Faculty Bibliography

RATIONALE/BACKGROUND:Microbiome studies of the lower airway based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS:Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. MEASUREMENTS/METHODS:We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole genome (WGS) and RNA metatranscriptome sequencing. Short chain fatty acids (SCFA) were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. MAIN RESULTS/RESULTS:Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFAs levels were compared with WGS and metatranscriptome. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS:Functional characterisation of the lower airway microbiota through metatranscriptome identify metabolically active organisms capable of producing metabolites with immunomodulatory capacity such as SCFAs.

Rationale Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with increased Th17 inflammatory phenotype. In this study we evaluated the microbial and host immune response dynamics after aspiration with a oral commensals using a preclinical mouse model. Methods Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of sacrifice. Genetic background of mice included WT, MyD88 knock out and STAT3C. Measurements 16S rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host transcriptome sequencing was used to characterize host immune phenotype. Main Results While MOC aspiration correlated with lower airway dysbiosis that resolved within five days, it induced an extended inflammatory response associated with IL17-producing T-cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration prior to a respiratory challenge with S. pneumoniae led to a decreased in host's susceptibility to this pathogen. Conclusions Thus, in otherwise healthy mice, a single aspiration event with oral commensals are rapidly cleared from the lower airways, but induce a prolonged Th17 response that secondarily decreased susceptibility to respiratory pathogens. Translationally, these data implicate an immuno-protective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower airway pathogens.

RATIONALE: Derived neutrophil-to-lymphocyte ratio (dNLR) of peripheral blood, a marker of host inflammation and cytokine activation, may be a surrogate for more aggressive disease. It is a biomarker that has been associated with survival and response to immunotherapy in non-small cell lung cancer (NSCLC), although an optimal threshold value has not been established. We had previously found in a NSCLC cohort that a dNLR cutoff of 2 was an optimal cutoff to predict survival at 6 months in patients with NSCLC; median survival was significantly shorter in patients with a >=2 dNLR (7.0 months) versus those with a <2 dNLR (64.5 months; p = 0.004). Here we present an interim analysis aiming to validate the use of this biomarker in a second cohort.
METHOD(S): A veteran cohort (n=42) from the VA New York Harbor Healthcare System, who underwent diagnostic bronchoscopy and found to have NSCLC, was used as a validation cohort. Peripheral blood was obtained pre-treatment and at or near the time of diagnosis. The dNLR was calculated as ANC/(W

Rational Recent investigations support that the gut microbiota influences anti-PD-1 cancer immunotherapy. Lower airway dysbiosis with enrichment with oral commensals are associated with lung cancer. Recently we had shown, in both a prospective human cohort and preclinical mice model, that lung dysbiotic signatures were associated with clinical lung cancer prognosis and progression. To further understand the role of lung dysbiosis in lung cancer, we examined the role of PD-1 expression and anti- PD treatment in a lung cancer and lung dysbiotic model. Method KrasLSL-G12D/+;p53fl/fl Non-small cell Lung Cancer mice (KP) were challenged with an oral commensal, Veillonella parvula, through intra-tracheal inoculation and exposed to immune inhibition (anti- PD-1). Measurements included tumor burden and lower airway inflammatory markers (PD-1 expression and neutrophils) by FACS. Results In a preclinical lung cancer model, inoculation with Veillonella parvula, a marker taxon for the dysbiotic signature found in humans, led to: 1) decrease survival with increase tumor burden; 2) dysbiosis with oral commensal is associated with elevated level of PD-1 expression and neutrophils level compared to control. With exposure to PD-1 inhibition we observe a reverse of tumor growth (at day 7); there was significant decrease in tumor growth compared with Isotype-control (p=0.030, day7-14) and observed that PD-1+ level (p=0.0007) and Neutrophil level (p=0.0027) were lower as well. Discussion Our study suggests that lower airway dysbiosis induced by microaspiration of oral commensals may affect lung carcinogenesis due to increase in inflammatory markers and increase in the checkpoint inhibitor tone in the lower airways that may lead to suboptimal immune surveillance. These effects of lower airway dysbiosis can be partially blunted by PD-1 blockade. These data supports that treatment in lung cancer may be influenced by lower airway dysbiosis and dynamic changes in the microbial-host interaction in the lower airways

Rationale: Chronic airway colonization and recurrent infections are common in advanced stage chronic obstructive pulmonary disease (COPD). However, changes in the lung microbiota in early stages of this disease remain unclear. Here, we characterized the upper and lower airway microbiota of patients with early stage COPD and smoker controls.
Method(s): Upper and lower airway samples (plus appropriate environmental and technical controls) were obtained from patients with GOLD 1-2 COPD (n = 26) and smoker controls (n = 31). Bacterial load was measured with droplet digital PCR while microbiota profiling was performed using 16S rRNA gene sequencing. Data was analyzed using QIIME, Phyloseq, Vegan and DESeq. Parallel RNA metatranscriptome sequencing and host Transcriptome approach were just completed and data is becoming available.
Result(s): Characterization of the lower airway microbial communities with 16S rRNA gene sequencing showed that compared to smoker controls, COPD patients exhibited lower alpha Shannon diversity (Fig.1a, p = 0.0037). Beta diversity analysis based on Bray Curtis Dissimilarity index showed that the composition of the microbial communities in the lower airway samples were clearly distinct from background and upper airway as a whole. Some samples overlapped with both of those areas suggesting that for some subjects their lower airway microbiota was enriched with taxa commonly found in the oral cavity. We then evaluated for differentially enriched taxa in BAL samples using DESeq. The lower airway microbiota of subjects with COPD was enriched with oral commensals such as Veillonella, Prevotella (Fig 1c). Comparison of bacterial load based on bacterial composition was performed based on cluster determination of lower airway samples enriched with oral commensals (SPT for supraglottic predominant taxa) or enriched with background taxa (BPT for background predominant taxa). The bacterial load of lower airway samples categorized as SPT was one log higher than those categorized as BPT among the COPD group but not among the smoker controls (Fig.1d, p < 0.001).
Conclusion(s): Our results suggest that lower airway exposure to oral commensals occurs more frequently among subjects with COPD. Further investigation with functional microbiome approaches such as metatranscriptomics are warranted. This may be of importance given significant data showing that these taxa may contribute to an increase in lower airway inflammatory tone (especially in the Th17 pathway) that may lead to airway/parenchymal inflammatory damage and/or affect treatment response and clinical outcome in this disease

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

In lung cancer, enrichment of the lower airway microbiota with oral commensals commonly occurs and ex vivo models support that some of these bacteria can trigger host transcriptomic signatures associated with carcinogenesis. Here, we show that this lower airway dysbiotic signature was more prevalent in group IIIB-IV TNM stage lung cancer and is associated with poor prognosis, as shown by decreased survival among subjects with early stage disease (I-IIIA) and worse tumor progression as measured by RECIST scores among subjects with IIIB-IV stage disease. In addition, this lower airway microbiota signature was associated with upregulation of IL-17, PI3K, MAPK and ERK pathways in airway transcriptome, and we identified Veillonella parvula as the most abundant taxon driving this association. In a KP lung cancer model, lower airway dysbiosis with V. parvula led to decreased survival, increased tumor burden, IL-17 inflammatory phenotype and activation of checkpoint inhibitor markers.

BACKGROUND:Emphysema in asymptomatic heavy smokers can be detected during CT-scan screening for lung cancer. Metalloproteinases (MMPs) have been found to play a role in the pathogenesis of chronic obstructive pulmonary disease and to possibly serve as biomarkers for emphysema. METHODS:The NYU Lung Cancer Biomarker Center enrolled study subjects over 50 years of age with lung cancer risk factors from January 1, 2010, to December 31, 2015. These subjects received chest multi-detector computed tomography, spirometry, and provided serum for immunoassays for metalloproteinases (MMP) -1, -2, -7, -9, -10 and tissue inhibitor of metalloproteinases (TIMP) -1 and -2. RESULTS:/FVC percent compared to smokers without emphysema (68 ± 11 (mean ± sd) versus 75 ± 8; p < 0.0001). Increased age and pack-years of smoking were associated with increased odds of emphysema. None of the metalloproteinases or tissue inhibitors of metalloproteinases were useful to predict the presence of emphysema in smokers. CONCLUSION/CONCLUSIONS:/FVC ratio).