Whittle, E. | bioRxiv Science

bioRxiv2018

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of adults with poorly controlled asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 17A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFa, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, assessment of the circulating \"blood microbiome\" was performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in the plasma in a condition-specific manner, and many more were found to be expressed at altered levels. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthma state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

molecular biology↗

bioRxiv2018

Multi-Method Characterisation of the Human Circulating Microbiome

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonisation of various body niches (e.g. the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other \"classically sterile\" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a \"blood microbiome\" by providing a comprehensive description of bacterially-derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilised a set of plasma samples from healthy subjects (n = 5) and asthmatic subjects (n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA / RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

molecular biology↗