The Lung Microbiome.

Although the lungs were once considered a sterile environment, advances in sequencing technology have revealed dynamic, low-biomass communities in the respiratory tract, even in health. Key features of these communities-composition, diversity, and burden-are consistently altered in lung disease, associate with host physiology and immunity, and can predict clinical outcomes. Although initial studies of the lung microbiome were descriptive, recent studies have leveraged advances in technology to identify metabolically active microbes and potential associations with their immunomodulatory by-products and lung disease. In this brief review, we discuss novel insights in airway disease and parenchymal lung disease, exploring host-microbiome interactions in disease pathogenesis. We also discuss complex interactions between gut and oropharyngeal microbiota and lung immunobiology. Our advancing knowledge of the lung microbiome will provide disease targets in acute and chronic lung disease and may facilitate the development of new therapeutic strategies.

The Oral-Lung Microbiome Axis in Connective Tissue Disease-Related Interstitial Lung Disease.

Connective tissue disease-related interstitial lung disease (CTD-ILD) is a frequent and serious complication of CTD, leading to high morbidity and mortality. Unfortunately, its pathogenesis remains poorly understood; however, one intriguing contributing factor may be the microbiome of the mouth and lungs. The oral microbiome, which is a major source of the lung microbiome through recurrent microaspiration, is altered in ILD patients. Moreover, in recent years, several lines of evidence suggest that changes in the oral and lung microbiota modulate the pulmonary immune response and thus may play a role in the pathogenesis of ILDs, including CTD-ILD. Here, we review the existing data demonstrating oral and lung microbiota dysbiosis and possible contributions to the development of CTD-ILD in rheumatoid arthritis, Sjögren's syndrome, systemic sclerosis, and systemic lupus erythematosus. We identify several areas of opportunity for future investigations into the role of the oral and lung microbiota in CTD-ILD.

Commensal Oral Microbiota, Disease Severity and Mortality in Fibrotic Lung Disease.

RATIONALE: Oral microbiota associate with diseases of the mouth and serve as a source of lung microbiota. However, the role of oral microbiota in lung disease is unknown. OBJECTIVES: To determine associations between oral microbiota and disease severity and death in idiopathic pulmonary fibrosis. METHODS: We analyzed 16S rRNA gene and shotgun metagenomic sequencing data of buccal swabs from 511 patients with idiopathic pulmonary fibrosis in the multicenter CleanUP-IPF trial. Buccal swabs were collected from usual care, and antimicrobial cohorts. Microbiome data was correlated with measures of disease severity using principal component analysis and linear regression models. Associations between the buccal microbiome and mortality were determined using Cox additive models, Kaplan Meier analysis and Cox proportional hazards models. MEASUREMENTS AND MAIN RESULTS: Greater buccal microbial diversity associated with lower forced vital capacity (FVC) at baseline [mean diff -3.60: 95% CI -5.92 to -1.29 percent predicted FVC per 1 unit increment]. The buccal proportion of Streptococcus correlated positively with FVC [mean diff 0.80: 95% CI 0.16-1.43 percent predicted per 10% increase] (n=490). Greater microbial diversity was associated with an increased risk of death [HR 1.73: 95% CI 1.03-2.90] while a greater proportion of Streptococcus was associated with a reduced risk of death [HR 0.85: 95% CI 0.73 to 0.99]. The Streptococcus genus was mainly comprised of Streptococcus mitis species. CONCLUSIONS: Increasing buccal microbial diversity predicts disease severity and death in IPF. The oral commensal Streptococcus mitis spp associates with preserved lung function and improved survival.

Risk factors for lung function decline in systemic sclerosis-associated interstitial lung disease in a large single-centre cohort.

OBJECTIVES: The aim of this study was to identify risk factors of percent predicted forced vital capacity (ppFVC) decline in patients with SSc-associated interstitial lung disease (SSc-ILD). METHODS: We identified 484 patients with SSc who had HRCT Chest, of which 312 with ILD. Those with serial pulmonary function tests were included in a longitudinal analysis (n = 184). Linear mixed effect models were fitted to assess the decline in ppFVC over time, and to explore the effect of demographics and baseline characteristics on ppFVC decline. RESULTS: The majority of SSc-ILD patients were female (76.3%) and 51.3% had diffuse cutaneous subset. The mean (s.d.) age was 53.6 (12.7) years, median disease duration since first non-RP symptoms was 2.6 years, and 48.4% of the patients had ILD extent >20% on HRCT. In the univariate analysis, longer disease duration (>2.37 years), ILD extent >20%, and anti-topoisomerase I (ATA) positivity were significantly associated with ppFVC decline. In the multivariate analysis, the only statistically significant variable associated with ppFVC decline was ATA positivity. The overall group's mean decline in ppFVC was -0.28% (P-value 0.029), with -0.13% (n = 163) in those who were alive and -8.28% (P-value 0.0002 for the change in ppFVC trajectory) in patients who died within 2 years. CONCLUSION: Our study confirms that ppFVC is a marker of survival in SSc-ILD, supporting its use for risk stratification to identify patients who may benefit from earlier interventions and treatment. Our study also supports the role of ATA positivity as a predictive marker for ppFVC decline in this population.

Candidate Role for Toll-like Receptor 3 L412F Polymorphism and Infection in Acute Exacerbation of Idiopathic Pulmonary Fibrosis.

Rationale: The Toll-like receptor 3 Leu412Phe (TLR3 L412F) polymorphism attenuates cellular antiviral responses and is associated with accelerated disease progression in idiopathic pulmonary fibrosis (IPF). The role of TLR3 L412F in bacterial infection in IPF or in acute exacerbations (AE) has not been reported. Objectives: To characterize the association between TLR3 L412F and AE-related death in IPF. To determine the effect of TLR3 L412F on the lung microbiome and on antibacterial TLR responses of primary lung fibroblasts from patients with IPF. Methods: TLR-mediated antibacterial and antiviral responses were quantitated in L412F wild-type and 412F-heterozygous primary lung fibroblasts from patients with IPF using ELISA, Western blot analysis, and quantitative PCR. Hierarchical heatmap analysis was employed to establish bacterial and viral clustering in nasopharyngeal lavage samples from patients with AE-IPF. 16S ribosomal RNA quantitative PCR and pyrosequencing were used to determine the effect of TLR3 L412F on the IPF lung microbiome. Measurements and Main Results: A significant increase in AE-related death in patients with 412F-variant IPF was reported. We established that 412F-heterozygous IPF lung fibroblasts have reduced antibacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5), and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Using 16S ribosomal RNA sequencing, we demonstrated that 412F-heterozygous patients with IPF have a dysregulated lung microbiome with increased frequencies of Streptococcus and Staphylococcus spp. Conclusions: This study reveals that TLR3 L412F dysregulates the IPF lung microbiome and reduces the responses of IPF lung fibroblasts to bacterial TLR agonists and live bacterial infection. These findings identify a candidate role for TLR3 L412F in viral- and bacterial-mediated AE death.

Myeloid- and Epithelial-derived Heparin-Binding Epidermal Growth Factor-like Growth Factor Promotes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood, progressive lethal lung disease with no known cure. In addition to alveolar epithelial cell (AEC) injury and excessive deposition of extracellular matrix proteins, chronic inflammation is a hallmark of IPF. Literature suggests that the persistent inflammation seen in IPF primarily consists of monocytes and macrophages. Recent work demonstrates that monocyte-derived alveolar macrophages (moAMs) drive lung fibrosis, but further characterization of critical moAM cell attributes is necessary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an important epidermal growth factor receptor ligand that has essential roles in angiogenesis, wound healing, keratinocyte migration, and epithelial-mesenchymal transition. Our past work has shown HB-EGF is a primary marker of profibrotic M2 macrophages, and this study seeks to characterize myeloid-derived HB-EGF and its primary mechanism of action in bleomycin-induced lung fibrosis using Hbegff/f;Lyz2Cre+ mice. Here, we show that patients with IPF and mice with pulmonary fibrosis have increased expression of HB-EGF and that lung macrophages and transitional AECs of mice with pulmonary fibrosis and humans all express HB-EGF. We also show that Hbegff/f;Lyz2Cre+ mice are protected from bleomycin-induced fibrosis and that this protection is likely multifactorial, caused by decreased CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter.

Toll-like receptors, environmental caging, and lung dysbiosis.

Recent studies have implicated lung microbiota in shaping local alveolar immune responses. Toll-like receptors are major sensors of microbiota and determinants of local epithelial homeostasis. The impact of toll-like receptor deficiency on lung microbiota is unknown. To determine whether the absence of toll-like receptors results in altered lung microbiota or dysbiosis, we compared lung microbiota in wild-type and toll-like receptor-deficient experimental mice using 16S ribosomal RNA gene quantification and sequencing. We used a randomized environmental caging strategy to determine the impact of toll-like receptors on lung microbiota. Lung microbiota are detectable in toll-like receptor-deficient experimental mice and exhibit considerable variability. The lung microbiota of toll-like receptor-deficient mice are altered in community composition (PERMANOVA P < 0.001), display reduced diversity (t test P = 0.0075), and bacterial burden (t test P = 0.016) compared with wild-type mice with intact toll-like receptors and associated signaling pathways. The lung microbiota of wild-type mice when randomized to cages with toll-like receptor-deficient mice converged with no significant difference in community composition (PERMANOVA P > 0.05) after 3 wk of cohousing. The lung microbiome of toll-like receptor-deficient mice is distinct from wild-type mice and may be less susceptible to the effects of caging as an environmental variable. Our observations support a role for toll-like receptor signaling in the shaping of lung microbiota.

Methods in Lung Microbiome Research.

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.

The evolving role of the lung microbiome in pulmonary fibrosis.

Mucosal surfaces are constantly exposed to a microbiome consisting of microorganisms that heavily influence human immunity and health. In the lung these microorganisms consist of bacteria, viruses, and fungi and exist in a relatively low biomass state. Bacterial communities of the lung modulate local inflammation and correlate with changes in pulmonary physiology and clinical outcomes in patients with lung disease. Instrumental to this progress has been the study of these bacterial communities in the pathogenesis of pulmonary fibrosis, a fatal and progressive disease culminating in respiratory failure. Key pathophysiological mechanisms in pulmonary fibrosis include recurrent idiopathic alveolar epithelial injury, unchecked collagen deposition, mucociliary dysfunction due to muc5b overexpression, hypoxia, and altered host defense. These key mechanisms and their related consequences promote severe progressive architectural lung destruction and loss of local homeostasis. As such, pulmonary fibrosis is an appropriate target disease for the study of the lung microbiome. Herein, we discuss recent advances in our understanding of the role of the lung microbiome in the pathogenesis of pulmonary fibrosis. We highlight fundamental clinical observations and mechanistic insights and identify crucial areas for further discovery science. An improved understanding of how the lung microbiome acts to influence outcomes in patients with pulmonary fibrosis will lead to enhanced therapies for this devastating lung disease.

A Germline Mutation in the C2 Domain of PLCγ2 Associated with Gain-of-Function Expands the Phenotype for PLCG2-Related Diseases.

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

First-Onset Herpesviral Infection and Lung Injury in Allogeneic Hematopoietic Cell Transplantation.

Rationale: "Noninfectious" pulmonary complications are significant causes of morbidity and mortality after allogeneic hematopoietic cell transplant. Early-onset viral reactivations or infections are common after transplant. Whether the first-onset viral infection causes noninfectious pulmonary complications is unknown. Objectives: To determine whether the first-onset viral infection within 100 days after transplant predisposes to development of noninfectious pulmonary complications. Methods: We performed a retrospective review of 738 allogeneic hematopoietic cell transplant patients enrolled from 2005 to 2011. We also established a novel bone marrow transplantation mouse model to test whether herpesviral reactivation after transplant causes organ injury. Measurements and Main Results: First-onset viral infections with human herpesvirus 6 or Epstein-Barr virus within 100 days after transplant increase the risk of developing idiopathic pneumonia syndrome (adjusted hazard ratio [aHR], 5.52; 95% confidence interval [CI], 1.61-18.96; P = 0.007; and aHR, 9.21; 95% CI, 2.63-32.18; P = 0.001, respectively). First infection with human cytomegalovirus increases risk of bronchiolitis obliterans syndrome (aHR, 2.88; 95% CI, 1.50-5.55; P = 0.002) and grade II-IV acute graft-versus-host disease (aHR, 1.59; 95% CI, 1.06-2.39; P = 0.02). Murine roseolovirus, a homolog of human herpesvirus 6, can also be reactivated in the lung and other organs after bone marrow transplantation. Reactivation of murine roseolovirus induced an idiopathic pneumonia syndrome-like phenotype and aggravated acute graft-versus-host disease. Conclusions: First-onset herpesviral infection within 100 days after allogeneic hematopoietic cell transplant increases risk of pulmonary complications. Experimentally reactivating murine roseolovirus causes organ injury similar to phenotypes seen in human transplant recipients.

Lung Microbiota Contribute to Pulmonary Inflammation and Disease Progression in Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

Lung Dysbiosis, Inflammation, and Injury in Hematopoietic Cell Transplantation.

RATIONALE: Hematopoietic cell transplant (HCT) is a common treatment for hematological neoplasms and autoimmune disorders. Among HCT recipients, pulmonary complications are common, morbid, and/or lethal, and they have recently been associated with gut dysbiosis. The role of lung microbiota in post-HCT pulmonary complications is unknown. OBJECTIVES: To investigate the role of lung microbiota in post-HCT pulmonary complications using animal modeling and human BAL fluid. METHODS: For animal modeling, we used an established murine model of HCT with and without postengraftment herpes virus infection. For human studies, we characterized lung microbiota in BAL fluid from 43 HCT recipients. Lung bacteria were characterized using 16S ribosomal RNA gene sequencing and were compared with lung histology (murine) and with alveolar inflammation and pulmonary function testing (human). MEASUREMENTS AND MAIN RESULTS: Both HCT and viral infection independently altered the composition of murine lung microbiota, but they had no effect on lung microbial diversity. By contrast, combined HCT and viral infection profoundly altered lung microbiota, decreasing community diversity with an associated pneumonitis. Among human HCT recipients, increased relative abundance of the Proteobacteria phylum was associated with impaired pulmonary function, and lung microbiota were significantly associated with alveolar concentrations of inflammatory cytokines. CONCLUSIONS: In animal models and human subjects, lung dysbiosis is a prominent feature of HCT. Lung dysbiosis is correlated with histologic, immunologic, and physiologic features of post-HCT pulmonary complications. Our findings suggest the lung microbiome may be an unappreciated target for the prevention and treatment of post-HCT pulmonary complications.

Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator that we have previously shown to be associated with an aggressive clinical phenotype in cystic fibrosis. It possesses unique tautomerase enzymatic activity. However, to date, no human-derived substrate has been identified that has the capacity to interact with this cytokine's unique tautomerase activity. This led us to hypothesize that MIF may have the capacity to interact with external substrates. We describe for the first time how Pseudomonas aeruginosa can utilize human recombinant MIF (rMIF) to significantly (P < 0.01) enhance its endogenous biofilm formation. Our in vivo studies demonstrate that utilizing a small-molecular-weight inhibitor targeting MIF's tautomerase activity (SCD-19) significantly reduces the inflammatory response in a murine pulmonary chronic P. aeruginosa model. In addition, we show that in in vitro experiments, pretreatment of P. aeruginosa with rMIF is associated with reduced bacterial killing by tobramycin. Our novel findings support the concept of an anti-MIF strategy that targets this enzymatic activity as a potential future antibacterial therapeutic approach.-Tynan, A., Mawhinney, L., Armstrong, M. E., O'Reilly, C., Kennedy, S., Caraher, E., Jülicher, K., O'Dwyer, D., Maher, L., Schaffer, K., Fabre, A., McKone, E. F., Leng, L., Bucala, R., Bernhagen, J., Cooke, G., Donnelly, S. C. Macrophage migration inhibitory factor enhances Pseudomonas aeruginosa biofilm formation, potentially contributing to cystic fibrosis pathogenesis.

The Lung Microbiome, Immunity, and the Pathogenesis of Chronic Lung Disease.

The development of culture-independent techniques for microbiological analysis has uncovered the previously unappreciated complexity of the bacterial microbiome at various anatomic sites. The microbiome of the lung has relatively less bacterial biomass when compared with the lower gastrointestinal tract yet displays considerable diversity. The composition of the lung microbiome is determined by elimination, immigration, and relative growth within its communities. Chronic lung disease alters these factors. Many forms of chronic lung disease demonstrate exacerbations that drive disease progression and are poorly understood. Mounting evidence supports ways in which microbiota dysbiosis can influence host defense and immunity, and in turn may contribute to disease exacerbations. Thus, the key to understanding the pathogenesis of chronic lung disease may reside in deciphering the complex interactions between the host, pathogen, and resident microbiota during stable disease and exacerbations. In this brief review we discuss new insights into these labyrinthine relationships.

Proteomics: Clinical and research applications in respiratory diseases.

The proteome is the study of the protein content of a definable component of an organism in biology. However, the tissue-specific expression of proteins and the varied post-translational modifications, splice variants and protein-protein complexes that may form, make the study of protein a challenging yet vital tool in answering many of the unanswered questions in medicine and biology to date. Indeed, the spatial, temporal and functional composition of proteins in the human body has proven difficult to elucidate for many years. Given the effect of microRNA and epigenetic regulation on silencing and enhancing gene transcription, the study of protein arguably provides more accurate information on homeostasis and perturbation in health and disease. There have been significant advances in the field of proteomics in recent years, with new technologies and platforms available to the research community. In this review, we briefly discuss some of these new technologies and developments in the context of respiratory disease. We also discuss the types of data science approaches to analyses and interpretation of the large volumes of data generated in proteomic studies. We discuss the application of these technologies with regard to respiratory disease and highlight the potential for proteomics in generating major advances in the understanding of respiratory pathophysiology into the future.

The role of periostin in lung fibrosis and airway remodeling.

Periostin is a protein that plays a key role in development and repair within the biological matrix of the lung. As a matricellular protein that does not contribute to extracellular matrix structure, periostin interacts with other extracellular matrix proteins to regulate the composition of the matrix in the lung and other organs. In this review, we discuss the studies exploring the role of periostin to date in chronic respiratory diseases, namely asthma and idiopathic pulmonary fibrosis. Asthma is a major health problem globally affecting millions of people worldwide with significant associated morbidity and mortality. Periostin is highly expressed in the lungs of asthmatic patients, contributes to mucus secretion, airway fibrosis and remodeling and is recognized as a biomarker of Th2 high inflammation. Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by progressive aberrant fibrosis of the lung matrix and respiratory failure. It predominantly affects adults over 50 years of age and its incidence is increasing worldwide. Periostin is also highly expressed in the lungs of idiopathic pulmonary fibrosis patients. Serum levels of periostin may predict clinical progression in this disease and periostin promotes myofibroblast differentiation and type 1 collagen production to contribute to aberrant lung fibrosis. Studies to date suggest that periostin is a key player in several pathogenic mechanisms within the lung and may provide us with a useful biomarker of clinical progression in both asthma and idiopathic pulmonary fibrosis.