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.

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.

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.

Horizontal transmission of gut microbiota attenuates mortality in lung fibrosis.

Pulmonary fibrosis is a chronic and often fatal disease. The pathogenesis is characterized by aberrant repair of lung parenchyma, resulting in loss of physiological homeostasis, respiratory failure, and death. The immune response in pulmonary fibrosis is dysregulated. The gut microbiome is a key regulator of immunity. The role of the gut microbiome in regulating the pulmonary immunity in lung fibrosis is poorly understood. Here, we determine the impact of gut microbiota on pulmonary fibrosis in substrains of C57BL/6 mice derived from different vendors (C57BL/6J and C57BL/6NCrl). We used germ-free models, fecal microbiota transplantation, and cohousing to transmit gut microbiota. Metagenomic studies of feces established keystone species between substrains. Pulmonary fibrosis was microbiota dependent in C57BL/6 mice. Gut microbiota were distinct by ß diversity and α diversity. Mortality and lung fibrosis were attenuated in C57BL/6NCrl mice. Elevated CD4+IL-10+ T cells and lower IL-6 occurred in C57BL/6NCrl mice. Horizontal transmission of microbiota by cohousing attenuated mortality in C57BL/6J mice and promoted a transcriptionally altered pulmonary immunity. Temporal changes in lung and gut microbiota demonstrated that gut microbiota contributed largely to immunological phenotype. Key regulatory gut microbiota contributed to lung fibrosis, generating rationale for human studies.

Fibrotic Lung Disease Alters Neutrophil Trafficking and Promotes Neutrophil Elastase and Extracellular Trap Release.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease characterized by collagen deposition within the interstitium of the lung. This impairs gas exchange and results in eventual respiratory failure. Clinical studies show a correlation between elevated neutrophil numbers and IPF disease progression; however, the mechanistic roles neutrophils play in this disease are not well described. In the present study, we describe alterations to the trafficking and function of neutrophils after the development of fibrosis. We observed increased numbers of total and aged neutrophils in peripheral tissues of fibrotic mice. This appeared to be driven by an upregulation of neutrophil chemokine Cxcl2 by lung cells. In addition, neutrophil recruitment back to the bone marrow for clearance appeared to be impaired, because we saw decreased aged neutrophils in the bone marrow of fibrotic mice. Neutrophils in fibrosis were activated, because ex vivo assays showed increased elastase and extracellular trap release by neutrophils from fibrotic mice. This likely mediated disease exacerbation, because mice exhibiting a progressive disease phenotype with greater weight loss and mortality had more activated neutrophils and increased levels of extracellular DNA present in their lungs than did mice with a nonprogressive disease phenotype. These findings further our understanding of the dynamics of neutrophil populations and their trafficking in progressive fibrotic lung disease and may help inform treatments targeting neutrophil function for patients with IPF experiencing disease exacerbation in the future.

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.

Fibrotic lung disease inhibits immune responses to staphylococcal pneumonia via impaired neutrophil and macrophage function.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations, and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized patients with IPF. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like humans with IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared with uninfected fibrotic mice. We determine that fibrosis conferred a defect in MRSA clearance compared with nonfibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibited neutrophil intracellular killing of MRSA through impaired neutrophil elastase release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments of antimicrobial responses upon pulmonary fibrosis development, and our findings suggest a possible mechanism for why patients with IPF are at greater risk of morbidity and mortality related to infection.

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.

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.

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.

Identification of a unique temporal signature in blood and BAL associated with IPF progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive and heterogeneous interstitial lung disease of unknown origin with a low survival rate. There are few treatment options available due to the fact that mechanisms underlying disease progression are not well understood, likely because they arise from dysregulation of complex signaling networks spanning multiple tissue compartments. To better characterize these networks, we used systems-focused data-driven modeling approaches to identify cross-tissue compartment (blood and bronchoalveolar lavage) and temporal proteomic signatures that differentiated IPF progressors and non-progressors. Partial least squares discriminant analysis identified a signature of 54 baseline (week 0) blood and lung proteins that differentiated IPF progression status by the end of 80 weeks of follow-up with 100% cross-validation accuracy. Overall we observed heterogeneous protein expression patterns in progressors compared to more homogenous signatures in non-progressors, and found that non-progressors were enriched for proteomic processes involving regulation of the immune/defense response. We also identified a temporal signature of blood proteins that was significantly different at early and late progressor time points (p < 0.0001), but not present in non-progressors. Overall, this approach can be used to generate new hypothesis for mechanisms associated with IPF progression and could readily be translated to other complex and heterogeneous diseases.

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.