Cytoplasmic DNA sensing by KU complex in aged CD4+ T cell potentiates T cell activation and aging-related autoimmune inflammation.

Aging is associated with DNA accumulation and increased homeostatic proliferation of circulating T cells. Although these attributes are associated with aging-related autoimmunity, their direct contributions remain unclear. Conventionally, KU complex, the regulatory subunit of DNA-dependent protein kinase (DNA-PK), together with the catalytic subunit of DNA-PK (DNA-PKcs), mediates DNA damage repair in the nucleus. Here, we found KU complex abundantly expressed in the cytoplasm, where it recognized accumulated cytoplasmic DNA in aged human and mouse CD4+ T cells. This process enhanced T cell activation and pathology of experimental autoimmune encephalomyelitis (EAE) in aged mice. Mechanistically, KU-mediated DNA sensing facilitated DNA-PKcs recruitment and phosphorylation of the kinase ZAK. This activated AKT and mTOR pathways, promoting CD4+ T cell proliferation and activation. We developed a specific ZAK inhibitor, which dampened EAE pathology in aged mice. Overall, these findings demonstrate a KU-mediated cytoplasmic DNA-sensing pathway in CD4+ T cells that potentiates aging-related autoimmunity.

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

A B-Cell-Specific Enhancer Orchestrates Nuclear Architecture to Generate a Diverse Antigen Receptor Repertoire.

The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.

Nuclear cGAS: guard or prisoner?

cGAS, an innate immune sensor of cellular stress, recognizes double-stranded DNA mislocalized in the cytosol upon infection, mitochondrial stress, DNA damage, or malignancy. Early models suggested that cytosolic localization of cGAS prevents autoreactivity to nuclear and mitochondrial self-DNA, but this paradigm has shifted in light of recent findings of cGAS as a predominantly nuclear protein tightly bound to chromatin. This has raised the question how nuclear cGAS is kept inactive while being surrounded by chromatin, and what function nuclear localization of cGAS may serve in the first place? Cryo-EM structures have revealed that cGAS interacts with nucleosomes, the minimal units of chromatin, mainly via histones H2A/H2B, and that these protein-protein interactions block cGAS from DNA binding and thus prevent autoreactivity. Here, we discuss the biological implications of nuclear cGAS and its interaction with chromatin, including various mechanisms for nuclear cGAS inhibition, release of chromatin-bound cGAS, regulation of different cGAS pools in the cell, and chromatin structure/chromatin protein effects on cGAS activation leading to cGAS-induced autoimmunity.

Combination of cytoplasmic and nuclear patterns on Hep-2 antinuclear antibody is useful as a screening test for anti-synthetase syndrome.

OBJECTIVE: This study aimed to establish a screening model for differentiating anti-synthetase syndrome (ASS) from other ANA-associated rheumatic diseases (AARDs) using a combination of cytoplasmic and non-cytoplasmic ANA (ncANA) patterns. METHODS: : This retrospective observational study included patients with AARDs such as SLE, SSc, SS, MCTD and PM/DM who underwent ANA screening between April 2012 and December 2021. Variables included age, sex, ANA patterns (Cytoplasmic and ncANA) and titres. Logistic regression analysis of Cytoplasmic and ncANA patterns was performed to differentiate ASS from other AARDs. RESULT: : The 981 diagnosed cases of AARDs consisted of SS (n = 451), SSc (n = 264), SLE (n = 201), PM/DM (n = 104), MCTD (n = 52) and ASS, including PM/DM (n = 64). Of these, 155 patients had ≥2 overlapping diseases; however, there was no overlap between AARDs and ASS. ASS is more likely to occur when the cytoplasmic titre is positive and the ncANA <320. Receiver operating characteristic analysis of the Cytoplasmic and ncANA range revealed an area under the receiver operating characteristic curve of 0.885 (95% CI: 0.844-0.927). CONCLUSION: : It is important to detect cytoplasmic patterns as an ANA screening test for ASS diagnosis, even if the titre is low. Additionally, combining the cytoplasmic and ncANA patterns yields more accurate ASS screening results.

IFI16 Isoforms with Cytoplasmic and Nuclear Locations Play Differential Roles in Recognizing Invaded DNA Viruses.

IFN-γ-inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5'-AGTGTT-3' DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-ß and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN-dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-ß and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN-dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Human Cytomegalovirus Protein UL94 Targets MITA to Evade the Antiviral Immune Response.

Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP), which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes the host antiviral immune response to establish latent infection. Here, we identified HCMV tegument protein UL94 as an inhibitor of the cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic double-stranded DNA (dsDNA)- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94 deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalsome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.IMPORTANCE Human cytomegalovirus (HCMV), a large double-stranded DNA (dsDNA) virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of the central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of the innate immune response, which functions in the late phase of HCMV infection.

Nuclear processing of nascent transcripts determines synthesis of full-length proteins and antigenic peptides.

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

A proof of evidence supporting abnormal immunothrombosis in severe COVID-19: naked megakaryocyte nuclei increase in the bone marrow and lungs of critically ill patients.

Coronavirus disease 2019 (COVID-19) is a global public health emergency with many clinical facets, and new knowledge about its pathogenetic mechanisms is deemed necessary; among these, there are certainly coagulation disorders. In the history of medicine, autopsies and tissue sampling have played a fundamental role in order to understand the pathogenesis of emerging diseases, including infectious ones; compared to the past, histopathology can be now expanded by innovative techniques and modern technologies. For the first time in worldwide literature, we provide a detailed postmortem and biopsy report on the marked increase, up to 1 order of magnitude, of naked megakaryocyte nuclei in the bone marrow and lungs from serious COVID-19 patients. Most likely related to high interleukin-6 serum levels stimulating megakaryocytopoiesis, this phenomenon concurs to explain well the pulmonary abnormal immunothrombosis in these critically ill patients, all without molecular or electron microscopy signs of megakaryocyte infection.

Nuclear carbonic anhydrase 6B associates with PRMT5 to epigenetically promote IL-12 expression in innate response.

Interleukin-12 (IL-12) is critical for induction of protective immunity against intracellular bacterial infection. However, the mechanisms for efficient induction of IL-12 in innate response remain poorly understood. Here we report that the B type of carbonic anhydrase 6 (Car6-b, which encoded CA-VI B) is essential for host defense against Listeria monocytogenes (LM) infection by epigenetically promoting IL-12 expression independent of its carbonic anhydrase activity. Deficiency of Car6-b attenuated IL-12 production upon LM infection both in vitro and in vivo. Car6-/- mice were more susceptible to LM infection with less production of IL-12. Mechanistically, the nuclear localized CA-VI B selectively promotes IL-12 expression by interaction with protein arginine N-methyltransferase 5 (PRMT5), which reduces symmetric dimethylation of histone H3 arginine 8 modification (H3R8me2s) at Il12 promoters to facilitate chromatin accessibility, selectively enhancing c-Rel binding to the Il12b promoter. Our findings add insights to the epigenetic regulation of IL-12 induction in innate immunity.

Activin-A in the regulation of immunity in health and disease.

The TGF-ß superfamily of cytokines plays pivotal roles in the regulation of immune responses protecting against or contributing to diseases, such as, allergy, autoimmunity and cancer. Activin-A, a member of the TGF-ß superfamily, was initially identified as an inducer of follicle-stimulating hormone secretion. Extensive research over the past decades illuminated fundamental roles for activin-A in essential biologic processes, including embryonic development, stem cell maintenance and differentiation, haematopoiesis, cell proliferation and tissue fibrosis. Activin-A signals through two type I and two type II receptors which, upon ligand binding, activate their kinase activity, phosphorylate the SMAD2 and 3 intracellular signaling mediators that form a complex with SMAD4, translocate to the nucleus and activate or silence gene expression. Most immune cell types, including macrophages, dendritic cells (DCs), T and B lymphocytes and natural killer cells have the capacity to produce and respond to activin-A, although not in a similar manner. In innate immune cells, including macrophages, DCs and neutrophils, activin-A exerts a broad range of pro- or anti-inflammatory functions depending on the cell maturation and activation status and the spatiotemporal context. Activin-A also controls the differentiation and effector functions of Th cell subsets, including Th9 cells, TFH cells, Tr1 Treg cells and Foxp3+ Treg cells. Moreover, activin-A affects B cell responses, enhancing mucosal IgA secretion and inhibiting pathogenic autoantibody production. Interestingly, an array of preclinical and clinical studies has highlighted crucial functions of activin-A in the initiation, propagation and resolution of human diseases, including autoimmune diseases, such as, systemic lupus erythematosus, rheumatoid arthritis and pulmonary alveolar proteinosis, in allergic disorders, including allergic asthma and atopic dermatitis, in cancer and in microbial infections. Here, we provide an overview of the biology of activin-A and its signaling pathways, summarize recent studies pertinent to the role of activin-A in the modulation of inflammation and immunity, and discuss the potential of targeting activin-A as a novel therapeutic approach for the control of inflammatory diseases.

Duck interferon regulatory factor 7 (IRF7) can control duck Tembusu virus (DTMUV) infection by triggering type I interferon production and its signal transduction pathway.

Human interferon regulatory factor 7 (IRF7) plays an important role in the innate antiviral immune response. To date, the characteristics and functions of waterfowl IRF7 have not been clarified. This study reports the cDNA sequence, tissue distribution, and antiviral function of duck IRF7. The duck IRF7 gene has a 1536-bp open read frame (ORF) and encodes a 511-amino acid polypeptide. IRF7 is highly expressed in the blood and pancreas of 5-day-old ducklings and in the small intestine, large intestine and liver of 60-day-old adult ducks. Indirect immunofluorescence assay (IFA) showed that over-expressed duck IRF7 was located in both the cytoplasm and nucleus of transfected duck embryo fibroblasts (DEFs), which was also observed in poly(I:C)-stimulated or duck Tembusu virus (DTMUV)-infected DEFs. Titres and copies of DTMUV were significantly reduced in DEFs overexpressing IRF7. Moreover, overexpression of duck IRF7 significantly induced IFNα/ß, but not IFNγ, mRNA expression, and transcription of downstream interferon-stimulated genes (ISGs), such as MX, OASL and IL-6, which were significantly induced by poly(I:C) co-stimulation, was enhanced. Additionally, duck IRF7 overexpression can significantly activate the IFNß promoter in DEFs. Collectively, duck IRF7 plays an important role in host anti-DTMUV immune regulation, which depends on type I interferons and associated signal transduction pathway(s).

Intranuclear delivery of the transcription modulation domain of Tbet-improved lupus nephritis in (NZB/NZW) F1 lupus-prone mice.

Excessive expression of Tbet and IFNγ is evidence of systemic lupus erythematosus (SLE) in lupus patients. In this study, the nucleus-transducible form of Transcription Modulation Domain (TMD) of Tbet (ntTbet-TMD), which is a fusion protein between Protein Transduction Domain Hph-1 (Hph-1-PTD) and the TMD of Tbet comprising DNA binding domain and isotype-specific domain, was generated to inhibit Tbet-mediated transcription in the interactomic manner. ntTbet-TMD was effectively delivered into the nucleus of the cells and specifically inhibited Tbet-mediated transcription without influencing the differentiation of other T cell subsets and signaling events for T cell activation. The severity of nephritis was significantly reduced by ntTbet-TMD as effectively as methylprednisolone in lupus-prone mice. The number of Th1, Th2 or Th17 cells and the secretion of their cytokines substantially decreased in the spleen and kidney of lupus-prone mice by ntTbet-TMD treatment. In contrast to methylprednisolone, the marked increase of Treg cells and the secretion of their immunosuppressive cytokine were detected in the spleen of (NZB/NZW) F1 mice treated with ntTbet-TMD. Thus, ntTbet-TMD can improve nephritis in lupus-prone mice by modulating the overall proinflammatory microenvironment and rebalancing T cell subsets, leading to new immune therapeutics for Th1-mediated autoimmune diseases.

Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells.

The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1 cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H2O2 or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1ß, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1 cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H2O2. Moreover, SNP or H2O2 exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1 cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1ß. Thus, hypoxia not only increases RAGE expression in THP-1 cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression.

Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.