Nuclear DNA Sensor IFI16 as Circulating Protein in Autoimmune Diseases Is a Signal of Damage that Impairs Endothelial Cells through High-Affinity Membrane Binding

Abstract

IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki) of 14.43 nM and half maximal inhibitory concentration (IC50) of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage, suggesting a new pathogenic and alarmin function through which this protein triggers the development of autoimmunity.

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…IFI16 is a target for autoantibodies and several studies have shown the presence of anti-IFI16 autoantibodies in chronic autoimmune diseases such as SLE [64], systemic sclerosis [65], rheumatoid arthritis [66] and Sjö gren's syndrome [67]. IFI16 is known to relocalise from the nucleus to the cytosol following UV treatment or viral DNA detection.…”

“…It is hypothesised that IFI16 can also be released into the extracellular milieu. This extracellular IFI16 can in turn bind to the plasma membrane of endothelial cells, and down regulate both their migratory activities and tube morphogenesis [66]. In this context IFI16 may be acting as an alarmin, propagating the stress signal and causing further damage.…”

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

“…In addition to its role in defense against foreign DNA, the aberrant activity of IFI16 is also associated with several autoimmune disorders, such as systemic lupus erythematosus and Sjögren syndrome. IFI16 is involved in the onset and progression of these diseases not only by generating abnormally high levels of the proinflammatory cytokines but also by being recognized as an autoantigen by the host adaptive immune system (4)(5)(6)(7)(8).…”

Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

“…Nuclear IFI16 is induced in the cytoplasm of stratified squamous epithelia in response to UVB exposure and acts as a mechanism of auto-antigen processing in SLE [19] . Meanwhile, endogenous IFI16 released by apoptotic cells acts as a novel alarmin, binding to neighbor cells and propagating the damaged-signal [32] . In addition, nuclear IFI16 is relocalized to the cytoplasm leading to proteasomal degradation by infection with HSV-1 [33] .…”