Paul W. Dempsey

IRF3 Mediates a TLR3/TLR4-Specific Antiviral Gene Program

We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

The signaling adaptors and pathways activated by TNF superfamily

Members of the TNF receptor superfamily play pivotal roles in numerous biological events in metazoan organisms. Ligand-mediated trimerization by corresponding homo- or heterotrimeric ligands, the TNF family ligands, causes recruitment of several intracellular adaptors, which activate multiple signal transduction pathways. While recruitment of death domain (DD) containing adaptors such as Fas associated death domain (FADD) and TNFR associated DD (TRADD) can lead to the activation of a signal transduction pathway that induces apoptosis, recruitment of TRAF family proteins can lead to the activation of transcription factors such as, NF-kappaB and JNK thereby promoting cell survival and differentiation as well as immune and inflammatory responses. Individual TNF receptors are expressed in different cell types and have a range of affinities for various intracellular adaptors, which provide tremendous signaling and biological specificities. In addition, numerous signaling modulators are involved in regulating activities of signal transduction pathways downstream of receptors in this superfamily. Most of the TNF receptor superfamily members as well as many of their signaling mediators, have been uncovered in the last two decades. However, much remains unknown about how individual signal transduction pathways are regulated upon activation by any particular TNF receptor, under physiological conditions.

Activation of noncanonical NF-κB requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2, TRAF3 and the kinase NIK

Recent studies suggest that nuclear factor κB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-κB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

Involvement of receptor-interacting protein 2 in innate and adaptive immune responses

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-κB, whereas dominant negative Rip2 inhibited NF-κB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-γ production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.

PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1−/− mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-γ- or IFN-β-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-γ or IFN-β was consistently enhanced by Pias1 disruption. Pias1−/− mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-γ- or IFN-β-mediated innate immune responses.

Rescue of TRAF3-null mice by p100 NF-κB deficiency

Proper activation of nuclear factor (NF)–κB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-κB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-κB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-κB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280–288). We demonstrate that loss of tumor necrosis factor (TNF) receptor–associated factor 3 (TRAF3) results in constitutive noncanonical NF-κB activity. Importantly, TRAF3−/− B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-κB–inducing kinase in TRAF3−/− cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-κB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-κB pathway and that constitutive activation of the noncanonical NF-κB pathway causes the lethal phenotype of TRAF3-deficient mice.

The NOD/RIP2 Pathway Is Essential for Host Defenses Against Chlamydophila pneumoniae Lung Infection

Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2−/− mice compared to wild-type (WT) mice at day 3. Rip2−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1−/− and Nod2−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.

The art of war: Innate and adaptive immune responses.

Research over the last several years has greatly advanced our understanding of the mechanisms by which the immune system functions. There exist two main branches of immunity, termed innate and adaptive immunity. Innate immunity uses the genetic memory of germline-encoded receptors to recognize the molecular patterns of common pathogens. Adaptive immunity, akin to somatic memory, is a complex system by which the body learns to recognize a pathogen’s unique antigens and builds an antigen specific response to destroy it. The effective development of the overall immune response depends on careful interplay and regulation between innate and adaptive immunity. Here we review our current understanding of how these integrated systems distinguish targets against which a response is appropriate and neutralize potentially pathogenic challenges.

Toll-like Receptor 3-Mediated Suppression of TRAMP Prostate Cancer Shows the Critical Role of Type I Interferons in Tumor Immune Surveillance

Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.