Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus.

Abstract

Genetic variations in <i>TNFAIP3</i> (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB 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. CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating <i>C103A</i> knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and <i>C103A</i> KI cells. Functional studies were performed in A20 <i>C103A</i> U937 cells and in immune cells from A20 <i>C103A</i> mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 <i>C103A</i> mice and SLE-patients with rs2230926. Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with <i>C103A</i> mutation or rs2230926 polymorphism presented an upregulated expression of <i>PADI4</i>, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 <i>C103A</i> 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. We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of <i>PADI4</i> with resultant protein citrullination and extracellular trap formation.