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

Abstract

<h3>Objectives</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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. <h3>Conclusions</h3> 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.