C5orf30 a Novel Regulator of Inflammation and Tissue Damage in Rheumatoid Arthritis

Munitta Muthana¹, Sarah Hawtree¹, Holly Davies², Hannah Roberts¹, Sachin Khetan¹, Mohammed Akil³, Fiona Wright¹, Barbara Ciani⁴, Ursula Fearon⁵, DJ Veale⁶ and Anthony G. Wilson⁷, ¹Infection and Immunity, University of Sheffield, Sheffield, United Kingdom, ²University of Sheffield, Sheffield, United Kingdom, ³Rheumatology Department, Sheffield South Yorkshire, United Kingdom, ⁴Chemistry, University of Sheffield, Sheffield, United Kingdom, ⁵Dublin Academic Medical Centre, Translational Rheumatology Research Group, Dublin, Ireland, ⁶Consultant Rheumatologist, St. Vincent’s University Hospital, Dublin, Ireland, ⁷Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin, Ireland

Meeting: 2014 ACR/ARHP Annual Meeting

Keywords: Animal models, arthritis and fibroblasts, Gene Expression

Session Information

Title: Cytokines, Mediators, Cell-cell Adhesion, Cell Trafficking and Angiogenesis I

Session Type: Abstract Submissions (ACR)

Background/Purpose

A recent genome wide association study identified the variant rs26232 in the first intron of the uncharacterized gene, C5orf30, as a rheumatoid arthritis (RA) susceptibility variant¹. In addition, it has been associated with severity of radiological joint damage suggesting a role in tissue breakdown². To date there is no function assigned for C5orf30 and neither the gene or protein show homology to any known functional sequences. However, C5orf30 is highly conserved in chimpanzee, dog, cow, mouse, chicken, and zebrafish (orthologs).

Purpose – To determine the biological roles of C5orf30 in rheumatoid arthritis.

Methods

Immunohistochemistry on synovial samples was used to determine expression of C5orf30 using anti-C5orf30 and antibodies to macrophages (CD68), fibroblasts (5B5), T (CD3) & B (CD19) cells. Real time PCR and western blotting were used to examine C5orf30 transcript and protein levels in RA PBMCs and fibroblast-like synovial cells (RASF) treated with TNF & hypoxia. To investigate gene function siRNA was used to knockdown (KD) either C5orf30 or a non-targeting control (NTC) in RASF in vitro. After knockdown cell migration, invasion, and global gene expression (Illumina BeadChip array) were assessed. The disease-modulating activity of siRNA designed to silence C5orf30 was also investigated in vivoin a mouse model of collagen-induced arthritis (CIA). Clinical scores of CIA were measured daily (days 0-49) and the bones were analysed using a microCT scanner.

Results

Confocal microscopy revealed C5orf30 to be strongly expressed in the cytoplasmic compartment of RA synovial lining cells including macrophages and RASF but not T & B cells. C5orf30 was undetectable in arthroscopy sections obtained from osteoarthritis (OA) or OASFs. C5orf30 was found to be up-regulated by hypoxia (8-fold) and down-regulated by TNF treatment (0.5-fold) in RASF. We found that C5orf30^KD increased the invasiveness of FLS assayed using Matrigel (N=6 p=0.01) and increased FLS migration in a scratch-wound assay (N=6 p=0.02). Gene profiling studies also revealed C5orf30^KD resulted in upregulation of cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Finally, in mice with CIA, siRNA designed to inhibit C5orf30 strongly induced joint inflammation (n=10, p=0.0001) and bone erosion compared to irrelevant NTC siRNA.

Conclusion

Our data, in both RA and murine inflammatory arthritis, reveals C5orf30 to be a novel negative regulator of tissue breakdown modulating the autoaggressive phenotype that is characteristic of rheumatoid synovial fibroblasts.

1. Stahl EA, Raychaudhuri S, Remmers EF, et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet 2010;42:508-14.

2. Teare MD, Knevel R, Morgan MD, et al. Allele-Dose Association of the C5orf30 rs26232 Variant With Joint Damage in Rheumatoid Arthritis. Arthritis Rheum. 2013;65:2555-61.

Disclosure:

M. Muthana,
None;

S. Hawtree,
None;

H. Davies,
None;

H. Roberts,
None;

S. Khetan,
None;

M. Akil,
None;

F. Wright,
None;

B. Ciani,
None;

U. Fearon,
None;

D. Veale,
None;

A. G. Wilson,
None.

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