Faculty Bibliography

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.

The diverse microbial communities within our bodies produce metabolites that modulate host immune responses. Even the microbiome at distal sites has an important function in respiratory health. However, the clinical importance of the microbiome in tuberculosis, the biggest infectious cause of death worldwide, is only starting to be understood. Here, we critically review research on the microbiome's association with pulmonary tuberculosis. The research indicates five main points: (1) susceptibility to infection and progression to active tuberculosis is altered by gut Helicobacter co-infection, (2) aerosol Mycobacterium tuberculosis infection changes the gut microbiota, (3) oral anaerobes in the lung make metabolites that decrease pulmonary immunity and predict progression, (4) the increased susceptibility to reinfection of patients who have previously been treated for tuberculosis is likely due to the depletion of T-cell epitopes on commensal gut non-tuberculosis mycobacteria, and (5) the prolonged antibiotic treatment required for cure of tuberculosis has long-term detrimental effects on the microbiome. We highlight knowledge gaps, considerations for addressing these knowledge gaps, and describe potential targets for modifying the microbiome to control tuberculosis.

With the advances of culture-independent techniques, we now know that microbes, which were present on this planet way before we were, have adapted to live in extreme conditions which were previously thought to be inhospitable. It is therefore no surprise that the lung is frequently exposed to microbes and can easily harbor a complex microbiota. After all, it is a moist mucosa, exposed to more than 10 thousand liters of air every day, and in direct communication with another mucosa that has one of the highest microbial burden in the body: the oral cavity. Therefore, it is time to eradicate the preconception of sterility of the lower airways which has been fostered by the rapid growth of data based on culture-independent methods. Recent recognition of this has led to improved understanding of the roles of these lower airway microbes to lung mucosal immunity. The lung has several features with ecological impact: a) lipid-rich biofilm, b) subjected to frequent episodic immigration of upper airway commensals through microaspiration; and c) is prepared to maintain low amounts of material in the airway lumen, and thus low biomass, to facilitate oxygen and CO2 exchange.1 While most of our understanding of the role of microbes in host immunity comes from studying the gut microbiota, the special conditions present in the lung are key pressures that determine the composition of the lower airway microbiota and its interactions with the host. Early development of the lung microbiota is a major determinant of lower airway mucosal immunity. There is now evidence that in early life, the lung microbiota affects the maturation of the host immunity, where a diverse microbial community signaled by enrichment with oral commensals contributes to the development of antimicrobial peptides and immunoglobulins.2 Delivery mode, Csection vs. vaginal, affects the composition of the lower airway microbiota in preterm births. Experimental preclinical data support that these changes are key to a host immune shift from a Th2- predominant phenotype to a Th1 phenotype. Germ-free mice models demonstrate that the lower airway microbiota is key to the development of immune tolerance to allergens, mechanisms likely triggered by induction of checkpoint inhibition.3 The upper airways are the gatekeepers of the microbes into the lower airways. Longitudinal studies of the upper airway microbiota have now shown that crosspollination of oral taxa (eg, Neisseria, Streptococcus, Prevotella and Fusobacterium) into the nasopharynx occurs prior and during respiratory tract infections in early childhood.4 In the adult lung, microaspiration commonly occurs and its rate is increased in many inflammatory respiratory diseases.5,6 The exposure of the lower airways to upper airway secretions can be identified using culture-independent techniques by the enrichment of oral commensals such as Prevotella, Veillonella and Streptococcus. In healthy individuals, this exposure contributes to a subclinical pro-inflammatory state characterized by increased inflammatory cells with a Th17 phenotype in bronchoalveolar lavage (BAL) and a blunted toll-like receptor (TLR) response of alveolar macrophages.7,8 Therefore, similar to the gut, specific lung microbiomes are associated with Th17 immunity.8 The molecular mechanisms determinant of this association still need to be elucidated, although there are multiple bacterial metabolites found in the lower airways that may exert immunomodulatory effects. For example, short chain fatty acids (SCFAs) are produced by oral anaerobes through fermentation.9 We have described that that high levels of SCFAs can be found in the lower airways associated with the enrichment of the lung microbiota enriched with oral anaerobes.10 Importantly, the levels of these SCFAs in the lower airways may affect the production of cytokines that are key to the innate immune response to pathogens such as IFN-gamma and IL-17A.10 Taken together, these data support that the lung microbiota exerts an immune-modulatory role that may affect a subject's lower airway immune tone and susceptibility to pathogens

With the advent of next generation sequencing (NGS), we now recognize that the lower airways harbor a complex microbial community. This community is influenced by the environment as well as by the host conditions. Changes in the lower airway microbial community structure and composition are associated with host immune tone, disease states and acquisition of pathogens. For a long time, we have recognized the presence of an infection by a pathogen. Now that we are starting to accept that the lower airways are not sterile, we need to identify changes in the lower airway microbial structure and composition that may lead to changes in host immunity and pathogen susceptibility. Investigators have adopted the term dysbiosis as a way to describe in broad terms changes in mucosal microbiota that occur in different pathogenic conditions. Even more obscure is the use of this term when referring to the lung microbiota. In early life, the lung microbiota develops under the influence of the environmental microbial exposure. For example, preterm babies born by C-section have a lower airway microbiota enriched with Staphylococcus, a common skin commensal, whereas preterm babies born via vaginal delivery have their lower airway microbiota enriched with Ureaplasma, a common vaginal commensal.1 These early dysbiotic signatures delay the maturation of the innate immune system in the lower airways, which requires the development of a more diverse lower airway microbiota. The main source of this diverse microbiota is the upper airways. Microaspiration of oral secretions commonly occur leading to one form of dysbiosis, under which the lower airways are enriched with oral commensals, which we termed pneumotypeSPT for presence of supraglottic predominant taxa.2,3 This form of dysbiosis is frequently found in health, is consistent with prior observations that microaspiration frequently occurs even in healthy subjects,4,5 and its prevalence is likely increased in many airway inflammatory diseases. In children with increased risk of aspiration, finding oral commensals in the lower airways is associated with inflammatory markers.6 The mechanisms by which this form of dysbiosis leads to specific host immune response are not clear. Some of these are likely mediated by pathogenassociated molecular patterns. Although oral commensals are not considered pathogens in the upper airways, they do express small molecules that can bind to pattern recognition receptors such as toll-like receptors. Further, this lower airway microbiota signature can also affect the lower airway metabolic environment with presence of metabolites with significant immunomodulatory effects.3 An example is the presence in the lower airways of short chain fatty acids, which are end-products of fermentation of anaerobes such as oral commensals and cannot be produced by mammalian cells. These molecules can affect T cell responses to pathogens.7 A different type of dysbiosis occurs during infections with pathogens. In this scenario, one would expect that the lower airway microbiota would be enriched and dominated by the pathogen. This situation has been described in the lower airways of children with chronic cough colonized with Haemophilus.6 However, in many other situations, pathogens are present in low abundance and coexisting with a very diverse microbiota. An example of this situation occurs during infections with non-tuberculous mycobacterium, where these pathogens are rarely found in high abundance.8 Importantly, some of the non-pathogenic microbes seem to have a stronger association with levels of inflammatory markers in the lower airways than the pathogen itself. This therefore suggests that other dysbiotic signatures beyond the presence of the pathogen might be contributing to the disease process. In conclusion, we are now starting to identify dysbiotic signatures in the lower airways by evaluating either host inflammatory profiles present or colonization with pathogens. These signatures will be key to uncover mechanisms by which the lower airway microbiota contributes to the disease process