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An ancient system of host defense
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An ancient system of host defense Ruslan Medzhitov and Charles A Janeway Jr Research over the past few years has begun to provide significant advances in our understanding of the interplay between the innate and adaptive immune systems. New findings in several model systems reveal remarkable parallels and conservation of ancient host defense pathways in organisms separated by over a billion years of evolution.
Addresses Section of Immunobiology, Yale UniversitySchool of Medicine and Howard Hughes Medical Institute, New Haven, CT 06520-8011, USA Current Opinion in Immunology 1998, 10:12-15 http :llbiomednet.coml el ecref1095 2 7 91501000012
© Current Biology Ltd ISSN 0952-7915 Abbreviations IL Interleukin IRAK IL-1 receptor-associated kinase LRR Leucine-richrepeats SIlK Serine/threonineinnate immunity kinase
Introduction
The field of innate immunity is quickly approaching its renaissance. T h e traditional distinction between the functions of innate and adaptive immune systems is becoming obsolete and sometimes misleading, while new advances clearly indicate the fundamental role of innate components of immunity in adaptive immune responses in vertebrates [1°,2]. Indeed, both initiation of the adaptive immune responses and induction of particular effector mechanisms appear to depend on the signals provided by the innate immune system [1°,2,3]. These signals, in turn, are induced upon recognition of pathogens by non-clonal receptors that are distinct from the clonally distributed antigen receptors expressed on lymphocytes [2,4]. Comparative analysis of the mechanisms of innate and adaptive immune recognition [3], as well as analyses of the molecular components involved [5], indicates that innate immunity preceded the development of adaptive immune system in the evolution of vertebrates. Indeed, all multicellular organisms are thought to have some form of innate host defenses. Moreover, recent studies of model organisms that lack adaptive immunity have revealed striking conservation of signaling pathways involved in innate host defense in organisms as diverse as human and fruit fly, and to certain extent even in plants. Here we review these recent findings. Drosophila
The availability of numerous mutants in Drosophila and of powerful methods of genetic analysis has made this organism a prototypic model of insect immunity [6,7°°,8]. In particular, insects respond to infection by rapid and
transient synthesis of antimicrobial peptides produced by the fat body and hemocytes [6]. T h e regulatory regions of these peptides contain multiple binding sites for transcription factors containing the Rel domain [9,10]. Three Rel family proteins, Dorsal, Dif and Relish, are induced in the fat body of Drosophila in response to infection [7"',11,12]. These transcription factors are homologs of the NF~cB family of transcriptional activators in mammals, where they are involved in the control of a wide variety of immune response genes important for both innate and adaptive immunity [7,13-15]. T h e signaling cascade that activates Rel family proteins in Drosophila is best understood in the case of Dorsal in respect of its function in the establishment of dorso-ventral pattern during early embryonic development [16]. The order of action of the components of this signaling pathway has been defined by double-mutant analysis and the genes encoding these components have been identified [16]. A spatially restricted series of proteolytic reactions generates an active form of a protein called Sp~itzle, which is a ligand for a transmembrane receptor protein called Toll. Binding of active Sp~itzle by Toll results in activation by Toll of two intracellular proteins--Tube and Pelle. Although the exact function of Tube has not yet been biochemically defined, it is thought to function as an adaptor protein which recruits the serine/threonine kinase Pelle to the membrane, thereby activating Pelle. Activation of Pelle ultimately results in phosphorylation, presumably via an intermediate kinase, of C a c t u s - - a homolog of mammalian I~¢B. Cactus is an inhibitory subunit in the cytoplasmic Cactus-Dorsal complex. Phosphorylation of Cactus targets it for rapid degradation, which releases Dorsal and unmasks its nuclear localization signal. This results in nuclear translocation of Dorsal where it activates transcription of its target genes. Homology of the cytoplasmic domain of Toll to the cytoplasmic domain of the receptor for interleukin-1 (IL-1) prompted many to speculate that similar pathways might be involved in the induction of an immune response in adult Drosophila. This idea, of course, was substantiated by the fact that the major immune response genes in the fruit fly (encoding antimicrobial peptides) are controlled by the Rel-like transactivators (see above). In an elegant study that used various immune deficiency mutants it was indeed demonstrated that the Sp/itzle/Toll/Cactus cascade controls the production of the antifungal peptide drosomycin in adult flies in response to fungal infection [17°°]. The known antimicrobial peptides in Drosophila form two functional classes: antibacterial peptides (cecropins, diptericin, defensin, attacin and drosocin) and the antifungal peptide, drosomycin [17°°]. Analysis of immunodeficient mutants of Drosophila suggests that antibacterial peptides can be further divided into two
An ancient system of host defense Medzhitov and Janewsy
groups, depending on the signaling pathways involved in their induction: while expression of cectopin, attacin and defensin is dependent on the Toll pathway (as is the expression of drosomycin), the induction of diptericin and drosocin is unaffected in Toll pathway mutants [17"']. Interestingly, in contrast to the Toll signaling pathway that establishes dorso-ventral pattern, Dorsal is not involved in the Toll-mediated activation of antimicrobial peptides in adult Drosophila; rather some other member(s) of the Rel family, possibly Dif, is the downstream transactivator [17"'1. In a complementary study, a distinct, albeit homologous, pathway responsible for the induction of several antibacterial peptides has been identified [18"']. In this work, the effect of mutations in the gene encoding 18-Wheeler (18W) on the immune response was analyzed. T h e protein 18W is homologous to Toll, and can therefore be expected to signal through the same or similar components as Toll [19]. Indeed, in larvae deficient for 18W protein, activation of Dif is inhibited and induction of several antibacterial genes is compromised. As with the Toll mutants, the activity of Dorsal is not affected [18"']. It is remarkable that even the seemingly simple and stereotyped immune responses in Drosophila can result in induction of distinct effector mechanisms (in this case antifungal versus antibacterial). This is reminiscent to the activation of various effector mechanisms in vertebrates, where induction of a particular type of effector response is controlled by cytokines induced upon infection and corresponds to the type of pathogen encountered. A major question remaining to be answered, however, is how the active form of ligand for Toll (that is, Sp~itzle) or 18W is generated upon infection? As activation of Sp~itzle requires proteolytic processing, it seems likely that the proteolytic cascade resulting in the generation of the active form of the ligand must be triggered by recognition of pathogens or of pathogen-associated molecular patterns. This suggests that the pattern recognition receptor involved would have to have the property of activating a proteolytic cascade. Such mechanisms exist in the mammalian immune system: induction of the lectin pathway of complement activation, for example, depends on the direct recognition of microbial pathogen-associated molecular patterns by several different pattern recognition receptors of the collectin family. This recognition event then triggers activation of serine proteases thus initiating the cascade that culminates in the destruction of the recognized pathogen [20"1. Mammals
The parallels in the signaling pathways between Toll/ Cactus/Dorsal in Drosophila and the IL-1 receptor (IL1R/NF~cB) in mammals have been appreciated for some time now [7"',13] and, to an extent, inspired some of the studies discussed above. However, discovery of a mammalian homolog of the Drosophila Toll protein, and the realization that the entire pathway is conserved
13
between insects and mammals [21], extends these parallels to several important points. First, a human Toll homolog analyzed in our own work has an expression profile (predominant expression in lymphoid tissues and leukocytes) consistent with a function in immunity. Second, activation of human Toll resulted in the induction of several major types of signals proposed to control the adaptive immune response through innate immune recognition, namely a pro-inflammatory cytokine (IL-1), an effector cytokine (IL-6), a chemokine (IL-8) and, most importantly, co-stimulatory molecules (B7.1 and B7.2). T h e co-stimulatory molecules, such as B7.1, are known to be absolutely required for the activation of naive T lymphocytes, and therefore for the induction of most types of adaptive immune responses [22]. Third, the homology of Drosophila and human Toll receptors suggests that the ligands recognized are likely to be homologous as well and, more importantly, that the mechanisms responsible for the generation of the active form of the ligands could be similar [4]. Finally, the discovery of the mammalian Toll and its properties not only reveals the conservation of the TolI/NF~B pathway for hundreds of millions years of evolution, but also illustrates what might be a general trend in the evolution of immunity: signaling pathways which, in the absence of adaptive immunity, function to activate innate immune responses. Later, with the development of adaptive immunity, these same signaling pathways are adopted to generate signals required for the activation of adaptive immune responses. Analysis of the downstream signaling components in the Toll pathway are currently underway, but it seems very likely that these components would be homologs of the corresponding proteins from Drosophila. The IL-1 receptor-associated kinase (IRAK), which is involved in signal transduction from the IL-1R, appears to be a homolog of Drosophila Pelle [23"']. As the cytoplasmic domain of human Toll is more similar to the equivalent domain from Drosophila than it is to that of the IL-1R, the kinase involved in the Toll pathway should be related to Pelle as well. In addition to Toll, the IL-1R and IL-1R-related proteins mammals have another protein containing the TolI/IL-1R signaling domain called MyD88. MyD88 was initially discovered as an early differentiation marker of myeloid cells induced by various differentiation stimuli, particularly IL-6 [24]. The domain structure and localization of MyD88, however, is distinct from that of both Toll and IL-1R: the extracellular portion of Toll consists of leucine-rich repeats (LRR) flanked on both sides by cysteine-rich domains, whereas the extracellular part of the IL-1R consists of three immunoglobulin domains. MyD88 represents a novel configuration of domains with a carboxy-terminal TolI/IL-1R homology domain and an amino-terminal death domain [25,26]. The death domain is found in a variety of signaling proteins, particularly, although not exclusively, in proteins involved in control of apoptosis, such as the tumor necrosis factor receptor T N F R - I and Fas. The death domain itself, however,
14
Innate immunity
functions as a protein-protein interaction module, rather than an effector in the induction of apoptosis [27]. It thus seems likely that the death domain in MyD88 is involved in specific interaction either with itself to form homodimers or oligomers, or with other death domain-containing proteins. In the latter case, the interaction partner for MyD88 could be a transmembrane receptor protein with a cytoplasmic death domain (Figure 1). If this is the case, one could speculate that, by analogy with other signaling pathways, MyD88 is recruited to the membrane by the interaction with its cognate transmembrane receptor, perhaps in a ligand-dependent manner. If activation of MyD88 depends on such an interaction, the activity of MyD88 could be controlled by an extracellular signal. Alternatively, the activity of MyD88 could be controlled at the level of expression and, once the protein is expressed, it would be capable of initiating a signaling event. At present there is no evidence directly proving or disproving either of these two possibilities. It may very well turn. out that both mechanisms control the activity of MyD88. T h e presence of the death domain seems, however, to favor the former scenario, whereas the fact that expression of MyD88 seems to be under control of several factors, such as IL-6, NF~cB-dependent signals, and presumably interferons [28], as well as the presence of three A T T T A motifs implicated in rapid mRNA turnover in the 3' untranslated region of the MyD88 mRNA [24], favors the latter possibility. Finally, the cytoplasmic localization of MyD88 suggests that it could be involved in detection of viral (or other intracellular) infection and may be able to signal the presence of such infection through an NF~B-dependent pathway. Whereas little is known about the function of MyD88, this protein does activate NFKB when overexpressed in a variety of cell types and can induce several immune response genes. It thus appears, on the basis of the available data, that MyD88 is yet another component of innate immunity that can signal the presence of infection by inducing the signals that activate the adaptive immune response (R Medzhitov and CA Janeway Jr., unpublished data).
Plants Molecular genetic analysis of the components of plant defense against pathogens has undergone a tremendous breakthrough in the last few years. Since several excellent reviews on the subject have been published recently by experts in the field [29,30°°,31°°], here we only consider those issues pertinent to the current discussion, namely the remarkable conservation of the protein modules involved in innate host defense. Indeed, a product of practically every plant-disease resistance gene cloned so far contains at least one protein domain also found in the proteins that function in host defense in insects and vertebrates. These domains include the TolI/IL-1R homology domain, the LRR domain, and the serine/threonine kinase domain [30°°]. T h e L R R domain is present in Toll and the endotoxin receptor CD14 expressed on macrophages in vertebrates. In plants, the L R R is found
Figure 1
L,0 th Vertebrates Toll
Insects Toll
Plants
ISgl Pgl Pgl MyD88
~ & N
L6 RPP5 Pelle
IRAK
IKB
Cactus
NFKB
Dorsal Dif
Relish/ I
pto pt,,er
?
D Immuneresponsegenes Current Opinion in Immunology
Conserved signaling modules found in the host defense pathways in mammals, insects and plants. DD, death domain; NBS, nucleotide-binding site. The TolI/IL-1R homology domain is represented by diagonally hatched ovals.
in a variety of resistance proteins (R proteins), such as N protein from tobacco, Cf-9 protein from tomato and Xa-21 protein from rice. Notably, in each of these three examples, the LRR domain occurs in a different configuration with other protein domains [30°°]. Moreover, N protein, as well as L6 protein from flax and RPP5 protein from Arabidopsis, not only contains LRR domains but also the Toll/IL-1R homology domain, much like Toll itself. However, the order of the domains in the polypeptide chain is different, and the plant proteins from these groups all appear to be intracellular proteins responsible for resistance to intracellular pathogens [30°°]. Another group of R genes, including pto, pti, and fen from tomato, encode cytoplasmic serine/threonine kinases that are most closely related to IRAK and Pelle [7°°,30 °°] and define a subgroup of kinases the that has been named the serine/threonine innate immunity kinases (the SIIK group kinases) [7°°]. Although the details of the interactions between R proteins from different structural classes are not yet known, the implications of this remarkable conservation are extremely informative.
Conclusions Analysis of the innate defense mechanisms in vertebrates, insects and plants has revealed the existence of a nonclonal host defense pathway with structural and functional homologies in phylogenetic lineages that diverged over a billion years ago. T h e limited set of conserved signaling modules found in these defense pathways, such as the
An ancient system of host defense Medzhitov and Janeway
ToI1/IL-1R homology domain, the SIIK domain, the Rel homology domain and perhaps the LRR domain, may represent the original 'building blocks' that have been put by evolution into various molecular contexts in the various host defense systems. These ancient components of innate defense have been preserved over millions of years of evolution and appear to have a crucial role in the control of the 'recently' developed adaptive immunity of vertebrates.
15
14.
Siebenlist U, Franzoso G, Brown K: Structure, regulation end function of NF-~
15.
Kopp EB, Ghosh S: NF-KB and rel proteins in innate immunity. Adv Immuno11995, 58:1-27.
16.
Morisato D, Anderson KV: Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu Rev Genet 1995, 29:371-399.
Acknowledgement
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA: The dorsoventral regulatory gene cassette sp~itzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996, 86:973-983. A seminal article on the role of the sp~ltzle, Toll and cactus genes in host defense against infection by fungal spores in adult Drosophila.
Supported by a grant from the Human Frontiers of Science, and by NIH grant AI-26810 (CAJ Jr).
18. •.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••
17. e,
Williams MJ, Rodriguez A, Kimbrell DA, Eldon ED: The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila host defense. EMBO J 1997, 16:6120-6130. This work very nicely complements findings reported in [17 °°] and points out the potential diversity of signaling pathways in inducing distinct effector responses in Drosophila. 19.
of special interest of outstanding interest
1. FearonDT, Locksley RM: The instructive role of innate immunity • in the acquired immune response. Science 1998, 272:50-54. This article provides a detailed and very useful review of the multiple points of control of the adaptive immune response by the innate immune system. 2.
Medzhitov RM, and Janeway, CA Jr: Innate immunity: impact on the adaptive immune response. Curt Opin Immunol 1997, 9:4-9.
3.
JanewayCA Jr: Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Syrup Ouant Biol 1989, 54:1-13.
4.
Medzhitov RM, Janeway CA Jr: Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997, 91:295-298.
5.
Hughes AL, Yeager M: Molecular evolution of the vertebrate immune system. BioEssays 1997, 19:?77-786.
6.
HoffmannJA: Innate immunity of insects. Curt Opin Immunol 1995, 7:4-10.
Eldon E, Kooyer S, D'Evelyn D, Duman M, Lawinger P, Botas J, Bellen H: The Drosophila 18 wheeler is required for morphogenesis and has striking similarities to Toll. Development 1994, 120:885-899.
20.
Lu J: Collectins: collectors of microorganisms for the innate immune system. BioEssays 1997, 19:509-518. A nice, up-to-date, and very informative review. 21.
Medzhitov RM, Preston-Hurlburt P, Janeway CA Jr: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997, 388:394-397.
22.
Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell costimulation. Annu Rev Immuno11996, 14:233-258.
23. ;is
Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 1996, 271:1128-1131. paper reports the cloning of the gene encoding IRAK, another homologous component in the TolI/NFKB pathway. 24.
Lord KA, Hoffman-Liebermann B, Liebermann DA: Nucleotide sequence and expression of • cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 1990, 5:1095-1097.
25.
HardimanG, Rock FL, Balasubramanian S, Kastelein RA, Bazan JF: Molecular characterization end modular analysis of human MyD88. Oncogene 1996, 13:2467-2475.
26.
Bonnert TP, Garka KE, Pamet P, Sonoda G, Testa JR, Sims JE: The cloning and characterization of human MyD88: a member of an IL-1 receptor related family. FEBS Lett 1997, 402:81-84.
27.
Baker SJ, Reddy EP: Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 1996, 12:1-9.
28.
Georgel P, Meister M, Kappler C, Lemaltre B, Reichhart JM, Hoffmann JA: Insect immunity: the diptericin promoter contains multiple functional regulatory sequences homologous to mammalian acute-phase response elements. Biochem Biophys Res Commun 1993, 197:508-517.
Harroch S, Gothelf Y, Revel M, Chebath J: 5" upstream sequences of MyD88, an IL-6 primary response gene in M1 cells: detection of functional IRF-1 and Stat factors binding sites. Nucleic Acids Res 1995, 23:3539-3546.
29.
Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JD: Molecular genetics of plant disease resistance. Science 1995, 268:661-667.
11.
DushayMS, Asling B, Hultmark D: Origins of immunity: relish, a compound Rel-like gene in the antibacterial defense of Drosophila. Proc Nat/Acad Sci USA 1996, 93:10343-10347.
30. ;is
12.
Petersen UM, Bjorklund G, Ip YT, Engstrom Y: The dorsal-related immunity factor, Dif, is a sequence-specific trans-activator of Drosophila cecropin gene expression. EMBO J 1995, 14:31463158.
7. .,,
Belvin MP, Anderson KV: A conserved signaling pathway: the Drosophila Toll-Dorsal pathway. Annu Rev Cell Dev Biol 1996, 12:393-416. An excellent review with detailed analysis of the conserved signaling pathway involved in dorso-ventral patterning in Drosophila development as well as in the innate immunity of Drosophila and mammals. 8. Hultmark D: Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet 1993, 9:178-183. 9. Engstrom Y, Kadalayil L, Sun SC, Samakovlis C, Hultmark D, Faye IJ: kappa B-like motifs regulate the induction of immune genes in Drosophila. Mo/Biol 1993, 232:327-333. 10.
13.
Wasserman SA: A conserved signal transduction pathway regulating the activity of the rel-like proteins dorsal and NF-kB. Mol Biol Cell 1993, 4:767-771.
Yang Y, Shah J, Klessig DF: Signal perception and transduction in plant defense responses. Genes Dev 1997, 11:1621-1639. article, together with [31°'], provides an extremely useful and comprehensive account of the current state of affairs in the biology of the plant defense systems. 31. ;is
Hammond-Kosack KE, Jones JDG: Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:575-607. article, together with [30"'], provides an extremely useful and comprehensive account of the current state of affairs in the biology of the plant defense systems.
An ancient system of host defense Ruslan Medzhitov and Charles A Janeway Jr Research over the past few years has begun to provide significant advances in our understanding of the interplay between the innate and adaptive immune systems. New findings in several model systems reveal remarkable parallels and conservation of ancient host defense pathways in organisms separated by over a billion years of evolution.
Addresses Section of Immunobiology, Yale UniversitySchool of Medicine and Howard Hughes Medical Institute, New Haven, CT 06520-8011, USA Current Opinion in Immunology 1998, 10:12-15 http :llbiomednet.coml el ecref1095 2 7 91501000012
© Current Biology Ltd ISSN 0952-7915 Abbreviations IL Interleukin IRAK IL-1 receptor-associated kinase LRR Leucine-richrepeats SIlK Serine/threonineinnate immunity kinase
Introduction
The field of innate immunity is quickly approaching its renaissance. T h e traditional distinction between the functions of innate and adaptive immune systems is becoming obsolete and sometimes misleading, while new advances clearly indicate the fundamental role of innate components of immunity in adaptive immune responses in vertebrates [1°,2]. Indeed, both initiation of the adaptive immune responses and induction of particular effector mechanisms appear to depend on the signals provided by the innate immune system [1°,2,3]. These signals, in turn, are induced upon recognition of pathogens by non-clonal receptors that are distinct from the clonally distributed antigen receptors expressed on lymphocytes [2,4]. Comparative analysis of the mechanisms of innate and adaptive immune recognition [3], as well as analyses of the molecular components involved [5], indicates that innate immunity preceded the development of adaptive immune system in the evolution of vertebrates. Indeed, all multicellular organisms are thought to have some form of innate host defenses. Moreover, recent studies of model organisms that lack adaptive immunity have revealed striking conservation of signaling pathways involved in innate host defense in organisms as diverse as human and fruit fly, and to certain extent even in plants. Here we review these recent findings. Drosophila
The availability of numerous mutants in Drosophila and of powerful methods of genetic analysis has made this organism a prototypic model of insect immunity [6,7°°,8]. In particular, insects respond to infection by rapid and
transient synthesis of antimicrobial peptides produced by the fat body and hemocytes [6]. T h e regulatory regions of these peptides contain multiple binding sites for transcription factors containing the Rel domain [9,10]. Three Rel family proteins, Dorsal, Dif and Relish, are induced in the fat body of Drosophila in response to infection [7"',11,12]. These transcription factors are homologs of the NF~cB family of transcriptional activators in mammals, where they are involved in the control of a wide variety of immune response genes important for both innate and adaptive immunity [7,13-15]. T h e signaling cascade that activates Rel family proteins in Drosophila is best understood in the case of Dorsal in respect of its function in the establishment of dorso-ventral pattern during early embryonic development [16]. The order of action of the components of this signaling pathway has been defined by double-mutant analysis and the genes encoding these components have been identified [16]. A spatially restricted series of proteolytic reactions generates an active form of a protein called Sp~itzle, which is a ligand for a transmembrane receptor protein called Toll. Binding of active Sp~itzle by Toll results in activation by Toll of two intracellular proteins--Tube and Pelle. Although the exact function of Tube has not yet been biochemically defined, it is thought to function as an adaptor protein which recruits the serine/threonine kinase Pelle to the membrane, thereby activating Pelle. Activation of Pelle ultimately results in phosphorylation, presumably via an intermediate kinase, of C a c t u s - - a homolog of mammalian I~¢B. Cactus is an inhibitory subunit in the cytoplasmic Cactus-Dorsal complex. Phosphorylation of Cactus targets it for rapid degradation, which releases Dorsal and unmasks its nuclear localization signal. This results in nuclear translocation of Dorsal where it activates transcription of its target genes. Homology of the cytoplasmic domain of Toll to the cytoplasmic domain of the receptor for interleukin-1 (IL-1) prompted many to speculate that similar pathways might be involved in the induction of an immune response in adult Drosophila. This idea, of course, was substantiated by the fact that the major immune response genes in the fruit fly (encoding antimicrobial peptides) are controlled by the Rel-like transactivators (see above). In an elegant study that used various immune deficiency mutants it was indeed demonstrated that the Sp/itzle/Toll/Cactus cascade controls the production of the antifungal peptide drosomycin in adult flies in response to fungal infection [17°°]. The known antimicrobial peptides in Drosophila form two functional classes: antibacterial peptides (cecropins, diptericin, defensin, attacin and drosocin) and the antifungal peptide, drosomycin [17°°]. Analysis of immunodeficient mutants of Drosophila suggests that antibacterial peptides can be further divided into two
An ancient system of host defense Medzhitov and Janewsy
groups, depending on the signaling pathways involved in their induction: while expression of cectopin, attacin and defensin is dependent on the Toll pathway (as is the expression of drosomycin), the induction of diptericin and drosocin is unaffected in Toll pathway mutants [17"']. Interestingly, in contrast to the Toll signaling pathway that establishes dorso-ventral pattern, Dorsal is not involved in the Toll-mediated activation of antimicrobial peptides in adult Drosophila; rather some other member(s) of the Rel family, possibly Dif, is the downstream transactivator [17"'1. In a complementary study, a distinct, albeit homologous, pathway responsible for the induction of several antibacterial peptides has been identified [18"']. In this work, the effect of mutations in the gene encoding 18-Wheeler (18W) on the immune response was analyzed. T h e protein 18W is homologous to Toll, and can therefore be expected to signal through the same or similar components as Toll [19]. Indeed, in larvae deficient for 18W protein, activation of Dif is inhibited and induction of several antibacterial genes is compromised. As with the Toll mutants, the activity of Dorsal is not affected [18"']. It is remarkable that even the seemingly simple and stereotyped immune responses in Drosophila can result in induction of distinct effector mechanisms (in this case antifungal versus antibacterial). This is reminiscent to the activation of various effector mechanisms in vertebrates, where induction of a particular type of effector response is controlled by cytokines induced upon infection and corresponds to the type of pathogen encountered. A major question remaining to be answered, however, is how the active form of ligand for Toll (that is, Sp~itzle) or 18W is generated upon infection? As activation of Sp~itzle requires proteolytic processing, it seems likely that the proteolytic cascade resulting in the generation of the active form of the ligand must be triggered by recognition of pathogens or of pathogen-associated molecular patterns. This suggests that the pattern recognition receptor involved would have to have the property of activating a proteolytic cascade. Such mechanisms exist in the mammalian immune system: induction of the lectin pathway of complement activation, for example, depends on the direct recognition of microbial pathogen-associated molecular patterns by several different pattern recognition receptors of the collectin family. This recognition event then triggers activation of serine proteases thus initiating the cascade that culminates in the destruction of the recognized pathogen [20"1. Mammals
The parallels in the signaling pathways between Toll/ Cactus/Dorsal in Drosophila and the IL-1 receptor (IL1R/NF~cB) in mammals have been appreciated for some time now [7"',13] and, to an extent, inspired some of the studies discussed above. However, discovery of a mammalian homolog of the Drosophila Toll protein, and the realization that the entire pathway is conserved
13
between insects and mammals [21], extends these parallels to several important points. First, a human Toll homolog analyzed in our own work has an expression profile (predominant expression in lymphoid tissues and leukocytes) consistent with a function in immunity. Second, activation of human Toll resulted in the induction of several major types of signals proposed to control the adaptive immune response through innate immune recognition, namely a pro-inflammatory cytokine (IL-1), an effector cytokine (IL-6), a chemokine (IL-8) and, most importantly, co-stimulatory molecules (B7.1 and B7.2). T h e co-stimulatory molecules, such as B7.1, are known to be absolutely required for the activation of naive T lymphocytes, and therefore for the induction of most types of adaptive immune responses [22]. Third, the homology of Drosophila and human Toll receptors suggests that the ligands recognized are likely to be homologous as well and, more importantly, that the mechanisms responsible for the generation of the active form of the ligands could be similar [4]. Finally, the discovery of the mammalian Toll and its properties not only reveals the conservation of the TolI/NF~B pathway for hundreds of millions years of evolution, but also illustrates what might be a general trend in the evolution of immunity: signaling pathways which, in the absence of adaptive immunity, function to activate innate immune responses. Later, with the development of adaptive immunity, these same signaling pathways are adopted to generate signals required for the activation of adaptive immune responses. Analysis of the downstream signaling components in the Toll pathway are currently underway, but it seems very likely that these components would be homologs of the corresponding proteins from Drosophila. The IL-1 receptor-associated kinase (IRAK), which is involved in signal transduction from the IL-1R, appears to be a homolog of Drosophila Pelle [23"']. As the cytoplasmic domain of human Toll is more similar to the equivalent domain from Drosophila than it is to that of the IL-1R, the kinase involved in the Toll pathway should be related to Pelle as well. In addition to Toll, the IL-1R and IL-1R-related proteins mammals have another protein containing the TolI/IL-1R signaling domain called MyD88. MyD88 was initially discovered as an early differentiation marker of myeloid cells induced by various differentiation stimuli, particularly IL-6 [24]. The domain structure and localization of MyD88, however, is distinct from that of both Toll and IL-1R: the extracellular portion of Toll consists of leucine-rich repeats (LRR) flanked on both sides by cysteine-rich domains, whereas the extracellular part of the IL-1R consists of three immunoglobulin domains. MyD88 represents a novel configuration of domains with a carboxy-terminal TolI/IL-1R homology domain and an amino-terminal death domain [25,26]. The death domain is found in a variety of signaling proteins, particularly, although not exclusively, in proteins involved in control of apoptosis, such as the tumor necrosis factor receptor T N F R - I and Fas. The death domain itself, however,
14
Innate immunity
functions as a protein-protein interaction module, rather than an effector in the induction of apoptosis [27]. It thus seems likely that the death domain in MyD88 is involved in specific interaction either with itself to form homodimers or oligomers, or with other death domain-containing proteins. In the latter case, the interaction partner for MyD88 could be a transmembrane receptor protein with a cytoplasmic death domain (Figure 1). If this is the case, one could speculate that, by analogy with other signaling pathways, MyD88 is recruited to the membrane by the interaction with its cognate transmembrane receptor, perhaps in a ligand-dependent manner. If activation of MyD88 depends on such an interaction, the activity of MyD88 could be controlled by an extracellular signal. Alternatively, the activity of MyD88 could be controlled at the level of expression and, once the protein is expressed, it would be capable of initiating a signaling event. At present there is no evidence directly proving or disproving either of these two possibilities. It may very well turn. out that both mechanisms control the activity of MyD88. T h e presence of the death domain seems, however, to favor the former scenario, whereas the fact that expression of MyD88 seems to be under control of several factors, such as IL-6, NF~cB-dependent signals, and presumably interferons [28], as well as the presence of three A T T T A motifs implicated in rapid mRNA turnover in the 3' untranslated region of the MyD88 mRNA [24], favors the latter possibility. Finally, the cytoplasmic localization of MyD88 suggests that it could be involved in detection of viral (or other intracellular) infection and may be able to signal the presence of such infection through an NF~B-dependent pathway. Whereas little is known about the function of MyD88, this protein does activate NFKB when overexpressed in a variety of cell types and can induce several immune response genes. It thus appears, on the basis of the available data, that MyD88 is yet another component of innate immunity that can signal the presence of infection by inducing the signals that activate the adaptive immune response (R Medzhitov and CA Janeway Jr., unpublished data).
Plants Molecular genetic analysis of the components of plant defense against pathogens has undergone a tremendous breakthrough in the last few years. Since several excellent reviews on the subject have been published recently by experts in the field [29,30°°,31°°], here we only consider those issues pertinent to the current discussion, namely the remarkable conservation of the protein modules involved in innate host defense. Indeed, a product of practically every plant-disease resistance gene cloned so far contains at least one protein domain also found in the proteins that function in host defense in insects and vertebrates. These domains include the TolI/IL-1R homology domain, the LRR domain, and the serine/threonine kinase domain [30°°]. T h e L R R domain is present in Toll and the endotoxin receptor CD14 expressed on macrophages in vertebrates. In plants, the L R R is found
Figure 1
L,0 th Vertebrates Toll
Insects Toll
Plants
ISgl Pgl Pgl MyD88
~ & N
L6 RPP5 Pelle
IRAK
IKB
Cactus
NFKB
Dorsal Dif
Relish/ I
pto pt,,er
?
D Immuneresponsegenes Current Opinion in Immunology
Conserved signaling modules found in the host defense pathways in mammals, insects and plants. DD, death domain; NBS, nucleotide-binding site. The TolI/IL-1R homology domain is represented by diagonally hatched ovals.
in a variety of resistance proteins (R proteins), such as N protein from tobacco, Cf-9 protein from tomato and Xa-21 protein from rice. Notably, in each of these three examples, the LRR domain occurs in a different configuration with other protein domains [30°°]. Moreover, N protein, as well as L6 protein from flax and RPP5 protein from Arabidopsis, not only contains LRR domains but also the Toll/IL-1R homology domain, much like Toll itself. However, the order of the domains in the polypeptide chain is different, and the plant proteins from these groups all appear to be intracellular proteins responsible for resistance to intracellular pathogens [30°°]. Another group of R genes, including pto, pti, and fen from tomato, encode cytoplasmic serine/threonine kinases that are most closely related to IRAK and Pelle [7°°,30 °°] and define a subgroup of kinases the that has been named the serine/threonine innate immunity kinases (the SIIK group kinases) [7°°]. Although the details of the interactions between R proteins from different structural classes are not yet known, the implications of this remarkable conservation are extremely informative.
Conclusions Analysis of the innate defense mechanisms in vertebrates, insects and plants has revealed the existence of a nonclonal host defense pathway with structural and functional homologies in phylogenetic lineages that diverged over a billion years ago. T h e limited set of conserved signaling modules found in these defense pathways, such as the
An ancient system of host defense Medzhitov and Janeway
ToI1/IL-1R homology domain, the SIIK domain, the Rel homology domain and perhaps the LRR domain, may represent the original 'building blocks' that have been put by evolution into various molecular contexts in the various host defense systems. These ancient components of innate defense have been preserved over millions of years of evolution and appear to have a crucial role in the control of the 'recently' developed adaptive immunity of vertebrates.
15
14.
Siebenlist U, Franzoso G, Brown K: Structure, regulation end function of NF-~
15.
Kopp EB, Ghosh S: NF-KB and rel proteins in innate immunity. Adv Immuno11995, 58:1-27.
16.
Morisato D, Anderson KV: Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. Annu Rev Genet 1995, 29:371-399.
Acknowledgement
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA: The dorsoventral regulatory gene cassette sp~itzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996, 86:973-983. A seminal article on the role of the sp~ltzle, Toll and cactus genes in host defense against infection by fungal spores in adult Drosophila.
Supported by a grant from the Human Frontiers of Science, and by NIH grant AI-26810 (CAJ Jr).
18. •.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••
17. e,
Williams MJ, Rodriguez A, Kimbrell DA, Eldon ED: The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila host defense. EMBO J 1997, 16:6120-6130. This work very nicely complements findings reported in [17 °°] and points out the potential diversity of signaling pathways in inducing distinct effector responses in Drosophila. 19.
of special interest of outstanding interest
1. FearonDT, Locksley RM: The instructive role of innate immunity • in the acquired immune response. Science 1998, 272:50-54. This article provides a detailed and very useful review of the multiple points of control of the adaptive immune response by the innate immune system. 2.
Medzhitov RM, and Janeway, CA Jr: Innate immunity: impact on the adaptive immune response. Curt Opin Immunol 1997, 9:4-9.
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Lu J: Collectins: collectors of microorganisms for the innate immune system. BioEssays 1997, 19:509-518. A nice, up-to-date, and very informative review. 21.
Medzhitov RM, Preston-Hurlburt P, Janeway CA Jr: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997, 388:394-397.
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Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 1996, 271:1128-1131. paper reports the cloning of the gene encoding IRAK, another homologous component in the TolI/NFKB pathway. 24.
Lord KA, Hoffman-Liebermann B, Liebermann DA: Nucleotide sequence and expression of • cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 1990, 5:1095-1097.
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Belvin MP, Anderson KV: A conserved signaling pathway: the Drosophila Toll-Dorsal pathway. Annu Rev Cell Dev Biol 1996, 12:393-416. An excellent review with detailed analysis of the conserved signaling pathway involved in dorso-ventral patterning in Drosophila development as well as in the innate immunity of Drosophila and mammals. 8. Hultmark D: Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet 1993, 9:178-183. 9. Engstrom Y, Kadalayil L, Sun SC, Samakovlis C, Hultmark D, Faye IJ: kappa B-like motifs regulate the induction of immune genes in Drosophila. Mo/Biol 1993, 232:327-333. 10.
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Yang Y, Shah J, Klessig DF: Signal perception and transduction in plant defense responses. Genes Dev 1997, 11:1621-1639. article, together with [31°'], provides an extremely useful and comprehensive account of the current state of affairs in the biology of the plant defense systems. 31. ;is
Hammond-Kosack KE, Jones JDG: Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:575-607. article, together with [30"'], provides an extremely useful and comprehensive account of the current state of affairs in the biology of the plant defense systems.