Laser capture microscopy (LCM)-RNAseq for topological mapping of synovial pathology during rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining, or synovium, becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessels were captured by LCM from seven RA and seven osteoarthritis (OA) patients. RNAseq was performed on RNA extracted from captured tissue. Principal component analysis (PCA) was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold-change and q-value parameters. Pathway analysis was performed using Reactome Pathway Database on differentially expressed genes (DEGs) between disease states. Significantly enriched pathways in each synovial region were selected based on false discovery rate (FDR). RESULTS: RA and OA transcriptomes were distinguishable by PCA. Pairwise comparisons of synovial lining, sublining, and vessels between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biological function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining was marked by anomalous immune cell signaling, RA sublining by aberrant cell cycle, and RA vessels by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between RA and OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Multifaceted immune dysregulation characterizes individuals at-risk for rheumatoid arthritis.

Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.