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Laboratory for Host Defense Host defense in mammals consists of innate and adaptive immunity. Innate immunity functions as a hard wired pathogen sensor and eradicator. Furthermore, innate immunity contributes both to the establishment and the features of an adaptive immune response. Dendritic cells (DCs) are antigen presenting cells critically involved in regulating these immune responses. DCs sense various pathogen-derived molecules and exert their immunostimulatory functions by producing inflammatory cytokines and/or upregulating expression of costimulatory molecules. The pathogen-derived components, termed immune adjuvants based on their DC activating abilities, are recognized by various types of pattern recognition receptors including Toll-like receptors (TLRs). Identification of new types of immune adjuvants and characterization of the mechanisms by which they activate DCs should contribute to development of novel immunoregulatory strategies. We are attempting to clarify how DCs are activated through pattern recognition receptors and to obtain essential information for effectively manipulating the immune response. Various immune adjuvants, TLR ligands, and gene targeted mice are important tools for this purpose. Team leader Tsuneyasu Kaisho Research Scientists : Katsuaki Hoshino, Takashi Tanaka Technical Staff : Yuri Fukuda, Emiri Haga, Nana Iwami, Miku Kawada, Nahoko Okita Student Trainees Assistant : Masuyoshi Saito, Izumi Sasaki, Takahiro Sugiyama, Chihiro Yamazaki, Takahiro Yano : Sachiko Haraguchi Immunoadjuvant effects of poly (A:U) A variety of immune adjuvants, which can be broadly categorized as lipids, proteins, or nucleic acids, can exert their own unique functions. Poly(I:C) is a synthetic mimic of double-stranded RNA (dsRNA) and a known ligand for TLR3 and cytosolic sensors. Another type of dsRNA, poly(A:U), can also act as an immune adjuvant, but it has been unclear how it exhibits its adjuvant effects. We have evaluated the effects of poly(A:U) on a various types of DCs from several gene targeted mice. Poly(A:U) could induce production of both IFN-α and IL-12p40 by murine bone marrow (BM) DCs. Poly(A:U)-induced IFN-α production depended on a particular DC subset, the plasmacytoid DC (pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7-dependent manner. In addition to pDC, conventional DC (cDC) also produced IL-12p40 in response to poly(A:U). This IL- 12p40 was derived from two cDC subsets, CD24 high and CD11b high , in a TLR3- and TLR7- dependent manner, respectively. Furthermore, in vivo injection of poly(A:U) with antigen led to CD8 + T cell responses, which were shown to 48
esult in clonal expansion and IFN-γ production by antigen-specific CD8 + T cells. Consistent with the previous in vitro findings, TLR3 and TLR7 were both required for the clonal T-cell expansion. Notably, TLR3 was more critical than TLR7, for generating IFN-γ-producing CD8 + T cells. Interestingly, type I IFNs, which are thought to play a major role in the generation of CD8 + T-cell responses, were dispensable for responses induced by poly(A:U). Our results demonstrate that poly(A:U) is an in vivo and in vitro immunoadjuvant functioning mainly through TLR3 and TLR7. The next important issue is to clarify the mechanisms by which TLR3 signaling induces CD8 + T-cell responses The regulation of innate immune responses A nuclear ubiquitin E3 ligase, PDLIM2/ SLIM, interacts with STAT4, the transcription factor essential for IL-12-mediated T-helper 1 (Th1) cell differentiation (Tanaka et al. Immunity 22, 729-736, 2005). PDLIM2 contains a PDZ domain at the N-terminus and a LIM domain at the C-terminus and belongs to a large family of LIM proteins. In CD4 + T cells, PDLIM2 can interact with STAT proteins in the nucleus and promote their polyubiquitination and subsequent proteasomal degradation. Thus, PDLIM2 negatively regulates STAT-dependent signaling. Consistent with these observations, Figure 1 Molecular mechanisms of poly(A:U)-induced adjuvant effects. Murine BM DCs can be generated in the presence of Flt3L or GM-CSF. Flt3L-induced BM DCs contain both pDC and cDC, the latter of which can be further divided into two subsets. By contrast, GM-CSF-induced BM DCs contain only cDC. Notably, all of these DC subsets are distinct in the expression pattern of and responsiveness to TLRs and cytosolic sensors. poly(A:U) fails to activate cytosolic sensors but can stimulate DCs through TLR3 or TLR7. Th1 cells from PDLIM2-deficient mice have increased levels of STAT protein and enhanced IFN-γ production Activation of the NF-κB transcription factor in innate immune cells is also tightly regulated to prevent excessive inflammatory responses. How NF-κB activation is terminated, however, is not fully understood. After stimulation with various TLR ligands, NF-κB activation is first upregulated, then downregulated and terminated. During the downregulating phase, an NF-kB subunit, p65, is ubiquitinated and degraded in the nucleus. Expression of PDLIM2 suppressed NF-κB activity. PDLIM2 bound to p65 and promoted p65 polyubiquitination in the nucleus. In addition, PDLIM2 targeted polyubiquitinated p65 to discrete intranuclear compartments where it was degraded in a proteasome-dependent manner. This intranuclear translocation of p65 depended on the PDZ domain of PDLIM2. Furthermore, PDLIM2-deficiency led to defective p65 ubiquitination and an increase in nuclear p65 levels. Consistent with these findings, PDLIM2- deficient DCs showed enhanced production of proinflammatory cytokines in response to TLR4 or TLR9 signaling and the mutant mice were hypersensitive to endotoxin shock. Our findings define a pathway by which PDLIM2 terminates NF-κB activation through nuclear sequestration and subsequent degradation. Recent publications K. Hoshino, T. Sugiyama, M. Matsumoto, T. Tanaka, M. Saito, H. Hemmi, O. Ohara, S. Akira, T. Kaisho. 2006. IκB kinase-α is critical for interferon-α production induced by Toll-like receptors 7 and 9. Nature. 440, 949-953 (2006). D. Kinoshita, F. Hirota, T. Kaisho, M. Kasai, K. Izumi, Y. Bando, Y. Mouri, A. Matsushima, S. Niki, H. Han, K. Oshikawa, N. Kuroda, M. Maegawa, M. Irahara, K. Takeda, S. Akira, M. Matsumoto. 2006. Essential role of IκB kinase α in thymic organogenesis required for the establishment of self-tolerance. J. Immunol. 176, 3995-4002 (2006). T. Kaisho, S. Akira. 2006. TLR function and signaling. J. Allergy Clin. Immunol. 117, 979-987 (2006). T. Tanaka, M. J. Grusby and T. Kaisho. PDLIM2-mediated termination of transcription factor NF-κB activation by intranuclear sequestration and degradation of the p65 subunit. Nature Immunol. 8, 584-591 (2007). T. Sugiyama, K. Hoshino, M. Saito, T. Yano, I. Sasaki, C. Yamazaki , S. Akira and T. Kaisho. Immunoadjubant effects of polyadenylic-polyuridylic acids through TLR3 and TLR7. Int. Immunol. 20, 1-9 (2008). Figure 2 PDLIM2-mediated regulation of NF-κB activity. PDLIM2 can bind and polyubiquitinate the p65 subunit of NF-κB in the nucleus. The p65 is then degraded in specialized nuclear regions in a proteasome-dependent manner. 49
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