Background The transcription factor interferon regulatory factor 5 (IRF5) is primarily expressed in human monocytes, B cells, and dendritic cells. Polymorphisms in the IRF5 gene have been significantly associated with susceptibility to the autoimmune disease systemic lupus erythematosus (SLE). In peripheral blood mononuclear cells (PBMC) from SLE patients, IRF5 expression was significantly elevated as compared to expression in healthy donor PBMC. The specific immune cell subset that IRF5 is (dys)functioning in to contribute to SLE pathogenesis is not known. SLE patients are characterised by increased levels of auto-reactive B cells and autoantibodies, and Irf5-/- mice were previously shown to have drastically reduced numbers of antibody secreting cells. Conclusions made from these mice, however, were compromised by the discovery of a secondary mutation in DOCK2. Important, multiple murine models of lupus show Irf5-/- mice are protected from pathogenic autoantibodies. To determine if IRF5 has similar function(s) in human primary B cells, we sought to characterise the role of IRF5 in plasmablast differentiation, antibody secretion, and B cell activation.
Materials and methods Knockdown of IRF5 was performed in primary human B cells using an Amaxa 4 D nucleofector, followed by in vitro culture to assay differentiation of naïve B cells to plasmablasts. B cell receptor cross-linking antibody and the TLR9 ligand CpG-B were used to activate B cells. Identification of IRF5 transcriptional targets was done through chromatin immunoprecipitation combined with next generation sequencing. B cell proliferation was assessed through reactive dye dilution.
Results Upon IRF5 knockdown in primary human B cells, a marked decrease in plasmablast differentiation was seen (Figure 1). Secreted levels of IgG1, IgG2, and IgG3 were severely decreased. Interestingly, upstream B cell activation pathways were also found to be decreased in B cells with the IRF5 knockdown. B cell proliferation was also found to be consistently decreased following IRF5 knockdown. ChIP-seq experiments identified IRF5 enrichment on the promoter regions of several plasmablast-associated genes as well as several genes known to impact B cell proliferation.
Conclusions These findings demonstrate for the first time that IRF5 is a critical regulator of human plasmablast differentiation. Furthermore, our work suggests that IRF5 knockdown impacts early stage B cell activation and proliferation. IRF5 ChIP-Seq results indicate that upon activation, IRF5 acts as a transcriptional regulator for a large group of genes that regulate both plasmablast differentiation and B cell function. Given that plasmablasts are over-represented in SLE, the therapeutic potential of IRF5 inhibition is supported.
Acknowledgements This work was supported by the Alliance for Lupus Research and NIAMS grant AR065959 to B.J.B. S.D. was supported in part by a predoctoral fellowship from the New Jersery Commission on Cancer Research.
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