Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system, and dysbiosis in its development may set the stage for subsequent lung disease.

Rationale: DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF). Objectives: To determine the DNA methylation changes in IPF and their effects on gene expression. Methods: Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genomewide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels. Measurements and Main Results: DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase expression without observed alterations in global DNA methylation. Genome-wide differences in DNA methylation status and RNA expression were demonstrated by array hybridization. Among the genes whose DNA methylation status and RNA expression were both significantly altered, 16 genes were hypermethylated in DNA associated with decreased mRNA expression or vice versa. We validated CLDN5, ZNF467, TP53INP1, and DDAH1 genes at the level of DNA methylation status, RNA, and protein-level expression. Conclusions: Changes in DNA methylation correspond to altered mRNA expression of a number of genes, some with known and others with previously uncharacterized roles in IPF, suggesting that DNA methylation is important in the pathogenesis of IPF.

Many extremely preterm infants continue to suffer from bronchopulmonary dysplasia, which results from abnormal saccular-stage lung development. Here, we show that fibroblast growth factor-10 (FGF-10) is required for saccular lung development and reduced in the lung tissue of infants with bronchopulmonary dysplasia. Although exposure to bacteria increases the risk of bronchopulmonary dysplasia, no molecular target has been identified connecting inflammatory stimuli and abnormal lung development. In an experimental mouse model of saccular lung development, activation of Toll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) inhibited FGF-10 expression, leading to abnormal saccular airway morphogenesis. In addition, Toll-mediated FGF-10 inhibition disrupted the normal positioning of myofibroblasts around saccular airways, similar to the mislocalization of myofibroblasts seen in patients with bronchopulmonary dysplasia. Reduced FGF-10 expression may therefore link the innate immune system and impaired lung development in bronchopulmonary dysplasia.

The sizes and anomers of the products formed during the hydrolysis of chitin oligosaccharides by the Family 18 chitinase A (ChiA) from Serratia marcescens were analysed by hydrophilic interaction chromatography using a novel approach in which reactions were performed at 0 degrees C to stabilize the anomer conformations of the initial products. Crystallographic studies of the enzyme, having the structure of the complex of the ChiA E315L (Glu315-->Leu) mutant with a hexasaccharide, show that the oligosaccharide occupies subsites -4 to +2 in the substrate-binding cleft, consistent with the processing of beta-chitin by the release of disaccharide at the reducing end. Products of the hydrolysis of hexa- and penta-saccharides by wild-type ChiA, as well as by two mutants of the residues Trp275 and Phe396 important in binding the substrate at the +1 and +2 sites, show that the substrates only occupy sites -2 to +2 and that additional N -acetyl-D-glucosamines extend beyond the substrate-binding cleft at the reducing end. The subsites -3 and -4 are not used in this four-site binding mode. The explanation for these results is found in the high importance of individual binding sites for the processing of short oligosaccharides compared with the cumulative recognition and processive hydrolysis mechanism used to digest natural beta-chitin.

Summary IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ecto-domain at 2.14Ǻ resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml−1) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6–100 nm at lower concentrations (50 μg ml−1), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml−1. Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.