Loss of Airway Phylogenetic Diversity Is Associated with Clinical and Pathobiological Markers of Disease Development in COPD.

RATIONALE: The airway microbiome has the potential to shape COPD pathogenesis, but its relationship to outcomes in milder disease is unestablished. OBJECTIVES: Identify sputum microbiome characteristics associated with markers of COPD in participants of the SubPopulations and InteRmediate Outcome Measures of COPD Study (SPIROMICS). METHODS: Sputum DNA from 877 participants were analyzed using 16S rRNA gene sequencing. Relationships between baseline airway microbiota composition and clinical, radiographic and muco-inflammatory markers, including longitudinal lung function trajectory, were examined. MEASUREMENTS AND MAIN RESULTS: Participant data represented predominantly milder disease (GOLD 0-2: N=732/877). Phylogenetic diversity (range of different species within a sample) correlated positively with baseline lung function, declined with higher GOLD stage, and correlated negatively with symptom burden, radiographic markers of airway disease, and total mucin concentrations (p<0.001). In co-variate adjusted regression models, organisms robustly associated with better lung function included members of Alloprevotella, Oribacterium, and Veillonella. Conversely, lower lung function, greater symptoms and radiographic measures of small airway disease associated with enrichment in members of Streptococcus, Actinobacillus, Actinomyces, and other genera. Baseline sputum microbiota features also associated with lung function trajectory during SPIROMICS follow up (stable/improved, decliner, or rapid decliner). The 'stable/improved' group (slope of FEV1 regression ≥ 66th percentile) had higher bacterial diversity at baseline, associated with enrichment in Prevotella, Leptotrichia, and Neisseria. In contrast, the 'rapid decliner' group (FEV1 slope ≤ 33rd percentile) had significantly lower baseline diversity, associated with enrichment in Streptococcus. CONCLUSIONS: In SPIROMICS baseline airway microbiota features demonstrate divergent associations with better or worse COPD-related outcomes.

Airway microbiota and immune mediator relationships differ in obesity and asthma.

BACKGROUND: Asthma and obesity are both complex conditions characterized by chronic inflammation, and obesity-related severe asthma has been associated with differences in the microbiome. However, whether the airway microbiome and microbiota-immune response relationships differ between obese persons with or without nonsevere asthma is unestablished. OBJECTIVE: We compared the airway microbiome and microbiota-immune mediator relationships between obese and nonobese subjects, with and without mild-moderate asthma. METHODS: We performed cross-sectional analyses of the airway (induced sputum) microbiome and cytokine profiles from blood and sputum using 16S ribosomal RNA gene and internal transcribed spacer region sequencing to profile bacteria and fungi, and multiplex immunoassays. Analysis tools included QIIME 2, linear discriminant analysis effect size (aka LEfSe), Piphillin, and Sparse inverse covariance estimation for ecological association inference (aka SPIEC-EASI). RESULTS: Obesity, irrespective of asthma status, was associated with significant differences in sputum bacterial community structure and composition (unweighted UniFrac permutational analysis of variance, P = .02), including a higher relative abundance of Prevotella, Gemella, and Streptococcus species. Among subjects with asthma, additional differences in sputum bacterial composition and fungal richness were identified between obese and nonobese individuals. Correlation network analyses demonstrated differences between obese and nonobese asthma in relationships between cytokine mediators, and these together with specific airway bacteria involving blood PAI-1, sputum IL-1ß, GM-CSF, IL-8, TNF-α, and several Prevotella species. CONCLUSION: Obesity itself is associated with an altered sputum microbiome, which further differs in those with mild-moderate asthma. The distinct differences in airway microbiota and immune marker relationships in obese asthma suggest potential involvement of airway microbes that may affect mechanisms or outcomes of obese asthma.

Effects of Fluticasone Propionate on Klebsiella pneumoniae and Gram-Negative Bacteria Associated with Chronic Airway Disease.

Inhaled corticosteroids (ICS) are commonly prescribed first-line treatments for asthma and chronic obstructive pulmonary disease (COPD). Recent evidence has shown that ICS use is associated with changes in the airway microbiome, which may impact clinical outcomes such as potential increased risk for pneumonia in COPD. Although the immunomodulatory effects of corticosteroids are well appreciated, whether ICS could directly influence the behavior of respiratory tract bacteria has been unknown. In this pilot study we explored the effects of fluticasone proprionate, a commonly prescribed inhaled corticosteroid, on respiratory bacteria with an expanded focus on Klebsiella pneumoniae, a species previously implicated in fluticasone-associated pneumonia in COPD. We observed significant effects of fluticasone proprionate on growth responses of K. pneumoniae, as well as other bacterial species isolated from asthmatic patients. Fluticasone-exposed K. pneumoniae displayed altered expression of several bacterial genes and reduced the metabolic activity of bronchial epithelial cells and their expression of human ß-defensin 2. Targeted assays identified a fluticasone metabolite from fluticasone-exposed K. pneumoniae cells, suggesting this species may be capable of metabolizing fluticasone proprionate. Collectively, these observations support the hypothesis that specific members of the airway microbiota possess the functional repertoire to respond to or potentially utilize corticosteroids in their microenvironment. These findings lay a foundation for novel research directions into the potential direct effects of ICS, often prescribed long term to patients, on the broader airway microbial community and on the behavior of specific microbial species implicated in asthma and COPD outcomes. IMPORTANCE Inhaled corticosteroids are widely prescribed for many respiratory diseases, including asthma and COPD. While they benefit many patients, corticosteroids can also have negative effects. Some patients do not improve with treatment and even experience adverse side effects. Recent studies have shown that inhaled corticosteroids can change the make-up of bacteria in the human respiratory tract. However, whether these medications can directly impact the behavior of such bacteria has been unknown. Here, we explored the effects of fluticasone propionate, a commonly prescribed inhaled corticosteroid, on Klebsiella pneumoniae and other airway bacteria of interest, including primary species isolated from adult asthma patients. We provide evidence of growth responses to direct fluticasone exposure in culture and further examined fluticasone's effects on K. pneumoniae, including gene expression changes and effects of fluticasone-exposed bacteria on airway cells. These findings indicate that members of the human airway bacterial community possess the functional ability to respond to corticosteroids, which may have implications for the heterogeneity of treatment response observed clinically.

Lung Microbiota and Metabolites Collectively Associate with Clinical Outcomes in Milder Stage Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is variable in its development. Lung microbiota and metabolites collectively may impact COPD pathophysiology, but relationships to clinical outcomes in milder disease are unclear. Objectives: Identify components of the lung microbiome and metabolome collectively associated with clinical markers in milder stage COPD. Methods: We analyzed paired microbiome and metabolomic data previously characterized from bronchoalveolar lavage fluid in 137 participants in the SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study), or (GOLD [Global Initiative for Chronic Obstructive Lung Disease Stage 0-2). Datasets used included 1) bacterial 16S rRNA gene sequencing; 2) untargeted metabolomics of the hydrophobic fraction, largely comprising lipids; and 3) targeted metabolomics for a panel of hydrophilic compounds previously implicated in mucoinflammation. We applied an integrative approach to select features and model 14 individual clinical variables representative of known associations with COPD trajectory (lung function, symptoms, and exacerbations). Measurements and Main Results: The majority of clinical measures associated with the lung microbiome and metabolome collectively in overall models (classification accuracies, >50%, P < 0.05 vs. chance). Lower lung function, COPD diagnosis, and greater symptoms associated positively with Streptococcus, Neisseria, and Veillonella, together with compounds from several classes (glycosphingolipids, glycerophospholipids, polyamines and xanthine, an adenosine metabolite). In contrast, several Prevotella members, together with adenosine, 5'-methylthioadenosine, sialic acid, tyrosine, and glutathione, associated with better lung function, absence of COPD, or less symptoms. Significant correlations were observed between specific metabolites and bacteria (Padj < 0.05). Conclusions: Components of the lung microbiome and metabolome in combination relate to outcome measures in milder COPD, highlighting their potential collaborative roles in disease pathogenesis.

The lung microbiome in end-stage Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease with mortality driven primarily by respiratory failure. Patients with LAM frequently have respiratory infections, suggestive of a dysregulated microbiome. Here we demonstrate that end-stage LAM patients have a distinct microbiome signature compared to patients with end-stage chronic obstructive pulmonary disease.

Real Time Breath Analysis Using Portable Gas Chromatography for Adult Asthma Phenotypes.

Asthma is heterogeneous but accessible biomarkers to distinguish relevant phenotypes remain lacking, particularly in non-Type 2 (T2)-high asthma. Moreover, common clinical characteristics in both T2-high and T2-low asthma (e.g., atopy, obesity, inhaled steroid use) may confound interpretation of putative biomarkers and of underlying biology. This study aimed to identify volatile organic compounds (VOCs) in exhaled breath that distinguish not only asthmatic and non-asthmatic subjects, but also atopic non-asthmatic controls and also by variables that reflect clinical differences among asthmatic adults. A total of 73 participants (30 asthma, eight atopic non-asthma, and 35 non-asthma/non-atopic subjects) were recruited for this pilot study. A total of 79 breath samples were analyzed in real-time using an automated portable gas chromatography (GC) device developed in-house. GC-mass spectrometry was also used to identify the VOCs in breath. Machine learning, linear discriminant analysis, and principal component analysis were used to identify the biomarkers. Our results show that the portable GC was able to complete breath analysis in 30 min. A set of nine biomarkers distinguished asthma and non-asthma/non-atopic subjects, while sets of two and of four biomarkers, respectively, further distinguished asthmatic from atopic controls, and between atopic and non-atopic controls. Additional unique biomarkers were identified that discriminate subjects by blood eosinophil levels, obese status, inhaled corticosteroid treatment, and also acute upper respiratory illnesses within asthmatic groups. Our work demonstrates that breath VOC profiling can be a clinically accessible tool for asthma diagnosis and phenotyping. A portable GC system is a viable option for rapid assessment in asthma.

Lung microbiota associations with clinical features of COPD in the SPIROMICS cohort.

Chronic obstructive pulmonary disease (COPD) is heterogeneous in development, progression, and phenotypes. Little is known about the lung microbiome, sampled by bronchoscopy, in milder COPD and its relationships to clinical features that reflect disease heterogeneity (lung function, symptom burden, and functional impairment). Using bronchoalveolar lavage fluid collected from 181 never-smokers and ever-smokers with or without COPD (GOLD 0-2) enrolled in the SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS), we find that lung bacterial composition associates with several clinical features, in particular bronchodilator responsiveness, peak expiratory flow rate, and forced expiratory flow rate between 25 and 75% of FVC (FEF25-75). Measures of symptom burden (COPD Assessment Test) and functional impairment (six-minute walk distance) also associate with disparate lung microbiota composition. Drivers of these relationships include members of the Streptococcus, Prevotella, Veillonella, Staphylococcus, and Pseudomonas genera. Thus, lung microbiota differences may contribute to airway dysfunction and airway disease in milder COPD.

Gut microbiota relationships to lung function and adult asthma phenotype: a pilot study.

INTRODUCTION: Despite strong evidence that maturation patterns of the gut microbiome in early life influence the risk for childhood asthma, very little is known about gut microbiota patterns in adults with established asthma, and of greater interest relationships to phenotypic features that characterise asthma heterogeneity. METHODS: Fifty-eight faecal samples from 32 adults with (n=24) and without (n=8) asthma were analysed using 16S ribosomal RNA gene sequencing methods to characterise intestinal bacterial composition. Compositional stability of paired samples was evaluated and features of gut bacterial community structure analysed in relation to extensive clinical characterisation data collected from subjects, who were enrolled in a prospective observational cohort study at the University of Michigan. RESULTS: Differences in gut bacterial community structure were associated with aeroallergen sensitisation and lung function as assessed by forced expiratory volume in 1 s (FEV1) %predicted. Associations with FEV1 were consistently observed across independent analytic approaches. k-means clustering of the gut microbiota data in subjects with asthma revealed three different clusters, distinguished most strongly by FEV1 (p<0.05) and trends in differences in other clinical and inflammatory features. CONCLUSION: In this pilot study of asthmatic and non-asthmatic subjects, significant relationships between gut microbiota composition, aeroallergen sensitisation and lung function were observed. These preliminary findings merit further study in larger cohorts to explore possible mechanistic links to asthma phenotype.

ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer.

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

Signaling mechanisms coupled to CXCL12/CXCR4-mediated cellular proliferation are PTEN-dependent.

A key difference between normal and malignant prostate cells in vitro and in vivo is that both alleles of PTEN are largely intact in normal benign prostate glands and cultured epithelial cells, whereas one or both alleles of PTEN are mutant or deleted in the majority of prostate tumors and malignant prostate cancer cell lines. Intact PTEN suppresses phosphorylation of Akt downstream of PI3K activation in non-transformed cells whereas Akt phosphorylation is unimpeded in malignant cells that are often PTEN-deficient. We have previously shown that activation of the CXCL12/CXCR4 axis transactivates the EGFR to promote pro-proliferative signaling preferentially through the Raf/MEK/Erk pathway in benign prostate epithelial cells. These cells demonstrate little basal pAkt and these levels do not increase with CXCL12 stimulation because PTEN is intact and fully functional. Thus, inactivation of PTEN may be the critical factor that modulates downstream signaling and the specific CXCL12-stimulated proliferative responses of non-transformed and transformed prostate epithelial cells. Based on these data, we hypothesize that the CXCL12/CXCR4-mediated activation of downstream pro-proliferative signaling through the Raf/MEK/Erk or PI3K/Akt pathways is modulated by PTEN status.

Leukocytic promotion of prostate cellular proliferation.

BACKGROUND: Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa. METHODS: An in vitro system designed to mimic the human prostatic microenvironment incorporating prostatic stroma (primary and immortalized prostate stromal fibroblasts), epithelium (N15C6, BPH-1, LNCaP, and PC3 cells), and inflammatory infiltrate (HL-60 cells, HH, and Molt-3 T-lymphocytes) was developed. Modified Boyden chamber assays were used to test the ability of prostate stromal and epithelial cells to attract leukocytes and to test the effect of leukocytes on prostate cellular proliferation. Antibody arrays were used to identify leukocyte-secreted cytokines mediating prostate cellular proliferation. RESULTS: Leukocytic cells migrated towards both prostate stromal and epithelial cells. CD4+ T-lymphocytes promoted the proliferation of both transformed and non-transformed prostate epithelial cell lines tested, whereas CD8+ T-lymphocytes as well as dHL-60M macrophagic and dHL-60N neutrophilic cells selectively promoted the proliferation of PCa cells. CONCLUSIONS: The results of these studies show that inflammatory cells can be attracted to the prostate tissue microenvironment and can selectively promote the proliferation of non-transformed or transformed prostate epithelial cells, and are consistent with differential role(s) for inflammatory infiltrate in the etiologies of benign and malignant proliferative disease in the prostate.

CXCL5 promotes prostate cancer progression.

CXCL5 is a proangiogenic CXC-type chemokine that is an inflammatory mediator and a powerful attractant for granulocytic immune cells. Unlike many other chemokines, CXCL5 is secreted by both immune (neutrophil, monocyte, and macrophage) and nonimmune (epithelial, endothelial, and fibroblastic) cell types. The current study was intended to determine which of these cell types express CXCL5 in normal and malignant human prostatic tissues, whether expression levels correlated with malignancy and whether CXCL5 stimulated biologic effects consistent with a benign or malignant prostate epithelial phenotype. The results of these studies show that CXCL5 protein expression levels are concordant with prostate tumor progression, are highly associated with inflammatory infiltrate, and are frequently detected in the lumens of both benign and malignant prostate glands. Exogenous administration of CXCL5 stimulates cellular proliferation and gene transcription in both nontransformed and transformed prostate epithelial cells and induces highly aggressive prostate cancer cells to invade through synthetic basement membrane in vitro. These findings suggest that the inflammatory mediator, CXCL5, may play multiple roles in the etiology of both benign and malignant proliferative diseases in the prostate.

Pilot and feasibility study of serum chemokines as markers to distinguish prostatic disease in men with low total serum PSA.

BACKGROUND: The incidence and prevalence of both benign prostatic hypertrophy (BPH) and prostate cancer (PCa) increase with the aging process. Our laboratory recently showed that the chemokines CXCL5 and CXCL12, which normally function as inflammatory mediators, are secreted at higher levels by aging prostate stromal fibroblasts and elicit proliferative responses from both prostate stromal fibroblast and epithelial cells. Because both CXCL5 and CXCL12 are secreted molecules, we hypothesized that their levels in patient serum might serve as biomarkers to distinguish between BPH and PCa. METHODS: Serum CXCL5 and CXCL12 levels were determined using sandwich ELISAs for 51 men demonstrating low serum PSA values of < or =10 ng/ml who underwent diagnostic needle biopsy for the detection of PCa. The bivariate relationship of circulating chemokine levels, age, and disease status in the prostate was tested using the Wilcoxon rank-sum test. RESULTS: Total serum CXCL12 levels were significantly higher for men who were biopsy positive compared to those who were biopsy negative for cancer and histological prostatitis (P = 0.050). Among men who were biopsy negative for PCa, total serum CXCL5 levels were inversely associated with prostate volume and were significantly higher in men with concomitant BPH and histological prostatitis compared to those without evidence of prostatic disease (P < 0.003). CONCLUSIONS: The results of this pilot and feasibility study suggest that serum or plasma CXCL5 and CXCL12 levels may potentially distinguish between BPH and PCa among patients presenting with low serum PSA, and may be useful toward facilitating decisions to perform diagnostic needle biopsy in this patient population.

CXCL12 activates a robust transcriptional response in human prostate epithelial cells.

CXCL12 is a CXC-type chemokine that plays important roles in hematopoiesis, development, and organization of the immune system and supports the survival or growth of a variety of normal or malignant cell types. Our laboratory recently showed that CXCL12 is secreted by aging stromal fibroblast cells and is a major paracrine factor that specifically stimulates the proliferation of prostate epithelial cells. The current study shows that this CXCL12-mediated proliferative response may be either ERK-dependent or ERK-independent. Moreover, CXCL12 initiates a previously undefined and complex global transcriptional response in prostate epithelial cells. This CXCL12-mediated transcriptional response directly stimulates the expression of genes encoding proteins that are involved in the promotion of cellular proliferation and progression through the cell cycle, tumor metastasis, and cellular motility, and directly represses the transcription of genes encoding proteins involved in cell-cell adhesion and resistance to apoptosis. Thus, CXCL12 may play a major role in the etiology of benign proliferative disease in the context of an aging tissue microenvironment.

Concordant copy number and transcriptional activity of genes mapping to derivative chromosomes 8 during cellular immortalization in vitro.

Deletion, rearrangement, or amplification of sequences mapping to chromosome 8 are frequently observed in human prostate and other tumors. However, it is not clear whether these events alter the transcriptional activity of the affected genes. To examine this question, we have utilized oligonucleotide microarray technology and compared the transcriptional patterns of normal human prostate tissues and five immortalized cell lines carrying either two normal chromosomes 8 or one normal and one derivative chromosome 8. Comparison of the transcriptional profiles of the tissues and cell lines identified 125 differentially expressed transcripts specific to chromosome 8, with 46 transcripts mapping to 8p and 79 transcripts mapping to 8q. The majority of genes mapping to 8p (44/46, 96%) were transcriptionally down-regulated in cells hemizygous for 8p, whereas the majority of genes mapping to 8q (58/79, 73%) were up-regulated in cells carrying three copies of 8q. Moreover, hemizygous alleles on 8p exhibited sub-haploinsufficient transcript levels for several genes that could be induced to haploinsufficient levels under hypomethylating conditions, suggesting that epigenetic regulation is a common mechanism for gene silencing in cells deleted for one copy of 8p. The results of these studies clearly demonstrate that alterations of gene copy number and transcriptional activity are directly correlated in cell lines harboring derivative chromosomes 8, and that these events are commonly observed during cellular immortalization in vitro.

CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro.

The direct relationship between the aging process and the incidence and prevalence of both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) implies that certain risk factors associated with the development of both diseases increase with the aging process. In particular, both diseases share an overly proliferative phenotype, suggesting that mechanisms that normally act to suppress cellular proliferation are disrupted or rendered dysfunctional as a consequence of the aging process. We propose that one such mechanism involves changes in the prostate microenvironment, which 'evolves' during the aging process and disrupts paracrine interactions between epithelial and associated stromal fibroblasts. We show that stromal fibroblasts isolated from the prostates of men 63-81 years of age at the time of surgery express and secrete higher levels of the CXCL12 chemokine compared with those isolated from younger men, and stimulate CXCR4-mediated signaling pathways that induce cellular proliferation. These studies represent an important first step towards a mechanistic elucidation of the role of aging in the etiology of benign and malignant prostatic diseases.

Evolution of 8p loss in transformed human prostate epithelial cells.

Deletion or rearrangement of sequences that map to the short arm of chromosome 8 (8p) are frequently associated with human prostate tumorigenesis. These losses often involve the entire short arm of chromosome 8 or very large regions of distal or proximal 8p, and several putative tumor suppressor genes mapping to 8p have been described. However, the mechanism responsible for 8p loss during prostate tumorigenesis has not been elucidated. In this study, we report data obtained using array comparative genomic hybridization and spectral karyotyping, which demonstrate successive translocation and deletion events responsible for loss of one copy of 8p in transformed human prostate epithelial cells. Moreover, this loss was accompanied by a pronounced transcriptional downregulation of genes mapping to the remaining copy of 8p and enhanced expression of traits associated with neoplastic transformation. Taken together, these studies illustrate a potential mechanism and functional role for 8p loss in human prostate tumorigenesis.