Background: Due to the disparity between genetic predisposition and rheumatoid arthritis (RA) incidence, the human microbiome is one factor hypothesized to contribute to RA pathogenesis. In addition, putative interactions occurring between microbiota and host cell receptors [a family of G protein-coupled receptors, protease-activated receptors (PARs)] may also be associated with RA. We will explore these factors through analyses of both a healthy cohort and one with established RA, for differences in oral and gastrointestinal microbial composition and inflammatory response.
As this is a new project, the initial aims were to (1) investigate different DNA extraction kits, and establish protocols for microbiome analyses, (2) gain an overall understanding of gut and oral microbial composition and (3) identify differences in PAR activity and MMP activity, between cohorts.
Methods: Agarose gel electrophoresis and qPCR were employed to determine quality and copy number of DNA extracted from human faeces. ARISA and qPCR are currently being employed for initial microbiome analyses. PARs and matrix metalloproteinases (MMPs) activity will be analysed using RT-qPCR and gelatin zymography, respectively, from plasma and PBMC samples.
Results: Of the DNA extraction kits tested, the PSP Spin Stool DNA Kit resulted in the highest DNA concentration, quality and 16s rRNA copy number. Microbial community composition, MMP upregulation and PAR analysis is currently underway with results forthcoming.
Conclusions: Investigation of the human microbiome and host interactions is necessary to develop our understanding of RA and other inflammatory arthritides. This research has the potential to identify novel biomarkers for disease development and progression.