Background/Purpose: rs26232 in the first intron of C5orf30 has been associated with risk of developing rheumatoid arthritis (RA) and severity of tissue damage. C5orf30 is highly expressed by RA synovial fibroblasts (RASF) and macrophages. Inhibition of C5orf30 in RASF results in increased cellular invasiveness and migration in vitro and inhibition in the collagen-induced arthritis model accentuated joint inflammation and damage. There is no published data on the biological activities of C5orf30 in macrophages.

Methods: Monocyte cell line (THP1) and primary monocyte-derived macrophages (MDM) were used. C5orf30 mRNA was assessed by qPCR and protein via Western blot. Transcript and protein half-lives were assessed using actinomycin D and cyclohexamide. Polarization to M1 and M2a phenotypes and stimulation with TNF and LPS on C5orf30 expression were compared. C5orf30 levels were manipulated using siRNA and functional effects on macrophage biology was assessed using ELISAs, invasion assays, pathogen phagocytosis assays, reactive oxygen species assays, gene expression and intracellular signalling assays. In vivo, antisense morpholino oligonucleotides were used to knockdown C5orf30 in zebrafish. Confocal imaging was used to assess the number of invading macrophages.

Results: C5orf30 has a half-life of 3.13 hours, which does not significantly change with the addition of inflammatory (LPS +IFNγ) or anti-inflammatory (IL-4) stimuli. Protein half-life is 19.54 hours, rising to 22.05 hours when pretreated with LPS+ IFNγ (M1-like) and decreasing to 15.07 hours when pretreated with IL-4 (M2a-like). Polarization to M2a increased C5orf30 protein expression whereas polarization to M1 resulted in phosphorylation of C5orf30 protein and decreased expression of C5orf30 (p=0.01). Treatment with TNF or LPS reduced C5orf30 expression (TNF p=0.001, LPS p=0.02). LPS phosphorylates C5orf30. Pretreatment of cells with the JNK inhibitor SP600125 retarded the phosphorylation of C5orf30 in response to LPS in a dose-dependent manner and prevented downregulation of C5orf30 gene expression. Knockdown of C5orf30 reduced the invasive capacity of macrophages (p=0.003) with an associated decrease in MMP1 (p=0.01), MMP3 (p=0.01) and MMP9 (p=0.03). Decrease in invasion was intensified upon incubation with either TNF (p=0.02) or LPS (p=0.01). C5orf30 knockdown increased phagocytosis when co-stimulated with LPS (p=0.01). C5orf30 knockdown also increased activation of the JNK pathway. In vivo, tail amputations in zebrafish with C5orf30 deficient embryos showed an increased macrophage infiltration at the wound site (p=0.01).

Conclusion: Stimulation with inflammatory mediators induces phosphorylation of C5orf30 and downregulates C5orf30 gene expression, whereas treatment with anti-inflammatory signals increases C5orf30 protein expression. C5orf30 knockdown enhanced the proinflammatory macrophage phenotypes of phagocytosis and JNK activation whilst diminishing the tissue-clearing (M2-like) phenotype of macrophage invasion. This data indicates an important role for C5orf30 in the immunomodulatory regulation of macrophages and is consistent with our previous findings in RASF.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-rheumatoid-arthritis-susceptibility-gene-c5orf30-is-an-immunomodulator-in-macrophages/