANOVA for repeated measurements was used for comparisons within and between groups. The interaction between conditions was included when significant. Tukey post hoc test was used for normally distributed data and Kruskal–Wallis test otherwise. The statistical significance of messenger RNA (mRNA) expression was calculated by a paired, two-tailed Student’s t test. Tests were two tailed and performed in R (R version 3.4.4). Significance was considered at P < 0.05.
Cardiopulmonary Function Is Compromised by Atelectasis and Exacerbated by Systemic Inflammation
Baseline cardiopulmonary function was normal (fig. 2 and Supplemental Digital Content 1, table S1, http://links.lww.com/ALN/C457). Eight hours of one-lung atelectasis resulted in decreased respiratory system compliance (fig. 2A) and Pao2/inspired oxygen fraction ratio (fig. 2B). This hypoxemia resulted per protocol in increased inspired oxygen fraction (P < 0.001) and positive end-expiratory pressure (P = 0.029) at 8 h with LPS exposure (Supplemental Digital Content 1, table S1, http://links.lww.com/ALN/C457). Addition of systemic LPS to one-lung atelectasis impaired cardiovascular function with high pulmonary artery pressure (fig. 2C), low mean arterial pressure (fig. 2D), low cardiac output (fig. 2E), and high pulmonary capillary wedge pressure (fig. 2F).
Metabolic Activity Is Affected by Atelectasis and Systemic Inflammation
To explore processes specific to atelectasis, we compared atelectatic to normally aerated regions of the same lung (fig. 3A). As expected, the atelectatic lung had a gas fraction less than 0.1 and no volumetric strain (fig. 3, B and C). The gas fraction of the aerated regions was consistent with normal aeration (LPS[–]: 0.61 ± 0.07, LPS[+]: 0.61 ± 0.09) (fig. 3B). Volumetric strain in those regions (LPS[–]: 0.58 ± 0.44, LPS[+]: 0.42 ± 0.20) (fig. 3C) was below levels proposed as injurious.24
The tissue-normalized 2-deoxy-2-[18F] fluoro-D-glucose uptake rate, an in vivo measure of cellular metabolic activity, was affected by aeration (fig. 3D) and systemic inflammation (fig. 3D). LPS exposure increased tissue-normalized net uptake rate by 358% in atelectatic regions compared to LPS-unexposed atelectasis. It also increased cellular phosphorylation rate in lung tissue (fig. 3E). The tissue-normalized volume of distribution of 2-deoxy-2-[18F] fluoro-D-glucose was lower in atelectatic than aerated regions (fig. 3F), and contributed most to the lower tissue-normalized net uptake rate in atelectasis with LPS.
The tissue-normalized distribution volume of 2-deoxy-2-[18F] fluoro-D-glucose not immediately for phosphorylation, a measure of edema, was larger in LPS-exposed than LPS-unexposed conditions (fig. 3G), without significant difference in atelectatic versus aerated regions (fig. 3G). LPS exposure increased that tissue-normalized distribution volume by 206% in atelectasis (LPS[+] vs. [–]: 0.316 ± 0.218 vs. 0.103 ± 0.025) and 101% in aerated regions (LPS[+] vs. [–]: 0.374 ± 0.140 vs. 0.186 ± 0.027). These results were corroborated by increased wet/dry ratios with LPS exposure (fig. 3H) and significant correlation of the tissue-normalized distribution volume of 2-deoxy-2-[18F] fluoro-D-glucose not immediately for phosphorylation with wet/dry ratios (R = 0.75, Supplemental Digital Content 1, fig. S1, http://links.lww.com/ALN/C457). Such increased wet/dry ratios and tissue-normalized distribution volume with LPS occurred in the absence of significant different fluid volume administered to each group (LPS[–]: 1,000 ± 316 ml, LPS[+]: 1,250 ± 524 ml, P = 0.341).
LPS exposure increased histological lung injury scores with significant capillary congestion and infiltration of neutrophils (Supplemental Digital Content 1, fig. S2 and table S2, http://links.lww.com/ALN/C457) as well as the densities of myeloperoxidase (Supplemental Digital Content 1, fig. S3, http://links.lww.com/ALN/C457) in atelectatic and aerated regions. Circulating peripheral white blood cells decreased after LPS exposure (Supplemental Digital Content 1, fig. S4, http://links.lww.com/ALN/C457).
Regional Transcriptomics Are Altered by Aeration State and Systemic LPS Exposure
Differential expression was tested in 26,867 transcripts. Relative to control, gene expression differed according to regional aeration (atelectasis vs. normal aeration) and to LPS exposure (fig. 4A). The number of differentially expressed genes in lung tissues increased with the presumed magnitude of injurious stimuli, i.e., level of local aeration and LPS exposure (fig. 4B). Accordingly, LPS-unexposed atelectatic lung presented 1,682 differentially expressed genes, while 4,618 were found in LPS-exposed atelectasis. The corresponding numbers for the aerated lung were 3,421 and 6,419. Without LPS, only 17% differentially expressed genes were common to both atelectatic and aerated regions (up: 318 and down: 533), while the overlap increased to 34% with LPS (up: 1,816 and down: 1,947).
Functional analysis in each condition versus control (fig. 4C and Supplemental Digital Content 2, tables S3–-S6, http://links.lww.com/ALN/C458) revealed transcriptomic changes involved in apoptosis, immunity, and barrier integrity. As expected, systemic LPS induced transcriptomic changes associated with immune response in both atelectatic and aerated lung regions, characterized by positive enrichment for gene sets such as cytokine production, response to LPS and cytokine, inflammatory response, and leukocyte-associated processes. Interestingly, immune-related gene sets were negatively enriched in LPS-unexposed atelectasis. Furthermore, transcriptomic patterns associated with alveolar–capillary barrier function were dysregulated in all lung regions, independent of aeration and LPS exposure. Such dysregulation was characterized by negatively enriched gene sets including cell–matrix adhesion, extracellular structure organization, and cell junction assembly. Of note, transcriptomic dysregulation in organization of actin cytoskeleton and cell junction occurred only in atelectasis, regardless of LPS exposure.
Atelectasis Is Associated with Transcriptomic Changes in Immune Regulation
Without LPS, atelectatic versus aerated regions of the same lung presented 2,363 differentially expressed genes (8.8% of total transcripts; fig. 5A and Supplemental Digital Content 1, fig. S5, http://links.lww.com/ALN/C457). Functional analysis revealed 206 of 226 significantly altered biologic processes negatively enriched in atelectatic tissue (fig. 5B and Supplemental Digital Content 2, table S7, http://links.lww.com/ALN/C458). For immune processes, normalized enrichment scores were negative (less than –1.23, adjusted P value less than 0.05, fig. 5C and Supplemental Digital Content 1, figs. S6A and, S7, http://links.lww.com/ALN/C457), consistent with negative enrichment in atelectatic versus control tissues (fig. 4C). Such processes included inflammatory response, cytokine production, and leukocyte migration (fig. 5C). Also negatively enriched in atelectasis were gene sets for cellular stress responses (e.g., response to cytokine, TNF, LPS and extracellular stimulus) and immune-associated signaling (e.g., mitogen-activated protein kinase [MAPK] cascade, extracellular signal-regulated kinase [ERK]1 and ERK2 cascade, and nuclear factor kappa B [NF-κB] signaling; fig. 5C).
Atelectasis Increases Sensitivity of Lung Tissue to Systemic Inflammation
Transcriptomic changes due to LPS exposure were more pronounced in atelectatic than aerated tissue. LPS exposure induced 3,767 differentially expressed genes (14% of total transcripts) in atelectasis (fig. 5A), which was more than threefold the 1,197 (4.5% of total transcripts) genes induced in aerated tissue. As expected, systemic LPS induced positive enrichment of gene sets associated with apoptosis and immune and stress response in both lung regions relative to the corresponding LPS-unexposed regions (fig. 6A and Supplemental Digital Content 2, tables S8 and S9, http://links.lww.com/ALN/C458). Interestingly, LPS produced positive enrichment of gene sets related to leukocyte function, NF-κB signaling, and hypoxia (i.e., response to oxygen levels and hypoxia) only in the atelectatic lung (fig. 6A).
Atelectatic versus aerated tissues with LPS yielded 1,293 differentially expressed genes (4.8% of total transcripts; fig. 5A). Functional analysis revealed 113 of 200 significantly altered biologic processes negatively enriched in atelectasis (fig. 6B and Supplemental Digital Content 2, table S10, http://links.lww.com/ALN/C458). Of note, LPS exposure led to a shift toward enhanced immune transcriptomic response in atelectatic tissue (normalized enrichment scores greater than 1.33, adjusted P value less than 0.05, fig. 6C and Supplemental Digital Content 1, figs. S6B and, S8, http://links.lww.com/ALN/C457), in contrast to the negatively enriched response without LPS (fig. 5C). Cellular stress responses and immune processes were positively enriched in LPS-exposed atelectasis (fig. 6C). These included leukocyte-related processes such as leukocyte migration, activation, mediated immunity, and leukocyte cell–cell adhesion (fig. 6C).
Atelectasis Dysregulates the Transcriptome Related to Pulmonary Barrier Function
Notably, atelectasis by itself resulted in remarkable transcriptomic changes involved in pulmonary barrier function (normalized enrichment scores less than –1.35, adjusted P value less than 0.05, fig. 7 and Supplemental Digital Content 1, fig. S9A, http://links.lww.com/ALN/C457). Processes including angiogenesis, vasculature development, and epithelial cell development, related to major cellular structures of the alveolar–capillary barrier, were negatively enriched. Also, gene sets for actin cytoskeleton and cell junction, structural elements for the architectural integrity of the pulmonary barrier, and for tissue repair function, such as wound healing and response to wounding, were negatively enriched in atelectatic versus aerated tissue. Additionally, transforming growth factor beta (TGF-β) signaling, relevant in wound healing and cell proliferation and differentiation, was dysregulated in atelectasis. LPS-exposed atelectatic regions showed negative enrichment for barrier integrity–related processes similar to LPS-unexposed atelectasis (normalized enrichment scores less than –1.51, adjusted P value less than 0.05, fig. 7 and Supplemental Digital Content 1, fig. S9B, http://links.lww.com/ALN/C457).
Interferon-stimulated Genes Are Related to the Response of Atelectatic Tissue to Systemic Inflammation
To explore the regulatory mechanisms triggering transcriptomic response to atelectasis and systemic inflammation, we performed transcription factor prediction analysis (fig. 8, A and B and Supplemental Digital Content 1, fig. S10, A and, B, http://links.lww.com/ALN/C457). This revealed enrichment for NF-κB with low gene expression in LPS-unexposed atelectasis (fig. 8A and Supplemental Digital Content 1, fig. S10A, http://links.lww.com/ALN/C457). While total protein levels for the NF-κB component p65 were not affected by lung aeration (P = 0.443, Supplemental Digital Content 1, fig. S11, http://links.lww.com/ALN/C457), NF-κB–related factors, such as chemokine (C-X-C motif) ligand 8 (CXCL8; P = 0.018) and coagulation factor II (thrombin) receptor-like 1 (F2RL1; P = 0.025), validated by real-time polymerase chain reaction, were less expressed in LPS-unexposed atelectatic tissue than in aerated regions (fig. 8C). Protein levels of CXCL8 tended to be lower in atelectatic than aerated regions in the absence of LPS (P = 0.086, Supplemental Digital Content 1, fig. S12A, http://links.lww.com/ALN/C457).
Predicted transcription factors differed according to systemic inflammation. LPS exposure produced significant enrichment for interferon regulatory factors (IRFs) and signal transducer and activator of transcription (STAT) 1/2 with relative higher gene expression in atelectasis (fig. 8B, Supplemental Digital Content 1, fig. S10B, http://links.lww.com/ALN/C457). Due to their essential function in regulation of interferon-stimulated genes,25 IRFs/STAT1/2 were targeted by a subset of interferon-stimulated genes, which were more strongly expressed in LPS-exposed atelectasis (fig. 8D and Supplemental Digital Content 1, fig. S10D, http://links.lww.com/ALN/C457). We also found changes in interferon-stimulated genes related to bacterium response (e.g., CXCL10, interferon-stimulated gene 15, IL12B, and CCL5) and leukocyte-associated processes, such as leukocyte migration (e.g., CXCL10, CXCL11, and CCL5) and activation (e.g., IL12B, CCL5, and RSAD2; Supplemental Digital Content 1, fig. S6B, http://links.lww.com/ALN/C457), consistent with their function in immune responses and host defenses.26
Real-time polymerase chain reaction validation confirmed the increase of mRNA expression for interferon-stimulated genes, including CXCL9, CXCL10, CCL5, IL12B, and MX1 and 2, in LPS-exposed atelectasis (fig. 8E). With LPS exposure, protein levels were larger for CXCL10 (P = 0.022) and numerically larger for CCL5 (P = 0.087) in atelectasis than in aerated regions (Supplemental Digital Content 1, fig. S12, B and, C, http://links.lww.com/ALN/C457). Furthermore, mRNA expression of interferon-stimulated gene activators was increased in LPS-exposed atelectasis (fig. 8F)—ZNF395 (P = 0.005), a hypoxia-induced transcription factor and interferon-stimulated gene activator,27 and NOD2 (P = 0.002), a proinflammatory receptor with roles in interferon response and subsequent induction of interferon-stimulated genes28 —while STAT1 protein levels were comparable in atelectatic versus aerated regions (P = 0.117, Supplemental Digital Content 1, fig. S11, http://links.lww.com/ALN/C457).
Yes-associated Protein Signaling Is Associated with Barrier Dysfunction in Atelectasis
Interestingly, transcription factor prediction analysis revealed that SRF, TEAD4, and AP-1 binding sites exhibited enrichment in the altered genes independent of systemic LPS (fig. 8, A and B; and Supplemental Digital Content 1, fig. S10, A and, B, http://links.lww.com/ALN/C457). These transcription factors were associated with a subset of differentially expressed genes, including CYR61/CCN1, CTGF/CCN2, ANKRD1, THBS1, SERPINE1, ESM1, and F3 (Supplemental Digital Content 1, fig. S10, C and, D, http://links.lww.com/ALN/C457). Moreover, the activity of these transcription factors is regulated by the transcriptional effector Yes-associated protein, which directly controls the expression of the identified genes.
Therefore, we examined Yes-associated protein and found that nuclear Yes-associated protein distribution was markedly reduced in atelectatic versus aerated regions (fig. 9, A and B). mRNA expression of Yes-associated protein–responsive genes, such as SERPINE1 and THBS1, was also lower in atelectasis independent of LPS exposure (fig. 9C). Furthermore, lower protein levels of SERPINE1 (P = 0.029) and numerically lower levels of THBS1 (P = 0.113) were found in atelectatic versus aerated regions (Supplemental Digital Content 1, fig. S12, D and, E, http://links.lww.com/ALN/C457).
Because Yes-associated protein’s role in barrier function relates to regulation and response to actin cytoskeleton dynamics,29 we further validated the mRNA expression of genes involved in cytoskeleton organization (ACTN1, FLNA, and FLNC). These were less expressed in atelectatic regions, even with LPS exposure (fig. 9D). Furthermore, SRF, associated with actin dynamics and Yes-associated protein signaling, and RHOD, the regulator of actin cytoskeleton dynamics, presented lower mRNA expression in atelectatic versus aerated regions (fig. 9E).
In a large animal model of unilateral lung collapse with global cardiopulmonary measurements consistent with clinical conditions, we documented transcriptomic changes in atelectasis indicative of dysregulated pulmonary immunity and alveolar–capillary barrier, two major processes in the development of acute lung injury. Exposure to systemic inflammation exacerbated lung injury in atelectatic tissue and enhanced its transcriptomic immune response, particularly leukocyte-related processes, which became larger than present in aerated lung and at least in part attributable to interferon-stimulated genes. Pulmonary barrier dysfunction was associated with defective distribution of Yes-associated protein independent of systemic inflammation. Therefore, our study of injurious transcriptomic profiles in atelectatic lung regions provides new data to inform clinical controversies on tolerance of atelectasis in patients7–9 and targets for mechanistic studies on the biology and molecular management of atelectasis.
While atelectasis is a major pathophysiological process in clinical anesthesia, there is poor understanding of its biologic and molecular mechanisms and their relationship to lung injury. Our experimental model aimed to mimic atelectasis and cardiopulmonary conditions comparable to those found in humans. Histological lung injury was mild in atelectatic and normally aerated regions and further exacerbated by LPS exposure. This is consistent with previous findings in atelectatic and aerated regions in large animals with either noninjurious17,30 or moderately injurious ventilation settings.31,32 The used tidal volume of 10 ml/kg aimed to increase the normally aerated lung.33 Our measurements of regional lung aeration and strain confirmed the absence of overdistension in sampled areas.
Gene expression profiles in atelectatic tissue were dramatically distinct from those in aerated regions despite their comparable histological findings. Independent of systemic inflammation, more than half of the biologic processes, including metabolic processes in RNA, lipid, and protein, were negatively enriched in atelectatic tissue. Together with previous findings34 and our in vivo 2-deoxy-2-[18F] fluoro-D-glucose measurement of regional lower metabolic activity (tissue-normalized net uptake rate), these findings indicate less cellular activation due to atelectasis.
In LPS-unexposed atelectasis, gene sets involved in cytokine production, leukocyte migration, and cellular stress, all fundamental immune responses, were negatively enriched. Functional and transcription factor prediction analyses identified NF-κB signaling, the classic pathway in inflammation, immune, and stress responses, as involved in such dysregulation. Additionally, NF-κB associated factors (CXCL8 and F2RL1) were less expressed in atelectatic than aerated regions. These findings support the concept of local immune dysregulation in atelectatic tissue likely through NF-κB signaling.
They are also consistent with previous findings of impaired alveolar macrophage function15 and less regional lung injury30,31 in atelectasis. However, they contrast with reports of atelectasis association with a proinflammatory state in rats.11,12 The discrepancy might be explained by the different species, the duration of atelectasis, and the unknown sterility of rodent preparation. Our large animal model allowed for mimicking of clinical settings preserving sterility, and including alterations in regional lung blood volume, and exposure to a systemic inflammatory trigger and circulating inflammatory cells and mediators.
LPS exposure resulted in positively enriched transcriptomic patterns of immune responses, particularly leukocyte-related processes, in atelectatic versus aerated tissue. Such findings contrasted with their negative enrichment in LPS-unexposed atelectasis. Our genomic findings are likely due to changes in cellular transcriptomic activity in atelectatic tissue by LPS exposure, rather than to changes in local cellular composition. This is because neutrophilic infiltrates assessed by counts and myeloperoxidase were similar in atelectatic and aerated regions during LPS exposure, and the fractions of parenchymal cells in sampled tissues were likely similar in the acute condition. Expanding a previous report of vulnerability to infection in the atelectatic lung exposed to Group B streptococci,35 our findings suggest that the altered transcriptomic response of the atelectatic lung could contribute to its susceptibility to inflammatory stimuli.
Interferon-stimulated genes are a broad gene family in immunity and host defenses,26 and with high expression in stimulated blood leukocytes.36 For instance, CXCL9 and CXCL10 promote the recruitment of inflammatory cells (i.e., neutrophils, T-lymphocytes, monocytes, and natural killers).37 Our findings of upregulated interferon-stimulated genes in LPS-exposed atelectasis suggest their association with increased sensitivity of atelectasis to systemic inflammation. Such association was reinforced by functional analysis showing positively enriched leukocyte migration and by higher CXCL10 protein levels in LPS-exposed atelectasis. Recently, interferon-stimulated genes have been related to acute respiratory distress syndrome severity and prognosis,38,39 further supporting their role in lung injury. Our data also suggest that interferon-stimulated genes in atelectatic tissue could be regulated through transcription factors IRFs and STATs25 and by regulators ZNF39527 and NOD2.28 Therefore, our results provide molecular support to the frequently stated but poorly demonstrated cause–effect relationship between atelectasis and pneumonia, ventilator-induced lung injury, and postoperative pulmonary complications.6 They simultaneously raise concerns on consequences of currently proposed management methods allowing for substantial lung collapse,8–10 particularly in the presence of significant inflammatory response.
We also found that atelectasis was associated with negatively enriched biologic processes related to structural cells (endothelium and epithelium), structural elements (cytoskeleton and cell junction), and tissue repair function. Of note, such processes were still negatively enriched in LPS-exposed atelectasis, implying that atelectasis dysregulated barrier function transcriptomics independent of LPS exposure. These results contrast with the local transcriptomic response associated with immunity, which shifted from inhibited without to enhanced with LPS. While wet/dry ratios and the tissue-normalized distribution volume of 2-deoxy-2-[18F] fluoro-D-glucose not immediately for phosphorylation were lower in atelectatic than aerated regions, the numerically larger increase of the tissue-normalized distribution volume in atelectasis by LPS relative to aerated regions suggests higher susceptibility of atelectasis to permeability increase, consistent with transcriptomic findings. The LPS-exposed atelectatic sample with highest tissue-normalized distribution volume clustered well with the transcriptomic data of other samples, suggesting no undue effect on transcriptomics analysis.
Our findings pointed to reduced Yes-associated protein signaling in atelectasis: less nuclear Yes-associated protein distribution, low levels of Yes-associated protein–responsive proteins SERPINE1 and THBS1, and less expression of cytoskeleton-associated genes (e.g., ACTN1, FLNA, FLNC, SRF, and RHOD), independent of LPS exposure. Yes-associated protein, the primary transcriptional effector of the Hippo pathway, is distributed in respiratory epithelial cells.40 With roles in lung homeostasis and development,41 Yes-associated protein was increased after murine lung injury due to hemorrhage and LPS,42 and recently associated with alveolar epithelium repair and regeneration after pneumonectomy43 or bacterial pneumonia.44 It also contributes to barrier function by regulating and responding to actin cytoskeleton dynamics.29 Lack of cyclic stretch45 and presence of cell contact46 during atelectasis could modulate the Hippo–Yes-associated protein pathway. Thus, our data suggest that reduced Yes-associated protein activity may participate in atelectasis-associated barrier dysfunction by compromising cytoskeleton organization and cell repair. Such findings constitute potential mechanisms for previous reports of increased permeability with pulmonary ultrastructural cell damage (i.e., microvascular disruption and epithelial damage)47,48 and vascular injury49 in atelectatic regions.
Our findings of reduced nuclear Yes-associated protein in atelectasis, and protein levels consistent with the immune and alveolar–capillary barrier function, provide plausibility to our transcriptomic data. The asynchrony between gene expression and protein synthesis is well documented, implying that transcriptomic changes are not necessarily simultaneous with corresponding protein levels.50 Future studies will need to address the effects of the reported early regional transcriptional patterns on regional cellular function, protein levels, and their time courses.
Our use of image-guided tissue sampling allowed not only for better characterization of the tissue samples with their corresponding in vivo imaging endophenotypes, but also for paired statistics (aerated × atelectatic lung). This approach reduces the number of studied animals and may be considered in future studies in the field.
Our study has limitations. We studied only female sheep. While this reduces biologic variability, it may limit the generalization of the results to males as it is unknown whether atelectasis-related lung injury presents sex dependence. Whereas nondependent single-lung collapse with pneumothorax reflects conditions during thoracic surgery, associated regional intrapulmonary tissue pressures and perfusion patterns likely differ from those encountered in the more common clinical presentation of dependent atelectasis, and could determine different inflammatory responses in the distinct atelectasis conditions. Our animal model describes acute atelectasis (8 h) as present in the first hours after intubation in critical care and surgical settings. Thus, results are expected to relate to mechanisms of lung injury in the early stages of atelectasis and did not address changes related to re-expansion. Our direct measurements for barrier damage were limited to wet/dry ratios. The exact cellular sources and functional roles of interferon-stimulated genes and Yes-associated protein in atelectasis could not be established in this large animal study. Mechanistic studies with pharmacologic modulation in models allowing for genetic manipulation will be required to establish the role of the identified pathways in atelectatic injury. While further investigation will be necessary to assess these topics, our study strongly indicates that the atelectatic lung is associated with marked transcriptomic changes in immune and barrier dysfunction.
In conclusion, our study identified a local differential transcriptomic response consistent with impairment of the acute pulmonary immune function in atelectatic versus aerated lung in the absence of any additional inflammatory stimulus. In contrast, a distinct inflammatory pattern with augmentation of the transcriptomic immune response was present in atelectatic tissue exposed to systemic LPS. Locally increased interferon-stimulated genes were potentially associated with the atelectatic tissue response to systemic LPS. Our results also documented the dysregulation of transcriptomic responses related to alveolar–capillary barrier integrity in atelectatic tissue irrespective of systemic inflammation. This dysregulation was likely associated with Yes-associated protein signaling. Therefore, modulators of these pathways might be novel candidate targets in atelectasis-associated lung injury.
The authors thank Steve Weise, B.S., Department of Radiology (Nuclear Medicine and Molecular Imaging), Massachusetts General Hospital, Boston, Massachusetts, for the expert technical support with computed tomographic and positron emission tomographic imaging. The authors also thank Dana-Farber/Harvard Cancer Center, Boston, Massachusetts, for the use of the Specialized Histopathology Core, which provided histology and immunohistochemistry service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant No. NIH 5 P30 CA06516.
This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute (Bethesda, Maryland) grant No. RO1 HL121228.
Dr. Hutchinson received funding from the Harvard Clinical and Translational Science Center (Cambridge, Massachusetts; National Center for Advancing Translational Sciences, National Institutes of Health Award No. UL 1TR002541, Bethesda, Maryland), the Harvard Medical School Foundry (Cambridge, Massachusetts), the National Institute of Environmental Health Sciences (NIEHS) Center for Environmental Science (Durham, North Carolina), the Harvard Center for AIDS Research (Cambridge, Massachusetts), Astra-Zeneca (Waltham, Massachusetts), and Boehringer-Ingelheim (Biberach an der Riß, Germany), and Dr. Baron received funding from Merck (Kenilworth, New Jersey) and Genetech (San Francisco, California) for projects not related to the current work. The other authors declare no conflicts of interest.