Pedro A. Dimitriu

The Lung Tissue Microbiome in Chronic Obstructive Pulmonary Disease

RATIONALE Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others. OBJECTIVES To extend these findings to human lung tissue samples. METHODS DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing. MEASUREMENT AND MAIN RESULTS Total bacterial populations within lung tissue were small (20-1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007). CONCLUSIONS There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.

Host Response to the Lung Microbiome in Chronic Obstructive Pulmonary Disease

RATIONALE The relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression. METHODS Culture-independent pyrosequencing microbiome analysis was used to examine the V3-V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1-V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro-computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression. MEASUREMENTS AND MAIN RESULTS Ten operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4(+) T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (P < 0.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1. CONCLUSIONS These results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.

Perinatal antibiotic-induced shifts in gut microbiota have differential effects on inflammatory lung diseases.

BACKGROUND Resident gut microbiota are now recognized as potent modifiers of host immune responses in various scenarios. Recently, we demonstrated that perinatal exposure to vancomycin, but not streptomycin, profoundly alters gut microbiota and enhances susceptibility to a TH2 model of allergic asthma. OBJECTIVE Here we sought to further clarify the etiology of these changes by determining whether perinatal antibiotic treatment has a similar effect on the TH1/TH17-mediated lung disease, hypersensitivity pneumonitis. METHODS Hypersensitivity pneumonitis was induced in C57BL/6 wild-type or recombination-activating gene 1-deficient mice treated perinatally with vancomycin or streptomycin by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Disease severity was assessed by measuring lung inflammation, pathology, cytokine responses, and serum antibodies. Microbial community analyses were performed on stool samples via 16S ribosomal RNA pyrosequencing and correlations between disease severity and specific bacterial taxa were identified. RESULTS Surprisingly, in contrast to our findings in an allergic asthma model, we found that the severity of hypersensitivity pneumonitis was unaffected by vancomycin, but increased dramatically after streptomycin treatment. This likely reflects an effect on the adaptive, rather than innate, immune response because the effects of streptomycin were not observed during the early phases of disease and were abrogated in recombination-activating gene 1-deficient mice. Interestingly, Bacteroidetes dominated the intestinal microbiota of streptomycin-treated animals, while vancomycin promoted the expansion of the Firmicutes. CONCLUSIONS Perinatal antibiotics exert highly selective effects on resident gut flora, which, in turn, lead to very specific alterations in susceptibility to TH2- or TH1/TH17-driven lung inflammatory disease.

Relationship between soil properties and patterns of bacterial β-diversity across reclaimed and natural boreal forest soils.

Productivity gradients in the boreal forest are largely determined by regional-scale changes in soil conditions, and bacterial communities are likely to respond to these changes. Few studies, however, have examined how variation in specific edaphic properties influences the composition of soil bacterial communities along environmental gradients. We quantified bacterial compositional diversity patterns in ten boreal forest sites of contrasting fertility. Bulk soil (organic and mineral horizons) was sampled from sites representing two extremes of a natural moisture-nutrient gradient and two distinct disturbance types, one barren and the other vegetation-rich. We constructed 16S rRNA gene clone libraries to characterize the bacterial communities under phylogenetic- and species-based frameworks. Using a nucleotide analog to label DNA-synthesizing bacteria, we also assessed the composition of active taxa in disturbed sites. Most sites were dominated by sequences related to the α-Proteobacteria, followed by acidobacterial and betaproteobacterial sequences. Non-parametric multivariate regression indicated that pH, which was lowest in the natural sites, explained 34% and 16% of the variability in community structure as determined by phylogenetic-based (UniFrac distances) and species-based (Jaccard similarities) metrics, respectively. Soil pH was also a significant predictor of richness (Chao1) and diversity (Shannon) measures. Within the natural edaphic gradient, soil moisture accounted for 32% of the variance in phylogenetic (but not species) community structure. In the boreal system we studied, bacterial β-diversity patterns appear to be largely related to “master” variables (e.g., pH, moisture) rather than to observable attributes (e.g., plant cover) leading to regional-scale fertility gradients.

Temporal stability of the mouse gut microbiota in relation to innate and adaptive immunity

Gut microbial community properties of mammals are thought to be partly shaped by a combination of host immunity and environmental factors, but their relative importance is not firmly established. To address this gap, we first characterized the faecal bacteria of mice with a functioning immune system (wild-type, WT), mice with defective immune responses (CD45), mice lacking an adaptive immune system (RAG), and mice with both immune dysfunctions (45RAG). Using fingerprinting of 16S rRNA genes, we observed significant differences in gut microbiota composition across all mouse strains (P < 0.001) and identified several mouse strain-specific genera via pyrosequencing, including Turicibacter sp. (in WT mice) and Allobaculum sp. (in CD45-deficient animals). To define the role of the host immune system in constraining gut microbiota stability after perturbation, we cohoused CD45-deficient and WT mice and monitored gut bacterial community dynamics during 8 weeks. Cohousing caused the WT bacterial communities to become indistinguishable from those of CD45 mice (P > 0.05). Time-series analysis indicated that the communities of cohoused mice changed directionally as opposed to the relatively stable communities of non-cohoused controls. When we considered only taxonomic membership, it was the communities of CD45 non-cohoused mice that experienced the highest rate of change. Rather than be governed by fluctuations in the relative abundance of taxa, we suggest that CD45-regulated immune responses either are stimulated by the presence of bacteria per se or promote temporal stability by selecting for the occurrence of specific taxa.

Associations between infant fungal and bacterial dysbiosis and childhood atopic wheeze in a nonindustrialized setting

Background: Asthma is the most prevalent chronic disease of childhood. Recently, we identified a critical window early in the life of both mice and Canadian infants during which gut microbial changes (dysbiosis) affect asthma development. Given geographic differences in human gut microbiota worldwide, we studied the effects of gut microbial dysbiosis on atopic wheeze in a population living in a distinct developing world environment. Objective: We sought to determine whether microbial alterations in early infancy are associated with the development of atopic wheeze in a nonindustrialized setting. Methods: We conducted a case‐control study nested within a birth cohort from rural Ecuador in which we identified 27 children with atopic wheeze and 70 healthy control subjects at 5 years of age. We analyzed bacterial and eukaryotic gut microbiota in stool samples collected at 3 months of age using 16S and 18S sequencing. Bacterial metagenomes were predicted from 16S rRNA data by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States and categorized by function with Kyoto Encyclopedia of Genes and Genomes ontology. Concentrations of fecal short‐chain fatty acids were determined by using gas chromatography. Results: As previously observed in Canadian infants, microbial dysbiosis at 3 months of age was associated with later development of atopic wheeze. However, the dysbiosis in Ecuadorian babies involved different bacterial taxa, was more pronounced, and also involved several fungal taxa. Predicted metagenomic analysis emphasized significant dysbiosis‐associated differences in genes involved in carbohydrate and taurine metabolism. Levels of the fecal short‐chain fatty acids acetate and caproate were reduced and increased, respectively, in the 3‐month stool samples of children who went on to have atopic wheeze. Conclusions: Our findings support the importance of fungal and bacterial microbiota during the first 100 days of life on the development of atopic wheeze and provide additional support for considering modulation of the gut microbiome as a primary asthma prevention strategy. Graphical abstract: Figure. No caption available.

Microbiome-driven allergic lung inflammation is ameliorated by short-chain fatty acids

The mammalian gastrointestinal tract harbors a microbial community with metabolic activity critical for host health, including metabolites that can modulate effector functions of immune cells. Mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated T helper type 2 cell (Th2) responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation with short-chain fatty acids (SCFAs) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFAs have fewer interleukin-4 (IL4)-producing CD4+ T cells and decreased levels of circulating immunoglobulin E (IgE). In addition, DCs exposed to SCFAs activate T cells less robustly, are less motile in response to CCL19 in vitro, and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can exacerbate allergic lung inflammation through both T cell- and DC-dependent mechanisms that are inhibited by SCFAs.

The early life gut microbiota and atopic disease

Background Asthma is the most prevalent of all childhood diseases and accounts for the majority of hospitalizations and school absences in children [1]. Current mouse model research has identified the early life gut microbiota as a potential therapeutic target for the prevention of asthma and atopic diseases [2-4]. We hypothesize that the early life gut microbiota could play a similar preventative role against atopic disease development in humans. Methods 1262 children enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) Study with complete skin prick test and wheeze data at one year were grouped into four clinically relevant phenotypes: atopy + wheeze, atopy only, wheeze only, and control. Bacterial 16S rDNA from 3-month and 1-year stool samples of 319 children in these four phenotypes was extracted, amplified, and subjected to high throughput Illumina sequencing. Quantitative polymerase chain reaction (qPCR) and short chain fatty acid (SCFA) analysis were also conducted on 44 children in the two extreme phenotypes (atopy + wheeze vs. control). Results 16S sequence analysis of our sample cohort (319 subjects) identified bacterial populations that differed in abundance in the atopy + wheeze group at 3-months of age but not at 1-year of age. Additionally, significant changes in the abundance of certain bacterial genera were found in the atopy + wheeze group when compared to controls by qPCR at 3-months of age only. Changes in stool short chain fatty acid production between the atopy + wheeze group and the control group were also observed at 3months of age only. Conclusions Shifts in the relative abundance of certain gut bacterial populations and differences in the levels of stool SCFAs before 3-months of age are associated with atopy and wheeze at one year of age.

The impact of the intestinal microbiome on human immune development and atopic disease.

Background Asthma is a chronic inflammatory disease characterized by bronchial hyper-responsiveness [1]. As the most endemic of all childhood diseases, asthma accounts for the majority of hospitalizations and school absences in children [2]. Exciting new research focuses on the involvement of the gut microbiome in asthma development. Murine studies support the hypothesis that the administration of probiotics or antibiotics during post-natal life alters the gut microbiome and ultimately the asthmatic symptoms of these mice [3-5]. This study will translate these findings into humans using stool samples obtained from the Canadian Healthy Infant Longitudinal Development (CHILD) study.