Biological functions and ubiquitin modification of TBK1 in innate immunity / 生物工程学报

The innate immune system initiates innate immune responses by recognizing pathogen-related molecular patterns on the surface of pathogenic microorganisms through pattern recognition receptors. Through cascade signal transduction, it activates downstream transcription factors NF-κB and interferon regulatory factors (IRFs), and then leads to the production of inflammatory cytokines and type Ⅰ interferon, which resists the infection of pathogenic microorganism. TBK1 is a central adapter protein of innate immune signaling pathway and can activate both NF-κB and IRFs. It is a key protein kinase in the process of anti-infection. The finetuning regulation of TBK1 is essential to maintain immune homeostasis and resist pathogen invasion. This paper reviews the biological functions and ubiquitin modification of TBK1 in innate immunity, to provide theoretical basis for clinical treatment of pathogenic infections and autoimmune diseases.

RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA): a new antiviral pathway

The innate immune response is the first line of host defense to eliminate viral infection. Pattern recognition receptors in the cytosol, such as RIG-I-like receptors (RLR) and Nod-like receptors (NLR), and membrane bound Toll like receptors (TLR) detect viral infection and initiate transcription of a cohort of antiviral genes, including interferon (IFN) and interferon stimulated genes (ISGs), which ultimately block viral replication. Another mechanism to reduce viral spread is through RIPA, the RLR-induced IRF3-mediated pathway of apoptosis, which causes infected cells to undergo premature death. The transcription factor IRF3 can mediate cellular antiviral responses by both inducing antiviral genes and triggering apoptosis through the activation of RIPA. The mechanism of IRF3 activation in RIPA is distinct from that of transcriptional activation; it requires linear polyubiquitination of specific lysine residues of IRF3. Using RIPA-active, but transcriptionally inactive, IRF3 mutants, it was shown that RIPA can prevent viral replication and pathogenesis in mice.

DNA sensor cGAS-mediated immune recognition

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.

Genetic characterization of bovine viral diarrhea virus strains in Beijing, China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-alpha (IFN-alpha), IFN-beta, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-alpha, and IFN-beta mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

Chronic Hepatitis C treatment for genotype 2 or 3 in Brazil: cost effectiveness analysis of peginterferon plus ribavirin as first choice treatment

Brazilian Guidelines to HCV treatment (2007) recommended that the first choice treatment for patients with chronic hepatitis C (CHC) and genotype 2 or 3 is interferon alpha (IFN) plus ribavirin (RBV) for 24 weeks. The aim of this study is compare the cost and effectiveness to Hepatitis C treatment in patients with genotype 2 or 3 of peginterferon alpha (PEG) as the first choice of treatment within PEG for those that do not respond to IFN. The target population is CHC patients with genotype 2 or 3 in Brazil. The interventions are: PEG-SEC (first IFN plus RBV for 24 weeks, after, for non-responders and relapsers subsequently PEG plus RBV for 48 weeks); PEG-FIRST24 (PEG+RBV for 24 weeks). The type of the study is cost-effectiveness analysis. The data sources are: Effectiveness data from meta-analysis conducted on the Brazilian population. Treatment cost from Brazilian micro costing study is converted into USD (2010). The perspective is the Public Health System. The outcome measurements are Sustained Viral Response (SVR) and costs. PEG-FIRST24 (SVR: 87.8%, costs: USD 8,338.27) was more effective and more costly than PEG-SEC (SVR: 79.2%, costs: USD 5,852.99). The sensitivity analyses are: When SVR rates with IFN was less than 30% PEG-FIRST is dominant. On the other hand, when SVR with IFN was more then 75% PEG-SEC is dominant (SVR=88.2% and costs USD $ 3,753.00). PEG-SEC is also dominant when SVR to PEG24 weeks was less than 54%. In the Brazilian context, PEG-FIRST is more effective and more expensive than PEG-SEC. PEG-SEC could be dominant when rates of IFN therapy are higher than 75% or rates of PEG24 therapy are lower than 54%.

O protocolo brasileiro de tratamento da Hepatite C (2007) recomendava como primeira escolha para pacientes com hepatite C crônica e portadores de genótipo 2 ou 3 o tratamento com interferona alfa (IFN) associada à ribavirina (RBV), por 24 semanas. O objetivo deste estudo é comparar o custo e a efetividade para pacientes com hepatite C crônica e portadores do genótipo 2 ou 3 o uso de peguinterferon (PEG) como primeiro escolha com o PEG como secunda escolha para aqueles que não responderam ao tratamento com IFN. A população alvo compreende pacientes com hepatite C crônica portadores de genótipo 2 ou 3 no Brasil. As intervenções são: PEG-SEC (IFN + RBV por 24 semanas, para os não respondedores e recidivantes tratamento subsequente com PEG + RBV por 48 semanas; PEG-FIRST24 (PEG + RBV por 24 semanas). O tipo de estudo envolvido é Análise de Custo Efetividade. Os dados de efetividade são provenientes de um metanálise de estudos brasileiros e os dados de custo do tratamento de um estudo de custo do contexto brasileiro. A perspectiva é o Sistema Público de Saúde. Os desfechos avaliados foram Resposta Viral Sustentada (RVS) e Custos. PEG-FIRST24 (RVS: 87,8%, costs: USD 8.338,27) foi mais efetivo e apresentou maior custo que PEG-SEC (RVS: 79,2%, custo USD 5.852,99). A análise de sensibilidade demonstrou que PEG-SEC é dominado por PEG-FIRST24 quando RVS com IFN for menor que 30%. Por outro lado, quando RVS com IFN for maior que 75% PEG-SEC é dominante (RVS=88.2% e custo USD $ 3.753,00). PEG-SEC é também dominante quando RVS para PEG24 for menor que 54%. Conclusão: No contexto brasileiro, PEG-FIRST é mais efetivo e mais custoso que PEG-SEC. PEG-SEC poderia ser dominante quando as taxas de RVS do tratamento com IFN forem superiores a 75% ou as taxas de PEG24 forem inferiores a 54%.

MicroRNA-548 down-regulates host antiviral response via direct targeting of IFN-λ1

Interferon (IFN)-mediated pathways are a crucial part of the cellular response against viral infection. Type III IFNs, which include IFN-λ1, 2 and 3, mediate antiviral responses similar to Type I IFNs via a distinct receptor complex. IFN-λ1 is more effective than the other two members. Transcription of IFN-λ1 requires activation of IRF3/7 and nuclear factor-kappa B (NF-κB), similar to the transcriptional mechanism of Type I IFNs. Using reporter assays, we discovered that viral infection induced both IFN-λ1 promoter activity and that of the 3'-untranslated region (UTR), indicating that IFN-λ1 expression is also regulated at the post-transcriptional level. After analysis with microRNA (miRNA) prediction programs and 3'UTR targeting site assays, the miRNA-548 family, including miR-548b-5p, miR-548c-5p, miR-548i, miR-548j, and miR-548n, was identified to target the 3'UTR of IFN-λ1. Further study demonstrated that miRNA-548 mimics down-regulated the expression of IFN-λ1. In contrast, their inhibitors, the complementary RNAs, enhanced the expression of IFN-λ1 and IFN-stimulated genes. Furthermore, miRNA-548 mimics promoted infection by enterovirus-71 (EV71) and vesicular stomatitis virus (VSV), whereas their inhibitors significantly suppressed the replication of EV71 and VSV. Endogenous miRNA-548 levels were suppressed during viral infection. In conclusion, our results suggest that miRNA-548 regulates host antiviral response via direct targeting of IFN-λ1, which may offer a potential candidate for antiviral therapy.

Heat shock cognate 71 (HSC71) regulates cellular antiviral response by impairing formation of VISA aggregates

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.

Calcium/Calmodulin-Dependent Protein Kinase is Involved in the Release of High Mobility Group Box 1 Via the Interferon-beta Signaling Pathway

Previously, we have reported that high mobility group box 1 (HMGB1), a proinflammatory mediator in sepsis, is released via the IFN-beta-mediated JAK/STAT pathway. However, detailed mechanisms are still unclear. In this study, we dissected upstream signaling pathways of HMGB1 release using various molecular biology methods. Here, we found that calcium/calmodulin-dependent protein kinase (CaM kinase, CaMK) is involved in HMGB1 release by regulating IFN-beta production. CaMK inhibitor, STO609, treatment inhibits LPS-induced IFN-beta production, which is correlated with the phosphorylation of interferon regulatory factor 3 (IRF3). Additionally, we show that CaMK-I plays a major role in IFN-beta production although other CaMK members also seem to contribute to this event. Furthermore, the CaMK inhibitor treatment reduced IFN-beta production in a murine endotoxemia. Our results suggest CaMKs contribute to HMGB1 release by enhancing IFN-beta production in sepsis.

Study on type I interferon and phospho-IRF3 in murine liver dendritic cells after intervened by HBV / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To detect the secretions of type I interferon and the expressions of phospho-IRF3 in murine liver dendritic cells intervened by HBV.</p><p><b>METHODS</b>The murine liver dendritic cells were isolated via anti-CD11c microbeads and were incubated in the presence of GM-CSF and IL-4 to induce the DC generation and proliferation in 24-well cell culture plates. HBV virions were isolated via ultracentrifugation and were detected by quantitative Realtime-PCR. The DCs were divided into two groups: one group was cultured with HBV virions for 24 hours, the other group was cultured without HBV as control group. The cells were harvested at Oh, 1h, 2h, 6h and 24h after being stimulated with poly I:C and the expressions of p-IRF3 and the concentration of IFN beta in supernatants were detected with western blot and ELISA respectively.</p><p><b>RESULTS</b>The IFN beta concentrations at 0 h, 6 h and 24 h in the supernatants of the HBV group and the control group were (12.38 +/- 3.71) pg/ml, (88.67 +/- 9.01) pg/ml and (69.89 +/- 5.80) pg/ml vs (10.83 +/- 4.11) pg/ml, (137.68 +/- 12.28) pg/ml and (72.25 +/- 8.61) pg/ml, respectively. No statistical differences found at 0 h (t = 0.8398, P > 0.05) and 24 h (t = 0.6820, P > 0.05) between the two groups except that at 6 h (t = 9.653, P < 0.01). The expressions of phospho-IRF3 in HBV group were lower than that in control group.</p><p><b>CONCLUSIONS</b>The type I interferon secretion and the phospho-IRF3 expression were decreased in murine liver dendritic cells when intervened by HBV.</p>

Retinoic acid inducible gene-I, more than a virus sensor

Retinoic acid inducible gene-I (RIG-I) is a caspase recruitment domain (CARD) containing protein that acts as an intracellular RNA receptor and senses virus infection. After binding to double stranded RNA (dsRNA) or 5'-triphosphate single stranded RNA (ssRNA), RIG-I transforms into an open conformation, translocates onto mitochondria, and interacts with the downstream adaptor mitochondrial antiviral signaling (MAVS) to induce the production of type I interferon and inflammatory factors via IRF3/7 and NF-κB pathways, respectively. Recently, accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events, such as inflammation and inflammation related diseases, cell proliferation, apoptosis and even senescence. Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.

Ribosomal Protein L19 and L22 Modulate TLR3 Signaling

BACKGROUND: Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) and induces inflammation. In this study we attempted to ascertain if there are endogenous host molecules controlling the production of cytokines and chemokines. Two candidates, ribosomal protein L19 and L22, were analyzed to determine if they influence cytokine production followed by TLR3 activation. In this study we report that L19 acts upon production of IP-10 or IL-8 differently in glioblastoma cells. METHODS: L19 or L22 was transfected into HEK293-TLR3, A549 or A172 cells. After treatment with several inhibitors of NF-kB, PI3K, p38 or ERK, production of IL-8 or IP-10 was measured by ELISA. siRNA was introduced to suppress expression of L19. After Vesicular stomatitis virus infection, viral multiplication was measured by western blot. RESULTS: L19 increased ERK activation to produce IL-8. In A172 cells, in which TLR3 is expressed at endosomes, L19 inhibited interferon regulatory factor 3 (IRF3) activation and IP-10 production to facilitate viral multiplication, whereas L19 inhibited viral multiplication in A549 cells bearing TLR3 on their cell membrane. CONCLUSION: Our results suggest that L19 regulates TLR3 signaling, which is cell type specific and may be involved in pathogenesis of autoimmune diseases and chronic inflammatory diseases.

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.

Liver X receptor alpha inhibits LPS-induced inflammation by down-regulating IFR3 and GRIP1 in mouse Kupffer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the possible mechanism of the inhibitory effect of liver X receptor alpha (LXRalpha) on lipopolysaccharide (LPS)-induced inflammation in mouse Kupffer cells (KCs).</p><p><b>METHODS</b>The KCs isolated from the liver of male KM mice and cultured in RPMI 1640 containing 20% FBS for 24 h were divided into control, LPS, T0901317, and LPS+T0901317 groups with corresponding treatments. The expressions of LXRalpha, interferon regulatory factor 3 (IRF3) and glucocorticoid receptor interacting protein 1 (GRIP1) in the KCs were detected by Western blotting. The levels of interferon beta (IFNbeta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The level of LXRalpha protein was highest in T0901317 group and lowest in LPS group, and was significantly higher in LPS+T0901317 group than in LPS group but lower than in T0901317 group (P<0.05). The levels of IRF3 and GRIP1 protein were the highest in LPS group, and significantly lowered by T0901317 treatment (P<0.05). The expression of IRF3 and GRIP1 proteins in LPS group and LPS+ T0901317 group were significantly higher than those in the control and T0901317 groups (P<0.05). The concentration of IFN-beta was significantly higher in LPS group than in the control and T0901317 group (P<0.05), and decreased in LPS+T0901317 group in comparison with that in LPS group (P<0.05). IFN-beta was the lowest in T0901317 group. The levels of TNF-alpha and IL-1beta were the highest in LPS group (P<0.05), and comparable between the other 3 groups (P>0.05).</p><p><b>CONCLUSION</b>Pre-treatment with T0901317 before LPS stimulation can suppress the expressions of IRF3 and GRIP1 to inhibit the inflammation and hence Kupffer cell activation.</p>

Interferon-Stimulated Genes Response in Endothelial Cells Following Hantaan Virus Infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta , IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta??(3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.