GSE116780
RNA sequencing of CRISPR/Cas9-generated A20 DUB-inative and A20 knock-out U937 cells to elucidate the role of human A20 DUB-domain
Objectives: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-?B but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. Methods: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A knock-in cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was adressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. Results: Genetic disruption of A20 DUB domain in human and murine myeoloid cells did not give rise to enhanced NF-?B signaling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. Conclusions: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation. Overall design: We have performed RNA sequencing on U937 cells with different genotypes generated by CRISPR-Cas9. Three U937 clones with homozygote A20 C103A knock-in (KI) mutation (A20 DUB-inactive) were generated (KI1-KI3), as well as four A20 knock-out clones.  Control cells (wt) were grown in parallel and underwent the same processes as the targeted cells but in absence of CRISPR/Cas9 plasmids. Cells where differentiated to macrophages and stimulated with LPS or vehicle and after 6 hours, RNA was extracted and RNA-sequencing was performed with the aim of analyzing differentially expressed genes between the genotypes.