Background Antibodies to double-stranded DNA are prevalent and pathogenic in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis. However, the physical nature and regulation of cell-free DNA (cfDNA) that becomes antigenic in SLE is poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity, whereas a hypomorphic variant of DNASE1L3 is associated with SLE and other autoimmune diseases. We sought to characterize the role of DNASE1L3 in the regulation of cfDNA and its relevance for sporadic forms of SLE that are not associated with DNASE1L3 variants or mutations.

Methods Activity of DNASE1L3 in the plasma of SLE patients was measured using a novel assay based on the digestion of intact cell nuclei. Antibodies to DNASE1L3 were measured using a novel ELISA with recombinant DNASE1L3 as an antigen. The fraction of microparticle-associated cell-free DNA was measured using differential centrifugation followed by genomic qPCR. Antibodies to DNASE1L3-sensitive antigens on microparticles were measured by flow cytometry using tissue culture-derived microparticles digested with recombinant DNASE1L3.

Results More than 50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation. This reduced activity was associated with the presence of neutralizing autoantibodies to DNASE1L3, but not to its close homolog DNASE1. Patients with reduced DNASE1L3 activity had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles released from apoptotic cells. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments and were stronger inducers of type I interferon. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens on microparticles included DNA-associated proteins such as HMGB1, a known self-antigen in SLE

Conclusions Our results suggest that autoantibody-mediated impairment of DNASE1L3 activity is a common non-genetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE. In particular, it leads to the accumulation of longer polynucleosomal DNA fragments in circulating microparticles, and to autoantibody responses to microparticle-associated DNA and protein antigens.

Acknowledgments Supported by the NIH grants AR071703, AR070591 and AR069515, the Lupus Research Alliance, the Colton Center for Autoimmunity and the German Research Foundation (DFG).