The host type I interferon response to viral and bacterial infections

Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNs are produced following viral infections, but until recently, the mechanisms of viral recognition leading to IFN production were largely unknown. Toll like receptors (TLRs) have emerged as key transducers of type I IFN during viral infections by recognizing various viral components. Furthermore, much progress has been made in defining the signaling pathways downstream of TLRs for type I IFN production. TLR7 and TLR9 have become apparent as universally important in inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells. New intracellular viral pattern recognition receptors leading to type I IFN production have been identified. Many bacteria can also induce the up-regulation of these cytokines. Interestingly, recent studies have found a detrimental effect on host cells if type Ⅰ IFN is produced during infection with the intracellular gram-positive bacterial pathogen, Listeria monocytogenes. This review will discuss the recent advances made in defining the signaling pathways leading to type I IFN production.     

猪I型与II型干扰素的克隆、表达及抗病毒活性比较

干扰素(IFN)是由多种细胞受病毒感染或其他生物诱导剂刺激而产生的天然蛋白质,主要功能为抗病毒增殖、调节免疫反应和激活免疫细胞等。本研究克隆并测序了猪干扰素(PoIFN)α、γ、αγ及ω基因。构建原核表达载体pET-His/PoIFN-α、pET-His/PoIFN-γ、pET-His/PoIFN-αγ和pET-His/PoIFN-ω,转化大肠杆菌Rosetta(DE3)进行表达,经纯化、复性得到具有生物学活性的蛋白。用细胞病变抑制法在Marc-145/PRRSV、Marc-145/VSV、PK-15/VSV、Vero/VSV、MDBK/VSV系统上进行抗病毒活性测定,结果表明猪α和αγ融合干扰素有较为显著的抗病毒活性,抗PRRSV活性高达108U/mg;猪γ干扰素活性效价约为α干扰素的1/2到1/3;猪ω干扰素几乎未检测到抗病毒活性,需进一步验证。本研究对干扰素在抗病毒、提高机体免疫方面的应用提供了理论依据。     

The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates embryonic development and tissue homeostasis; however, aberrations of its activity occur in cancer. TGF-beta signals through its Type II and Type I receptors (TbetaRII and TbetaRI) causing phosphorylation of Smad proteins. TGF-beta-associated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, was originally identified as an effector of TGF-beta-induced p38 activation. However, the molecular mechanisms for its activation are unknown. Here we report that the ubiquitin ligase (E3) TRAF6 interacts with a consensus motif present in TbetaRI. The TbetaRI-TRAF6 interaction is required for TGF-beta-induced autoubiquitylation of TRAF6 and subsequent activation of the TAK1-p38/JNK pathway, which leads to apoptosis. TbetaRI kinase activity is required for activation of the canonical Smad pathway, whereas E3 activity of TRAF6 regulates the activation of TAK1 in a receptor kinase-independent manner. Intriguingly, TGF-beta-induced TRAF6-mediated Lys 63-linked polyubiquitylation of TAK1 Lys 34 correlates with TAK1 activation. Our data show that TGF-beta specifically activates TAK1 through interaction of TbetaRI with TRAF6, whereas activation of Smad2 is not dependent on TRAF6.     

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.     

NMR backbone dynamics of the human type I interferon binding subunit, a representative cytokine receptor

The antiviral and antiproliferative activities of type I interferons (IFNs) are mediated by a common receptor, and its second subunit (IFNAR2) exhibits nanomolar affinity to both IFNalpha and IFNbeta subtypes. We have previously determined the structure of the IFN-binding extracellular domain of IFNAR2 (IFNAR2-EC) using multidimensional NMR [Chill, J. H., Quadt, S. R., Levy, R., Schreiber, G. E., and Anglister, J. (2003) Structure 11, 791-802], showing it to comprise two fibronectin domains linked by a hinge. As the first cytokine receptor structure determined in the unliganded state and in solution, IFNAR2-EC offers an opportunity to characterize the dynamics of the cytokine receptor family and their correlation to biological function. Backbone dynamics of IFNAR2-EC were investigated using 15N relaxation at 11.74 and 18.79 T, and measurements of residual dipolar couplings (RDCs). Dynamics of the binding site distinguish between rigid structural domains, which stabilize the binding site conformation, and a more flexible binding interface which interacts with the ligand. Measurements of diffusional anisotropy and RDCs and model-free analysis all show that the backbone of the hinge interdomain region of IFNAR2-EC is rigid on the picosecond to nanosecond time scale. Signal transduction in cytokines receptors is initiated by ligand-induced juxtaposition of the two receptor subunits, triggering the mutual phosphorylation of kinases associated to their cytoplasmic domains. The rigidity of the hinge ensures correct positioning of the receptor subunits in the ternary signaling complex and modulates the interaction between kinases in the cytoplasm, thereby controlling the rate and efficiency of phosphorylation.     

The role of cell membrane in the antiviral effect of interferon

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I · C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells. In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.     

The role of TRAF6 in signal transduction and the immune response

Signals from the IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily are critical for regulating the function of antigen-presenting cells such as dendritic cells (DCs). It has been revealed that TNF receptor-associated factor 6 (TRAF6), a signaling adapter molecule common to the IL-1R/TLR family and TNFR superfamily, is important not only for DC maturation, cytokine production, and T cell stimulatory capacity of DCs in response to TLR ligands (e.g. lipopolysaccharide) or CD40 ligand, but also for the homeostasis of splenic DC subsets.     

Ligand-induced association of the type I interferon receptor components.

Two transmembrane polypeptides, IFNAR and IFN-alpha/Beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein. Host cells expressing IFN-alpha/beta R bound IFN-alpha 2 with a high affinity (Kd of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha 2 was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha 2 to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.     

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.     

The role of type I interferon production by dendritic cells in host defense

Type I interferons (IFN) and dendritic cells (DC) share an overlapping history, with rapidly accumulating evidence for vital roles for both production of type 1 IFN by DC and the interaction of this IFN both with DC and components of the innate and adaptive immune responses. Within the innate immune response, the plasmacytoid DC (pDC) are the "professional" IFN producing cells, expressing specialized toll-like receptors (TLR7 and -9) and high constitutive expression of IRF-7 that allow them to respond to viruses with rapid and extremely robust IFN production; following activation and production of IFN, the pDC subsequently mature into antigen presenting cells that help to shape the adaptive immune response. However, like most cells in the body, the myeloid or conventional DC (mDC or cDC) also produce type I IFNs, albeit typically at a lower level than that observed with pDC, and this IFN is also important in innate and adaptive immunity induced by these classic antigen presenting cells. These two major DC subsets and their IFN products interact both with each other as well as with NK cells, monocytes, T helper cells, T cytotoxic cells, T regulatory cells and B cells to orchestrate the early immune response. This review discusses some of the converging history of DC and IFN as well as mechanisms for IFN induction in DC and the effects of this IFN on the developing immune response.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.