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: An increased ratio of pulmonary artery to aorta (PA/A) diameter, as a marker of vascular injury, measured by computed tomography (CT) predicts future exacerbations in patients with chronic obstructive pulmonary disease (COPD) Wells et. al. NEJM. World Trade Center (WTC) exposed fire fighters have developed respiratory symptoms and a subset had a decline in pulmonary function. Our group has previously shown that systemic biomarkers of inflammation and cardiovascular disease predict this decline. We hypothesize that a PA/A ratio >/= to 1 will be associated with a decline in FEV1. Methods: From a baseline cohort of never smokers with normal spirometry pre 9/11, cases and controls were selected. Cases had FEV1 fall to less than the lower limit of normal (< LLN), and controls had preserved lung function, see figure 1. Spirometry was performed according to ATS/ERS guidelines. Inspiratory series CT images, collected contemporaneously with spirometry, were retrospectively assessed using iSite PACS, (Philips iSite Enterprise, Version 3.6.114; www.healthcare.philips.com). The diameter of the main PA at the level of its bifurcation and the diameter of the ascending aorta in its maximum dimension were recorded using the same image. ES, who was blinded to group assignment, made all measurements. Statistics and data management were performed using SPSS. Results: CT images were available for 91 patients in the case/control cohort. Body mass index (BMI), age, exposure and pulmonary function data are shown in Table 1. Exposure intensity, age at exposure, time from 9/11 to spirometry and to CT were similar. BMI was increased in cases compared to controls. The mean PA diameter and PA/A ratio were increased in cases (p=0.05, 0.09), the mean A diameter was similar. Using binary logistic regression the odds ratio of having an FEV1< LLN if the PA/A ratio was >/= 1 was 3.6 (p=0.047), when corrected for exposure, age at 9/11 and BMI. Conclusions: In this preliminary study a PA/A ratio >/= 1 was associated with WTC related decline in FEV1. There are several potential confounders. Data on the presence of congestive heart failure, sleep apnea and other comorbidities are presenting lacking. Additionally, patient effort and respiratory system compliance may influence inspiratory measurements of the PA. In future studies, we plan to analyze expiratory CT images and correlate with other markers of heart disease. Increased PA/A represents another potentially useful non-invasive tool to assess for obstructive lung dysfunction and warrants further study. (Table Presented)

RATIONALE: Analysis of local in vivo inflammation is relevant to the understanding of pathogenesis and disease progression in emphysema. Bronchial reactivity is an early marker of disease in asthma but the relevance of reactivity to the natural history of emphysema is not understood. We hypothesize that bronchial reactivity is a phenotype of early emphysema that might be related to the degree of inflammation in the lung. METHODS: Normal subjects were enrolled as part of a normal volunteer protocol. Emphysema subjects were identified from the NYU Lung Cancer Biomarker Center CT-scan screening cohort. All patients underwent spirometry, plethysmography, diffusion, and oscillometry, as well as bronchoscopy with bronchoalveolar lavage (BAL). Bronchial reactivity was assessed by changes in FEV1, V50 and R5 . From the BAL fluid, cell count differential was obtained, as well as measurement of 39 cytokines in concentrated BAL fluid with Luminex using Human Cytokine Panel I (Millipore). Results amongst the groups were compared with ANOVA and post-hoc LSD comparison. RESULTS: Twenty patients were available for analysis: Six subjects in the control group, 6 emphysema subjects without bronchial reactivity (BR-), and 8 emphysema subjects with bronchial reactivity (BR+). Baseline demographics and pertinent spirometry/oscillometry are listed in Table 1. Emphysema subjects were all GOLD stage 0 or 1. Post-bronchodilator spirometric and oscillometric parameters were not significantly different between BR- and BR+ emphysema groups. There were 28/39 cytokines with reliably measurable levels. Both emphysema groups had elevated neutrophils and higher degree of inflammation as compared to controls (significant data shown Table 1). However, the BR+ emphysema group evidenced higher degree of neutrophils, IL-6, IL-8, G-CSF, Eotaxin, GRO and Fractalkine as compared with the BR- emphysema group. CONCLUSION: These data suggest that in early emphysema a phenotype of proximal and/or distal bronchial reactivity is associated with an increased degree of inflammation as assessed by neutrophils and in vivo inflammatory cytokines. In contrast with early asthma, the phenotype of bronchial reactivity in early emphysema may be characterized by neutrophilic inflammation produced by increased IL-8 in the lung. The role of IL-6, G-CSF, Eotaxin, GRO and Fractalkine in producing emphysema related bronchial reactivity requires further investigation. (Table Presented)

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)

BACKGROUND: The lung microbiome of healthy individuals frequently harbors oral organisms. Despite evidence that microaspiration is commonly associated with smoking-related lung diseases, the effects of lung microbiome enrichment with upper airway taxa on inflammation has not been studied. We hypothesize that the presence of oral microorganisms in the lung microbiome is associated with enhanced pulmonary inflammation. To test this, we sampled bronchoalveolar lavage (BAL) from the lower airways of 29 asymptomatic subjects (nine never-smokers, 14 former-smokers, and six current-smokers). We quantified, amplified, and sequenced 16S rRNA genes from BAL samples by qPCR and 454 sequencing. Pulmonary inflammation was assessed by exhaled nitric oxide (eNO), BAL lymphocytes, and neutrophils. RESULTS: BAL had lower total 16S than supraglottic samples and higher than saline background. Bacterial communities in the lower airway clustered in two distinct groups that we designated as pneumotypes. The rRNA gene concentration and microbial community of the first pneumotype was similar to that of the saline background. The second pneumotype had higher rRNA gene concentration and higher relative abundance of supraglottic-characteristic taxa (SCT), such as Veillonella and Prevotella, and we called it pneumotypeSCT. Smoking had no effect on pneumotype allocation, alpha, or beta diversity. PneumotypeSCT was associated with higher BAL lymphocyte-count (P= 0.007), BAL neutrophil-count (P= 0.034), and eNO (P= 0.022). CONCLUSION: A pneumotype with high relative abundance of supraglottic-characteristic taxa is associated with enhanced subclinical lung inflammation.

OBJECTIVE: After 9/11/2001, some Fire Department of New York (FDNY) workers had excessive lung function decline. We hypothesized that early serum matrix metalloproteinases (MMP) expression predicts World Trade Center-Lung Injury (WTC-LI) years later. METHODS: This is a nested case-control analysis of never-smoking male firefighters with normal pre-exposure Forced Expiratory Volume in one second (FEV1) who had serum drawn up to 155 days post 9/11/2001. Serum MMP-1, 2,3,7,8, 9, 12 and 13 were measured. Cases of WTC-LI (N = 70) were defined as having an FEV1 one standard deviation below the mean (FEV1