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.

Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane Binding

GUGLIESI, Francesca;DE ANDREA, Marco;DELL'OSTE, Valentina;LANDOLFO, Santo Giuseppe
2013

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.
8
5
1
11
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0063045
Francesca Gugliesi ; Mandar Bawadekar ; Marco De Andrea ; Valentina Dell’Oste ; Valeria Caneparo; Angela Tincani; Marisa Gariglio; Santo Landolfo
File in questo prodotto:
File Dimensione Formato  
Gugliesi et al 2013.pdf

Accesso aperto

Tipo di file: PDF EDITORIALE
Dimensione 1.3 MB
Formato Adobe PDF
1.3 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/134106
Citazioni
  • ???jsp.display-item.citation.pmc??? 16
  • Scopus 31
  • ???jsp.display-item.citation.isi??? 32
social impact