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

et al. (2013) Nuclear DNA Sensor IFI16 as Circulating Protein in Autoimmune Diseases Is a Signal of Damage that Impairs Endothelial Cells through High-Affinity Membrane Binding. PLoS ONE 8(5): e63045. doi:10.1371/journal.pone.0063045 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 0 Mandar Bawadekar 0 Marco De Andrea 0 Valentina Dell'Oste 0 Valeria Caneparo 0 Angela Tincani 0 Marisa Gariglio 0 Santo Landolfo 0 Michael P. Bachmann, Carl-Gustav Carus Technical University-Dresden, Germany 0 1 Department of Public Health and Pediatric Sciences, University of Turin, Medical School , Turin , Italy , 2 Department of Translational Medicine, University of Piemonte Orientale ''Amedeo Avogadro'', Medical School , Novara , Italy , 3 Interdisciplinary Research Center of Autoimmune Diseases, Department of Translational Medicine, University of Piemonte Orientale ''Amedeo Avogadro'', Medical School , Novara , Italy , 4 Rheumatology and Clinical Immunology, Spedali Civili and University of Brescia , Brescia , Italy 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. - Funding: This study was supported by MIUR PRIN 2008 (Ministero dellIstruzione, dellUniversita` e della Ricerca - Progetti di Ricerca di Interesse Nazionaleto) to SL, MG and AT, and research funding from the University of Turin 2011 to SL. VC acknowledges a grant for the Lagrange Project-CRT Foundation. MB is a recipient of an international PhD fellowship in Innovative Biomedical Technologies (IBT) funded by Cariplo Foundation-Milan, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. A wealth of data now exists demonstrating the critical role of interferons (IFNs) in the pathogenesis and perpetuation of autoimmunity [15]. Genomic studies have revealed that type I IFN inducible genes are markedly overexpressed in the peripheral blood of patients with systemic autoimmune diseases including Systemic Lupus Erythematosus (SLE), Systemic Sclerosis (SSc), and Sjogrens Syndrome (SjS) [68]. 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 [1012]. One family of IFN-inducible genes is the HIN200/Ifi200 gene family, which encodes evolutionary related human (IFI16, IFIX, MNDA, and AIM2) and murine (Ifi202a, Ifi202b, Ifi203, Ifi204, Ifi205/D3, and Ifi206) proteins. The common domain architecture of this protein family consists of one or two copies of the HIN domain (a 200 amino acid repeat) and an N-terminal PYD domain, also named PAAD, DAPIN, or Pyrin. 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]. The IFI16 protein is specifically expressed in vascular endothelial cells, keratinocytes, and hematopoietic cells [15] and has been recently shown to act as a foreign DNA sensor [1619]. We have previously demonstrated that oxidative stress and various proinflammatory cytokines can also trigger IFI16 nuclear expression [20] and [21]. In addition, a role of IFI16 as an inducer of proinflammatory molecules (e.g. ICAM-1, RANTES, and CCL20) and apoptosis in endothelial cells has also been observed, supporting its role in the initial steps of the inflammatory processes that precede the onset of autoimmune syndromes [2224]. IFI16 protein is also a target for autoantibodies. Anti-IFI16 autoantibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases including SLE, SSc, and SjS. [25 28]. To explain this observation, we hypothesized that its overexpression and extranuclear appearance during cell death contribute to its release into the extracellular milieu and eventually to the induction of specific autoantibodies. Consistent with this hypothesis, we have recently demonstrated in vitro that the IFI16 protein, normally detected in the nucleus of human keratinocytes, can be induced to appear in the cytoplasm under conditions of UV light-induced cell injury and then released in the culture media. A similar situation was also found in tissue sections of skin biopsies from patients with SLE. In this model, IFI16 expression was upregulated and mislocalized to the cytoplasm, suggesting that aberrant expression of IFI16 in epithelial and inflammatory cells can also play a role in triggering an autoimmune response in vivo [29]. However, since IFI16 wa (...truncated)