Background/Purpose:  Rheumatoid Arthritis (RA) is a chronic, inflammatory condition that affects more than 1% population globally. Several studies suggest RA to be controlled by a combination of different genetic & environmental factors. While non-genetic factors remains elusive, a large number of genes/loci have been identified to confer susceptibility to RA. In the first ever GWAS on RA in north Indian populations, we had identified a novel gene ARL15 (5p15.2) in addition to the already established HLA locus. ARL 15 is a small G Protein belonging to ADP ribosyl family (ARF). The inhibition of this protein family in the Collagen induced Arthritis model of RA has been shown to improve the arthritis score and inflammation. But importantly, expression of either ARL15 or any other ARF genes in synovial fibroblast or in blood samples of RA patients has never been reported. Based on this background, the present study was aimed at functional characterization of ARL15, the novel GWAS hit to understand its implications, if any, for RA biology.

Methods:  Synovial fluid and tissue samples were collected from each of the RA patients undergoing total knee replacements at AIIMS (As per American College of rheumatology criteria 2010), New Delhi with institutional ethical committee clearance and informed consent. Homogeneity of cells was checked by Fluorescence-Activated Cell Sorting. Presence of ARL15 gene and protein in RASF cells were tested by RT-PCR and western blots. TNF induction and siRNA knock down was carried out to find the expression of genes of interest by RT-PCR. Global expression profiling in knock down and control cells was captured by transcriptome sequencing. Apoptosis of ARL15 knock down cells were detected by FACS using Annexin V staining. A549 cancer monolayer cells were used for checking the expression of ARL15under hypoxic condition.

Results:  FACS experiment confirmed the cultures of synovial fibroblasts. RT-PCR showed the presence of ARF2, ARF6 and ARL15. Western blot confirmed the presence of ARL15 protein in cell lysates. SiRNA mediated knock down experiments showed differential expression of GAPDH, Adiponectin, Adiponectin receptorI and IL6 genes in RASF. RNAseq validated the above findings and also revealed differential expression of several additional genes involved in IL6 signaling, RNAseq also indicated the possible role of ARL15 in hypoxia which was then confirmed using A549 cancer cell line. Annexin V staining of ARL15knock down cells showed increase in apoptosis of RASF cells compared to control.

Conclusion:  Our results confirm for the first time the expression of ARF genes including ARL15 in the synovial fluid and tissue derived synovial fibroblast cultures of RA patients and its connection with IL6. Transcriptome sequencing data validated these findings in addition to identifying several other differentially expressed genes related to hypoxia and apoptosis. These preliminary findings taken together with previously available literature indicate the use of ARL15 as potential therapeutic target for RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/arl15-expressed-by-rheumatoid-arthritis-synovial-fibroblasts-regulates-il6-and-plays-a-role-in-apoptosis-and-hypoxia/