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Santo Landolfo - One of the best experts on this subject based on the ideXlab platform.
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Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding.
PLOS Pathogens, 2020Co-Authors: Andrea Iannucci, Valeria Caneparo, Marisa Gariglio, Santo Landolfo, Stefano Raviola, Isacco Debernardi, Donato Colangelo, Riccardo Miggiano, Gloria Griffante, Marco De AndreaAbstract:Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4. IFI16 is a nuclear protein involved in a variety of physiological processes, including cell cycle regulation, tumor suppression, and virus sensing. Emerging evidence indicates that IFI16 is released in the extracellular milieu under injury or stress conditions. Here we show that extracellular IFI16 acts as a damage-associated molecular pattern (DAMP), triggering inflammation through Toll-like receptor 4 (TLR4) activation. Furthermore, we demonstrate that IFI16 activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating lipopolysaccharide (LPS) variants, which are known to be present in various pathological settings other than gram-negative infections. Our study provides new insights into the role of extracellular IFI16 during low-grade endotoxemia.
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Distinct Anti-IFI16 and Anti-GP2 antibodies in inflammatory bowel disease and their variation with infliximab therapy
Inflammatory Bowel Diseases, 2016Co-Authors: Valeria Caneparo, Santo Landolfo, Luca Pastorelli, Laura Francesca Pisani, Barbara Bruni, Flavia Prodam, Renzo Boldorini, Dirk Roggenbuck, Maurizio Vecchi, Marisa GariglioAbstract:BACKGROUND Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the expression and function of pattern recognition receptors, including the IFI16 protein. Because this protein is a target for autoantibodies and its aberrant expression was reported in colonic mucosa from active patients with ulcerative colitis, we studied its expression and specific seroresponse in patients with IBD before and after infliximab (IFX) therapy. METHODS Anti-IFI16 antibodies (IgG and IgA subtypes) were measured in the sera of 74 patients with IBD: 48 patients with Crohn's disease (CD) and 26 patients with ulcerative colitis, prospectively harvested before and after IFX therapy. Anti-GP2 antibodies (both IgG and IgA subtypes) were also tested for comparison. The patient antibody statuses were qualitatively and quantitatively associated with disease phenotype and response to IFX therapy. RESULTS Significantly higher titers of anti-IFI16 IgG were found in both CD and ulcerative colitis patients compared with healthy controls. Anti-IFI16 IgA titers were also present in patients with CD. Anti-GP2 IgG subtype titers were significantly increased in patients with CD, as were IgA subtype titers. Significant changes in anti-IFI16 IgG subtype titers were observed after IFX in patients with CD who correlated with clinical remission or response. CONCLUSIONS Our results highlight the importance of IFI16 in IBD pathogenesis showing that its de novo overexpression in the gut epithelial cells leads to a breakdown in immune tolerance and the subsequent development of specific autoantibodies. Anti-IFI16 IgG antibodies hold the potential to serve as a biomarker of response to IFX therapy.
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Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability
Journal of Virology, 2016Co-Authors: Matteo Biolatti, Marco De Andrea, Marisa Gariglio, Valentina Dell'oste, Sara Pautasso, Jens Von Einem, Manfred Marschall, Bodo Plachter, Santo LandolfoAbstract:UNLABELLED A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.
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Circulating Interferon-Inducible Protein IFI16 Correlates With Clinical and Serological Features in Rheumatoid Arthritis.
Arthritis Care and Research, 2016Co-Authors: Alessia Alunno, Valeria Caneparo, Marisa Gariglio, Santo Landolfo, Onelia Bistoni, Sara Caterbi, Riccardo Terenzi, Elena Bartoloni, Antonio Manzo, Roberto GerliAbstract:OBJECTIVE The interferon-inducible protein 16 (IFI16) has been detected in sera from patients with autoimmune/inflammatory diseases, but not in healthy subjects. This leaking leads to loss of tolerance toward this self-protein and the development of autoantibodies. In this study, clinical significance of both IFI16 protein and anti-IFI16 antibodies in rheumatoid arthritis (RA) was investigated. METHODS IFI16 protein and anti-IFI16 antibody levels were assessed by enzyme-linked immunosorbent assay in serum samples from 154 RA patients and 182 healthy controls, and in synovial fluid (SF) samples from 21 RA patients and 25 patients with osteoarthritis (OA). RESULTS Mean serum levels for both IFI16 and anti-IFI16 antibodies were higher in RA patients than in healthy controls, with a direct correlation between IFI16 concentration and anti-IFI16 antibody titer. The majority of RA patients with detectable circulating IFI16 protein were also positive for rheumatoid factor (RF)/anti-cyclic citrullinated peptide antibody (anti-CCP). The latter group was found to be positive for anti-IFI16 antibodies as well. The mean SF concentrations of both IFI16 protein and anti-IFI16 antibodies were higher in RA patients when compared with control OA patients. Interestingly, the presence of circulating IFI16 protein, but not anti-IFI16 antibodies, significantly correlated with RA-associated pulmonary disease. This correlation was not dependent on the presence of anti-IFI16 antibodies, sex, and smoking habit. CONCLUSION Our data demonstrate that the high levels of circulating IFI16 in RA are more frequent in RF/anti-CCP-positive RA patients and significantly associated with pulmonary involvement. The relevance of circulating IFI16 protein as a new clinical biomarker of RA should be verified with additional studies.
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ID: 40: The circulating interferon-inducible protein IFI16 correlates with clinical and serological features in Rheumatoid Arthritis
Cytokine, 2015Co-Authors: Valeria Caneparo, Marisa Gariglio, Alessia Alunno, Onelia Bistoni, Sara Caterbi, Riccardo Terenzi, Elena Bartoloni, Roberto Gerli, Santo LandolfoAbstract:Objectives The IFN-inducible protein 16 (IFI16) has been detected in sera from patients with autoimmune/inflammatory diseases but not in healthy subjects. This leaking leads to loss of tolerance towards this self-protein and development of autoantibodies. In this study, the clinical significance of both IFI16 protein and anti-IFI16 antibodies (Abs) in rheumatoid arthritis (RA) was investigated. Methods IFI16 protein and anti-IFI16 Abs levels were assessed by ELISA in serum samples from 154 RA patients and 182 healthy controls (HC), and in synovial fluid (SF) samples from 21 RA patients and 25 patients with osteoarthritis (OA). Results Mean serum levels for both IFI16 and anti-IFI16 Abs were higher in RA than in HC with a direct correlation between IFI16 concentration and anti-IFI16 Abs titer. The majority of RA patients with detectable circulating IFI16 protein were also positive for RF/ACPA. The latter group was found positive for anti-IFI16 Abs as well. The mean SF concentrations of both IFI16 protein and anti-IFI16 Abs were higher in RA when compared with control OA. Interestingly, the presence of circulating IFI16 protein, but not anti-IFI16 Abs, significantly correlated with RA-associated pulmonary disease. This correlation was not dependent on the presence of anti-IFI16 Abs, gender and smoking habit. Conclusions Our data demonstrate that the high levels of circulating IFI16 in RA are more frequent in RF/ACPA-positive RA patients and significantly associated with pulmonary involvement. The relevance of circulating IFI16 protein as new clinical biomarker of RA should be verified with additional studies.
Marisa Gariglio - One of the best experts on this subject based on the ideXlab platform.
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Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding.
PLOS Pathogens, 2020Co-Authors: Andrea Iannucci, Valeria Caneparo, Marisa Gariglio, Santo Landolfo, Stefano Raviola, Isacco Debernardi, Donato Colangelo, Riccardo Miggiano, Gloria Griffante, Marco De AndreaAbstract:Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4. IFI16 is a nuclear protein involved in a variety of physiological processes, including cell cycle regulation, tumor suppression, and virus sensing. Emerging evidence indicates that IFI16 is released in the extracellular milieu under injury or stress conditions. Here we show that extracellular IFI16 acts as a damage-associated molecular pattern (DAMP), triggering inflammation through Toll-like receptor 4 (TLR4) activation. Furthermore, we demonstrate that IFI16 activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating lipopolysaccharide (LPS) variants, which are known to be present in various pathological settings other than gram-negative infections. Our study provides new insights into the role of extracellular IFI16 during low-grade endotoxemia.
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Distinct Anti-IFI16 and Anti-GP2 antibodies in inflammatory bowel disease and their variation with infliximab therapy
Inflammatory Bowel Diseases, 2016Co-Authors: Valeria Caneparo, Santo Landolfo, Luca Pastorelli, Laura Francesca Pisani, Barbara Bruni, Flavia Prodam, Renzo Boldorini, Dirk Roggenbuck, Maurizio Vecchi, Marisa GariglioAbstract:BACKGROUND Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the expression and function of pattern recognition receptors, including the IFI16 protein. Because this protein is a target for autoantibodies and its aberrant expression was reported in colonic mucosa from active patients with ulcerative colitis, we studied its expression and specific seroresponse in patients with IBD before and after infliximab (IFX) therapy. METHODS Anti-IFI16 antibodies (IgG and IgA subtypes) were measured in the sera of 74 patients with IBD: 48 patients with Crohn's disease (CD) and 26 patients with ulcerative colitis, prospectively harvested before and after IFX therapy. Anti-GP2 antibodies (both IgG and IgA subtypes) were also tested for comparison. The patient antibody statuses were qualitatively and quantitatively associated with disease phenotype and response to IFX therapy. RESULTS Significantly higher titers of anti-IFI16 IgG were found in both CD and ulcerative colitis patients compared with healthy controls. Anti-IFI16 IgA titers were also present in patients with CD. Anti-GP2 IgG subtype titers were significantly increased in patients with CD, as were IgA subtype titers. Significant changes in anti-IFI16 IgG subtype titers were observed after IFX in patients with CD who correlated with clinical remission or response. CONCLUSIONS Our results highlight the importance of IFI16 in IBD pathogenesis showing that its de novo overexpression in the gut epithelial cells leads to a breakdown in immune tolerance and the subsequent development of specific autoantibodies. Anti-IFI16 IgG antibodies hold the potential to serve as a biomarker of response to IFX therapy.
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Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability
Journal of Virology, 2016Co-Authors: Matteo Biolatti, Marco De Andrea, Marisa Gariglio, Valentina Dell'oste, Sara Pautasso, Jens Von Einem, Manfred Marschall, Bodo Plachter, Santo LandolfoAbstract:UNLABELLED A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.
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Circulating Interferon-Inducible Protein IFI16 Correlates With Clinical and Serological Features in Rheumatoid Arthritis.
Arthritis Care and Research, 2016Co-Authors: Alessia Alunno, Valeria Caneparo, Marisa Gariglio, Santo Landolfo, Onelia Bistoni, Sara Caterbi, Riccardo Terenzi, Elena Bartoloni, Antonio Manzo, Roberto GerliAbstract:OBJECTIVE The interferon-inducible protein 16 (IFI16) has been detected in sera from patients with autoimmune/inflammatory diseases, but not in healthy subjects. This leaking leads to loss of tolerance toward this self-protein and the development of autoantibodies. In this study, clinical significance of both IFI16 protein and anti-IFI16 antibodies in rheumatoid arthritis (RA) was investigated. METHODS IFI16 protein and anti-IFI16 antibody levels were assessed by enzyme-linked immunosorbent assay in serum samples from 154 RA patients and 182 healthy controls, and in synovial fluid (SF) samples from 21 RA patients and 25 patients with osteoarthritis (OA). RESULTS Mean serum levels for both IFI16 and anti-IFI16 antibodies were higher in RA patients than in healthy controls, with a direct correlation between IFI16 concentration and anti-IFI16 antibody titer. The majority of RA patients with detectable circulating IFI16 protein were also positive for rheumatoid factor (RF)/anti-cyclic citrullinated peptide antibody (anti-CCP). The latter group was found to be positive for anti-IFI16 antibodies as well. The mean SF concentrations of both IFI16 protein and anti-IFI16 antibodies were higher in RA patients when compared with control OA patients. Interestingly, the presence of circulating IFI16 protein, but not anti-IFI16 antibodies, significantly correlated with RA-associated pulmonary disease. This correlation was not dependent on the presence of anti-IFI16 antibodies, sex, and smoking habit. CONCLUSION Our data demonstrate that the high levels of circulating IFI16 in RA are more frequent in RF/anti-CCP-positive RA patients and significantly associated with pulmonary involvement. The relevance of circulating IFI16 protein as a new clinical biomarker of RA should be verified with additional studies.
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ID: 40: The circulating interferon-inducible protein IFI16 correlates with clinical and serological features in Rheumatoid Arthritis
Cytokine, 2015Co-Authors: Valeria Caneparo, Marisa Gariglio, Alessia Alunno, Onelia Bistoni, Sara Caterbi, Riccardo Terenzi, Elena Bartoloni, Roberto Gerli, Santo LandolfoAbstract:Objectives The IFN-inducible protein 16 (IFI16) has been detected in sera from patients with autoimmune/inflammatory diseases but not in healthy subjects. This leaking leads to loss of tolerance towards this self-protein and development of autoantibodies. In this study, the clinical significance of both IFI16 protein and anti-IFI16 antibodies (Abs) in rheumatoid arthritis (RA) was investigated. Methods IFI16 protein and anti-IFI16 Abs levels were assessed by ELISA in serum samples from 154 RA patients and 182 healthy controls (HC), and in synovial fluid (SF) samples from 21 RA patients and 25 patients with osteoarthritis (OA). Results Mean serum levels for both IFI16 and anti-IFI16 Abs were higher in RA than in HC with a direct correlation between IFI16 concentration and anti-IFI16 Abs titer. The majority of RA patients with detectable circulating IFI16 protein were also positive for RF/ACPA. The latter group was found positive for anti-IFI16 Abs as well. The mean SF concentrations of both IFI16 protein and anti-IFI16 Abs were higher in RA when compared with control OA. Interestingly, the presence of circulating IFI16 protein, but not anti-IFI16 Abs, significantly correlated with RA-associated pulmonary disease. This correlation was not dependent on the presence of anti-IFI16 Abs, gender and smoking habit. Conclusions Our data demonstrate that the high levels of circulating IFI16 in RA are more frequent in RF/ACPA-positive RA patients and significantly associated with pulmonary involvement. The relevance of circulating IFI16 protein as new clinical biomarker of RA should be verified with additional studies.
Ileana M Cristea - One of the best experts on this subject based on the ideXlab platform.
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Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA.
Mbio, 2019Co-Authors: Krystal K Lum, Timothy R. Howard, Catherina Pan, Ileana M CristeaAbstract:The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.
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viral dna sensors IFI16 and cyclic gmp amp synthase possess distinct functions in regulating viral gene expression immune defenses and apoptotic responses during herpesvirus infection
Mbio, 2016Co-Authors: Benjamin A Diner, Krystal K Lum, Jared E Toettcher, Ileana M CristeaAbstract:ABSTRACT The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. IMPORTANCE How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.
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interactions of the antiviral factor interferon gamma inducible protein 16 IFI16 mediate immune signaling and herpes simplex virus 1 immunosuppression
Molecular & Cellular Proteomics, 2015Co-Authors: Benjamin A Diner, Aaron Javitt, Ileana M CristeaAbstract:The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antiviral cytokines during infection with herpesviruses. The knowledge of the mechanisms and protein interactions through which IFI16 exerts its antiviral functions remains limited. Here, we provide the first characterization of endogenous IFI16 interactions following infection with the prominent human pathogen herpes simplex virus 1 (HSV-1). By integrating proteomics and virology approaches, we identified and validated IFI16 interactions with both viral and host proteins that are involved in HSV-1 immunosuppressive mechanisms and host antiviral responses. We discover that during early HSV-1 infection, IFI16 is recruited to sub-nuclear puncta and subsequently targeted for degradation. We observed that the HSV-1 E3 ubiquitin ligase ICP0 is necessary, but not sufficient, for the proteasom e-mediated degradation of IFI16 following infection. We substantiate that this ICP0-mediated mechanism suppresses IFI16-dependent immune responses. Utilizing an HSV-1 strain that lacks ICP0 ubiquitin ligase activity provided a system for studying IFI16-dependent cytokine responses to HSV-1, as IFI16 levels were maintained throughout infection. We next defined temporal IFI16 interactions during this immune signaling response. We discovered and validated interactions with the viral protein ICP8 and cellular ND10 nuclear body components, sites at which HSV-1 DNA is present during infection. These interactions may be critical for IFI16 to bind to nuclear viral DNA. Altogether, our results provide critical insights into both viral inhibition of IFI16 and interactions that can contribute to IFI16 antiviral functions.
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cgas mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Megan H. Orzalli, Benjamin A Diner, Ileana M Cristea, Nicole M. Broekema, Dustin C Hancks, Nels C Elde, David M. KnipeAbstract:Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to detect herpesviral DNA directly in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required for this response. We therefore investigated their relative roles in human foreskin fibroblasts (HFFs) infected with HSV or transfected with plasmid DNA. siRNA depletion studies showed that both are required for the production of IFN in infected HFFs. We found that cGAS shows low production of cGMP-AMP in infected cells, but instead cGAS is partially nuclear in normal human fibroblasts and keratinocytes, interacts with IFI16 in fibroblasts, and promotes the stability of IFI16. IFI16 is associated with viral DNA and targets to viral genome complexes, consistent with it interacting directly with viral DNA. Our results demonstrate that IFI16 and cGAS cooperate in a novel way to sense nuclear herpesviral DNA and initiate innate signaling.
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acetylation modulates cellular distribution and dna sensing ability of interferon inducible protein IFI16
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Tuo Li, Benjamin A Diner, Jin Chen, Ileana M CristeaAbstract:Detection of pathogenic nucleic acids is essential for mammalian innate immunity. IFN-inducible protein IFI16 has emerged as a critical sensor for detecting pathogenic DNA, stimulating both type I IFN and proinflammatory responses. Despite being predominantly nuclear, IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasm and the nucleus. However, the mechanisms regulating its localization and sensing ability remain uncharacterized. Here, we propose a two-signal model for IFI16 sensing. We first identify an evolutionarily conserved multipartite nuclear localization signal (NLS). Next, using FISH and immunopurification, we demonstrate that IFI16 detects HSV-1 DNA primarily in the nucleus, requiring a functional NLS. Furthermore, we establish a localization-dependent IFN-β induction mediated by IFI16 in response to HSV-1 infection or viral DNA transfection. To identify mechanisms regulating the secondary cytoplasmic localization, we explored IFI16 posttranslation modifications. Combinatorial MS analyses identified numerous acetylations and phosphorylations on endogenous IFI16 in lymphocytes, in which we demonstrate an IFI16-mediated IFN-β response. Importantly, the IFI16 NLS was acetylated in lymphocytes, as well as in macrophages. Mutagenesis and nuclear import assays showed that NLS acetylations promote cytoplasmic localization by inhibiting nuclear import. Additionally, broad-spectrum deacetylase inhibition triggered accumulation of cytoplasmic IFI16, and we identify the acetyltransferase p300 as a regulator of IFI16 localization. Collectively, these studies establish acetylation as a molecular toggle of IFI16 distribution, providing a simple and elegant mechanism by which this versatile sensor detects pathogenic DNA in a localization-dependent manner.
Marco De Andrea - One of the best experts on this subject based on the ideXlab platform.
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Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding.
PLOS Pathogens, 2020Co-Authors: Andrea Iannucci, Valeria Caneparo, Marisa Gariglio, Santo Landolfo, Stefano Raviola, Isacco Debernardi, Donato Colangelo, Riccardo Miggiano, Gloria Griffante, Marco De AndreaAbstract:Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4. IFI16 is a nuclear protein involved in a variety of physiological processes, including cell cycle regulation, tumor suppression, and virus sensing. Emerging evidence indicates that IFI16 is released in the extracellular milieu under injury or stress conditions. Here we show that extracellular IFI16 acts as a damage-associated molecular pattern (DAMP), triggering inflammation through Toll-like receptor 4 (TLR4) activation. Furthermore, we demonstrate that IFI16 activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating lipopolysaccharide (LPS) variants, which are known to be present in various pathological settings other than gram-negative infections. Our study provides new insights into the role of extracellular IFI16 during low-grade endotoxemia.
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Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability
Journal of Virology, 2016Co-Authors: Matteo Biolatti, Marco De Andrea, Marisa Gariglio, Valentina Dell'oste, Sara Pautasso, Jens Von Einem, Manfred Marschall, Bodo Plachter, Santo LandolfoAbstract:UNLABELLED A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.
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ID: 216: The nuclear Interferon-inducible IFI16 protein and the innate sensing to HPV18 replication
Cytokine, 2015Co-Authors: Marco De Andrea, Santo Landolfo, Bala Chandran, Karen E Johnson, Irene Lo Cigno, Cinzia Borgogna, Silvia Albertini, Alberto Peretti, Marisa GariglioAbstract:Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as sensors of foreign DNA and antiviral restriction factors. It is a multifunctional nuclear protein involved in transcriptional regulation, induction of Interferon- β (IFN- β ) , and activation of the inflammasome response. It interacts with the sugar-phosphate backbone of dsDNA and modulates viral and cellular transcription through largely undetermined mechanisms. IFI16 is a restriction factor for Human Cytomegalovirus (HCMV) and Herpes Simplex virus (HSV-1). We have recently demonstrated that IFI16 has also a profound effect on HPV18 replication in human keratinocytes (NIKS cells transfected with religated HPV18 genome) and the osteosarcoma cell line U2OS, transfected by electroporation with HPV18 minicircles (Lo Cigno et al., J. Virol. (2015)). ChIP studies demonstrated that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Type I IFN response was not involved in the observed phenotypes. Experiments are underway with EdU-labeled HPV18 minicircles to visualize any interaction with IFI16 at the early stages of infection, and to clarify which molecular events regulate the IFI16 immune-sensing to HPV. Altogether, these results argue that IFI16 restricts chromatinised HPV DNA through epigenetic modifications and executes a broad surveillance role against viral DNA in the nucleus that is not restricted to Herpesviruses.
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ID: 37: The IFI16 restriction factor cooperates with HCMV pUL83 to down-regulate UL54 gene expression and viral DNA synthesis
Cytokine, 2015Co-Authors: Matteo Biolatti, Marco De Andrea, Marisa Gariglio, Valentina Dell'oste, Sara Pautasso, Jens Von Einem, Manfred Marschall, Bodo Plachter, Santo LandolfoAbstract:During the early phase of human cytomegalovirus (HCMV) infection, the Interferon- γ -Inducible factor 16 (IFI16) behaves as a pattern recognition receptor (PRR) sensing viral DNA and triggering antiviral cytokine release. Later on, it restricts virus replication by down-regulating expression of viral genes committed to DNA synthesis including UL54 and UL44. These activities are modulated by viral proteins including pUL83, a tegument protein involved in viral evasion. Here, we demonstrate that pUL83 interacts with IFI16 relieving its inhibitory activity on UL54 gene transcription. We also establish that, starting from 48 h post-infection, IFI16 is stabilized and protected from degradation by pUL83 as observed infecting human foreskin fibroblasts with the wild type HCMV strain (v65Rev) or the v65Stop lacking pUL83 expression. Upon infection with an HCMV mutant virus (RV-VM1) expressing a pUL83 lacking the nuclear egression signal (NES), IFI16 is retained in the nucleus and does not migrate into the cytoplasm. Interestingly, accumulation of nuclear pUL83 preventes the formation of discrete puncta and dissipates aggregation of IFI16 filaments. We observe that IFI16 shows an half-life of less than 1 h in the absence of pUL83 compared with 2 h in the presence of pUL83 demonstrating that IFI16 is less stable in the absence of pUL83. Consistent with this, we observe restoration of IFI16 protein in v65Stop-infected cells compared to v65Rev-infected cells in presence of the proteasome inhibitor MG132. Our results demonstrate a novel role for the pUL83 protein that stabilizes and protects IFI16 from proteasome degradation during HCMV infection and modulates suppression of UL54 gene activity.
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The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters
Journal of Virology, 2015Co-Authors: Irene Lo Cigno, Marco De Andrea, Santo Landolfo, Bala Chandran, Karen E Johnson, Cinzia Borgogna, Silvia Albertini, Manuela M. Landini, Alberto Peretti, Marisa GariglioAbstract:ABSTRACT The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.
Divaker Choubey - One of the best experts on this subject based on the ideXlab platform.
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IFI16, an Amplifier of DNA-damage Response: Role in Cellular Senescence and Aging-Associated Inflammatory Diseases
Ageing Research Reviews, 2016Co-Authors: Divaker Choubey, Ravichandran PanchanathanAbstract:DNA-damage induces a DNA-damage response (DDR) in mammalian cells. The response, depending upon the cell-type and the extent of DNA-damage, ultimately results in cell death or cellular senescence. DDR-induced signaling in cells activates the ATM-p53 and ATM-IKKα/β-interferon (IFN)-β signaling pathways, thus leading to an induction of the p53 and IFN-inducible IFI16 gene. Further, upon DNA-damage, DNA accumulates in the cytoplasm, thereby inducing the IFI16 protein and STING-dependent IFN-β production and activation of the IFI16 inflammasome, resulting in the production of proinflammatory cytokines (e.g., IL-1β and IL-18). Increased expression of IFI16 protein in a variety of cell-types promotes cellular senescence. However, reduced expression of IFI16 in cells promotes cell proliferation. Because expression of the IFI16 gene is induced by activation of DNA-damage response in cells and increased levels of IFI16 protein in cells potentiate the p53-mediated transcriptional activation of genes and p53 and pRb-mediated cell cycle arrest, we discuss how an improved understanding of the role of IFI16 protein in cellular senescence and associated inflammatory secretory phenotype is likely to identify the molecular mechanisms that contribute to the development of aging-associated human inflammatory diseases and a failure to cancer therapy.
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A Pathologic Link between Wilms Tumor Suppressor Gene, WT1, and IFI16
Neoplasia, 2014Co-Authors: Marianne Kh Kim, Divaker Choubey, Jacqueline M. Mason, Windy Berkofsky-fessler, Paul E. Grundy, Benjamin Tycko, Le Jiang, Jonathan D. LichtAbstract:The Wilms tumor gene ( WT1 ) is mutated or deleted in patients with heredofamilial syndromes associated with the development of Wilms tumors, but is infrequently mutated in sporadic Wilms tumors. By comparing the microarray profiles of syndromic versus sporadic Wilms tumors and WT1-inducible Saos-2 osteosarcoma cells, we identified interferon-inducible protein 16 ( IFI16 ), a transcriptional modulator, as a differentially expressed gene and a candidate WT1 target gene. WT1 induction in Saos-2 osteosarcoma cells led to strong induction of IFI16 expression and its promoter activity was responsive to the WT1 protein. Immunohistochemical analysis showed that IFI16 and WT1 colocalized in WT1-replete Wilms tumors, but not in normal human midgestation fetal kidneys, suggesting that the ability of WT1 to regulate IFI16 in tumors represented an aberrant pathologic relationship. In addition, endogenous IFI16 and WT1 interacted in vivo in two Wilms tumor cell lines. Furthermore, IFI16 augmented the transcriptional activity of WT1 on both synthetic and physiological promoters. Strikingly, short hairpin RNA (shRNA)-mediated knockdown of either IFI16 or WT1 led to decreased growth of Wilms tumor cells. These data suggest that IFI16 and WT1, in certain cellular context including sporadic Wilms tumors, may support cell survival.
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Identification of a negative feedback loop between cyclic di-GMP-induced levels of IFI16 and p202 cytosolic DNA sensors and STING:
Innate Immunity, 2013Co-Authors: Ravichandran Panchanathan, Hongzhu Liu, Duan Xin, Divaker ChoubeyAbstract:A host type I IFN response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP (c-di-GMP) by STING (stimulator of IFN genes). Because the STING, an adaptor protein, links the cytosolic detection of DNA by the cytosolic DNA sensors such as the IFN-inducible human IFI16 and murine p202 proteins to the TBK1/IRF3 axis, we investigated whether c-di-GMP-induced signaling could regulate expression of IFI16 and p202 proteins. Here, we report that activation of c-di-GMP-induced signaling in human and murine cells increased steady-state levels of IFI16 and p202 proteins. The increase was c-di-GMP concentration- and time-dependent. Unexpectedly, treatment of cells with type I IFN decreased levels of the adaptor protein STING. Therefore, we investigated whether the IFI16 or p202 protein could regulate the expression of STING and activation of the TBK1/IRF3 axis. We found that constitutive knockdown of IFI16 or p202 expression in cells increased steady-state levels of STING. Additionally, the knockdown of IFI16 resulted in activation of the TBK1/IRF3 axis. Accordingly, increased levels of the IFI16 or p202 protein in cells decreased STING levels. Together, our observations identify a novel negative feedback loop between c-di-GMP-induced levels of IFI16 and p202 cytosolic DNA sensors and the adaptor protein STING.
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Interferon-inducible p200-family protein IFI16, an innate immune sensor for cytosolic and nuclear double-stranded DNA: regulation of subcellular localization.
Molecular Immunology, 2011Co-Authors: Sudhakar Veeranki, Divaker ChoubeyAbstract:Abstract The interferon (IFN)-inducible p200-protein family includes structurally related murine (for example, p202a, p202b, p204, and Aim2) and human (for example, AIM2 and IFI16) proteins. All proteins in the family share a partially conserved repeat of 200-amino acid residues (also called HIN-200 domain) in the C-terminus. Additionally, most proteins (except the p202a and p202b proteins) also share a protein–protein interaction pyrin domain (PYD) in the N-terminus. The HIN-200 domain contains two consecutive oligosaccharide/oligonucleotide binding folds (OB-folds) to bind double stranded DNA (dsDNA). The PYD domain in proteins allows interactions with the family members and an adaptor protein ASC. Upon sensing cytosolic dsDNA, Aim2, p204, and AIM2 proteins recruit ASC protein to form an inflammasome, resulting in increased production of proinflammatory cytokines. However, IFI16 protein can sense cytosolic as well as nuclear dsDNA. Interestingly, the IFI16 protein contains a nuclear localization signal (NLS). Accordingly, the initial studies had indicated that the endogenous IFI16 protein is detected in the nucleus and within the nucleus in the nucleolus. However, several recent reports suggest that subcellular localization of IFI16 protein in nuclear versus cytoplasmic (or both) compartment depends on cell type. Given that the IFI16 protein can sense cytosolic as well as nuclear dsDNA and can initiate different innate immune responses (production of IFN-β versus proinflammatory cytokines), here we evaluate the experimental evidence for the regulation of subcellular localization of IFI16 protein in various cell types. We conclude that further studies are needed to understand the molecular mechanisms that regulate the subcellular localization of IFI16 protein.
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IFI16 protein mediates the anti-inflammatory actions of the type-I interferons through suppression of activation of caspase-1 by inflammasomes.
PLOS ONE, 2011Co-Authors: Sudhakar Veeranki, Ravichandran Panchanathan, Xin Duan, Hongzhu Liu, Divaker ChoubeyAbstract:Background: Type-I interferons (IFNs) are used to treat certain inflammatory diseases. Moreover, activation of type-I IFNsignaling in immune cells inhibits the production of proinflammatory cytokines and activation of inflammasomes. However, the molecular mechanisms remain largely unknown. Upon sensing cytosolic double-stranded DNA, the AIM2 protein forms the AIM2-ASC inflammasome, resulting in activation of caspase-1. Given that the IFI16 and AIM2 proteins are IFN-inducible and can heterodimerize with each other, we investigated the regulation of IFI16, AIM2, and inflammasome proteins by typeI and type-II IFNs and explored whether the IFI16 protein could negatively regulate the activation of the AIM2 (or other) inflammasome. Methodology/ Principal Findings: We found that basal levels of the IFI16 and AIM2 proteins were relatively low in peripheral blood monocytes (CD14 + ) and in the THP-1 monocytic cell line. However, treatment of THP-1 cells with type-I (IFN-a or b) or type-II (IFN-c) IFN induced the expression levels of IFI16, AIM2, ASC and CASP1 proteins. The induced levels of IFI16 and AIM2 proteins were detected primarily in the cytoplasm. Accordingly, relatively more IFI16 protein bound with the AIM2 protein in the cytoplasmic fraction. Notably, increased expression of IFI16 protein in transfected HEK-293 cells inhibited activation of caspase-1 by the AIM2-ASC inflammasome. Moreover, the constitutive knockdown of the IFI16 expression in THP-1 cells increased the basal and induced [induced by poly(dA:dT) or alum] activation of the caspase-1 by the AIM2 and NLRP3 inflammasomes. Conclusions/Significance: Our observations revealed that the type-I and type-II IFNs induce the expression of IFI16, AIM2, and inflammasome proteins to various extents in THP-1 cells and the expression of IFI16 protein in THP-1 cells suppresses the activation of caspase-1 by the AIM2 and NLRP3 inflammasomes. Thus, our observations identify the IFI16 protein as a mediator of the anti-inflammatory actions of the type-I IFNs.
