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Rationale: Acute cellular rejection (ACR) after lung transplant is a leading risk factor for chronic lung allograft dysfunction. Prior studies have demonstrated dynamic microbial changes occurring within the allograft and gut that influence local adaptive and innate immune responses. However, the lung microbiome's overall impact on ACR risk remains poorly understood. Objectives: To evaluate whether temporal changes in microbial signatures were associated with the development of ACR. Methods: We performed cross-sectional and longitudinal analyses (joint modeling of longitudinal and time-to-event data and trajectory comparisons) of 16S rRNA gene sequencing results derived from lung transplant recipient lower airway samples collected at multiple time points. Measurements and Main Results: Among 103 lung transplant recipients, 25 (24.3%) developed ACR. In comparing samples acquired 1 month after transplant, subjects who never developed ACR demonstrated lower airway enrichment with several oral commensals (e.g., Prevotella and Veillonella spp.) than those with current or future (beyond 1 mo) ACR. However, a subgroup analysis of those who developed ACR beyond 1 month revealed delayed enrichment with oral commensals occurring at the time of ACR diagnosis compared with baseline, when enrichment with more traditionally pathogenic taxa was present. In longitudinal models, dynamic changes in α-diversity (characterized by an initial decrease and a subsequent increase) and in the taxonomic trajectories of numerous oral commensals were more commonly observed in subjects with ACR. Conclusions: Dynamic changes in the lower airway microbiota are associated with the development of ACR, supporting its potential role as a useful biomarker or in ACR pathogenesis.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

This report presents the proceedings from a workshop titled "Microbiome, Metabolism and Immunoregulation of Asthma" that was held virtually May 13 and 14, 2021. The workshop was jointly sponsored by the American Thoracic Society (Assembly on Allergy, Immunology, and Inflammation) and the National Institute of Allergy and Infectious Diseases. It convened an interdisciplinary group of experts with backgrounds in asthma immunology, microbiome science, metabolomics, computational biology, and translational pulmonary research. The main purpose was to identify key scientific gaps and needs to further advance research on microbial and metabolic mechanisms that may contribute to variable immune responses and disease heterogeneity in asthma. Discussions were structured around several topics, including 1 ) immune and microbial mechanisms of asthma pathogenesis in murine models, 2 ) the role of microbes in pediatric asthma exacerbations, 3 ) dysregulated metabolic pathways in asthma associated with obesity, 4 ) metabolism effects on macrophage function in adipose tissue and the lungs, 5 ) computational approaches to dissect microbiome-metabolite links, and 6 ) potential confounders of microbiome-disease associations in human studies. This report summarizes the major points of discussion, which included identification of specific knowledge gaps, challenges, and suggested directions for future research. These include questions surrounding mechanisms by which microbiota and metabolites shape host health versus an allergic or asthmatic state; direct and indirect influences of other biological factors, exposures, and comorbidities on these interactions; and ongoing technical and analytical gaps for clinical translation.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype. Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model. Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica , Veillonella parvula , and Streptococcus mitis ) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds. Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen. Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae . Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

Rationale : Chronic hypersensitivity pneumonitis (CHP) is a condition that arises after repeated exposure and sensitization to inhaled antigens. The lung microbiome is increasingly implicated in respiratory disease, but, to date, no study has investigated the composition of microbial communities in the lower airways in CHP. Objectives: To characterize and compare the airway microbiome in subjects with CHP, subjects with idiopathic pulmonary fibrosis (IPF), and control subjects. Methods: We prospectively recruited individuals with a CHP diagnosis ( n  = 110), individuals with an IPF diagnosis ( n  = 45), and control subjects ( n  = 28). Subjects underwent BAL and bacterial DNA was isolated, quantified by quantitative PCR and the 16S ribosomal RNA gene was sequenced to characterize the bacterial communities in the lower airways. Measurements and Main Results: Distinct differences in the microbial profiles were evident in the lower airways of subjects with CHP and IPF. At the phylum level, the prevailing microbiota of both subjects with IPF and subjects with CHP included Firmicutes , Bacteroidetes , Proteobacteria , and Actinobacteria . However, in IPF, Firmicutes dominated, whereas the percentage of reads assigned to Proteobacteria in the same group was significantly lower than the percentage found in subjects with CHP. At the genus level, the Staphylococcus burden was increased in CHP, and Actinomyces and Veillonella burdens were increased in IPF. The lower airway bacterial burden in subjects with CHP was higher than that in control subjects but lower than that of those with IPF. In contrast to IPF, there was no association between bacterial burden and survival in CHP. Conclusions: The microbial profile of the lower airways in subjects with CHP is distinct from that of IPF, and, notably, the bacterial burden in individuals with CHP fails to predict survival.

Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease. Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers. Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, 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. Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the 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 the 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.

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 hopefully provide the foundation for formulating consensus approaches in lung microbiome research.

Rationale: Obstructive sleep apnea (OSA) is associated with recurrent obstruction, subepithelial edema, and airway inflammation. The resultant inflammation may influence or be influenced by the nasal microbiome., Objectives: To evaluate whether the composition of the nasal microbiota is associated with obstructive sleep apnea and inflammatory biomarkers., Methods: Two large cohorts were used: 1) a discovery cohort of 472 subjects from the WTCSNORE (Seated, Supine and Post-Decongestion Nasal Resistance in World Trade Center Rescue and Recovery Workers) cohort, and 2) a validation cohort of 93 subjects rom the Zaragoza Sleep cohort. Sleep apnea was diagnosed using home sleep tests. Nasal lavages were obtained from cohort subjects to measure: 1) microbiome composition (based on 16S rRNA gene sequencing), and 2) biomarkers for inflammation (inflammatory cells, IL-8, and IL-6). Longitudinal 3-month samples were obtained in the validation cohort, including after continuous positive airway pressure treatment when indicated., Measurements and Main Results: In both cohorts, we identified that: 1) severity of OSA correlated with differences in microbiome diversity and composition; 2) the nasal microbiome of subjects with severe OSA were enriched with Streptococcus, Prevotella, and Veillonella; and 3) the nasal microbiome differences were associated with inflammatory biomarkers. Network analysis identified clusters of cooccurring microbes that defined communities. Several common oral commensals (e.g., Streptococcus, Rothia, Veillonella, and Fusobacterium) correlated with apnea-hypopnea index. Three months of treatment with continuous positive airway pressure did not change the composition of the nasal microbiota., Conclusions: We demonstrate that the presence of an altered microbiome in severe OSA is associated with inflammatory markers. Further experimental approaches to explore causal links are needed.

Rationale: In lung cancer, upregulation of the PI3K (phosphoinositide 3-kinase) pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals., Objectives: We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways., Methods: Airway brushings were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with noncancer diagnoses. In addition, samples from 10 healthy control subjects were included. 16S ribosomal RNA gene amplicon sequencing and paired transcriptome sequencing were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed., Measurements and Main Results: The composition of the lower airway transcriptome in the patients with cancer was significantly different from the control subjects, which included up-regulation of ERK (extracellular signal-regulated kinase) and PI3K signaling pathways. The lower airways of patients with lung cancer were enriched for oral taxa (Streptococcus and Veillonella), which was associated with up-regulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways., Conclusions: The data presented here show that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

The development of culture-independent techniques has revolutionized our understanding of how our human cells interact with the even greater number of microbial inhabitants of our bodies. As part of this revolution, data are increasingly challenging the old dogma that in health, the lung mucosa is sterile. To understand how the lung microbiome may play a role in human health, we identified five major questions for lung microbiome research: (1) Is the lung sterile? (2) Is there a unique core microbiome in the lung? (3) How dynamic are the microbial populations? (4) How do pulmonary immune responses affect microbiome composition? and (5) Are the lungs influenced by the intestinal immune responses to the gut microbiome? From birth, we are exposed to continuous microbial challenges that shape our microbiome. In our changing environment, perturbation of the gut microbiome affects both human health and disease. With widespread antibiotic use, the ancient microbes that formerly resided within us are being lost, for example, Helicobacter pylori in the stomach. Animal models show that antibiotic exposure in early life has developmental consequences. Considering the potential effects of this altered microbiome on pulmonary responses will be critical for future investigations.

Microbes are readily cultured from epithelial surfaces of the skin, mouth, and colon. In the last 10 years, culture-independent DNA-based techniques demonstrated that much more complex microbial communities reside on most epithelial surfaces; this includes the lower airways, where bacterial culture had failed to reliably demonstrate resident bacteria. Exposure to a diverse bacterial environment is important for adequate immunological development. The most common microbes found in the lower airways are also found in the upper airways. Increasing abundance of oral characteristic taxa is associated with increased inflammatory cells and exhaled nitric oxide, suggesting that the airway microbiome induces an immunological response in the lung. Furthermore, rhinovirus infection leads to outgrowth of Haemophilus in patients with chronic obstructive pulmonary disease, and human immunodeficiency virus-infected subjects have more Tropheryma whipplei in the lower airway, suggesting a bidirectional interaction in which the host immune defenses also influence the microbial niche. Quantitative and/or qualitative changes in the lung microbiome may be relevant for disease progression and exacerbations in a number of pulmonary diseases. Future investigations with longitudinal follow-up to understand the dynamics of the lung microbiome may lead to the development of new therapeutic targets.

Community-acquired pneumonia affects approximately 4 million people in the United States, with 40,000 deaths per year. The incidence is increased about 35-fold in HIV-infected individuals, and this rate has decreased since the antiretroviral era has begun. Bacterial pneumonia has decreased from 5 to 20 cases per 100 person-years to less than 1 to 5 cases per 100 person-years in the era of antiretroviral therapy. HIV-1 infection impairs the function of neutrophils in the lung and infects CD4⁺ cells and alveolar macrophages. Opportunistic infections dramatically increase local HIV replication in the lung cells, especially alveolar macrophages and CD4⁺ cells. This enhanced replication increases viral mutations and provides opportunities for viral escape from latent reservoirs. Mortality is increased with more comorbidities in this highly susceptible population. Immunization with vaccines is recommended, especially pneumococcal vaccines, although the vaccine itself may stimulate viral replication. Recent studies show that the lower respiratory tract is a microbial reservoir in HIV-infected individuals rather than being a sterile environment, as originally thought. This may provide new opportunities for preventing opportunistic infections in HIV-infected subjects. Bacterial pneumonia presents an ongoing challenge in these high-risk individuals, particularly in studying the functions of the innate and acquired immune response.