Francesca Gugliesi

Up-regulation of the interferon-inducible IFI16 gene by oxidative stress triggers p53 transcriptional activity in endothelial cells

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), induces injury of endothelium in a variety of pathophysiological conditions, such as inflammation, aging, and cancer. In our study, we characterized the signaling pathway linking oxidative stress induced by sublethal concentrations of H2O2 to p53 in primary human endothelial cells through the interferon (IFN)‐inducible gene IFI16. Induction of IFI16 by H2O2 was concentration‐ and time‐dependent (maximum at 50 μM, 6 h after treatment) and down‐regulated by pretreatment with N‐acetyl‐L‐cysteine, which acts as an antioxidant. This pathway is a general response to ROS and not specific to H2O2 treatment, as two other ROS‐generating compounds, i.e., S‐nitroso‐N‐acetylpenicillamine and tert‐butyl hydroperoxide, were equally capable to induce IFI16. Moreover, IFI16 up‐regulation is a result of protein accumulation, as expression of corresponding mRNA, assessed by real‐time polymerase chain reaction, was not affected. To investigate the mechanism of IFI16 accumulation, cells were incubated for 6 h in the presence of H2O2 or IFN‐β, and then cycloheximide was added to inhibit further protein synthesis. The half‐life of IFI16 protein was found to be significantly increased in H2O2‐treated cells compared with IFN‐β‐treated cells (t1/2=120 min vs. >30 min in H2O2‐ vs. IFN‐β‐treated cells, respectively). An increase of IFI16 was accompanied by interaction with p53 phosphorylated at its N terminus, as shown by immunoprecipitation experiments. Moreover, binding to IFI16 resulted in its transcriptional activation as shown by an increase in the activity of a reporter gene driven by p53‐responsive sequences derived from the p21WAF1 promoter, along with an increase in the p21 mRNA and protein levels. Altogether, these results demonstrate a novel role of IFI16 in the signal transduction pathway that leads to p53 activation by oxidative stress in endothelial cells.

A Novel Role of the Interferon-inducible Protein IFI16 as Inducer of Proinflammatory Molecules in Endothelial Cells

The human IFI16 gene is an interferon-inducible gene implicated in the regulation of endothelial cell proliferation and tube morphogenesis. Immunohistochemical analysis has demonstrated that this gene is highly expressed in endothelial cells in addition to hematopoietic tissues. In this study, gene array analysis of human umbilical vein endothelial cells overexpressing IFI16 revealed an increased expression of genes involved in immunomodulation, cell growth, and apoptosis. Consistent with these observations, IFI16 triggered expression of adhesion molecules such as ICAM-1 and E-selectin or chemokines such as interleukin-8 or MCP-1. Treatment of cells with short hairpin RNA targeting IFI16 significantly inhibited ICAM-1 induction by interferon (IFN)-γ demonstrating that IFI16 is required for proinflammatory gene stimulation. Moreover, functional analysis of the ICAM-1 promoter by deletion- or site-specific mutation demonstrated that NF-κB is the main mediator of IFI16-driven gene induction. NF-κB activation appears to be triggered by IFI16 through a novel mechanism involving suppression of IκBα mRNA and protein expression. Support for this finding comes from the observation that IFI16 targeting with specific short hairpin RNA down-regulates NF-κB binding activity to its cognate DNA and inhibits ICAM-1 expression induced by IFN-γ. Using transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrate indeed that activation of the NF-κB response is mediated by IFI16-induced block of Sp1-like factor recruitment to the promoter of the IκBα gene, encoding the main NF-κB inhibitor. Activation of NF-κB accompanied by induction of proinflammatory molecules was also observed when IκBα expression was down-regulated by specific small interfering RNA, resulting in an outcome similar to that observed with IFI16 overexpression. Taken together, these data implicate IFI16 as a novel regulator of endothelial proinflammatory activity and provide new insights into the physiological functions of the IFN-inducible gene IFI16.

The interferon-inducible HIN-200 gene family in apoptosis and inflammation: Implication for autoimmunity

The Ifi-200/HIN-200 gene family encodes highly homologous human (IFI16, myeloid cell nuclear differentiation antigen, absent in melanoma 2, and IFIX) and murine proteins (Ifi202a, Ifi202b, Ifi203, Ifi204, Ifi205, and Ifi206), which are induced by type I and II interferons (IFN). These proteins have been described as regulators of cell proliferation and differentiation and, more recently, several reports have suggested their involvement in both apoptotic and inflammatory processes. The relevance of HIN-200 proteins in human disease is beginning to be clarified, and emerging experimental data indicate their role in autoimmunity. Autoimmune disorders are sustained by perpetual activation of inflammatory process and a link between autoimmunity and apoptosis has been clearly established. Moreover, the interferon system is now considered as a key player in autoimmune disorders such as systemic lupus erythemathosus, systemic sclerosis, and Sjögrens syndrome, and it is therefore conceivable to hypothesize that HIN-200 may be among the pivotal mediators of IFN activity in autoimmune disease. In particular, the participation of HIN-200 proteins in apoptosis and inflammation could support their potential role in autoimmunity.

Innate Nuclear Sensor IFI16 Translocates into the Cytoplasm during the Early Stage of In Vitro Human Cytomegalovirus Infection and Is Entrapped in the Egressing Virions during the Late Stage

ABSTRACT Intrinsic immune mechanisms mediated by constitutively expressed proteins termed “restriction factors” provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.

The proapoptotic activity of the Interferon-inducible gene IFI16 provides new insights into its etiopathogenetic role in autoimmunity

Several lines of evidence link Interferons (IFNs) with autoimmune disorders. Autoantibodies against the Interferon-inducible IFI16 protein, a member of the HIN-200 family constitutively expressed in endothelial cells and keratinocytes, have been identified in patients affected by autoimmune diseases including Systemic Lupus Erythematosus (SLE), Sjogren Syndrome (SjS), and Scleroderma (SSc). These findings point to a role for IFI16 in the etiopathogenesis of autoimmune diseases, but the exact mechanisms involved in the development of autoimmunity remain obscure. In this study, we report for the first time that endothelial cells overexpressing IFI16 undergo apoptosis via the activation of caspase 2 and caspase 3, and that a positive feedback loop appears to link these two caspases. The relevance of IFI16-mediated apoptosis is highlighted by the observation that IFI16 knock down by RNA interference in endothelial cells inhibits the activation of both caspase 2 and caspase 3 by IFN-beta priming and synthetic double-stranded RNA treatment. Expression of a dominant-negative mutant of IKK2 kinase or treatment with AS602868, an inhibitor of IKK2 activity, results in a strong reduction of NF-kB activation along with absence of caspase 2 and caspase 3 activation and apoptosis induction. Collectively, our findings provide new insights into the role of IFI16 in the pathogenesis of autoimmune diseases by demonstrating that in addition to the stimulation of pro-inflammatory molecules, IFI16 also leads to apoptosis in endothelial cells.

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

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.

Role of the interferon-inducible IFI16 gene in the induction of ICAM-1 by TNF-α

The Interferon-inducible gene IFI16, a member of the HIN200 family, is activated by oxidative stress and cell density, in addition to Interferons, and it is implicated in the regulation of endothelial cell proliferation and vessel formation in vitro. We have previously shown that IFI16 is required for proinflammatory gene stimulation by IFN-gamma through the NF-kappaB complex. To examine whether IFI16 induction might be extended to other proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, we used the strategy of the RNA interference to knock down IFI16 expression, and analyze the capability of TNF-alpha to stimulate intercellular adhesion molecule-1 (ICAM-1 or CD54) expression in the absence of functional IFI16. Our studies demonstrate that IFI16 mediates ICAM-1 stimulation by TNF-alpha through the NF-kappaB pathway, thus reinforcing the role of the IFI16 molecule in the inflammation process.

The epithelial-mesenchymal transition induced by keratinocyte growth conditions is overcome by E6 and E7 from HPV16, but not HPV8 and HPV38: Characterization of global transcription profiles

The aim of this study was to evaluate the growth properties of primary human keratinocytes expressing E6 and E7 proteins, which are from either the beta- or alpha-genotypes, under different culture conditions. We demonstrated that keratinocytes expressing E6 and E7, from both HPV8 and 38, irreversibly underwent the epithelial-mesenchymal transition (EMT) when grown on plastic with FAD medium (F12/DMEM/5%FBS). Expression of E6/E7 from HPV16 was capable of fully overcoming the FAD-induced EMT. Immortalization was only observed in HPV16-transduced cell lines, while the more proliferating phenotype of both KerHPV8 and 38 was mainly related to FAD-induced EMT. Microarray analysis of exponentially growing cells identified 146 cellular genes that were differentially regulated in HPV16 compared to HPV8- and 38-transduced cells. A large accumulation of transcripts associated with epidermal development and differentiation was observed in HPV16-transduced cells, whereas transcripts of genes involved in the extracellular matrix, multicellular organismal processes, and inflammatory response were affected in HPV8 and 38-transduced cells.

Tumor-Derived Endothelial Cells Evade Apoptotic Activity of the Interferon-Inducible IFI16 Gene

The human interferon (IFN)-inducible IFI16 protein is a member of the 200-amino acid repeat family encoded by the HIN-200 genes. Forced IFI16 expression in normal human endothelial cells (ECs) inhibits cell growth and tube morphogenesis of ECs through the triggering of apoptosis by caspase-2 and caspase-3 via nuclear factor-κB (NF-κB) complex activation. Accumulating evidence suggests that tumor-derived ECs (TECs) possess a distinct and unique phenotype compared with normal ECs, and they may be able to acquire resistance to antiangiogenic agents such as IFNs. However, few functional studies are available on cultured TEC. In the present study, we have demonstrated that TEC obtained from tumors of various histological origin, namely kidney (Eck25), breast (B-TEC), and head and neck (HN4), continued to proliferate and generate microtubules on Matrigel following IFI16 overexpression. In contrast to normal ECs, they were resistant to apoptosis triggered by caspase-2 and caspase-3 activation via the NF-κB complex. At the molecular level, when overexpressed in TEC, IFI16 appeared unable to regulate NF-κB activity and lead to caspase activation. Altogether, these results indicate that TECs display abnormal responses, in terms of survival and angiogenic properties, to an antiproliferative and antiangiogenic IFN-inducible gene such as IFI16.

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activity

Interleukin-1β (IL-1β) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1β into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1β production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1β compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1β. Furthermore, we show that enhanced IL-1β production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-κB. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1β activation pathway where caspase-8 promotes IL-1β maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.