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

Francesca Gugliesi,Valentina Dell’Oste,Marisa Gariglio,Santo Landolfo,Angela Tincani,Mandar Bawadekar,Valeria Caneparo,Marco De Andrea,Marco De Andrea,Michael P Bachmann

doi:10.1371/journal.pone.0063045

Francesca Gugliesi, Valentina Dell’Oste + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0063045

Copy DOI

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.

Highlights

A wealth of data exists demonstrating the critical role of interferons (IFNs) in the pathogenesis and perpetuation of autoimmunity [1,2,3,4,5]
With the cut-off value set to the 95th percentile of the control population (27 ng/ml), mean IFI16 levels were significantly increased in patients with Systemic Sclerosis (SSc), Systemic Lupus Erythematosus (SLE), RA, and Sjogren’s Syndrome (SjS) compared to the control group (4.7 ng/ml) (SSc: 25.4 ng/ml, p,0.001; SLE: 23.5 ng/ml, p,0.001; RA: 222 ng/ml, p,0.001; SjS: 88.2 ng/ml, p,0.001)
We demonstrate for the first time: i) the presence of significant levels of extracellular IFI16 protein in the sera of patients affected by systemic autoimmune diseases, including SSc, SjS, SLE and RA but not in non-SLE GN as compared to healthy controls, and ii) that the extracellular IFI16 exerts biological effects on endothelial cells upon binding to a specific cell surface receptor

Summary

Introduction

A wealth of data exists demonstrating the critical role of interferons (IFNs) in the pathogenesis and perpetuation of autoimmunity [1,2,3,4,5]. In SLE patients, this so-called ‘‘IFN signature’’ is generally associated with active disease states, renal, and CNS involvement [9]. Together, these findings have led to the hypothesis that type I IFNs (IFN-a and IFN-b) may be the master cytokines responsible for the initiation and progression of the autoimmune process [10,11,12]. The PYD domain, commonly found in death-family proteins, like Pyrin and ASC, is present in the N terminus of most HIN200 proteins, suggesting a role of these proteins in inflammation and apoptosis [13,14].

Methods

Results

Conclusion