Post-Doctoral Fellow Boston Children's Hospital Boston, Massachusetts, United States
Introduction/Rationale: Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disease driven by a complex interplay of genetic and environmental factors. Emerging evidence highlights the contribution of alternative splicing and isoform dysregulation to SLE pathogenesis, although the underlying mechanisms require further elucidation.
Methods: We utilised a publicly available RNA-seq dataset to examine isoform usage in B cells from patients with SLE. Our analysis focused on five distinct B cell subsets to identify isoform alterations. We further investigated the role of Interferon Regulatory Factor 5 (IRF5) in double-negative 2 (DN2) B cells through CRISPR-Cas9 knockout and pharmacological inhibition experiments in primary B cells. Additionally, we integrated genome-wide association study fine-mapping, co-localisation analyses and CRISPR base-editing in the GM12878 cell line to identify a putative causal variant in the IRF5 locus.
Results: Our analysis identified extensive isoform alterations, notably within DN B cells, which displayed significant changes across hundreds of genes. IRF5, a known SLE risk locus, exhibited significant isoform dysregulation. By knocking out IRF5, we observed a substantial reduction in DN2 B cells, plasmablasts, and MKI67⁺ proliferating cells, accompanied by decreased production of IL-6 and IL-12. Furthermore, we identified a putative causal variant near the IRF5 transcription start site. Conversion of the non-risk allele to the risk allele induced significant upregulation of the E1B isoform. These findings were reproducible in primary B cells homozygous for the risk haplotype, which also displayed enhanced expansion of DN2-differentiating B cells on day 6 in vitro.
Conclusion: Our findings underscore a critical role for IRF5 in the differentiation of DN2 B cells and demonstrate how a genetic risk variant associated with lupus leads to isoform-specific dysregulation in B cells. Together, these provide new mechanistic insights into the molecular pathways involved in SLE pathogenesis.
