Faculty Bibliography

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

OBJECTIVES/OBJECTIVE:Four machine manufacturing facility workers had a novel occupational lung disease of uncertain aetiology characterised by lymphocytic bronchiolitis, alveolar ductitis and emphysema (BADE). We aimed to evaluate current workers' respiratory health in relation to job category and relative exposure to endotoxin, which is aerosolised from in-use metalworking fluid. METHODS:decline. RESULTS:) endotoxin exposure (aPR=10.5 (95% CI 1.3 to 83.1)) at baseline were associated with excessive decline. One production worker with excessive decline had BADE on subsequent lung biopsy. CONCLUSIONS:Lung function loss and BADE were associated with production work. Relationships with relative endotoxin exposure indicate work-related adverse respiratory health outcomes beyond the sentinel disease cluster, including an incident BADE case. Until causative factors and effective preventive strategies for BADE are determined, exposure minimisation and medical surveillance of affected workforces are recommended.

OBJECTIVE:To characterize the ecological effects of biologic therapies on the gut bacterial and fungal microbiome of psoriatic arthritis (PsA)/spondyloarthritis (SpA) patients. METHODS:Fecal samples from PsA/SpA patients pre- and post-treatment with tumor necrosis factor inhibitors (TNFi; n=15) or an anti-interleukin (IL)-17A monoclonal antibody inhibitor (IL-17i; n=14) underwent sequencing (16S, ITS and shotgun metagenomics) and computational microbiome analysis. Fecal levels of fatty acid metabolites and cytokines/proteins implicated in PsA/SpA pathogenesis or intestinal inflammation were correlated with sequence data. Additionally, ileal biopsies obtained from SpA patients who developed clinically overt Crohn's disease (CD) after treatment with IL-17i (n=5) were analyzed for expression of IL-23/Th-17 related cytokines, IL-25/IL-17E-producing cells and type-2 innate lymphoid cells (ILC2s). RESULTS:There were significant shifts in abundance of specific taxa after treatment with IL-17i compared to TNFi, particularly Clostridiales (p=0.016) and Candida albicans (p=0.041). These subclinical alterations correlated with changes in bacterial community co-occurrence, metabolic pathways, IL-23/Th17-related cytokines and various fatty acids. Ileal biopsies showed that clinically overt CD was associated with expansion of IL-25/IL-17E-producing tuft cells and ILC2s (p<0.05) compared to pre-IL-17i treatment levels. CONCLUSION/CONCLUSIONS:In a subgroup of SpA patients, the initiation of IL-17A blockade correlated with features of subclinical gut inflammation and intestinal dysbiosis of certain bacterial and fungal taxa, most notably C. albicans. Further, IL-17i-related CD was associated with overexpression of IL-25/IL-17E-producing tuft cells and ILC2s. These results may help to explain the potential link between inhibition of a specific IL-17 pathway and the (sub)clinical gut inflammation observed in SpA.

The lung microbiome is associated with host immune response and health outcomes in experimental models and patient cohorts. Lung microbiome research is increasing in volume and scope; however, there are no established guidelines for study design, conduct and reporting of lung microbiome studies. Standardized approaches to yield reliable and reproducible data that can be synthesized across studies, will ultimately improve the scientific rigor and impact of published work and greatly benefit microbiome research. In this review, we identify and address several key elements of microbiome research: conceptual modeling and hypothesis framing, study design, experimental methodology and pitfalls, data analysis and reporting considerations. Finally, we explore possible future directions and research opportunities. Our goal is to aid investigators who are interested in this burgeoning research area and will hopefully provide the foundation for formulating consensus approaches in lung microbiome research.

BACKGROUND:A cluster of severe lung disease occurred at a manufacturing facility making industrial machines. We aimed to describe disease features and workplace exposures. METHODS:Clinical, functional, radiologic, and histopathologic features were characterized. Airborne concentrations of thoracic aerosol, metalworking fluid, endotoxin, metals, and volatile organic compounds were measured. Facility airflow was assessed using tracer gas. Process fluids were examined using culture, polymerase chain reaction, and 16S ribosomal RNA sequencing. RESULTS: = 44% predicted) and reduced diffusing capacity (mean = 53% predicted); and radiologic centrilobular emphysema. Lung tissue demonstrated a unique pattern of bronchiolitis and alveolar ductitis with B-cell follicles lacking germinal centers, and significant emphysema for never-smokers. All had chronic dyspnea, three had a progressive functional decline, and one underwent lung transplantation. Patients reported no unusual nonoccupational exposures. No cases were identified among nonproduction workers or in the community. Endotoxin concentrations were elevated in two air samples; otherwise, exposures were below occupational limits. Air flowed from areas where machining occurred to other production areas. Metalworking fluid primarily grew Pseudomonas pseudoalcaligenes and lacked mycobacterial DNA, but 16S analysis revealed more complex bacterial communities. CONCLUSION/CONCLUSIONS:This cluster indicates a previously unrecognized occupational lung disease of yet uncertain etiology that should be considered in manufacturing workers (particularly never-smokers) with airflow obstruction and centrilobular emphysema. Investigation of additional cases in other settings could clarify the cause and guide prevention.