20 Years Later

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Approaching the Asymptote: 20 Years Later

Ruslan Medzhitov1,* 1Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA *Correspondence: [email protected] DOI 10.1016/j.immuni.2009.06.004

The pattern recognition theory proposed by the late Charles Janeway, Jr. 20 years ago provided a conceptual framework for our current understanding of the innate immune recognition and its role in the activation of adaptive immunity. Discovery of several families of pattern recognition receptors and their roles in mammalian immunity provided experimental support for the Janeway’s theory. In addition to pattern recognition, there are other forms of innate immune sensing, which presumably work in speciﬁc combinations depending on the pathogen class and the type of the immune response they elicit. Here, the development of the Jane- way’s theory is discussed in the context of the advances made in ﬁeld of innate immune recognition over the past two decades.

‘‘It is amateurs who have one big bright beautiful idea that respond to any antigens they may encounter because these by they can never abandon. Professionals know that they definition would be nonself. The concept was later modified by have to produce theory after theory before they are likely the two-signal model of Bretscher and Cohn (Bretscher and to hit the jackpot.’’ Francis Crick, What Mad Pursuit: A Cohn, 1970) and by the costimulatory model suggested by Personal View of Scientific Discovery (1988) Lafferty and Cunningham (Lafferty and Cunningham, 1975). The first experimental evidence for the two-signal requirement This year marks the 20th anniversary of a publication (Janeway, for T cell activation was later provided by Marc Jenkins and 1989) that in many ways revolutionized our understanding of Ronald Schwartz (Jenkins and Schwartz, 1987), although the the immune system. As part of the proceedings of the Cold nature of the second signal remained unknown for a few more Spring Harbor Symposium on Immune Recognition, the paper years. Importantly, however, in an unrelated line of studies, An- authored by the late Charles A. Janeway, Jr. was not a standard tonio Coutinho and Go¨ ran Mo¨ ller discovered the distinct roles peer-reviewed publication. In fact, Charlie used to refer to it as of antigen-specific and polyclonal signals in B cell activation ‘‘the best paper I’ve never published.’’ According to the Francis (Coutinho and Moller, 1974). The polyclonal (or mitogenic) signal Crick’s quotation above, Charlie was definitely a ‘‘professional’’ in their experiments was provided by lipopolysaccharide (LPS), who had many ideas and who hit more than one jackpot. The and thus their studies made one of the first critical connections pattern recognition theory was the biggest one. In addition to between antigen-specific receptor and nonclonal microbial suggesting a general principle of innate immune recognition, it receptors in activation of B cell responses. Other studies estab- provided a conceptual framework for the integration of the two lished adjuvant properties of LPS (Condie et al., 1955; Skidmore types of immunity—innate and acquired. It also suggested et al., 1976). In the meantime, Ralph Steinman, working in Zanvil a new set of ideas that explained the initiation of the immune Cohn’s group, discovered dendritic cells and characterized their responses, the mechanism of adjuvant effects, and the evolution unique immunostimulatory activity (Steinman and Cohn, 1973, of different forms of immune recognition. Collectively, these 1974). A critical connection between the initiation of T and B ideas provided guiding principles for our current understanding cell responses was provided by the discovery of antigen-specific of the functioning of the innate immune system and its connec- T and B cell interactions by Antonio Lanzavecchia (Lanzavec- tion with adaptive immunity. chia, 1985). Together, these and many other pioneering discov- eries, including analyses of the specificity of antibody responses Early Studies on the Initiation of Innate and Adaptive by Karl Landsteiner (Landsteiner, 1945), provided the essential Immunity background for future analyses of how the adaptive immune In the late 80s and early 90s, immunologists focused much of response is initiated. their attention on the mechanisms of adaptive immune recogni- In parallel, fundamental advances were being made in studies tion, including the structure and function of the antigen recep- of the innate host defense mechanisms. During the 1970s, anal- tors, the mechanisms of MHC restriction, and lymphocyte devel- yses of antimicrobial mechanisms of neutrophils and macro- opment and activation. However, little was known about the phages, particularly by Zanvil Cohn and his many pupils and requirements for the activation of adaptive immune responses. collaborators, including James Hirsch, Seymour Klebanoff, Carl Although the question of self-nonself discrimination has preoc- Nathan, Siamon Gordon, Ralph Steinman, Alan Aderem, Ira cupied the minds of immunologists for decades, the solution Mellman, Bill Muller, Gilla Kaplan, and many others, had estab- was thought to be largely provided by the pioneering works of lished many basic principles of macrophage and neutrophil func- Lederberg, Burnet, and Medawar (Billingham et al., 1953; tions, including phagocytic process, degranulation, secretory Burnet, 1959; Lederberg, 1959). The central idea at the time function, arachidonic acid metabolism, and oxygen-dependent was that the removal or inactivation of autoreactive clones during and -independent bacteriocidal functions (reviewed in Steinman lymphocyte development allowed the remaining lymphocytes to and Moberg, 1994). Many of these functions were found to be

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inducible in macrophages by microbial stimuli, including LPS, of immune recognition must be evolutionarily related to the Bacillus Calmette-Gue´ rin (BCG), and zymosan. Thus, the idea of immune systems of invertebrates, which lack adaptive immunity. microbial stimulation of innate effector functions was well appre- Finally, most adjuvants were suggested to work in part by ciated by that time. In addition, the discovery of antimicrobial mimicking microbial infections, by triggering the receptors of peptides by Hans Boman revealed a powerful microbicidal the innate immune system and inducing costimulatory signals, system, which is now known to be used by most and perhaps thus ‘‘tricking’’ the adaptive immune system into action. These all organisms for antimicrobial defense. In the field of antiviral and other ideas provided an elegant explanation of many funda- immunity, the crucial advances were the discovery of type I inter- mental aspects of the functioning of the immune system. feron (IFN) activity (Isaacs and Lindenmann, 1957; Isaacs et al., Remarkably, all of them turned out to be fundamentally correct. 1957; Nagano and Kojima, 1958), cloning of the first type I IFN Nevertheless, the new theory did not seem to attract a lot of gene (IFN-b) by Tadatsugu Taniguchi (Taniguchi et al., 1980), attention at first. The situation started to change about a decade and the isolation of the IFN-a gene by Shigekazu Nagata and later, when new studies began elucidating the pathways of Charles Weismann (Nagata et al., 1980). Importantly, type I IFNs innate immune recognition. were known to be inducible by poly IC, presumably because it mimics viral dsRNA—another important target of innate immune Toll-like Receptors recognition. Finally, characterization of two mouse strains defi- By the mid-90s, several receptors involved in innate immune cient in LPS responsiveness, C3H/HeJ (Heppner and Weiss, recognition were already known. These included CD14, a compo- 1965) and C57BL/10Cr (Coutinho et al., 1977), and demonstration nent of the LPS receptor complex (Wright et al., 1990); Mannan- of their susceptibility to a bacterial infection (O’Brien et al., 1980) binding lectin (MBL), an activator of the lectin pathway of comple- demonstrated that LPS recognition plays a critical role in host ment (Takahashi et al., 2006); macrophage mannose receptor defense against at least some Gram-negative pathogens. (Stahl and Ezekowitz, 1998); and MARCO, a member of the scav- The two lines of study briefly outlined above were developing enger receptor family (Elomaa et al., 1995). MBL was particularly largely independently of each other, as reflected in the way the well characterized in terms of its specificity and function. Its role innate and adaptive immune systems were covered in textbooks. in complement activation and microbial opsonization was remi- The critical role of innate immune recognition in the control of niscent of immunoglobulins, suggesting that MBL functioned as adaptive immunity was not well appreciated and a new concep- a nonclonal version of antibodies. To emphasize this point, Alan tual framework was clearly needed to integrate innate and adap- Ezekowitz referred to MBL as ‘‘ante-antibody,’’ highlighting the tive immune recognition to explain the fundamental properties of evolutionary ancestry of the former (Ezekowitz, 1991). However mammalian immunity. MBL, as well as pentraxins, although known at the time to function as pattern recognition molecules, were not the type of recep- Janeway’s Hypothesis tors that would be expected to control the expression of costimu- Charles Janeway’s unique contribution was in developing a new latory signals on APCs. Therefore, Janeway and his colleagues synthesis that placed many immunological phenomena in a clear were looking for cell-surface receptors, expressed on macro- biological context. First, he proposed that the costimulatory phages and dendritic cells (DCs), that would be able to induce signal required for lymphocyte activation was inducible (on signaling pathways resulting in expression of B7.1 and B7.2 antigen-presenting cells [APCs]). Whether and how the costimu- (now known as CD80 and CD86, respectively). Because many latory signals are regulated was not well understood at the time, PAMPs are either carbohydrates or glycolipids, the new pattern although expression of interleukin-1 (IL-1) by macrophages was recognition receptors were thought to probably contain a C- found by Emil Unanue and colleagues to be one of them (Weaver type lectin domain, similar to MBL and macrophage mannose et al., 1988). Second, the costimulatory signal was suggested to receptor. In addition, the intracellular portion was expected to be inducible by conserved microbial products, thus placing the be linked to the activation of the NF-kB signaling pathway. This activation of adaptive immunity under the control of pathogen- pathway was already well established to play a pivotal role in sensing mechanisms. This also explained the adjuvant proper- innate immunity (Kopp and Ghosh, 1995), and studies by Stepha- ties of certain microbial stimuli. The fact that many microbial nie Vogel, Doug Golenbock, Richard Ulevitch, and many others molecules, such as LPS, had immunostimulatory properties have demonstrated the role of NF-kB in LPS signaling. The was known before, but it was not clear why some microbial best-characterized receptors known at the time to activate NF- molecules have these properties, including adjuvant activity, kB were TNF and IL-1 receptors. The IL-1 receptor was particu- whereas others do not. Neither was it known how these microbial larly interesting in this regard because it contained a cytoplasmic structures exerted their adjuvant effects. Janeway suggested domain, which was known to occur in Drosophila Toll, identified in that the actual detection of infection was mediated by the recep- 1988 by Carl Hashimoto and Kathryn Anderson (Hashimoto et al., tors of the innate immune system, rather than the antigen recep- 1988), and in a resistance protein from tobacco known as N tors. Specifically, he proposed that innate immune system deter- protein, which gave it the name TIR (for Toll, IL-1R, and Resis- mined the origin of antigens recognized by T and B cells and tance protein) domain (Gay and Keith, 1991; Whitham et al., instructed the latter to initiate the response if antigen was of 1994). The idea therefore was to find a new transmembrane microbial origin. Pathogen sensing, in turn, was proposed to protein that would contain a C-type lectin ectodomain and a cyto- be mediated by a set of germline-encoded pattern recognition plasmic TIR domain. receptors that detect conserved products of microbial biosyn- In early 1996, a piece of an expressed sequence tag sequence thetic pathways (known as pathogen-associated molecular was identified in the NCBI database as containing similarity to patterns [PAMPs]). Janeway further pointed out that this form a portion of a TIR domain and used to screen a splenic cDNA

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library. To initial disappointment, the full-length clone did not required for LPS responsiveness, thus providing the first genetic contain a C-type lectin domain. Instead, it was a homolog of evidence for TLR4 function in microbial recognition. The same Drosophila Toll, which was known to have an endogenous result was later reported by Danielle Malo and colleagues (Qure- protein ligand Spatzle (Schneider et al., 1994). Moreover, one shi et al., 1999). Subsequent studies by Shizuo Akira and many sequence with similarity to Drosophila Toll was deposited in others have elucidated the specificities of other TLRs for various the database as ‘‘randomly sequenced cDNA 786’’ (rsc786) microbial ligands (Takeda et al., 2003). Thanks to these contribu- (Nomura et al., 1994), and it was reported in early 1996 that the tions, we now know that TLRs detect a variety of bacterial, viral, protein it encoded did not activate NF-kB(Mitcham et al., fungal, and protozoal ligands. TLR signaling pathways have also 1996). The reason for this negative result was that rsc786 is been elucidated in some detail, again in large part because of the what is now known as TLR1, which was subsequently shown numerous gene ‘‘knockout’’ mice generated by Shizuo Akira and by Alan Aderem and colleagues to signal only as a heterodimer colleagues. The latest breakthrough in the TLR field was the with TLR2. However, the clone isolated in the Janeway lab en- elucidation of the structures of several TLRs, in some cases in coded what is now known as TLR4, which functions as a homo- complex with their ligands (Jin and Lee, 2008). These structures dimer, and accordingly, its constitutively active form was able to allowed us to see in great detail the structural principles of induce NF-kB. Furthermore, a few months later, in the summer of pattern recognition. 1996, we learned from Jules Hoffmann about their stunning discovery of the essential role of Drosophila Toll in antifungal Other Pattern Recognition Receptors defense. This finding (Lemaitre et al., 1996) further reinforced and Their Functions the possibility that a mammalian Toll homolog may also have Elucidation of the functions of TLRs in mammalian immunity has an immune function, and indeed it turned out to induce expres- provided experimental support for the concept proposed by sion of genes encoding B7 and several cytokines (Medzhitov Charlie Janeway two decades ago. However, TLRs are not the et al., 1997), suggesting that TLR may couple innate immune only sensors of microbial infection. In May of 1999, two indepen- recognition with activation of adaptive immunity (Fearon, dent studies reported identification and initial characterization of 1997). On the basis of this, and given the immune function of NOD1, a founding member of a family of cytosolic receptors Drosophila Toll discovered by Bruno Lemaitre and Jules Hoff- (Bertin et al., 1999; Inohara et al., 1999). NOD1 and a related mann, human Toll was suggested to be involved in recognition protein, NOD2, were subsequently found to sense fragments of microbial PAMPs (Medzhitov and Janeway, 1997). The of bacterial peptidoglycans (Chamaillard et al., 2003; Girardin assumption at the time was that similar to its fly counterpart it et al., 2003a; Girardin et al., 2003b; Inohara et al., 2003). The would function downstream of a proteolytic cascade triggered role of NOD proteins in host defense is still being elucidated, by microbial recognition. However, the identity of the TLR but genetic studies have revealed their role in intestinal immunity ligands remained unknown for another year, despite numerous (Kobayashi et al., 2005), inflammation (Maeda et al., 2005), and attempts by multiple groups to test whether human TLR4 was the pathogenesis of Crohn’s disease (Hugot et al., 2001; Ogura a receptor for LPS. All these attempts failed because the other et al., 2001). In 2001, Gordon Brown and Siamon Gordon identi- key component of the LPS receptor complex, MD2, was not fied Dectin-1 as a receptor for b-glucans (Brown and Gordon, known at the time and was discovered by Kensuke Miyake and 2001). In subsequent studies, Dectin-1 was found to play impor- colleagues only two years later (Shimazu et al., 1999). In the tant roles in antifungal immunity (Gross et al., 2006; Leibundgut- meantime, several additional members of the Toll-like receptor Landmann et al., 2007; Saijo et al., 2007; Taylor et al., 2007). In family were identified (Chaudhary et al., 1998; Rock et al., 1998). 2004, Takashi Fujita and colleagues identified RIG-I and MDA-5 The first evidence that TLRs may indeed be involved in micro- as intracellular sensors for double-stranded RNA (Yoneyama bial recognition was provided by Paul Godowsky and colleagues et al., 2004). RIG-I was also found in a screen for mutations (Yang et al., 1998). This study indicated that TLRs could directly permissive for hepatitis C virus replication (Sumpter et al., respond to bacterial products. Specifically, it reported that TLR2 2005). RIG-I and MDA-5 were later shown to play essential roles conferred responsiveness to crude preparations of LPS, which in sensing RNA viruses and initiating antiviral immunity (Gitlin also contained bacterial lipoproteins. The same result was also et al., 2006; Kato et al., 2005; Kato et al., 2006). Definition of reported a few months later by a group from Tularik (Kirschning the RNA ligands recognized by RIG-I revealed the molecular et al., 1998). We now know that TLR2 in these experiments pattern sensed by this receptor (Hornung et al., 2006; Pichlmair responded to lipoproteins rather than to LPS (Hirschfeld et al., et al., 2006). Finally, in 2002, Ju¨ rg Tschopp and colleagues 2000). Nevertheless, the demonstration that a TLR recognized described the inflammasomes-multiprotein complexes that acti- a microbial stimulator had its impact because it immediately sug- vate caspase-1 and other inflammatory caspases to generate gested that all mammalian TLRs may recognize different micro- and secrete mature forms of IL-1 family members (Martinon bial ligands and that unlike Drosophila Toll, they may recognize et al., 2002). Although it is not yet clear how all the different in- them directly. In a commentary piece accompanying Godow- flammasomes are activated, at least some of them may function sky’s paper, Craig Gerard pointed out that the gene encoding as components of a pattern recognition system (Meylan et al., human TLR4 was located in a chromosomal region (9q32-33) 2006). syntenic to mouse chromosome 4, in which LPS unresponsive- Together with pentraxins, collectins, and ficolins (Bottazzi ness in the C3H/HeJ strain was previously mapped, suggesting et al., 2006; Holmskov et al., 2003), these newly identified pattern that TLR4 may be involved in LPS recognition (Gerard, 1998). recognition receptors account for most of the pathogen-sensing Positional cloning of the lps locus by Bruce Beutler and capabilities of mammalian hosts (Figure 1). However, there are colleagues (Poltorak et al., 1998) demonstrated that TLR4 was clearly additional pathogen recognition systems that remain to

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Figure 1. Pattern Recognition Receptors Major classes of mammalian pattern recognition receptors and some of their representative microbial ligands are shown. Secreted pattern recognition molecules activate complement and function as opsonins. Transmembrane and intracellular receptors trigger signaling pathways for innate host defense and activate adaptive immune responses. Mechanisms of inﬂammasome activation are still being deﬁned. NLRs, NOD-like receptors; CRD, carbohydrate recognition domain; ITAM, immunoreceptor tyrosine-based activation motif.

Rather, different mechanisms of immune recognition may sometimes work in speciﬁc combinations to trigger optimal immune responses. Therefore, different theories need not be competing, so long as they explain distinct phenomena. Thus, in addition to pattern recognition theory, three other concepts have been proposed to explain the mechanisms of activation of immune responses. These are the missing self theory, the guard theory, and the danger theory.

Missing Self Recognition The missing self concept was proposed by Klas Ka¨ rre to explain the specificity of tumor cell recognition by NK cells (Karre et al., 1986). The molecular basis be identified. One example is the intracellular receptors involved for missing self recognition by NK cells is the engagement of in recognition of viral and bacterial DNA that trigger the type I a class of inhibitory receptors (initially discovered by Wayne Yo- interferon response (Ishii et al., 2006; Okabe et al., 2005; Stetson koyama and colleagues), typically containing immunoreceptor and Medzhitov, 2006). It is likely that there is more than one tyrosine-based inhibitory motifs (ITIMs) (Karlhofer et al., 1992). receptor involved in intracellular sensing of microbial DNA They recruit SHP-1 tyrosine phosphatase to block Syk-72 (Takaoka et al., 2007). Another example is the receptor(s) dependent NK activating pathways, including cytotoxicity (Rau- involved in the recognition of chitin. Chitin is an abundant poly- let and Vance, 2006). The missing self concept also applies to saccharide produced by arthropods and parasitic worms, and many other forms of innate immune recognition. Activation of it was recently demonstrated that chitin can trigger a type 2 the alternative complement pathway (Gotze and Muller-Eber- inflammatory response (Reese et al., 2007). At least some of hard, 1971), for example, is negatively regulated, among other the activities of chitin are mediated by TLR2 (Da Silva et al., things, by Factor H, which can distinguish between microbial 2008), although additional receptors are likely to exist. Other and host cells by binding to host-specific sialic acids and pre- receptors involved in recognition of parasitic worms remain to venting the alternative pathway of complement activation on be identified. Innate immune recognition of helminthes is poorly host cells (Meri and Pangburn, 1990). There is growing evidence understood, and it is not even clear to what extent pattern recog- that phagocytosis is also negatively regulated by a variety of nition plays a role here. Indeed, multicellular parasites may not ITIMs containing inhibitory receptors, including SIRPa and prob- be evolutionarily distant enough from mammalian hosts to ably Siglec proteins. Thus, recognition of CD47 on target cells by produce reliable targets for pattern recognition receptors. With SIRPa prevents their phagocytosis by macrophages (Oldenborg the exception of chitin, there are no other known molecular pro- et al., 2000). Siglecs may play a similar role by recognizing host- ducts that are conserved among parasites and distinct from the specific sialic acids, analogously to Factor H (Crocker and Varki, mammalian hosts. Therefore, pattern recognition may not play 2001). Sialic acid recognition on antigens by CD22 may play a major role in the detection of multicellular parasites. Instead, a similar role in controlling B cell activation (Doody et al., 1995). other forms of innate immune recognition may play a predomi- It should be noted that missing self recognition is essentially nant role (see below). a negative control of some activating signal and thus always Pattern recognition also does not explain immune responses works in conjunction with an activating pathway. The activating to transplants and to most allergens. However, there is no other signal itself can be triggered by a variety of mechanisms. Thus, theory (and there may never be) that can explain all immune the alternative complement pathway is activated by properdin, responses, simply because they do not all follow the same rules. which was discovered in the early 1950s by Louis Pillemer

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(Pillemer et al., 1954). Properdin itself has features of a pattern can trigger (and therefore is sensed by) the Spatzle-activating recognition protein (Kemper and Hourcade, 2008). Phagocytosis proteolytic cascade resulting in the induction of antifungal immu- is positively regulated by a variety of phagocytic receptors nity (Gottar et al., 2006). In mammals, proteolytic activity of (Underhill and Ozinsky, 2002), some of which are pattern recog- protease allergens is detected by unknown sensors expressed nition receptors that directly recognize microbial cells. Finally, on basophils, resulting in the initiation of a T helper 2 cell response NK cell cytotoxic activity is triggered by activating receptors, (Sokol et al., 2007). This mechanism of allergen sensing presum- which recognize ligands that are induced on target cells upon ably evolved to detect proteases secreted by parasitic worms, viral infection and by some forms of stress (Lanier, 2005). How whereas allergens happen to trigger the same pathway inadver- these ligands are induced in virally infected cells is not yet well tently. As mentioned above, the pattern recognition strategy may defined, but it is possible that the intracellular sensors of viral not be sufficient or even practical for the detection of multicellular infections, such as RIG-I and MDA-5, may play a role. Thus it parasites. The host may have evolved instead a set of sensors appears that in some (but certainly not all) cases, the positive that detect abnormal biochemical activities associated with signals that are opposed by the ‘‘missing self’’ pathways may helminth infections, much like guard proteins in plants detect be inducible by the pattern recognition receptors. In other cases, activities of virulence factors. Cell-autonomous sensing of they may be triggered by mechanisms that are explained by the cellular perturbations associated with viral infections may also guard theory. be coupled with the induction of positive signals for NK cell activation. Guard Theory Interestingly, in some contexts, inflammasome activation and Another theory of immune recognition, known as ‘‘guard theory,’’ IL-1 production are critical for the induction of T cell responses was proposed by Dangl and Jones to explain the genetics of host (Ben-Sasson et al., 2009; Ichinohe et al., 2009), just as sensing resistance in plants (Dangl and Jones, 2001). Individual resis- of allergen protease activity by basophils is critical for Th2 cell tance genes in plants are often required to confer protection responses (Sokol et al., 2007). This suggests that indirect from a specific strain of pathogen carrying a particular virulence sensing of ‘‘virulence activity’’ can indeed be linked with the acti- factor. (In keeping with the overly confusing terminology in vation of adaptive immunity. animal immunity, ‘‘virulence factors’’ in plant immunity are referred to as ‘‘avirulence factors.’’) The idea of the guard theory Danger Theory is that rather than detecting pathogens directly, the products The danger theory was proposed by Polly Matzinger in 1994 as of at least some individual resistance genes monitor, or ‘‘guard,’’ an alternative to self-nonself discrimination theories (Matzinger, certain cellular processes that are often targeted by the virulence 1994). The main point of the danger model is that the immune factors of pathogens. Disturbances in these processes are system is not concerned with the origin of the antigen. Rather, sensed by resistance gene products, which become activated the immune response was suggested to be initiated by ‘‘danger and trigger a host defense response, for example, the hypersen- signals’’ released from damaged tissues. Thus, anything that can sitivity response. This form of microbial detection thus does not cause tissue damage, be it a pathogen or a surgical procedure, require direct molecular recognition of the pathogen, but rather is was proposed to induce an immune response. Benign stimuli not based on sensing the results of virulence factor activity by moni- associated with tissue damage, whether microbial or not, were toring their effects on the host. Because plants also use pattern proposed to be ignored by the immune system. Thus, according recognition for pathogen sensing, it is likely that the two mecha- to the danger model, initiation of the immune response is not nisms normally work together (Jones and Dangl, 2006). dependent on microbial recognition, but rather on the ability of Do the principles of guard theory apply to animal immunity? pathogens (or a transplantation procedure) to cause tissue Two lines of recent evidence suggest that they do. First, at least damage. Moreover, according to the danger model, pattern some inflammasomes (specifically the NALP3 inflammasome) recognition receptors (for example, TLRs) did not evolve to appear to function by monitoring cellular membrane integrity recognize their microbial ligands (for example, LPS); rather, the (Ogura et al., 2006). As it happens, cellular membrane integrity microbial ligands evolved to bind to and activate TLRs (Mat- is often targeted by pathogens. Many Gram-positive pathogens, zinger, 2002). for example, produce pore-forming exotoxins that can be se- Although the danger theory gained popularity in the past nsed by the NALP3 inflammasome (Mariathasan et al., 2006). decade, there appears to be a lot of confusion as to what exactly The NALP3 inflammasome can also be activated by noninfec- is considered to be ‘‘danger signals.’’ The term ‘‘danger signal’’ is tious stimuli that affect cellular membranes, although the mech- now often used to refer to microbial TLR ligands, which is ironic anisms involved remain poorly defined (Martinon et al., 2009). because the implication is the exact opposite of the main postu- Interestingly, NALP3 and other NOD-like receptors are structur- late of the danger theory. In other cases, PAMPs are being distin- ally similar to the products of many plant resistance genes, guished from DAMPs (damage-associated molecular patterns). although it is too early to say whether this similarity extends to There are certainly signals released from damaged tissues that their mechanisms of action. trigger inflammatory and tissue repair responses. Tissue damage The second example of sensing virulence activity is based on of course initiates the inflammatory response, but in the absence the detection of a proteolytic activity. In Drosophila, the proteo- of infection, this response leads to tissue repair, rather than adap- lytic cascade that leads to processing of Spatzle and activation tive immunity, which in this case should be autoreactive. It is clear of Toll is normally triggered by pattern recognition molecules that microbial ligands of pattern recognition receptors can trigger (Hoffmann, 2003), but can also be triggered by a virulence factor the immune response even in isolation from the pathogens. Simi- (a protease) secreted by a fungal pathogen. This protease, in turn, larly, nonpathogenic microbes that do not cause tissue damage

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can also induce immune responses, as evidenced, for example, with the type of antigen used in immunizations. Unlike the by the generation of IgA antibodies specific to commensal previous publications (Pasare and Medzhitov, 2004, 2005), in bacteria (Macpherson and Slack, 2007). Thus, microbial recogni- which native protein antigens were used for immunizations, tion appears to be sufficient to initiate the immune response, even Gavin et al. exclusively examined immune responses to hapten- in the absence of tissue damage. ated antigens. However, haptenated antigens, unlike native Microbial recognition, however, does not explain all adaptive protein antigens, have intrinsic immunogenic activity (Palm and immune responses. Some types of cell death are thought to be Medzhitov, 2009a). Thus, the results reported by Gavin et al. immunogenic in some contexts (Green et al., 2009; Rock and are not directly comparable to the previous reports. Second, Kono, 2008), and receptors that can sense immunogenic forms adjuvants have at least two types of activities: First, they trigger of cell death are beginning to be identified (Sancho et al., 2009; innate immune recognition pathways, commonly by engaging Yamasaki et al., 2008). Certain aspects of the danger model one or more pattern recognition receptors, and second, mineral may indeed be valid; tissue damage is likely to play a role in the oil (a component of Freund’s adjuvants) and alum have a depot regulation of immune responses, although it is unlikely to be effect that is critical when soluble antigens are used (but prob- necessary or sufficient to induce an adaptive immune response. ably dispensable for particulate antigens). Thus the TLR-inde- But then again, this depends on the definition of adaptive immune pendent, adjuvant-mediated enhancement of the antibody re- responses. If there are adaptive immune responses that play sponse to haptenated antigens reported by Gavin et al. most roles other than protection from microbial infections, than we likely reflects the depot effect, as previously reported for will all have to reconsider many of the arguments on which we MyD88-independent Th2 cell responses (Schnare et al., 2001). currently rely. Immunogenicity, on the other hand, was provided as a result of The Janeway model and the Matzinger model started off as the use of haptenated antigens. The fact that TLRs are not alternative views on the mechanisms of initiation of immunity. required for all immune responses have been well documented The two models have now coexisted for 15 years. As Francis before (Rivera et al., 2006; Schnare et al., 2001; Way et al., Crick once noted: ‘‘The basic trouble is that nature is so complex 2003), as is the fact that TLRs and several other pattern recogni- that many quite different theories can go some way to explaining tion receptors are sufficient to induce adaptive immunity, the results.’’ Eventually, the differences in our views will un- thereby making the requirement for any particular receptor doubtedly degenerate into purely semantic arguments, at which dependent on the pathogen or antigen and adjuvant used in point they will become irrelevant. immunization.

Innate Control of Adaptive Immunity TLRs in Human Immunity The principles of innate control of adaptive immunity have been Recent studies have uncovered an important role of TLRs in discussed before (Fearon and Locksley, 1996; Iwasaki and human immunity. Thus, individuals carrying mutations in TLR5 Medzhitov, 2004) and will not be covered here in detail. However, were found to be susceptible to Legionnaires’ disease (Hawn some aspects of this important subject have recently become et al., 2003). Surprisingly, TLR3 deficiency in humans resulted controversial, thus generating a great deal of confusion. in susceptibility to herpes simplex encephalitis (Zhang et al., As discussed above, several families of pattern recognition 2007). In addition, patients with null mutations in IRAK4 and receptors are now known and a few more remain to be identified. MyD88 are highly susceptible to several bacterial infections, In addition, other strategies of innate immune recognition, in which often result in lethality early in life (Ku et al., 2007; Medve- particular sensing of virulence activities and missing self recog- dev et al., 2003; von Bernuth et al., 2008). If the patients passed nition, are likely to play an important role in the control of adap- the critical period in the first few years of life, however, compen- tive immunity. It is now well established that several pattern satory host defense mechanisms, presumably in large part recognition receptors are coupled with the induction of adaptive antibody-mediated, afford protection from some pathogens, immunity, including TLRs, NLRs, Dectin-1, RIG-I, and MDA-5 although the patients still suffer from recurrent pyogenic bacte- (Palm and Medzhitov, 2009b). Additional, currently unknown rial infections. Although these findings were interpreted as indi- receptors that can induce adaptive immune responses must cating a narrowly restricted and redundant role of TLRs in human exist as well because some immune responses (for example, immunity (Ku et al., 2007), this interpretation is incongruent with some Th2 cell responses) cannot be accounted for by any of the fact that almost half of the patients with IRAK4 deficiency die the known innate immune-sensing mechanisms. It should also from infections, even though a majority of the patients are pre- be self-evident that if more than one pathway is sufficient to sumably on antibiotic medications. In a ‘‘natural’’ environment, induce an immune response, then none of the individual path- without modern medical interference, presumably all of them ways is required in general, except in the absence of stimuli for would be dead from infections early in life. Clearly, however, in any of the alternative pathways. Numerous studies have now some of the patients examined so far, the adaptive immune demonstrated that some adaptive immune responses are response can partially compensate for TLR signaling defects. It dependent on the TLR-MyD88 pathway, whereas others are would be interesting to learn in the future the rules of immunolog- not (Palm and Medzhitov, 2009b). However, a recent report ical compensation, which presumably operate in most types of (Gavin et al., 2006) claimed that TLRs are not involved in the nonlethal immunodeficiencies. control of adaptive immunity and that common adjuvants do not work through TLRs. This caused a great deal of confusion, Conclusions given that the reasons for these discrepant results were unclear. The 20 years that passed since Janeway’s publication have seen That main reason for the discrepancy, as it turns out, has to do a tremendous progress in our understanding of the innate

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immune system and its role in mammalian host defense. Still, to Coutinho, A., and Moller, G. (1974). Immune activation of B cells: Evidence for be sure, we will continue ‘‘approaching the asymptote’’ for many ‘‘one non-specific triggering signal’’ not delivered by the Ig receptors. Boll. Ist. Sieroter. Milan. suppl. 53, 131–143. more years. No theory is ever complete, and pattern recognition concept will continue to evolve and eventually integrate with Crocker, P.R., and Varki, A. (2001). Siglecs, sialic acids and innate immunity. Trends Immunol. 22, 337–342. other concepts into a more general theory. We soon will know the entire repertoire of pattern recognition receptors and will Da Silva, C.A., Hartl, D., Liu, W., Lee, C.G., and Elias, J.A. (2008). TLR-2 and learn more about other innate immune-sensing mechanisms. IL-17A in chitin-induced macrophage activation and acute inflammation. J. Immunol. 181, 4279–4286. We will then be able to learn all possible pathways for the activation of adaptive immunity. Perhaps at that point we will be able to Dangl, J.L., and Jones, J.D. (2001). Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. address far more daunting questions of the requirements for the generation of protective immunity. Here we know very little, and Doody, G.M., Justement, L.B., Delibrias, C.C., Matthews, R.J., Lin, J., this in part explains our continuous failures in developing new Thomas, M.L., and Fearon, D.T. (1995). A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 269, 242–244. effective vaccines. Once the principles of protective immunity are understood, we may finally be able to deliver on the promise Elomaa, O., Kangas, M., Sahlberg, C., Tuukkanen, J., Sormunen, R., Liakka, A., Thesleff, I., Kraal, G., and Tryggvason, K. (1995). Cloning of a novel of defeating major infectious diseases with vaccination. But then bacteria-binding receptor structurally related to scavenger receptors and again, pathogens may have a different opinion on this matter. expressed in a subset of macrophages. Cell 80, 603–609.

Ezekowitz, A.R.B. (1991). Ante-antibody immunity. Curr. Biol. 1, 60–62. ACKNOWLEDGMENTS Fearon, D.T. (1997). Seeking wisdom in innate immunity. Nature 388, 323–324.

This review is not meant to be a comprehensive account of the history of innate Fearon, D.T., and Locksley, R.M. (1996). The instructive role of innate immunity immunity, and therefore many important contributions are not discussed or in the acquired immune response. Science 272, 50–53. cited here. Some of the views expressed here reflect my personal opinion and should not be taken as representing a consensus in the field. I would Gavin, A.L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B., and Nema- like to thank T. Ichinohe for helping with the figure and my friends and zee, D. (2006). Adjuvant-enhanced antibody responses in the absence of toll- colleagues for reading the manuscript. This article is dedicated to the memory like receptor signaling. Science 314, 1936–1938. of C. Janeway. Gay, N.J., and Keith, F.J. (1991). Drosophila Toll and IL-1 receptor. Nature 351, 355–356.

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