Faculty Bibliography

Rationale: The use of culture-independent techniques to evaluate resident microbial communities in the lung has opened opportunities to evaluate host response phenotype in health and disease. Background environmental microbiome found in saline and bronchoscope prior to bronchoscopy is characterized by high relative abundance of Propionibacterium. Our preliminary data from the lung microbiome project suggest that substitution of background environmental microbiome by Prevotella or Streptococcus microbiome is associated with higher inflammation. We hypothesize that patients with emphysema who have asymmetric disease on CT will have disappearance of background environmental microbiome in the more affected lung segments. We will also evaluate whether background environmental microbiome is associated with lower inflammation (neutrophil counts, and chemo-attractant cytokines). Methods: Subjects with emphysema were enrolled for research bronchoscopy from NYU/EDRN cohort and CT scans were classified as symmetrical or asymmetrical lung disease. Broncho-alveolar lavages (BAL) were obtained from the right and left lung. Sequencing of 300 bp 16S rDNA included V1-V2 region, performed with 454 pyrosequence. Propionibacterium was used as a marker of background environmental microbiome. Cytokines in BAL fluid will be assayed using Human Cytokine Panel I (Millipore). Results: To date, 15 subjects had sequence data from two segments of different lungs (5 normal volunteers, 6 symmetrical lung disease patients, and 4 asymmetrical lung disease patients). Healthy volunteers were younger than subjects with emphysema (41 +/- 11, 61 +/- 6 respectively, p = 0.003). Although no significant difference in FEV1 was observed, emphysema groups trended to have lower FEV1/FVC (p=ns). There were no differences in high relative abundant taxa (greater than 0.05) between the right and left lung of normal volunteers and emphysema subjects with symmetrical lung disease. However, despite the small n, emphysema subjects with asymmetrical lung disease trended to have higher relative abundance of background environmental microbiome in lung segments with less disease ( Propionibacterium relative abundance = 0.13 +/- 0.04 for segments with less disease as compared with 0.03 +/- 0.04 in the more disease segments, p < 0.08). We will complete sequence in 5 more emphysema subjects and compare microbiota with in-vivo BAL cytokines. Conclusions: Patients with observable asymmetrical lung disease have lower background environmental microbiome in more diseased lung when compared to the less diseased side. This difference was not observed in normal volunteers and patients with symmetrical lung disease. Disappearance of background environmental microbiome in more diseased lung segments suggests higher airway colonization, which might be associated with subclinical inflammation

Rationale: Cigarette smoke causes a field of injury and molecular changes in the airways even in histologically normal areas termed "field cancerization" which describes the site(s) of neoplasia and adjacent normal tissue with molecular abnormalities in common. MicroRNAs ( miRNAs) are small, non-coding RNAs that act as post-transcriptional regulators of gene expression by recognizing target sites in the 3' untranslated regions (3'UTRs) via incomplete base-pairing and induce mRNA degradation or translational repression. Deregulation of miRNAs has been linked to cancer initiation and progression, and miRNAs may act as tumor suppressor genes or oncogenes. We hypothesized that miRNA expression in the peripheral airways of smokers with lung cancer is distinct from that of smokers without lung cancer and therefore, miRNAs can be used as biomarkers for the early detection of lung cancer. Methods: We collected human peripheral airway epithelial cells by bronchoscopic brushing from the unaffected lung of thirteen smokers with lung adenocarcinoma and twelve control smokers. Total RNA was extracted from the peripheral airway epithelial cells by miRNAeasy and miRNA profiling was performed using the TaqMan Quantitative qRT-PCR miRNA Assay. Results: Comparison of miRNA levels in peripheral airway epithelial cells from smokers with or without lung cancer demonstrated 53 miRNAs that were significantly different (p<0.05) between the two groups. The majority of miRNAs were up-regulated (41 miRNAs) in lung cancer patients, including miR-21, miR-26a, miR-31, miR-34c, and miR-205. Down-regulated miRNAs included let-7b, let-7e, and miR-126. Several of the miRNAs with increased expression are of interest: miR-21 inhibits tumor suppressor protein PTEN, miR-26a suppresses PTEN and increases AKT phosphorylation and nuclear factor kappaB (NFkappaB) activation, miR-31 represses tumor suppressor genes LATS2 and PPP2R2A, miR-205 is associated with cancer relapses, and miR-34c, a p53 target induced by DNA damage, suggests the involvement of p53 pathway in field carcinogenesis. Down-regulated let-7b leads to higher expression of CYP2J2 and decreased miR-126 enhances adhesion, migration and invasion through increased Crk protein. Further gene expression and pathway analyses will corroborate the relationship between miRNAs and predicted pathways in real time. Conclusion: We discovered a profile of miRNAs in the contralateral lung of patients with lung cancer as biomarkers of field cancerization in smokers with lung adenocarcinoma. Further knowledge of field cancerization may lead to better understanding of tumorigenesis and development of biomarkers for early lung cancer detection

Rationale: While bacterial colonization leads to airway inflammation and accelerated airway obstruction in advanced COPD, its role in early disease is not well understood. Major limitations have been the ability to study airway colonization in early COPD and measurements of in vivo cytokines levels in the lung. Here, we used a pyrosequence approach for 16s rDNA to study the airway microbiome and a concentration technique to achieve measurable levels of cytokines in BAL fluid from early emphysema and normal volunteers. We hypothesize that the microbiome of early emphysema will be characterized by the presence of potential pathogens that are associated with subclinical inflammation. Methods: CT-scan defined emphysema. Supraglotic and broncho-alveolar lavage (BAL) samples were obtained with two different bronchoscopes. Bacteria speciation was performed with 454 sequencing of rDNA. BAL differential was performed on diff quick stained cytospine slides. BAL was concentrated 50 fold by lyophilization and cytokes were measured using Luminex. Results: 24 subjects were available for analysis (8 healthy normal volunteers and 16 with emphysema). All emphysema subjects were ex-smokers (normal 1+/-0.5 vs. 39+/-20 pack-yr., p=0.019). Lung function of emphysema subjects was characterized by lower FEV1/FVC (78.9+/-5.1 vs. 69.6+/-6.1, p=0.001) and hyperinflation (TLC=87.7+/-10.3 vs. 104.3+/-15.2% predicted, p=0.016). Background environmental microbiome, present in "sterile" saline, is enriched with Staphylococcus and Propionobacterium rDNA (Figure 1 right panel). Supraglotic microbiome is enriched with Prevotella and Streptococcus (left panel). Individuals' BAL microbiome varied from a microbiome more similar to supraglotic with higher relative abundance of Prevotella to a background microbiome (middle panel). In this small sample there was a non-significant trend to higher relative abundance of Prevotella in emphysema vs. normal (p=0.078). There was significant positive correlation between Prevotella and BAL inflammatory cytokines (r2=0.48,p<0.001 and r2=0.22,p<0.02 for IL-1alpha and IL-8 respectively, Figure 2 panel A and B). Furthermore, Prevotella directly correlates with Neutrophils in lung (r2=0.24,p=0.02, Panel C). Conclusions: This data shows that inflammatory cytokines are produced and neutrophils recruited to the alveolar space when the lung and supraglotic microibome are similar. In patients with Prevotella, the bacteria has been present long enough to produce subclinical inflammation. It is therefore unlikely that the Prevotella microbiome is due to carryover during bronchoscopy. In individuals with a BAL microbiome similar to saline, poor sinal-to-noise ratio prevents investigation of the lung microbiome. The Prevotella microbiome more common in emphysema is associated with increased bronco-alveolar cytokines and neutrophils, suggesting an immunological response to this lung microbiome. (Figure Presented)