IFN-β Gene Research Articles

Krüppel-like factor 4 negatively regulates cellular antiviral immune response.

Viral infection triggers activation of the transcription factors NF-κB and IRF3, which collaborate to induce the expression of type I interferons (IFNs) and elicit innate antiviral response. In this report, we identified Krüppel-like factor 4 (KLF4) as a negative regulator of virus-triggered signaling. Overexpression of KLF4 inhibited virus-induced activation of ISRE and IFN-β promoter in various types of cells, while knockdown of KLF4 potentiated viral infection-triggered induction of IFNB1 and downstream genes and attenuated viral replication. In addition, KLF4 was found to be localized in the cytosol and nucleus, and viral infection promoted the translocation of KLF4 from cytosol to nucleus. Upon virus infection, KLF4 was bound to the promoter of IFNB gene and inhibited the recruitment of IRF3 to the IFNB promoter. Our study thus suggests that KLF4 negatively regulates cellular antiviral response.

Isolation and characterization of bioactive compounds from Lepisorus thunbergianus (Kaulf.)

Lepisorus thunbergianus (Kaulf.) (LET) is an evergreen fern found on rocks and tree trunks that is distributed in East and Southeast Asia. Our previous study showed that the methanol extract from LET had significant anti-oxidant activity, but the active components of LET are still unclear. In the present study, isovitexin, orientin, isoorientin and chlorogenic acid were isolated from LET under the guidance of antioxidant activity. In addition, the structure of isovitexin, orientin, isoorientin and chlorogenic acid was characterized using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance spectroscopy (NMR). Orientin and isoorientin presented similar activities toward the DPPH, with half maximal inhibitory concentrations (IC50) of 15–17μmol, and inhibition of reactive oxygen species (ROS) production by 50% at 100μmol. Chlorogenic acid significantly inhibited intracellular ROS and nitric oxide (NO) production and had a strong effect toward DPPH. Furthermore, chlorogenic acid demonstrated decreased iNOS, COX-2, IFN-β and TNF-α gene expression. These findings demonstrate the potential anti-inflammatory effects of chlorogenic acid. Isoorientin, at a concentration of 100μmol, showed 50% inhibition of human liver cancer cells (Huh7 and HepG2). These results suggest that compounds isolated from LET have potential to prevent liver cancer cell lines.

335 GENE THERAPEUTIC EFFECT OF ENGINEERED STEM CELLS EXPRESSING CYTOSINE DEAMINASE AND INTERFERON-B AGAINST ENDOMETRIAL CANCER IN A XENOGRAFT MOUSE MODEL

To generate an animal model of endometrial cancer, Ishikawa cells (2 × 106 cells) were implanted via subcutaneous injection into SCID mice. In order to evaluate the therapeutic effect on tumour growth, human neural stem cells (HB1.F3), transduced with genes expressing cytosine deaminase (CD) and interferon-β (IFN-b), were labelled with the membrane dye stained with CM-DiI and were injected nearby into the tumour masses (250 mm3) in these SCID mice (n = 24). Two days after stem cell injections, 5-fluorocytosine (5-FC) was intraperitoneally injected (500 mg kg–1 per day) for 14 consecutive days. Mice were sacrificed at 48 h after the last treatment with 5-FC delivered by intraperitoneal injection for 14 days. Results demonstrated that tumour mass growth in mice treated with stem cells and 5-FC was significantly inhibited (450 mm3 ± 52) in comparison to that of nontreated mice (1400 mm3 ± 124; P < 0.01). We confirmed that injected stem cells migrated to the tumour masses by visualising the red-coloured cells in the endometrial cancer masses. The RT-PCR analysis showed that the VEGF gene was highly expressed in endometrial cancer cells, while migration-induced uPA, SDF-1, SCF, MCP-1, IL-6, and IL-8 genes were moderately expressed in these tumour cells. Histological analysis of tumour masses showed that the aggressive nature of endometrial tumour masses, determined by labelled stem cells, was inhibited in treated mice. A further analysis showed that expression of proliferating nuclear antigen (PCNA) was significantly decreased in tumour masses from treated mice. To evaluate the effect of 5-FU on Ishikawa cells, 5-FU (1.0 mg mL–1) was added to in vitro-cultured cells and the levels of IFN-a/b receptor 2 (IFNAR2) and BAX, a proapoptotic genes, were detected by RT-PCR. In these cells, IFNAR2 and BAX genes were significantly increased by the addition of 5-FU. Taken together, these results indicate that co-expression of CD and IFN-b significantly inhibited the growth of endometrial tumour masses in the presence of 5-FC, and 5-FU and IFN-b inhibits tumour growth by inhibition of DNA synthesis and enhancing the apoptotic cascade, respectively. We provide evidence to support the efficacy of therapeutic stem cell-based immuno-therapy involving the targeted expression of CD and IFN-b genes at endometrial cancer sites.This work was supported by a National Research Foundation of Korea (NRF) grant (2013R1A1A2059092) funded by the Ministry of Education, Science and Technology (MEST) of the Republic of Korea government.

The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.

Dysregulated Toll-Like Receptor (TLR) signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test) and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.

Polymerase discordance in novel swine influenza H3N2v constellations is tolerated in swine but not human respiratory epithelial cells.

Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection.

Gcn5 and PCAF negatively regulate interferon-β production through HAT-independent inhibition of TBK1.

Viral infection triggers innate immune signaling, which in turn induces interferon-β (IFN-β) production to establish innate antiviral immunity. Previous studies showed that Gcn5 (Kat2a), a histone acetyltransferase (HAT) with partial functional redundancy with PCAF (Kat2b), and Gcn5/PCAF-mediated histone H3K9 acetylation (H3K9ac) are enriched on the active IFNB gene promoter. However, whether Gcn5/PCAF and H3K9ac regulate IFN-β production is unknown. Here, we show that Gcn5/PCAF-mediated H3K9ac correlates well with, but is surprisingly dispensable for, the expression of endogenous IFNB and the vast majority of active genes in fibroblasts. Instead, Gcn5/PCAF repress IFN-β production and innate antiviral immunity in several cell types in a HAT-independent and non-transcriptional manner: by inhibiting the innate immune signaling kinase TBK1 in the cytoplasm. Our results thus identify Gcn5 and PCAF as negative regulators of IFN-β production and innate immune signaling.

RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms.

Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.

64: Identification of two distinct mechanisms that control the duration of the interferon-mediated antiviral response

The innate antiviral response mainly relies on the production of type I interferons (IFN α / β ) . IFNs establish a robust antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs), which restrict virus replication and spreading. The inability of the host to sustain an antiviral response leads to failure in eradicating the infection. Conversely, uncontrolled duration of the antiviral response is associated with the development of autoimmune disorders. To reach the ideal duration for efficient fighting of the infection without generation of side effects, the antiviral response needs to be strictly regulated. However, these mechanisms are still barely characterized. We are studying the duration of the antiviral response against Paramyxoviruses in human airway epithelial cells. We performed a thorough analysis of the temporal involvement RIG-I and MDA5 in the regulation of IRF-3, a key regulator of the IFNB gene. Based on specific RNAi-mediated knockdown of RIG-I or MDA5, we unveiled that while RIG-I is critical for the initiation of IRF-3 activation, MDA5 rather prevents active IRF-3 degradation, thereby sustaining IRF-3 activation and downstream gene expression. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Once IFNs are produced, they promote the expression of ISGs. The proinflammatory cytokine TNF α is secreted concomitantly to IFN β and was shown to act synergistically to trigger a delayed specific antiviral transcriptional program. We identified the DUOX2 and DUOXA2 genes amongst the late genes specifically induced by IFN β /TNF α . These genes encode for the H 2 O 2 -producing DUOX2 enzyme and its activator DUOXA2. Using a RNAi approach, we found that DUOX2-mediated extracellular H 2 O 2 release in human airway epithelial cells is required to sustain the level of IFN β and IFN λ at late time of infection. Altogether, our results identified two novel mechanisms that contribute to the duration of the IFN-mediated antiviral response in response to Paramyxoviruses.

The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM−/− murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM−/− cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

MUC1 Regulates Epithelial Inflammation and Apoptosis by PolyI:C through Inhibition of Toll/IL-1 Receptor-Domain–Containing Adapter-Inducing IFN-β (TRIF) Recruitment to Toll-like Receptor 3

MUC1/Muc1 (MUC1 in humans, Muc1 in animals) is a membrane-tethered mucin expressed by airway epithelial cells and plays an antiinflammatory role during airway bacterial infection. We previously demonstrated that MUC1/Muc1 is a negative regulator of Toll-like receptor (TLR) inflammatory signaling mediated through the myeloid differentiation primary response gene 88 (MyD88) adaptor protein. In the present study, we determined whether MUC1 regulates MyD88-independent TLR signaling mediated through the TLR3-Toll/IL-1 receptor-domain-containing adapter-inducing IFN-β (TRIF) pathway in response to poly(I:C). Compared with MUC1/Muc1-expressing controls, cells deficient in MUC1/Muc1 were more prone to poly(I:C)-induced apoptosis; had increased poly(I:C)-driven activation of caspase-3, caspase-8, IFN regulatory factor-3, and NF-κB; and displayed heightened IFN-β gene expression. MUC1 overexpression by these cells had the opposite effects. Reciprocal coimmunoprecipitation experiments established constitutive TLR3/MUC1-CT (cytoplasmic tail) protein interaction in human embryonic kidney (HEK)293T cells overexpressing the two proteins and in lung epithelial cells expressing the endogenous proteins, the latter of which was confirmed by immunofluorescence colocalization of TLR3 with MUC1-CT. Coimmunoprecipitation studies also revealed that MUC1 overexpression by HEK293T cells reduced poly(I:C)-induced TLR3/TRIF protein interaction. Finally, MUC1 overexpression had no effect on TRIF-dependent auto-activation of TLR3 signaling, suggesting that the site of action of the MUC1-CT in TLR3 signaling is not downstream of TRIF. These data indicate that MUC1-CT counter-regulates apoptotic and inflammatory responses of airway epithelial cell through constitutive association with TLR3, thereby inhibiting poly(I:C)-induced recruitment of TRIF to TLR3.

Lysine 63-linked TANK-binding kinase 1 ubiquitination by mindbomb E3 ubiquitin protein ligase 2 is mediated by the mitochondrial antiviral signaling protein.

Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.

Function of duck RIG-I in induction of antiviral response against IBDV and avian influenza virus on chicken cells

The avian influenza (AI) H9N2 virus and IBDV are two major problems in the poultry industry. They have been prevalent among domestic poultry in Asia for many years and have caused considerable economic losses. Retinoic-acid-induced gene I (RIG-I) is a cytoplasmic sensor of dsRNA and ssRNA. It can detect Encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) in human cells, influenza virus in duck leads to production of IFN-β and IFN-stimulated antiviral genes and reductions in the replication of RNA virus. Chickens, which lack RIG-I, are more sensitive to influenza virus than ducks. However, little is known about the roles of duck RIG-I (dRIG-I) in the detection of IBDV and AI H9N2 in chicken cells DF-1. The purpose of this study was to examine the function of dRIG-I in the recognition of IBDV Ts strain and H9N2 A/Chicken/Shandong/ZB/2007(ZB07) and in the induction of antiviral gene expression to gain an understanding of antiviral ability of dRIG-I in chicken cells against dsRNA virus IBDV and ssRNA virus ZB07. After challenge with the IBDV Ts strain and ZB07 the expression levels of Type I IFN (IFN-β and IFN-α) and IFN-induced antiviral genes (Mx and PKR) were significantly up-regulated in dRIG-I-transfected DF-1cells compared with the empty-vector-transfected control. dRIG-I knockdown experiments further proved that dRIG-I is essential to sensing IBDV and ZB07 in duck embryo fibroblasts (DEF). Growth curves showed that dRIG-I repressed the replication of IBDV and almost blunted the growth of ZB07 in DF-1. Apoptosis analysis revealed that dRIG-I increase the number of the survival cells after IBDV Ts strain or ZB07 infection relative to the empty-vector-transfected control. These results indicate that dRIG-I can up-regulates type I IFN and reduce viral gene expression and viral replication and protect chicken cells from virus-induced apoptosis during ZB07 and IBDV infection.

Activated STING in a Vascular and Pulmonary Syndrome

BackgroundThe study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation.MethodsWe analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls.ResultsWe identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors.ConclusionsSTING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.)

DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-β production. Macrophages lacking DOK3 were impaired in IFN-β synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-β gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-β promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-β production.

An essential role for IFN-β in the induction of IFN-stimulated gene expression by LPS in macrophages.

TLR agonists such as LPS and poly(I:C) induce expression of type I IFNs, such as IFN-α and -β, by macrophages. To examine the role of IFN-β in the induction of ISGs by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from WT and Ifnb1(-/-) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I), and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1(-/-) mice or Ifnar1(-/-) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1(-/-) macrophages correlated with the failure of LPS to induce activation of STAT1 and -2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 KO mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1(-/-) and Ifnar1(-/-) macrophages, activation of NF-κB and induction of NF-κB-responsive genes, such as Tnf (TNF-α) and Il1b (IL-1β), were not affected by deletion of either the IFN-β or IFN-αR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-β and autocrine signaling through type I IFN receptors.

The mechanism of double-stranded DNA sensing through the cGAS-STING pathway

Microbial nucleic acids induce potent innate immune responses by stimulating the expression of type I interferons. Cyclic GMP-AMP synthase (cGAS) is a cytosolic dsDNA sensor mediating the innate immunity to microbial DNA. cGAS is activated by dsDNA and catalyze the synthesis of a cyclic dinucleotide cGAMP with 2′,5′ and 3′,5′phosphodiester linkages. cGAMP binds to the adaptor STING located on the endoplasmic reticulum membrane and mediates the recruitment and activation of the protein kinase TBK1 and transcription factor IRF3. Phosphorylated IRF3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The crystal structures of cGAS and its complex with dsDNA, STING and its complex with various cyclic dinucleotides have been determined recently. Here we summarize the results from these structural studies and provide an overview about the mechanism of cGAS activation by dsDNA, the catalytic mechanism of cGAS, and the structural basis of STING activation by cGAMP.

Crucial role of the MAPKAP kinases 2 and 3 for pathogen-induced inflammation and their relevance for the immune response of the liver

Crucial role of the MAPKAP kinases 2 and 3 for pathogen-induced inflammation and their relevance for the immune response of the liver

TLR3 mediated innate immune response in mice brain following infection with Chikungunya virus

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world. Many unusual clinical severities including neurological complications and death were reported in recent outbreaks. The mechanism underlying the host immune response to CHIKV in the brain is poorly characterized. In this study, the neuropathogenesis of CHIKV with E1:A226V mutation was elucidated in 1 week old BALB/c mice. The virus was found to replicate in mice brain with peak titer of 104 on 6th day post infection. Immunohistochemical analysis revealed preferential virus localization in neuronal cells of cerebellum. The expression profiling of TLR, antiviral genes and cytokines in mice brain revealed significant up regulation of TLR3, TRAF-6, TICAM-1, MCP-1, CXCL-10, IL-6, IL-4, ISG-15, MX-2, IFN-β, OAS-3 genes that ultimately resulted in virus clearance from brain by day 9–10 suggesting activation of innate immune pathway. Further the effect of poly I: C (Polyinosinic: Polycytidylic acid), a TLR-3 agonist and potent IFN inducer on CHIKV neuropathogenesis was studied. Pretreatment of mice with Poly I: C caused reduction of CHIKV titer in brain and offered 100% protection of animals. The protection was mediated by an increased induction of TLR3, IFN-β and antiviral genes in mice brain. Our result demonstrates that pre immune stimulation of animals by Poly I: C is effective inhibitor of CHIKV replication and might be a promising prevention agent against this virus.

Uncoupling of the dynamics of host-pathogen interaction uncovers new mechanisms of viral interferon antagonism at the single-cell level.

Antiviral defence in mammals is mediated through type-I interferons (IFNs). Viruses antagonise this process through expression of IFN antagonist proteins (IAPs). Understanding and modelling of viral escape mechanisms and the dynamics of IAP action has the potential to facilitate the development of specific and safe drugs. Here, we describe the dynamics of interference by selected viral IAPs, NS1 from Influenza A virus and NS3/4A from Hepatitis C virus. We used Tet-inducible IAP gene expression to uncouple this process from virus-driven dynamics. Stochastic activation of the IFN-β gene required the use of single-cell live imaging to define the efficacy of the inhibitors during the virus-induced signalling processes. We found significant correlation between the onset of IAP expression and halted IFN-β expression in cells where IFN-β induction had already occurred. These data indicate that IAPs not only prevent antiviral signalling prior to IFN-β induction, but can also stop the antiviral response even after it has been activated. We found reduced NF-κB activation to be the underlying mechanism by which activated IFN expression can be blocked. This work demonstrates a new mechanism by which viruses can antagonise the IFN response.

Bacterial amyloids promote type I interferon production and accelerate autoimmunity (BA3P.206)

Abstract Infection is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Curli is a bacterial amyloid that serves as a component of enteric bacterial biofilms. Patients are exposed to curli during urinary tract infections, gastrointestinal infections, and sepsis. Here we studied the immune response to curli amyloids in the context of murine SLE. We found that curli amyloids from bacterial cultures contain abundant nucleic acids and that exogenous DNA enhances amyloid fibrillization. Bone marrow-derived myeloid DCs strongly respond to curli-DNA composites, producing large quantities of IL-12, IL-6, and IL-10. Curli potently induces expression of IFN-β and IFN-stimulated genes. Using both natural and synthetic curli, we found that curli and nucleic acids synergize to activate DCs. Young, pre-disease lupus-prone NZBW-F1 mice injected i.p. with nucleic acid-containing curli produce high titers of anti-dsDNA and anti-chromatin autoantibodies within 2 weeks of the first injection, much earlier than mice treated with PBS. In conclusion, the bacterial amyloid curli contains nucleic acids which are integral to the immune response to bacterial biofilms. Curli is a potent activator of dendritic cells and can rapidly accelerate autoimmunity in lupus-prone mice. This work suggests that nucleic acid-containing bacterial amyloids are an important environmental trigger for lupus and that curli injection may be a novel method to accelerate murine lupus.