DNA Methylome Alterations Are Associated with Airway Macrophage Differentiation and Phenotype during Lung Fibrosis.

Rationale: Airway macrophages (AMs) are key regulators of the lung environment and are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal respiratory disease with no cure. However, knowledge about the epigenetics of AMs in IPF is limited. Objectives: To assess the role of epigenetic regulation of AMs during lung fibrosis. Methods: We undertook DNA methylation (DNAm) profiling by using Illumina EPIC (850k) arrays in sorted AMs from healthy donors (n = 14) and donors with IPF (n = 30). Cell-type deconvolution was performed by using reference myeloid-cell DNA methylomes. Measurements and Main Results: Our analysis revealed that epigenetic heterogeneity was a key characteristic of IPF AMs. DNAm "clock" analysis indicated that epigenetic alterations in IPF AMs were not associated with accelerated aging. In differential DNAm analysis, we identified numerous differentially methylated positions (n = 11) and differentially methylated regions (n = 49) between healthy and IPF AMs, respectively. Differentially methylated positions and differentially methylated regions encompassed genes involved in lipid (LPCAT1 [lysophosphatidylcholine acyltransferase 1]) and glucose (PFKFB3 [6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3]) metabolism, and importantly, the DNAm status was associated with disease severity in IPF. Conclusions: Collectively, our data identify that changes in the epigenome are associated with the development and function of AMs in the IPF lung.

The Respiratory Microbiome in Chronic Hypersensitivity Pneumonitis Is Distinct from That of Idiopathic Pulmonary Fibrosis.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is a condition that arises after repeated exposure and sensitization to inhaled antigens. The lung microbiome is increasingly implicated in respiratory disease, but, to date, no study has investigated the composition of microbial communities in the lower airways in CHP.Objectives: To characterize and compare the airway microbiome in subjects with CHP, subjects with idiopathic pulmonary fibrosis (IPF), and control subjects.Methods: We prospectively recruited individuals with a CHP diagnosis (n = 110), individuals with an IPF diagnosis (n = 45), and control subjects (n = 28). Subjects underwent BAL and bacterial DNA was isolated, quantified by quantitative PCR and the 16S ribosomal RNA gene was sequenced to characterize the bacterial communities in the lower airways.Measurements and Main Results: Distinct differences in the microbial profiles were evident in the lower airways of subjects with CHP and IPF. At the phylum level, the prevailing microbiota of both subjects with IPF and subjects with CHP included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. However, in IPF, Firmicutes dominated, whereas the percentage of reads assigned to Proteobacteria in the same group was significantly lower than the percentage found in subjects with CHP. At the genus level, the Staphylococcus burden was increased in CHP, and Actinomyces and Veillonella burdens were increased in IPF. The lower airway bacterial burden in subjects with CHP was higher than that in control subjects but lower than that of those with IPF. In contrast to IPF, there was no association between bacterial burden and survival in CHP.Conclusions: The microbial profile of the lower airways in subjects with CHP is distinct from that of IPF, and, notably, the bacterial burden in individuals with CHP fails to predict survival.

Itaconate controls the severity of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

Bacterial burden in the lower airways predicts disease progression in idiopathic pulmonary fibrosis and is independent of radiological disease extent.

Increasing bacterial burden in the lower airways of patients with idiopathic pulmonary fibrosis confers an increased risk of disease progression and mortality. However, it remains unclear whether this increased bacterial burden directly influences progression of fibrosis or simply reflects the magnitude of the underlying disease extent or severity.We prospectively recruited 193 patients who underwent bronchoscopy and received a multidisciplinary diagnosis of idiopathic pulmonary fibrosis. Quantification of the total bacterial burden in bronchoalveolar lavage fluid was performed by 16S rRNA gene qPCR. Imaging was independently evaluated by two readers assigning quantitative scores for extent, severity and topography of radiographic changes and relationship of these features with bacterial burden was assessed.Increased bacterial burden significantly associated with disease progression (HR 2.1; 95% CI 1.287-3.474; p=0.0028). Multivariate stepwise regression demonstrated no relationship between bacterial burden and radiological features or extent of disease. When specifically considering patients with definite or probable usual interstitial pneumonia there was no difference in bacterial burden between these two groups. Despite a postulated association between pleuroparenchymal fibroelastosis and clinical infection, there was no relationship between either the presence or extent of pleuroparenchymal fibroelastosis and bacterial burden.We demonstrate that bacterial burden in the lower airways is not simply secondary to the extent of the underlying architectural destruction of the lung parenchyma seen in idiopathic pulmonary fibrosis. The independent nature of this association supports a relationship with the underlying pathogenic mechanisms and highlights the urgent need for functional studies.

The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets during Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive disease with limited therapeutic options. Airway macrophages (AMs) are key components of the defense of the airways and are implicated in the pathogenesis of IPF. Alterations in iron metabolism have been described during fibrotic lung disease and in murine models of lung fibrosis. However, the role of transferrin receptor 1 (CD71)-expressing AMs in IPF is not known. Objectives: To assess the role of CD71-expressing AMs in the IPF lung. Methods: We used multiparametric flow cytometry, gene expression analysis, and phagocytosis/transferrin uptake assays to delineate the role of AMs expressing or lacking CD71 in the BAL of patients with IPF and of healthy control subjects. Measurements and Main Results: There was a distinct increase in proportions of AMs lacking CD71 in patients with IPF compared with healthy control subjects. Concentrations of BAL transferrin were enhanced in IPF-BAL, and furthermore, CD71- AMs had an impaired ability to sequester transferrin. CD71+ and CD71- AMs were phenotypically, functionally, and transcriptionally distinct, with CD71- AMs characterized by reduced expression of markers of macrophage maturity, impaired phagocytosis, and enhanced expression of profibrotic genes. Importantly, proportions of AMs lacking CD71 were independently associated with worse survival, underlining the importance of this population in IPF and as a potential therapeutic target. Conclusions: Taken together, these data highlight how CD71 delineates AM subsets that play distinct roles in IPF and furthermore show that CD71- AMs may be an important pathogenic component of fibrotic lung disease.

RIPOSTE: a framework for improving the design and analysis of laboratory-based research.

Lack of reproducibility is an ongoing problem in some areas of the biomedical sciences. Poor experimental design and a failure to engage with experienced statisticians at key stages in the design and analysis of experiments are two factors that contribute to this problem. The RIPOSTE (Reducing IrreProducibility in labOratory STudiEs) framework has been developed to support early and regular discussions between scientists and statisticians in order to improve the design, conduct and analysis of laboratory studies and, therefore, to reduce irreproducibility. This framework is intended for use during the early stages of a research project, when specific questions or hypotheses are proposed. The essential points within the framework are explained and illustrated using three examples (a medical equipment test, a macrophage study and a gene expression study). Sound study design minimises the possibility of bias being introduced into experiments and leads to higher quality research with more reproducible results.

Inter-laboratory evaluation of the response of primary human hepatocyte cultures to model CYP inducers - a European Centre for Validation of Alternative Methods (ECVAM) - funded pre-validation study.

The aim of the current work was to harmonise protocols between three laboratories by performing independent isolations and cultures of human hepatocytes and to assess their responses to prototypical cytochrome P450 (CYP) enzyme inducers, beta-naphthoflavone (BNF), rifampicin (RIF) or phenobarbital (PB). The magnitudes of the induction responses were CYP and donor-dependent but there was a good reproducibility between laboratories. CYP1A2 activity was evident in all cultures treated with BNF but not RIF or PB. Likewise, CYP3A4/5 activity was induced to the same extent by RIF and PB, while BNF did not affect this CYP in any of the cultures tested. All three compounds caused a concentration-dependent increase in CYP2B6 in cultures from 2 of the 3 laboratories and the response to PB was at least twice that of the other two inducers. In conclusion, the harmonised protocols used to study the response of primary cultures of human hepatocytes to prototypical inducers are transferable, reproducible within a given laboratory and between laboratories. The results obtained will support setting up a definitive validation study of the harmonised protocols.

Regulation of human hepatocytes by P2Y receptors: control of glycogen phosphorylase, Ca2+, and mitogen-activated protein kinases.

In the rat both short-term liver function, such as glycogen metabolism, and long-term events such as proliferation after partial hepatectomy, are in part controlled by release of nucleotides such as ATP acting on hepatocyte P2Y(1) and P2Y(2) receptors (members of a family of P2Y receptors for extracellular nucleotides such as ATP and UTP). Here, we have studied P2Y receptor regulation of signaling pathways involved in glycogen phosphorylase activation and proliferation of primary human hepatocytes. Stimulation of cultured hepatocytes with either ATP and UTP, but not UDP or 2-methylthio ADP, led to concentration-dependent increases in cytosolic free Ca(2+) concentration ([Ca(2+)](c); EC(50) for ATP = 3.3 microM, for UTP = 2.3 microM) and [(3)H]inositol (poly)phosphates (EC(50) for ATP = 9.4 microM, for UTP = 15.4 microM). ATP and UTP also stimulated glycogen phosphorylase in human hepatocytes, each with a threshold for activation of less than 1 microM. Application of 2-methylthio ADP up to 100 microM was ineffective. Phosphorylation of both extracellular signal-related kinase and c-Jun N-terminal kinase was stimulated by ATP and UTP, but not by 2-methylthio ADP or UDP, either alone or when costimulated with epidermal growth factor. In conclusion, in human hepatocytes P2Y receptors control both glycogen metabolism and proliferation-associated responses such as increased [Ca(2+)](c) and mitogen-activated protein kinase cascades. Regulation seems to be primarily through P2Y(2) receptors. In contrast with previous studies on rat hepatocytes, there is an absence of responses mediated by P2Y(1) receptors.

Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study.

BACKGROUND: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P-450 induction. AIMS AND METHODS: As first stage of this prevalidation study, the purpose of the present work was to set criteria for optimization and harmonization of hepatocyte isolation from human tissue among laboratories to establish a routine procedure. This was achieved by combining and/or comparing the data generated by the two independent European laboratories (France and UK). RESULTS: The results confirmed that surgical waste material is a valuable source for obtaining high quality hepatocytes under certain pre-, intra- and post-operative conditions: cell yield of viable hepatocytes was not significantly affected by age and sex of patients, nor indications for resection, steatosis or cholestasis. Cold ischeamia up to 5 hours did not influence viable cell yield allowing transport of material. CONCLUSION: The use of biopsy sizes between 50-100 g, cannulation with 2-4 cannulae, digestion with collagenase-containing digestion medium at a flow rate of 25 ml/cannula for 20 minutes, with cut surface being glued in order to reform Glisson's capsule, should optimize the total yield of viable human hepatocytes obtained per preparation of waste liver surgical resections.

Synthesis, in vitro formation, and behavioural effects of glutathione regioisomers of alpha-methyldopamine with relevance to MDA and MDMA (ecstasy).

Administration of 3,4-methylenedioxymethamphetamine (MDMA) or 3,4-methylenedioxyamphetamine (MDA) to rats produces serotonergic nerve terminal degeneration. However, they are not neurotoxic when injected directly into the brain, suggesting the requirement for peripheral metabolism of MDMA to a neurotoxic metabolite. Alpha-methyldopamine (alpha-MeDA) is a major metabolite of MDA. There are indications that a glutathione metabolite of alpha-MeDA and/or 3,4-dihydroxymethamphetamine may be responsible for the neurotoxicity and some of the behavioural effects produced by MDMA and/or MDA. The present study details the synthesis, purification and separation of the 5-(glutathion-S-yl)-alpha-MeDA and 6-(glutathion-S-yl)-alpha-MeDA regioisomers of alpha-MeDA. Incubation of MDA with human liver microsomes demonstrated that production of both glutathione adducts are related to cytochrome P450 2D6 isoform activity. Following intracerebroventricular administration (180 nmol) of either GSH adduct into Dark Agouti or Sprague-Dawley rats only 5-(glutathion-S-yl)-alpha-MeDA produced behavioural effects characterised by hyperactivity, teeth chattering, tremor/trembling, head weaving, splayed posture, clonus and wet dog shakes. Pre-treatment with a dopamine receptor antagonist (haloperidol, 0.25 mg/kg; i.p.) attenuated hyperactivity, teeth chattering, low posture and clonus and potentiated splayed postural effects. These results indicate that MDA can be converted into two glutathione regioisomers by human liver microsomes, but only the 5-(glutathion-S-yl)-alpha-MeDA adduct is behaviourally active in the rat.