An Investigation into C5orf30 and Immune Cell Expression
Stephanie Merrigan, Michelle Trenkmann, Emma Dorris and Gerry Wilson
School of Medicine University College Dublin
Rheumatoid arthritis (RA) is a chronic inflammatory condition affecting the joints, resulting in pain, stiffness, mobility restrictions and often systemic effects. It affects around 50,000 people in Ireland. The cause of RA is multifactorial including genetic and environmental components
We identified a variant in C5orf30 linked with both risk, and severity, of RA. Subsequently we revealed C5orf30 to encode a negative regulator of tissue damage mediated by RA synovial fibroblasts (RASFs) and have recently identified it as a regulator of the resolution of macrophage-mediated inflammation. Neutrophils constitute over 90% of cells found in the synovial fluid of RA patients, however the potential expression and biological roles of C5orf30 have not been determined this cell type. Our aim was to investigate C5orf30 expression in varies immune cells including neutrophils.
Cell populations (neutrophils, monocytes and macrophages) were isolated from healthy volunteer blood donors. Rheumatoid arthritis synovial fibroblasts (RASFs) were derived from patient biopsies. The neutrophil cell line model HL-60 was differentiated to neutrophil phenotype with all-trans retinoic acid (ATRA) or DMSO treatment. Inflammatory stimulations, 20 ng/µl TNF, 100 ng/µl LPS, and 20 ng/µl IL-4, were performed for 4 and 24 h. C5orf30 and variant expression was investigated by QRT-PCR.
C5orf30 was differentially expressed between cell types; RASFs had the highest level of total (all variant) C5orf30 expression, followed by neutrophils, macrophage, PBMCs and monocytes. C5orf30 transcript variants displayed cell-specific expression. Variant-1 expression was greatest in PBMCs, variant-2 in RASFs and neutrophils and variant-3 in PBMCs, macrophage and RASF. Variant 2 was the predominant variant expressed in neutrophils, macrophage and monocytes.
HL-60 cell differentiation resulted in augmented CD11b (neutrophil marker) and attenuated C5orf30 expression. Preliminary data shows C5orf30 expression to be regulated by anti- and pro- inflammatory stimuli TNFα, LPS and IL-4 in differentiated HL-60 cells.
C5orf30 is expressed by immune cells including neutrophils and may play a role in inflammatory responses. Future studies will investigate the requirement of C5orf30 in neutrophil invasion and phagocytosis