Faculty Bibliography

Background: Aspiration is associated with non-tuberculous mycobacterial (NTM) pulmonary disease and airway dysbiosis is associated with increased inflammation. We examined whether NTM disease was associated with a distinct airway microbiota and immune profile.Methods: 297 oral wash and induced sputum samples were collected from 106 participants with respiratory symptoms and imaging abnormalities compatible with NTM. Lower airway samples were obtained in 20 participants undergoing bronchoscopy. 16S rRNA gene and a nested mycobacteriome sequencing approaches characterised microbiota composition. Inflammatory profiles of lower airway samples were also examined.Results: The prevalence of NTM+ cultures was 58%. Few changes were noted in microbiota characteristic or composition in oral wash and sputum samples among groups. Among NTM+ samples, 27% of the lower airway samples were enriched with Mycobacterium A mycobacteriome approach identified Mycobacterium in a greater percentage of samples, including some non-pathogenic strains. In NTM+ lower airway samples, taxa identified as oral commensals were associated with increased inflammatory biomarkers.Conclusions: The 16S rRNA gene sequencing approach is not sensitive in identifying NTM among airway samples which are culture positive. However, associations between lower airway inflammation and microbiota signatures suggest a potential role for these microbes in the inflammatory process in NTM disease.

BACKGROUND:The role of commensal viruses in humans is poorly understood, and the impact of the virome on lung health and smoking-related disease is particularly understudied. METHODS:Genetic material from acellular bronchoalveolar lavage fluid was sequenced to identify and quantify viral members of the lower respiratory tract which were compared against concurrent bronchoalveolar lavage bacterial, metabolite, cytokine and cellular profiles, and clinical data. Twenty smoker and 10 nonsmoker participants with no significant comorbidities were studied. RESULTS:Viruses that infect bacteria (phages) represented the vast majority of viruses in the lung. Though bacterial communities were statistically indistinguishable across smokers and nonsmokers as observed in previous studies, lung viromes and metabolic profiles were significantly different between groups. Statistical analyses revealed that changes in viral communities correlate most with changes in levels of arachidonic acid and IL-8, both potentially relevant for chronic obstructive pulmonary disease (COPD) pathogenesis based on prior studies. CONCLUSIONS:Our assessment of human lung DNA viral communities reveals that commensal viruses are present in the lower respiratory tract and differ between smokers and nonsmokers. The associations between viral populations and local immune and metabolic tone suggest a significant role for virome-host interaction in smoking related lung disease.

In pediatric patients with chronic cough, respiratory culture techniques commonly yield negative results. Studies using culture-independent methods have found a high relative abundance of oral microbes in the lower airways, suggesting that the topographical continuity, and dynamics of the intraluminal contents of the aerodigestive system likely influence the lower airway microbiota. We hypothesize that in subjects with chronic cough, clinical diagnosis will correlate with distinct microbial signatures detected using culture-independent methods.

The advent of next generation sequencing (NGS) has enabled the characterization of the microbiome with unprecedented resolution and throughput. We now recognize that each one of us harbors 10 - 100 trillion microbial cells, with 99% of the microbial mass contained in the gut, the most characterized body site in microbiome studies (1). The microbiome suffers many changes over our lifetime, where the first major exposure occurs in the newborn during the birthing process. This exposure is highly dependent on mode of delivery (2-4). However, most of our understanding of the microbial challenges that we experience comes from studying the gut microbiota. The upper airway microbiota is also influenced by mode of delivery (5) and longitudinal compositional changes can be associated with respiratory tract infections (6). In early stages of cystic fibrosis (CF), the upper airway microbiome has distinct patterns characterized by enrichment with S. aureus, S. mitis, and Corynebacterium accolens (7). Much less is known about the lower airway microbiota, in part due to difficulties in obtaining reliable samples. Studies of lower airway samples using NGS have now demonstrated that the lung microbiota is characterized by low biomass and frequent enrichment with taxa that represent normal upper airway commensals (8). In fact, the lungs of infants with CF seem to also have a unique microbiota seeded by the upper respiratory tract microbiome (9). Thus, the upper airway represents the gatekeeper for episodic microbial challenges to the lower airways through microaspiration that will determine the composition of the lower airway microbiota. This immigration is counterbalanced by the emigration rate determined by mechanical clearance (eg, mucous ciliary clearance) and by host immune response, mechanisms that are affected in CF. In addition, microaspiration of oral commensals may contribute to the lower airway immune tone. Our group has identified two distinct lung microbiomes that we named "pneumotypes": a) pneumotypeSPT characterized by high bacterial load and supraglottic predominant taxa (SPT) such as Streptococcus, Prevotella and Veillonella; and b) pneumotypeBPT with low bacterial burden and background predominant taxa (BPT) (8). This is consistent with prior observations that microaspiration frequently occurs even in healthy subjects (10,11). Importantly, the NGS data also suggested that these compositionally distinct microbiota should also have distinct functional capacity based on inferred metagenome (the sum of the genes contain in a microbial population) (12). A way to further explore this requires evaluation of the host inflammatory and metabolic environment. We therefore showed that pneumotypeSPT is associated with increased inflammatory cells with a Th17 phenotype in bronchoalveolar lavage (BAL) and a blunted toll-like receptor (TLR) response of alveolar macrophages. Thus, similar to the gut, specific lung microbiomes are associated with Th17 immunity (12). Other emerging data also show that the lower airways contain complex bacterial communities that correlate with immune competence and levels of inflammation (13-16). Importantly, in a longitudinal investigation using macaques, development of chronic inflammatory obstructive lung disease was associated with increase in abundance of oral anaerobes such as Fusobacterium, Prevotella, Veillonella, Neisseria, and Porphyromonas (17). The mechanisms by which these microbial communities contribute to the lower airway host immune tone are much less understood. One of the possible ways microbes interact with the host is through immunoactive metabolites. Multiple bacterial metabolites are found in the lower airways consistent with active bacterial metabolism (12,18). One resalting characteristic of the lower airway microbiota is the presence of anaerobes and facultative anaerobes. Anaerobes can survive the oxygen stress of the lower airway by forming multicellular complexes within biofilm that enables hypoxic microenvironments, a condition that is enhanced in the CF lung (19,20). Short chain fatty acids (SCFAs) produced by fermentation, such as propionate and butyrate, are one energy source for anaerobes (21). We recently demonstrated that high levels of SCFAs in the lower airways, detected by targeted gas chromatography- mass spectroscopy (GC/MS), are associated with a lung microbiota enriched with anaerobes such as Prevotella, Veillonella, and Haemophilus (18). Importantly, in a prospective cohort of HIV-infected subjects followed over three years, high serum SCFA levels were associated with increased risk of developing tuberculosis. Butyrate reduces secretion of IFN-gand IL-17A, cytokines responsible for mucosal immune response to pathogens, in CD4⁺and CD8⁺lymphocytes (18). In sum, the upper airway functions as a gatekeeper for microbes that reach the lower airways through microaspiration. The dynamics that determine the lower airway microbiota are frequently affected in CF. Recognition of the immuno-modulatory role that the lung microbiota has is important to understand a subject's susceptibility to pathogens

Background/Purpose: The microbiome serves a number of important functions, including modulation of the immune system and protection from pathogenic microorganisms1. Many autoimmune diseases have been associated with intestinal microbial dysbiosis1. Recent studies have also demonstrated that microbiota can affect the lifetime, bioavailability and efficacy of drugs2. Conversely, even drugs designed to specifically target human cells have been associated with changes in microbial composition3. To date, most research has focused on bacterial microorganisms and little is known about the role that fungal microorganisms (the mycobiome) play, including their interactions with bacteria. In this study, we characterized the ecological effects of biologic therapies on the intestinal mycobiome.
Method(s): Fecal samples were collected from SpA patients pre- and post-treatment with either tumor necrosis factor inhibitors (TNFi; n=15) or secukinumab (n=14), an anti-IL-17A monoclonal antibody (IL-17i). Subjects treated with TNFi were naive to biologic therapy, whereas those treated with secukinumab previously failed or had incomplete response to TNFi. Samples underwent DNA extraction, amplification, and gene sequencing of the ITS1 region conserved in fungi. In parallel, gene sequencing of the 16S rRNA gene region conserved in bacteria was also performed. Sequences were analyzed with R and Quantitative Insights into Microbial Ecology (QIIME).
Result(s): ITS fungal data reveled that, on average, subjects treated with TNFi and IL-17i did not have major differences in overall microbial alpha or beta diversity pre- and post-treatment. However, there were dramatic shifts in the relative abundance of specific taxa, such as Candida albicans, which were more prominent in the IL-17i cohort compared to the TNFi cohort (p=0.04). The IL-17i cohort also demonstrated similar changes in certain 16S bacterial taxa, including Clostridia (p=0.02) and Clostridiales (p=0.02).
Conclusion(s): We characterized, for the first time, the effects of two biologic therapies on human intestinal fungal and bacterial microbiota composition. Treatment with biologics, particularly IL-17i, leads to a gut microbial dysbiosis characterized by significant changes in abundance of C. albicans and Clostridia in a subgroup of SpA patients. This is in line with the known increased risk of candidiasis seen with IL-17i, and may at least partially explain the potential link between IL-17 blockade, intestinal dysbiosis, and the subclinical and clinical gut inflammation observed in some patients treated with these molecules. Further studies to understand the downstream effects of these perturbations may allow for the development of precision medicine approaches in PsA and SpA

The diagnosis and treatment of chronic obstructive pulmonary disease (COPD) has been based largely on a one-size-fits-all approach. Diagnosis of COPD is based on meeting the physiologic criteria of fixed obstruction in forced expiratory flows and treatment focus on symptomatic relief, with limited effect on overall prognosis. However, patients with COPD have distinct features that determine very different evolutions of the disease. In this review we highlight distinct subgroups of COPD characterized by unique pathophysiologic derangements, response to treatment, and disease progression. It is likely that identification of subgroups of COPD will lead to discovery of much needed disease-modifying therapeutic approaches. We argue that a precision approach that integrates multiple dimensions (clinical, physiologic, imaging, and endotyping) is needed to move the field forward in the treatment of this disease.

Objective: To characterize the gut microbiota and host gene interactions in Reactive Arthritis (ReA) and postinfectious SpA. Methods: 32 patients with ReA and 32 controls with preceding Gastrointestinal (GI) and/or Genitourinary (GU) infections that did not develop arthritiswere prospectively recruited in Guatemala, a highly prevalent geographic region for this disease. Clinical variables, HLA status and 16S ribosomal RNA gene sequencing of intestinal microbiota were analysed. Results: Subjects with ReA showed no significant differences from controls in gut bacterial richness, alpha or beta diversity. However there was a higher abundance of Erwinia and Pseudomonas, and increased prevalence of typical enteropathogens associated with ReA. In Fact, at least one enteropathogen was present in 71.9% of ReA subjects vs 46.8% of controls (p<0.05). Subjects with ultrasound evidence of enthesitiswere enriched with Campylobacter,while subjects with Uveitis and radiographic sacroiliitis were enriched with Erwinia and unclassified Ruminococcaceae, respectively. Both were enriched in Dialister (log LDA>2). Host genetics, particularly HLA-A24 were associated with differences in gut microbiota diversity irrespective of disease status. We identified several co-occurring taxa that were also predictive of HLA-A24 status, including Ruminococcaceae-Rikenellaceae-Coriobacteriacea and Prevotellaceaeunclassified Sphingobacteriales-Elusimicrobiaceae. Conclusion: This is the first culture-independent study characterizing the gut microbial community of ReA. Although bacterial factors correlated with disease presence and clinical features of ReA, host genetics also appeared to be a major independent driver of intestinal community composition. Understanding of these gut microbiota host-genetic relationships may further clarify the pathogenesis of ReA and related Spondyloarthritis

Changes in lung microbiota associated with smoking might contribute to the development of acute respiratory distress syndrome in subjects with blunt trauma.