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Bronchoalveolar Lavage Fluid Protein Expression in Acute Respiratory Distress Syndrome Provides Insights into Pathways Activated in Subjects with Different Outcomes.

ABSTRACT: Acute respiratory distress syndrome (ARDS) is associated with high mortality. We sought to identify biological pathways in ARDS that differentiate survivors from non-survivors. We studied bronchoalveolar lavage fluid (BALF) from 36 patients with ARDS (20 survivors, 16 non-survivors). Each sample, obtained within seven days of ARDS onset, was depleted of high abundance proteins and labeled for iTRAQ LC-MS/MS separately. Protein identification and relative quantification was performed employing a target-decoy strategy. A variance weighted t-test was used to identify differential expression. Ingenuity Pathway Analysis was used to determine the canonical pathways that differentiated survivors from non-survivors. We identified 1115 high confidence proteins in the BALF out of which 142 were differentially expressed between survivors and non-survivors. These proteins mapped to multiple pathways distinguishing survivors from non-survivors, including several implicated in lung injury and repair such as coagulation/thrombosis, acute phase response signaling and complement activation. We also identified proteins assigned to fibrosis and ones involved in detoxification of lipid peroxide-mediated oxidative stress to be different in survivors and non-survivors. These results support our previous findings demonstrating early differences in the BALF protein expression in ARDS survivors vs. non-survivors, including proteins that counter oxidative stress and canonical pathways associated with fibrosis.

SUBMITTER: Bhargava M

PROVIDER: S-EPMC5547130 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Bronchoalveolar Lavage Fluid Protein Expression in Acute Respiratory Distress Syndrome Provides Insights into Pathways Activated in Subjects with Different Outcomes.

Bhargava Maneesh M Viken Kevin K Wang Qi Q Jagtap Pratik P Bitterman Peter P Ingbar David D Wendt Chris C

Scientific reports 20170807 1

Acute respiratory distress syndrome (ARDS) is associated with high mortality. We sought to identify biological pathways in ARDS that differentiate survivors from non-survivors. We studied bronchoalveolar lavage fluid (BALF) from 36 patients with ARDS (20 survivors, 16 non-survivors). Each sample, obtained within seven days of ARDS onset, was depleted of high abundance proteins and labeled for iTRAQ LC-MS/MS separately. Protein identification and relative quantification was performed employing a ...[more]

PMID: 28785034

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Project description:INTRODUCTION:Pulmonary vascular endothelial activation has been implicated in acute respiratory distress syndrome (ARDS), yet little is known about the presence and role of endothelial activation markers in the alveolar space in ARDS. We hypothesized that endothelial activation biomarkers would be differentially expressed in bronchoalveolar lavage fluid from patients with ARDS compared with healthy volunteers, and that biomarker concentrations would be associated with ARDS severity. METHODS:We performed a cross-sectional analysis of data from 26 intubated patients with ARDS undergoing evaluation for clinically suspected ventilator-associated pneumonia and five healthy volunteers. Patients underwent bronchoalveolar lavage a median of five days after intubation. Healthy volunteers also underwent bronchoalveolar lavage. Endothelial activation biomarkers (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble endothelial selectin [sESEL], angiopoietin-1 [Ang-1] and angiopoietin-2 [Ang-2]) were measured in bronchoalveolar lavage fluid. Clinically suspected ventilator-associated pneumonia was confirmed with microbiologic culture data. RESULTS:Patients with ARDS had significantly higher median sVCAM-1 concentrations in the bronchoalveolar lavage fluid compared with healthy volunteers (985 vs 119 pg/mL, p = 0.03). Additionally, there was a trend toward greater bronchoalveolar lavage fluid sVCAM-1 concentrations among patients with moderate/severe compared to mild ARDS (1395 vs 209 pg/mL, p = 0.06). We did not detect significant differences in bronchoalveolar lavage fluid levels of sESEL, Ang-1 or Ang-2 between patients with ARDS and healthy volunteers. Median bronchoalveolar lavage fluid biomarker levels did not differ between patients with and without microbiologically-confirmed ventilator-associated pneumonia. CONCLUSIONS:sVCAM-1 concentrations were significantly higher in the bronchoalveolar lavage fluid of patients with ARDS compared to healthy controls, and tended to be higher in moderate/severe ARDS compared to mild ARDS. Our findings add to the growing evidence supporting the concept that endothelial activation plays an important mechanistic role in the pathogenesis of ARDS. Further studies are necessary to characterize the role and/or clinical significance of sVCAM-1 and other endothelial activation markers present in the alveolar space in ARDS.

Project description:Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.

Project description:BackgroundIt is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data.ResultsUsing three mock communities across a range of densities ranging from 8E+?02 to 8E+?09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+?05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+?04 and 8E+?06 16S copies/ml. For the lowest densities, below 8E+?03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples.ConclusionRelative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+?04 16S copies/ml.

Dataset Information

Bronchoalveolar Lavage Fluid Protein Expression in Acute Respiratory Distress Syndrome Provides Insights into Pathways Activated in Subjects with Different Outcomes.

Publications

Bronchoalveolar Lavage Fluid Protein Expression in Acute Respiratory Distress Syndrome Provides Insights into Pathways Activated in Subjects with Different Outcomes.

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