Faculty Bibliography

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.

INTRODUCTION/BACKGROUND:Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease. As part of a public health investigation of a manufacturing facility, we performed paired environmental and human sampling to evaluate cross-pollination of microbes between environment and host and possible effects on lung pathology present among workers. METHODS:Workplace environmental microbiota was evaluated in air and MWF samples. Human microbiota was evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, lung tissue controls, and in skin, nasal and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B-cells were assessed. RESULTS:Increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared to controls. Among workers in different locations within the facility, those that worked in machine shop area had skin, nasal and oral microbiota more closely related to the microbiota present in MWF samples. Lung samples from four index cases, and skin and nasal samples from workers in machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in pathology of index cases. CONCLUSIONS:Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

INTRODUCTION: Bronchiectasis is a common chronic pulmonary condition characterized by inflammation and recurrent infections. There is evidence that gastroesophageal reflux disease (GERD) is associated with bronchiectasis and can increase the severity of pulmonary disease. Data regarding esophageal function in this population is sparse. We aimed to assess whether patients with bronchiectasis have an increased prevalence of esophageal motility disturbances and GERD.
METHOD(S): We conducted a single-center matched cohort study of all adult patients with confirmed bronchiectasis who underwent esophageal high-resolution manometry (HRM) between 11/ 2014-3/2018. All cases were randomly matched with a control by age (65 years) and sex. Chicago Classification 3.0 was used to characterize HRM findings. Combined multichannel intraluminal impedance-pH (pH-MII) was utilized to assess reflux burden. Statistical relationships between proportions were evaluated by Chi-square or Fisher's exact test and continuous variables were compared using t-test or rank sum test.
RESULT(S): 63 bronchiectasis patients underwent HRM, of which 54 underwent pH-MII. Of the controls, 63 underwent HRM, of which 39 underwent pH-MII. Baseline characteristics between cases and controls were similar. Mean age of bronchiectasis patients was 65 (SD 12.73), mean body mass index was 25.51 (SD 8.50), 70% were female, and 48% had a smoking history (Table). HRM did not demonstrate any significant differences between cases and controls. pH-MII trended towards a greater reflux burden among controls. However, nearly half of cases had conclusive evidence of pathologic reflux by esophageal acid exposure on pH-MII. On endoscopy, no significant differences were noted.
CONCLUSION(S): Esophageal motility and acid exposure did not significantly differ among patients with bronchiectasis and controls, which may indicate that esophageal physiology in bronchiectasis is not unique. Nevertheless, more than half of the bronchiectasis group had evidence of abnormal esophageal motility and almost half of patients had conclusive evidence of pathologic reflux. Small differences are likely due to the high prevalence of GERD and associated motility disorders in the control group. Larger studies are warranted to further characterize esophageal physiology in these patients and the potential impact on pulmonary pathology. (Table Presented)

INTRODUCTION/BACKGROUND:Neutrophilic inflammation is a major driver of bronchiectasis pathophysiology, and neutrophil elastase activity is the most promising biomarker evaluated in sputum to date. How active neutrophil elastase correlates with lung microbiome in bronchiectasis is still unexplored. We aimed at understanding if active neutrophil elastase is associated with low microbial diversity and distinct microbiome characteristics. METHODS:An observational, cross-sectional study was conducted at the Bronchiectasis Program of the Policlinico Hospital in Milan, Italy, where adults with bronchiectasis were enrolled between March 2017 and March 2019. Active neutrophil elastase was measured on sputum collected during stable state, microbiota analysed through 16S rRNA gene sequencing, molecular assessment of respiratory pathogens through real time PCR and clinical data collected. MEASUREMENTS AND MAIN RESULTS/RESULTS:with elevated active neutrophil elastase was found based on standard culture and targeted real-time PCR. CONCLUSIONS:infection.

Our understanding of the existence and role of the lung microbiome has grown at a slower pace than other microbiome research areas. This is likely a consequence of the original dogma that the lung was a sterile environment although there are other barriers that are worth discussing. Here we will not be conducting an exhaustive review of the current literature on the lung microbiome, but rather we will focus on what we see as some important challenges that the field needs to face in order to improve our mechanistic understanding of the lung microbiome and its role on human health.

BACKGROUND:Platelets are effector cells of the innate and adaptive immune system, however understanding their role during inflammation-driven pathologies can be challenging due to several drawbacks associated with current platelet depletion methods. The generation of antisense oligonucleotides (ASO)s directed to thrombopoietin (Tpo) mRNA represents a novel method to reduce circulating platelet count. OBJECTIVE:To understand if Tpo-targeted ASO treatment represents a viable strategy to specifically reduce platelet count in mice. METHODS:Female and male mice were treated with TPO-targeted ASOs and platelet count and function assessed, in addition to circulating blood cell counts and hematopoietic stem and progenitor cells. The utility of the platelet-depletion strategy was assessed in a murine model of lower airway dysbiosis. RESULTS AND CONCLUSIONS/CONCLUSIONS:Herein, we describe how in mice, ASO-mediated silencing of hepatic TPO expression reduces platelet, megakaryocyte, and megakaryocyte progenitor count, without altering platelet activity. TPO ASO-mediated platelet depletion can be achieved acutely and sustained chronically in the absence of adverse bleeding. TPO ASO-mediated platelet depletion allows for the reintroduction of new platelets, an advantage over commonly used antibody-mediated depletion strategies. Using a murine model of lung inflammation, we demonstrate that platelet depletion, induced by either TPO ASO or anti-CD42b treatment, reduces the accumulation of inflammatory immune cells, including monocytes and macrophages, in the lung. Altogether, we characterize a new platelet depletion method that can be sustained chronically and allows for the reintroduction of new platelets highlighting the utility of the TPO ASO method to understand the role of platelets during chronic immune-driven pathologies.

Background: Platelets are effector cells of the innate and adaptive immune system; however, understanding their role during inflammation can be challenging due to drawbacks associated with current platelet depletion methods. The generation of antisense oligonucleotides (ASO) directed to thrombopoietin (Thpo) mRNA represents a novel method to reduce platelet count to aid in elucidating the role of platelets during inflammation.
Aim(s): To understand if Thpo-targeted ASOs represent a viable strategy to reduce platelet count in mice, and to delineate the role of platelets to inflammation resolution during lower airway dysbiosis.
Method(s): Mice were treated with a Thpo-targeted ASO and the abundance of platelets, blood cells and bone marrow hematopoietic progenitor cells assessed. Additionally, platelet responsiveness to agonists and surface expression of P-selection and JON/A was measured. To assess the contribution of platelets to inflammation resolution Thpo-ASO and control-ASO treated mice were challenged with a bacteria cocktail to model lower airway dysbiosis. Thpo-ASO mediated platelet depletion was compared to anti-CD42b platelet depletion in the lung dysbiosis model.
Result(s): ASO-mediated silencing of hepatic Thpo reduces platelet, megakaryocyte, and megakaryocyte progenitor count by 50% relative to control ASO treated mice. Thpo-ASO treatment does not alter platelet reactivity to agonists, or platelet size. Following bacterial inoculation, we found a significant increase in lung platelet-leukocyte aggregates and consistent with a response to inflammation an increase in lung Ly6C^hi monocytes and macrophages in inoculated mice. Platelet depletion, by either Thpo-ASO or anti-CD42b treatment, reduces the accumulation of lung inflammatory immune cells, including monocytes (Ly6C^hi) and macrophages (CD45⁺CD11b⁺F4/80⁺). Furthermore, we found that in contrast to anti-CD42b platelet depletion, Thpo-ASO mediated depletion allows for introduction of new platelets.
Conclusion(s): Thpo ASO-mediated platelet depletion represents a viable approach to reduce platelet count. Platelet count directly impacts lung inflammation resolution during lower airway dysbiosis demonstrating the essential role of platelets in pulmonary immune defense