Faculty Bibliography

Malignant pleural effusions (MPE) complicate malignancies and portend worse outcomes. MPE is comprised of various components, including immune cells, cancer cells, and cell-free DNA/RNA. There have been investigations into using these components to diagnose and prognosticate MPE. We hypothesize that the microbiome of MPE is unique and may be associated with diagnosis and prognosis. We compared the microbiota of MPE against microbiota of pleural effusions from non-malignant and paramalignant states. We collected a total of 165 pleural fluid samples from 165 subjects; Benign (n = 16), Paramalignant (n = 21), MPE-Lung (n = 57), MPE-Other (n = 22), and Mesothelioma (n = 49). We performed high throughput 16S rRNA gene sequencing on pleural fluid samples and controls. We showed that there are compositional differences among pleural effusions related to non-malignant, paramalignant, and malignant disease. Furthermore, we showed differential enrichment of bacterial taxa within MPE depending on the site of primary malignancy. Pleural fluid of MPE-Lung and Mesothelioma were associated with enrichment with oral and gut bacteria that are commonly thought to be commensals, including Rickettsiella, Ruminococcus, Enterococcus, and Lactobacillales. Mortality in MPE-Lung is associated with enrichment in Methylobacterium, Blattabacterium, and Deinococcus. These observations lay the groundwork for future studies that explore host-microbiome interactions and their influence on carcinogenesis.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

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

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