Paula M. Pitha

LPS-TLR4 Signaling to IRF-3/7 and NF-κB Involves the Toll Adapters TRAM and TRIF

Toll–IL-1–resistance (TIR) domain–containing adaptor-inducing IFN-β (TRIF)–related adaptor molecule (TRAM) is the fourth TIR domain–containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-κB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-α/β, regulated on activation, normal T cell expressed and secreted (RANTES), and γ interferon–inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor–like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

ABSTRACT The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-α/β) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor α and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-κB. However, TLR8 activation by R-848 and TLR2 activation by {S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)} were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

The growing family of interferon regulatory factors

Interferons (IFN) exert their multiple biological effects through the induction of expression of over 30 genes encoding proteins with antiviral, antiproliferative and immunomodulatory functions. Among the many IFN-inducible proteins are the Interferon Regulatory Factors (IRFs), a family of transcription regulators, originally consisting of the well-characterized IRF-1 and IRF-2 proteins; the family has now expanded to over 10 members and is still growing. The present review provides a detailed description of recently characterized IRF family members. Studies analyzing IRF-expressing cell lines and IRF knockout mice reveal that each member of the IRF family exerts distinct roles in biological processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. Understanding the molecular mechanisms by which the IRFs affect these important cellular events and IFN expression will contribute to a greater understanding of events leading to various viral, immune and malignant disease states and will suggest novel strategies for antiviral and immune modulatory therapy.

Cutting Edge: Activation of Toll-Like Receptor 2 Induces a Th2 Immune Response and Promotes Experimental Asthma

Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1β, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNα, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.

Virus-specific Activation of a Novel Interferon Regulatory Factor, IRF-5, Results in the Induction of Distinct Interferon α Genes

Interferon regulatory factor (IRF) genes encode DNA-binding proteins that are involved in the innate immune response to infection. Two of these proteins, IRF-3 and IRF-7, serve as direct transducers of virus-mediated signaling and play critical roles in the induction of type I interferon genes. We have now shown that another factor, IRF-5, participates in the induction of interferon A (IFNA) and IFNB genes and can replace the requirement for IRF-7 in the induction of IFNA genes. We demonstrate that, despite the functional similarity, IRF-5 possesses unique characteristics and does not have a redundant role. Thus, 1) activation of IRF-5 by phosphorylation is virus-specific, and its in vivo association with the IFNA promoter can be detected only in cells infected with NDV, not Sendai virus, while both viruses activate IRF-3 and IRF-7, and 2) NDV infection of IRF-5-overexpressing cells preferentially induced the IFNA8 subtype, while IFNA1 was primarily induced in IRF-7 expressing cells. These data indicate that multiple signaling pathways induced by infection may be differentially recognized by members of the IRF family and modulate transcription of individual IFNA genes in a virus and cell type-specific manner.

The Interferon Regulatory Factor, IRF5, Is a Central Mediator of Toll-like Receptor 7 Signaling

Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF3 and IRF7 are activated in response to a variety of viruses or after engagement of Toll-like receptor (TLR) 3 and TLR4 by double-stranded RNA and lipopolysaccharide, respectively. The activation of IRF5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, interleukin 1 receptor-associated kinase 1, and tumor necrosis factor receptor-associated factor 6 are required for the activation of IRF5 and IRF7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF5 enabled type I interferon production in response to TLR7 signaling, whereas knockdown of IRF5 by small interfering RNA reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF5 and IRF7, therefore, emerge from these studies as critical mediators of TLR7 signaling.

Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte‐derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV‐1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV‐1. All three populations constitutively expressed most of the IRF genes. However, real‐time RT‐PCR demonstrated increased levels of IRF‐7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF‐7 protein than the other populations while IRF‐3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus‐stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus‐dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN‐α production due to the high levels of constitutively expressed IRF‐7.

Review: On the Role of IRF in Host Defense

Transcription factors of the interferon (IFN) regulatory factor (IRF) family have been shown to play an essential role in the regulated expression of type I IFN genes, IFN-stimulated genes (ISG), and other cytokines and chemokines. Three members of the IRF family, IRF-3, IRF-5, and IRF-7, have been identified as acting as direct transducers of virus-mediated signaling. In infected cells, these factors are activated by phosphorylation on the serine residues, transported to the nucleus, where they bind to the promoters of IFNA and IFNB genes and tether histone transacetylases to the transcription complex enhanceosome. IFNB and IFNA subtypes are expressed at different levels in infected cells. The ratio between the relative levels of IRF-3 and IRF-7 was shown to play an essential role in the inducible expression of type I IFN genes, whereas IRF-3 alone is sufficient for expression of the IFNB gene. IRF-5 was identified recently as another inducer of IFNA genes, which has two unique properties: (1) its activation is virus specific, and (2) the profile of IFNA genes induced by IRF-5 is distinct from that induced by IRF-7. Several viruses target functions of IRF to eliminate the early inflammatory response. Kaposis sarcoma herpesvirus (KSHV) encodes a cluster of four genes with homology to cellular IRF. Three of these vIRF were shown to inhibit induction of IFN genes and ISG in infected cells and function as dominant negative mutants of cellular IRF. The unique properties of previously uncharacterized vIRF-2 and vIRF-3 are discussed.

Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15

The goal of this study was to elucidate the molecular mechanism by which type I IFN inhibits assembly and release of HIV-1 virions. Our study revealed that the IFN-induced ubiquitin-like protein ISG15 mimics the IFN effect and inhibits release of HIV-1 virions without having any effect on the synthesis of HIV-1 proteins in the cells. ISG15 expression specifically inhibited ubiquitination of Gag and Tsg101 and disrupted the interaction of the Gag L domain with Tsg101, but conjugation of ISG15 to Gag or Tsg101 was not detected. The inhibition of Gag-Tsg101 interaction was also detected in HIV-1 infected, IFN-treated cells. Elimination of ISG15 expression by small interfering RNA reversed the IFN-mediated inhibition of HIV-1 replication and release of virions. These results indicated a critical role for ISG15 in the IFN-mediated inhibition of late stages of HIV-1 assembly and release and pointed to a mechanism by which the innate antiviral response targets the cellular endosomal trafficking pathway used by HIV-1 to exit the cell. Identification of ISG15 as the critical component in IFN-mediated inhibition of HIV-1 release advances the understanding of the IFN-mediated inhibition of HIV-1 replication and uncovers a target for the anti HIV-1 therapy.