Faculty Bibliography

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.

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)

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.

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

Introduction: Airway dysfunction has been detected by oscillometry in obese subjects despite normal large airway function as assessed by spirometry. This has been attributed to lung/airway compression as reflected by reduced FRC; we previously demonstrated improvement of abnormalities towards normal upon voluntary inflation to predicted FRC (AJRCCM 2010; 181:A2532). However, other causes of airway dysfunction such as inflammation or concomitant intrinsic airway disease may coexist and could not be excluded. The present study re-evaluated these subjects following bariatric surgery induced weight loss to evaluate for residual abnormality. Methods: 22 morbidly obese subjects without history of smoking and/or cardiopulmonary disease, underwent evaluation pre/post bariatric surgery (20% reduction in weight). Spirometry, plethysmography and impulse oscillometry (IOS) were performed. IOS parameters included resistance at 5Hz (R5), resistance at 20Hz (R20) frequency dependence of resistance (R5-20) and reactance at 5Hz (X5).IOS was also performed at an elevated lung volume (~1 liter) targeted to restore FRC to predicted values. All IOS measurements were repeated post bronchodilator. Results: Baseline weight and BMI were 256+/-43 kg and 46+/-7 kg/m², respectively. All subjects lost >20% of body weight, but obesity persisted in all subjects (weight 182 kg, BMI 33 kg/ m2). FEV /FVC was normal at baseline and remained unchanged post weight loss 1 (81+/-3% vs 83+/-4%) indicating normal large airway function. FRC and ERV improved post weight loss but values remained abnormal (FRC from 60+/-12 to 77+/-21% predicted, ERV from 46+/-16 to 75+/-38% predicted, p<0.05). Although IOS parameters improved following weight loss, data remained above the upper limit of normal (R5 from 6.8+/-1.8 to 5.1+/-1.4 cmH₂O/l/s, R₂₀ from 4.7+/-1.1 to 3.9+/-0.9 cmH₂O/l/s, R_5-20 from 2.1+/-1.1 to 1.2+/-0.9 cmH_2O /l/s, X₅ from -3.2+/-1.7 to -1.8+/-0.9 cmH₂O /l/s, p<0.05). Since FRC remained abnormal following weight loss, IOS was repeated following voluntary lung inflation (FRC 142+/-30%). While R20 corrected to normal at the elevated FRC (R₂₀ 3.1+/-1.0 cmH₂ O/l/s), R5, R5-20 and X5 remained abnormal indicating residual distal airway dysfunction (R₅ 4.2+/-1.4 cmH₂O/l/s, , R_5-20 1.1+/-0.7 cmH₂O /l/s, X₅ -2.0+/-0.8 cmH₂O /l/s); these residual oscillometric abnormalities were present in 11/22 subjects. Residual airway dysfunction was demonstrated by low specific conductance (assessed at 5HZ) despite restoration of FRC to supranormal values. Conclusions: Distal airway dysfunction persisted following weight loss and was not attributable to persistent mass loading in a subgroup of patients without clinical evidence of airway disease. These abnormalities may represent either functional abnormalities due to persistent obesity and/or intrinsic airway disease

Introduction: Macrolides are associated with a significant reduction in exacerbations compared with placebo in both chronic obstructive lung disease and cystic fibrosis. However, this may be due anti-bacterial and/or anti-inflammatory effects. The mechanism of the anti-inflammatory effects of macrolides in the lung are poorly understood. Alveolar macrophages (AM) have a central role on innate immune homeostasis of the lung; AM have developed many mechanisms to prevent autonomous induction and excess production of inflammatory cytokines through an increased expression of immune inhibitors such as C/EBPbeta and MafB. Interferon induces inhibitory C/EBPbeta, is regulated by MafB and has anti-inflammatory effect in the lung. Here, we test the hypothesis that azithromycin suppresses innate immune response by induction of inhibitory transcription factors. Methods: Alveolar macrophages were obtained from five subjects and incubated with lipopolysaccharide (LPS), interferon (IFN)-alpha, azithromycin (AZM) and AZM+LPS for 24hrs. Thirty nine supernatant cytokines were measured using Luminex. Protein extracts were obtained from AM and used for immunoblots for C/EBPbeta and MafB. Results: LPS strongly induced TNF-alpha production by AM, while the combination AZM and LPS inhibited LPS induced TNF-alpha production (Figure 1, p=0.02). AZM by itself had minimal effect. AZM also inhibited LPS induced CCL22, MDC, IL-1b, IL-6, IL10, G-GSF and GM-CSF. The inhibitory C/EBPbeta transcription factor and MafB expression was induced when AZM was added with LPS (Figure 2). Conclusions: AZM inhibits inflammatory cytokine production after LPS stimulation and induces inhibitory C/EBPbeta and MafB in AM. We will investigate if induction of innate immune inhibitors that blocks TLR signaling pathways underlies the broad anti-inflammatory effect achieved by macrolides. (Figure presented)

Introduction:Regulatory T cells (Treg) play important roles in immunological self-tolerance, and are functionally immunosuppressive subsets of T cells. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. It has been shown that the balance between Treg and Th17 cells is a key factor that regulates helper T-cell (CD4⁺ or Th) function. However, there is limited information on the balance between Treg and Th17 cells in bronchoalveolar lavage (BAL). We investigated the distribution of Th17 cells in relation to Treg in PBMC and BAL of healthy volunteers, emphysema patients and HIV-1 infected patients. Methods:BAL lymphocytes were obtained by plating BAL cells for >=1 hour to remove adherent alveolar macrophages. PBMC were isolated by Ficoll gradient fractionation. BAL and PB lymphocytes were stimulated with PMA and ionomycin, treated with monensin, permeabilized, then labeled with PerCP-anti-CD4, PE-anti-IL-17 and Alexa Fluor 647 anti-Foxp3, and analyzed on a FACSCalibur to determine the percentage of CD4⁺ lymphocytes expressing IL-17 and Foxp3 (mean+/-SD). Results:Ten subjects were available for analysis (2 healthy volunteers, 5 emphysema and 3 HIV-1 infected subjects). Both PB and BAL Th cells presented a wide range of FoxP3⁺ cells (19.2+/-18.6 and 6.3+/-5.9 respectively) and IL-17⁺ cells (9.5+/-8.1 and 8.8+/-4.6 respectively). There was a greater percentage of FoxP3⁺ in the HIV-infected group compared to the healthy volunteer and emphysema groups (11.7+/-6.4 vs. 1.7+/-1.1 and 4.9+/-4.7 respectively). Similarly, there was a trend towards higher percentage of IL-17⁺ cells in the BAL of HIV-infected subjects compared to the healthy volunteer and emphysema subjects (10.8+/-3.6 vs. 5.6+/-0.9 and 8.9+/-5.8 respectively). When the expression of FoxP3⁺ and IL-17⁺ cells in BAL was compared for each subject, a direct correlation with an r2 of 0.36 and a p-value of 0.06 was found Conclusions:We observed a relative preservation of FoxP3⁺ and IL-17⁺ cells in BAL CD4⁺ lymphocytes, despite prior evidence suggesting a preferential loss of both Treg and Th17 subsets in PBMC of HIV-1 infected subjects. The co-ordinated expression of these subsets in BAL Th cells warrants further investigation to evaluate its immune significance in the alveolar compartment

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R-associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-alpha fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IkappaBalpha, or induce TNF-alpha. CASP-6(-/-) mice subjected to cecal ligation and puncture have impaired TNF-alpha production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6-mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces