Toll-like Receptors and Innate Immunity

Shizuo Akirasakira@biken.osaka-u.ac.jp    Department of Host Defense, Research Institute for Microbial Diseases, Osaka University; CREST of Japan Science and Technology Corporation, Osaka, Japan 565-0871

I Introduction

Immunity in higher organisms can be broadly categorized into adaptive immunity and innate immunity. Adaptive immunity is mediated by clonally distributed T and B lymphocytes which provide immunological specificity and memory. In contrast, innate immunity is mediated by the action of other cells such as macrophages and neutrophils, and traditionally has been characterized as nonspecific. However, recent findings have shown that innate immunity has some capacity for specific recognition. For example, insects, which lack the high level of immune specificity and memory characteristics of vertebrates, can nevertheless combat the invasion of microbes rapidly and efficiently by producing specific antimicrobial peptides. The synthesis of these specific antimicrobial peptides is triggered by the differential activation of different Toll family receptors present on the cell surface. More recently, various studies have demonstrated that many aspects of innate immune systems are shared between insects and mammals, and indeed have shown that such systems play a crucial role in the immune response in higher organisms such as mammals (Hoffmann et al., 1999). Microbial invasion in mammals is first handled by the innate immune system, which is designed to control and eventually resolve the infection. The cells of this system are not only responsible for the first-line mechanism of bacterial clearance, but also play an instructive role in adaptive immunity through the action of soluble factors or costimulatory signals (Fearon and Locksley, 1996). Janeway (1992) has hypothesized that recognition of a pathogen is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors would recognize conserved molecular patterns (pathogen-associated molecular patterns [PAMPs]) shared by large groups of microorganisms. Recognition of these patterns would allow the innate immune system not only to detect the presence of an infectious microbe, but also to determine the type of the infecting pathogen. Recently, the family of Toll-like receptors has been found to be present in mammals and to function in a manner similar to the PRRs envisioned by Janeway (1992). I will discuss the role of a group of the Toll receptors, which are phylogenically conserved mediators of innate immunity essential for microbial recognition.

II Toll Receptors in Drosophila Development

Toll was originally identified as a transmembrane receptor that is critical to the determination of dorsoventral polarity in Drosophila embryo (Anderson et al., 1985; Belvin and Anderson, 1996). Toll is characterized by the presence of a leucine-rich repeat (LRR) in the extracellular domain and homology within the cytoplasmic domain to the interleukin-1 receptor (IL-1R) cytoplasmic domain. Stimulation of Toll activates the Rel family transcription factor, Dorsal, by inducing the degradation of cactus, via a process that involves the adaptor protein Tube and the serine-threonine protein kinase Pelle.

Toll is expressed over the entire surface of the embryo, but is activated only in the ventral region during early embryogenesis by interaction with Spatzel, a specially restricted, extracellular ligand. Spatzel is a member of the cysteine-knot family of growth factors and cytokine-like proteins (Mizuguchi et al., 1998). Production of Spatzel requires activation by an extracellular serine protease cascade that is confined to the ventral side of the embryo. Spatzel is cleaved by the protease, Easter, which in turn is activated by another protease known as Snake (Anderson, 1998). This activation of the Spatzel–Toll pathway in the ventral region results in the generation of a nuclear gradient of Dorsal along the dorsovental axis of the embryo. This gradient regulates the localized expression of a set of zygotic genes that in turn specify ventral and dorsal fate. Certain dominant mutant alleles of Toll encode proteins that behave as partially ligand-independent receptors, and embyos expressing these proteins become ventralized. Toll is required for proper motoneuron and muscle specification in the late embryonic stage.

III Toll Receptors in Innate Immunity of Drosophila

The Toll pathway also controls the production of potent antifungal peptides in the adult fly. In microbial infection of Drosophila, a battery of antimicrobial peptides are rapidly synthesized in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. Antimicrobial peptides are categorized into two groups: one group consists of several antibacterial peptides including cecropin, diptericin, drosocin, attacin, and insect defensin, and the other group consists of the major antifungal peptide, drosomycin (Hultmark, 1993; Hoffmann et al., 1996). The genes encoding these antibacterial and antifungal peptides are differentially expressed following infection by distinct microorganisms. Drosophila that are infected by insect-pathogenic fungi produce only peptides with antifungal activities as a result of the selective activation of the Toll pathway. Toll-deficient flies are defective in the induction of Drosomycin, and are poorly resistant to fungal infection (Lemaitre et al., 1996). The Toll pathway is also involved in the control of some of the antibacterial peptide genes (e.g., cecropin and attacin). However, the role of Dorsal in regulating the antimicrobial response is unclear, in contrast to its well-established and essential role in the regulation of the dorsoventral target genes. Indeed, the drosomycin gene remains fully inducible following immune challenge in Dorsal-deficient mutants, indicating that the control of antimicrobial peptide genes is redundant and that other Rel proteins can substitute for Dorsal in Dorsal-deficient mutants. In addition to Dorsal, Drosophila expresses two other Rel family proteins, Dif (for Dorsal-related immunity factor) and Relish (Ip et al., 1993; Dushay et al., 1996). Recently, Dif has been shown to act downstream in the Toll signaling pathway in induction of drosomycin (Meng et al., 1999; Manfruelli et al., 1999; Han and Ip, 1999; Rutschmann et al., 2000). Relish mutants are completely defective for the induction of all antimicrobial peptides and are more susceptible to fungal infection than wild-type Drosophila. Taken together, these observations indicate that Dorsal is essential to the regulation of the dorsoventral target genes, whereas the two other Rel transcription factors Dif and Relish apparently act downstream in the Toll signaling pathway for the induction of drosomycin.

Another Drosophila gene, 18-wheeler (18 W), exhibits homology to Toll, having both the intracellular IL-1R-like domain and the extracellular LRRs. 18 W is required for Drosophila morphogenesis and is thought to function as a cell adhesion or receptor molecule that faciliates cell movements. 18 W has also been shown to be involved in the host defense in Drosophila, since embryos carrying a mutated 18 W gene are compromised in their antibacterial response (Williams et al., 1997). In 18 W mutants, induction of attacin is reduced 95% and cecropin is reduced 65%, while diptericin expression is only slightly affected. However, drosomycin expression seems completely unaffected in 18 W mutants. Nuclear localization of Dif is also blocked, whereas that of dorsal is normal. These results would suggest that 18 W activates Dif, which in turn mediates the induction of attacin. However, other studies show that attacin induction is not affected by the absence of Dif. Thus, induction of attacin by the 18 W pathway may involve another Rel transcription factor, possibly Relish, that can compensate for the loss of Dif.

The imd mutants exhibit a severely reduced survival rate when injected with E. coli, compared to Toll deficient or wild-type flies. The mutants are also completely defective in the synthesis of antibacterial peptides, including cecropin, attacin, and diptericin, following challenge by pathogens (Lemaitre et al., 1995a). On the other hand, these mutants are more or less normal with respect to induction of the antifungal peptide gene drosomycin, and accordingly, their resistance to fungal infection is similar to that of wild-type flies. Recently, Relish was found to be a key factor in the induction of both antibacterial and antifungal peptides (Hedengren et al., 1999). Diptericin induction shows an absolute requirement for the Relish gene, suggesting that Relish is likely to be part of the imd pathway. Thus, the genes encoding the various antimicrobial peptides seem to be controlled by different combinations of Rel-transcription factors that, in turn, are activated via distinct signaling cascades elicited by specific microbial populations.

IV Mammalian IL-1R-Signaling Pathway: Its Similarity with Drosophila Toll Signaling

Recently, remarkable advances have been made in the field of IL-1 signal transduction (Fig. 1). The IL-1R...