Investigating the Ability of Anti-Dense Fine Speckled 70 (DFS70) Autoantibodies to Inhibit the Formation of Neutrophil Extracellular Traps

Abstract

Introduction: Autoantibodies to dense fine speckled 70 (DFS70) are absent or detected at low titers in patients with systemic autoimmune rheumatic diseases. Multivariate regression analysis showed serum DFS70 autoantibodies were differentially higher in survivors and predicted 60-day mortality. Given that DFS70 is a chromatin-associated nuclear antigen, we sought to investigate its direct impact on NETosis—a process that contributes to both severe infection outcomes and the development of autoimmunity.

Methodology: Anti-DFS70 autoantibodies in sera (healthy donors) were quantified using a line-immunoassay (Human Diagnostics, Germany) and isolated by magnetic co-immunoprecipitation with recombinant protein. An in vitro NETosis model was developed to assess the ability of various stimuli (immunoglobulins, immune complexes, calcium ionophore, and PMA) to induce NETs in neutrophils from healthy donors. Platelet-rich plasma was added to evaluate platelet activation, and NET formation with platelet aggregation to the NET scaffold was imaged via scanning electron microscopy and quantified using MATLAB software. Resting neutrophils and NETs were immunostained to colocalize DFS70 antigen with histones, DNA, and cytosolic proteins using confocal microscopy. Western blot quantified DFS70 antigen in neutrophils using the Abby Automated Western system. Neutrophils were stained for Annexin-V and 7AAD to evaluate cell viability.

Results: Serum isolated anti-DFS70 IgG significantly attenuated autoantibody-mediated NETosis and platelet aggregation in vitro (p<0.01). Serum-isolated DFS70 antibodies demonstrated this effect in a dose-dependent manner. Anti-DFS70 IgG inhibits NETosis downstream in the NET formation cascade, as it also attenuates NETosis induced by non-FcR triggers such as PMA and calcium ionophore. Culture supernatants in conditions with DFS70 autoantibodies demonstrate a decrease in beta-thrombomodulin, indicating reduced platelet activation. However, anti-DFS70 IgG does not reduce platelet activation independent of NETs. Immunostaining of neutrophils with a commercial anti-DFS70 antibody demonstrates nuclear localization of the DFS70 antigen in resting neutrophils and cytoplasmic relocation post-stimulation of NETs. Anti-DFS70 IgG and immunoglobulin-stimulated neutrophils show diffused nuclei instead of the characteristic multi-lobes. Although NETs are not formed, these neutrophils stain with 7AAD and Annexin-V, indicating cell death.

Conclusions: Our data suggest endogenous DFS70 autoantibodies limit NETosis and subsequent platelet aggregation and activation. The autoantibody-mediated inhibition of NETosis is associated with a non-lytic cell death. These findings may have implications in the management or treatment of infectious diseases, sepsis and other conditions where uncontrolled NETosis exacerbates pathology. However, further investigations are required to fully elucidate the mechanisms involved and the potential clinical relevance of these findings.