Faculty Bibliography

Rationale: Macrolide antibiotics, specifically azithromycin, have antimicrobial and immunomodulatory effects and, despite not having proven effect on spirometry, have been shown to prevent exacerbations in patients with moderate to severe chronic obstructive disease (COPD). We have previously shown that in asymptomatic smokers with early emphysema identified by computed tomography, distal lung dysfunction is an early marker of subclinical lung inflammation. Thus, we hypothesized that in early emphysema, treatment with azithromycin will impact both distal lung function and biomarkers of airway inflammation. Methods: Emphysema subjects were identified from the NYU Lung Cancer Biomarker Center CT-Scan Screening Cohort. Ten subjects (7M/3F) with emphysema were enrolled for pulmonary function evaluation and research bronchoscopy pre and post eight weeks 250mg/day azithromycin therapy. Physiologic assessment included spirometry, plethysmography, and diffusing capacity. Distal lung function was assessed (pre and post bronchodilator) with impulse oscillometry (IOS). Pre and post bronchodilator exhaled nitric oxide (NO) was measured at variable flow rates to determine airway and alveolar NO concentration. Results: Subjects were 65+/-4 years age. All had history of smoking with emphysema identified on computed tomography. Subjects were asymptomatic with GOLD 0 spirometry in 9/10. Lung volumes (FRC, RV and TLC) and diffusing capacity were within normal limits in all subjects. In contrast, baseline IOS revealed abnormal resistance spectrum in 5/10 and abnormal reactance spectrum in 8/10, consistent with dysfunction in the distal lung. Post bronchodilator there was significant reduction in frequency dependence of resistance and in the reactance spectrum (R5-20 = 3.88 [3.39, 5.85] vs. 3.39 [3.26, 5.06] cmH2O/L/s, p = 0.022; X5 = -1.40 [-2.02, -1.01] vs. -1.03 [-1.47, -0.90] cmH2 O/L/s, p = 0.022; resonant frequency 16.2 [13.2, 20.1] vs. 13.6 [10.9, 16.2] Hz, p = 0.007). Following azithromycin therapy, IOS demonstrated no change in resistance; however, improved reactance was seen in 8 patients (p<0.04) and bronchodilator responsiveness was no longer present. Alveolar NO normalized in all subjects post azithromycin (baseline range 1.2-9.9 vs. 0-3.6 PPB post azithromycin, p=0.06 ) despite lack of change in airway NO. (Figure presented) Conclusions: In patients with early emphysema, azithromycin administration was associated with improved oscillometry reactance but not resistance parameters and improved alveolar rather than airway NO. These data support a beneficial effect of azithromycin on distal lung function and inflammation that may not be detected by routine tests

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.

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: 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: 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)

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

OBJECTIVES/SPECIFIC AIMS: The lung is classically thought to be sterile although molecular techniques for microbial identification are now suggesting the existence of a human airway microbiota. The study of the microbiome has now opened an opportunity to characterize resident microbial flora without the need for bacterial culture although the lung microbiome in smokers has not been characterized. We therefore tested the hypothesis that in smokers, the upper and lower airways contain a distinct microbiota. METHODS/STUDY POPULATION: We obtained supraglotic and broncho-alveolar lavage (BAL) samples in 8 subjects. Bacterial quantification of supraglotic aspirate and BAL was determined by qPCR using universal primers for 16S rDNA. Dilution was corrected by urea. We defined bacteria OTU by 454 sequencing. We performed cluster analysis, principal coordinate analysis and weighted UniFrac to determine microbiome. RESULTS/ANTICIPATED RESULTS: Subjects were 55 +/- 13 yo, 4/8 female and 7/8 significant smokers (>10 pack/year). FEV1 was 94 +/- 11%, FVC 108 +/- 10% and FEV1/FVC was 71 +/- 8. Bacteria rDNA was higher in the supraglotic area than BAL (1.5e9 +/- 3.2e9 vs. 1e7 +/- 8.2e6 copies/mL of adjusted fluid, p < 0.001). Clustering of the bacterial community at the family level showed distinct microbiome in the upper airway and the lung (BAL). While Prevotellaceae, Veillonellaceae and Fusobacteriaceae predominated in the supraglotic sample, Micrococcaceae, Propionibacteriaceae and Staphylococcaceae among others predominated in BAL. UniFrac calculated distances showed no overlapping circle of inertia between supraglotic and BAL (p < 0.0001 for first principal axis). DISCUSSION/SIGNIFICANCE OF IMPACT: In the absence of signs or symptoms of infection, subjects had significant airway resident bacteria. Th is supports the existence of a bacterial reservoir in the lung, which might be influenced by smoking and/or innate immunity

BACKGROUND: The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to approximately 50 mm were recovered from rescue workers' lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. METHODOLOGY/PRINCIPAL FINDINGS: Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM(10-53) or WTC-PM(2.5) at concentrations of 10, 50 or 100 microg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM(10-53) and PM(2.5). GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM(10-53) and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM(10-53) consistently induced more cytokine release than WTC-PM(2.5) at 100 microg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. CONCLUSIONS: WTC-PM(10-53) induced a stronger inflammatory response by human AM than WTC-PM(2.5). This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.

Introduction: The lung is classically thought to be sterile although molecular techniques for microbial identification are now suggesting the existence of a human airway microbiota. The microbiome of emphysema patients remains largely uncharacterized. We hypothesize that lung microbiota differs in early CT-defined emphysema subjects when compared with normal volunteers and that microbial load is associated with host cytokine production. Methods: Emphysema subjects were identified from the NYU Lung Cancer Center CT-scan screening cohort. Supraglotic and broncho-alveolar lavage (BAL) samples were obtained with bronchoscopy. Bacterial quantificationofepithelial lining fluid (ELF) was determined by qPCR using universal primers for eubacteria 16S rDNA. Bacteria speciation will be performed with 454 sequencing. Alveolar macrophages obtained after centrifugation of BAL were incubated with media alone or lipopolysaccharide (LPS). Thirty-ninecytokines were measured in BAL 24-hr supernatants using Luminex. Cytokine production was clustered based on the protein production post LPS stimulation compared with the media alone. Results: 24 subjects were enrolled (8 healthy normal volunteers and 16 with emphysema). Mean age was 37+/-10 and 62+/-6 yr. (p=0.001). All emphysema subjects were ex-smokers (normal 1+/-0.5 vs. 39+/-20 pack-yr., p=0.019). There were no differences in the %BAL cells between the two groups (Alveolar macrophages 89.5+/-4 vs. 88.1+/-8.2%). 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). Bacteria rDNA was higher in the supraglotic area than lower respiratory tract (1.5e9+/-3.2e9 vs. 1e7+/-8.2e6 copies/mL of ELF, p<0.001). Bacterial load was similar in normal volunteers and emphysema subjects (8.4e6+/-8.8e6 vs. 1.1e7+/-8.1e6 copies/mL of ELF, p=ns). Cytokines'response to TLR stimulation grouped all study subjects into two clusters, and those with higher pro-inflammatory cytokine production were associated with higher amounts of bacteria in ELF (1.4e7+/-8.6e6 vs. 5.7e6+/-1.8e6 copies/mL, p<0.03). Conclusions: In the absence of signs or symptoms of infection both emphysema patients and normal controls had significant airway resident bacteria. Surprisingly, bacterial load was not increased in early CT-defined emphysema. Higher bacterial load was associated with higher TLR2/4-induced cytokine production. Next generation rDNA sequence data will define if microbiota is altered by emphysema or associated with TLR response

RATIONALE: The destruction of the World Trade Center (WTC) led to the exposure of over 300,000 rescue workers and residents to particulate matter (WTC-PM) and other products of combustion. Victims have suffered from a wide array of adverse health effects. Particulates up to 50 microns have been recovered from the airways of rescue workers 10 months after the event. Our study focuses on understanding the cytokine and chemokines elaboration from the ex-vivo exposure of human alveolar macrophages (AM) from 15 subjects exposed to WTC-PM₅₃ and PM_2.5. METHODS: AM from 15 volunteers with normal chest radiographs were incubated in media alone (MA) to measure spontaneous cytokine release, LPS 40 ng/ml as a positive control, or suspensions of WTC-PM₅₃ and WTC-PM_2.5 at concentrations of 10, 50 and 100 mug/mL to assess the impact of WTC dust. Supernatants were assayed for 39 pro-inflammatory chemokines and cytokines 24 hours after exposure. RESULTS: PM₅₃ and PM_2.5 produced dose dependent analyte induction for a majority of the chemokines and cytokines. WTC-PM53 induced significantly more GM-CSF, G-CSF, IL-6, TNF-alpha, IL-10, IL-1beta, IP-10 and GRO than fine WTC-PM_2.5, Table 1. PM₅₃ 100mug/mL induced more GM-CSF than LPS 40ng/ml. For all other cytokines and chemokines tested PM₅₃ induced less analyte than LPS. Baseline GM-CSF and MDC elaboration varied in AM preparations; the median GM-CSF concentration was 16.4(IQR: 7-66) pg/mL, and MDC was 1803(483-3348). There was a strong correlation between baseline elaboration and dust induced GM-CSF and MDC; the correlation coefficients between baseline and dust-induced GM-CSF and MDC expression are shown in Table 2. CONCLUSIONS: Ex-vivo exposure of human AM to coarse WTC-PM53 stimulated production of significantly more inflammatory mediators than fine WTC-PM_2.5, suggesting that inhalation of coarse PM at the WTC produced high incidence of lung injury in those exposed. There was patient specific variation in response to dust that may contribute to individual susceptibility to lung injury after irritant dust exposure. (Table presented)