Tsuneyasu Kaisho

Toll-Like Receptors

The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

Toll-like receptors: critical proteins linking innate and acquired immunity.

Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R–associated protein kinase and tumor necrosis factor receptor–activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.

Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway

The imidazoquinoline compounds imiquimod and R-848 are low-molecular-weight immune response modifiers that can induce the synthesis of interferon-α and other cytokines in a variety of cell types. These compounds have potent anti-viral and anti-tumor properties; however, the mechanisms by which they exert their anti-viral activities remain unclear. Here we show that the imidazoquinolines activate immune cells via the Toll-like receptor 7 (TLR7)-MyD88–dependent signaling pathway. In response to the imidazoquinolines, neither MyD88- nor TLR7-deficient mice showed any inflammatory cytokine production by macrophages, proliferation of splenocytes or maturation of dendritic cells. Imidazoquinoline-induced signaling events were also abolished in both MyD88- and TLR7-deficient mice.

Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4.

Signal transduction through Toll-like receptors (TLRs) originates from their intracellular Toll/interleukin-1 receptor (TIR) domain, which binds to MyD88, a common adaptor protein containing a TIR domain. Although cytokine production is completely abolished in MyD88-deficient mice, some responses to lipopolysaccharide (LPS), including the induction of interferon-inducible genes and the maturation of dendritic cells, are still observed. Another adaptor, TIRAP (also known as Mal), has been cloned as a molecule that specifically associates with TLR4 and thus may be responsible for the MyD88-independent response. Here we report that LPS-induced splenocyte proliferation and cytokine production are abolished in mice lacking TIRAP. As in MyD88-deficient mice, LPS activation of the nuclear factor NF-κB and mitogen-activated protein kinases is induced with delayed kinetics in TIRAP-deficient mice. Expression of interferon-inducible genes and the maturation of dendritic cells is observed in these mice; they also show defective response to TLR2 ligands, but not to stimuli that activate TLR3, TLR7 or TLR9. In contrast to previous suggestions, our results show that TIRAP is not specific to TLR4 signalling and does not participate in the MyD88-independent pathway. Instead, TIRAP has a crucial role in the MyD88-dependent signalling pathway shared by TLR2 and TLR4.

TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll–interleukin 1 receptor (TIR) domain–containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain–containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-β production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.

Stat3 Activation Is Responsible for IL-6-Dependent T Cell Proliferation Through Preventing Apoptosis: Generation and Characterization of T Cell-Specific Stat3-Deficient Mice

Stat3, a member of STAT, is activated by a variety of cytokines such as IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, and leptin. A recent study with mice genetically deficient in the Stat3 gene has revealed its important role in the early embryogenesis. To assess the function of Stat3 in adult tissues, we disrupted the Stat3 gene specifically in T cells by conditional gene targeting using Cre-loxP system. In Stat3-deficient T cells, IL-6-induced proliferation was severely impaired. IL-6 did not enhance cell cycle progression, but prevented apoptosis of normal T cells. In contrast, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Antiapoptotic protein, Bcl-2, was normally up-regulated in response to IL-6 even in Stat3-deficient T cells. These results demonstrate that Stat3 activation is involved in IL-6-dependent T cell proliferation through prevention of apoptosis independently of Bcl-2.

Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8α+ DC correlates with unresponsiveness to imidazoquinolines

Toll‐like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub‐types, including PDC, but that TLR3 is preferentially expressed by CD8α+ DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8α– DC and PDC, but not CD8α+ DC as measured by survival ex vivo, up‐regulation of surface markers and production of IL‐12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non‐plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8α+ DC in recognition of certain mouse pathogens.

Endotoxin-Induced Maturation of MyD88-Deficient Dendritic Cells

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-κB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not maturate in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c+ DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstrem of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.

T Cell-Specific Loss of Pten Leads to Defects in Central and Peripheral Tolerance

PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.

Toll-like receptors as adjuvant receptors

The mammalian Toll-like receptors (TLRs) are expressed on macrophages and dendritic cells, which are primarily involved in innate immunity. At present, ligands for several of the TLRs, such as TLR2, TLR3, TLR4, TLR5, TLR6, and TLR9, have been identified. Most of these ligands are derived from pathogens, but not found in the host, suggesting that the TLRs are critical to sensing invading microorganisms. Pathogen recognition by TLRs provokes rapid activation of innate immunity by inducing production of proinflammatory cytokines and upregulation of costimulatory molecules. Activated innate immunity subsequently leads to effective adaptive immunity. In this regard, the TLRs are considered to be adjuvant receptors. Distinct TLRs can exert distinct, but overlapping sets of biological effects. Accumulating evidence indicates that this can be attributed to both the common and unique aspects of the signaling mechanisms that mediate TLR family responses. For example, TLR2 and TLR9 require MyD88 as an essential signal transducer, whereas TLR4 can induce costimulatory molecule upregulation in a MyD88-independent manner. Understanding the TLR system should offer invaluable opportunity for manipulating host immune responses.