Immunology_Janeway

Immunology_Janeway. This chapter has described the preexisting, constitutive components of innate immunity. The body’s epithelial surfaces are a constant barrier to pathogen entry and have specialized adaptations, such as cilia, various antimicrobial molecules and mucus, that provide the simplest form of innate immunity. The complement system is a more specialized system that combines direct recognition of microbes with a complex effector system. Of the three pathways that can activate complement, two are devoted to innate immunity. The lectin pathway relies on pattern recognition receptors that detect microbial membranes, while the alternative pathway relies on spontaneous complement activation that is down-regulated by host molecules expressed on self membranes. The main event in complement activation is accumulation of C3b on microbial membranes, which is recognized by complement receptors on phagocytic cells to promote microbial clearance by cells recruited to sites of infection by C3a and C5a. In


Immunology_Janeway

Immunology_Janeway. accumulation of C3b on microbial membranes, which is recognized by complement receptors on phagocytic cells to promote microbial clearance by cells recruited to sites of infection by C3a and C5a. In addition, C5b initiates the membrane-attack complex that is able to lyse some microbes directly. The complement cascade is under regulation to prevent attack on host tissues, and genetic variation in regulatory pathways can result in autoimmune syndromes and age-related tissue damage.


Immunology_Janeway

Immunology_Janeway. Questions. 2.1 Multiple Choice: The widely used β-lactam antibiotics are mainly active against Gram-positive bacteria. These inhibit the transpeptidation step in synthesis of peptidoglycan, a major component of the bacterial cell wall that is critical for the survival of the microorganism. Which of the following is an antimicrobial enzyme that functions to disrupt the same bacterial structure that β-lactams ultimately target? A. Phospholipase A B. Lysozyme C. Defensins D. Histatins 2.2 Short Answer: Why is the capacity of mannose-binding lectin (MBL) trimers to oligomerize important for their function? 2.3 Multiple Choice: Choose the option that correctly describes ficolins. A. C-type lectin domain, affinity for carbohydrates such as fucose and N-acetylglucosamine (GlcNAc), synthesized in the liver B. Fibrinogen-like domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver


Immunology_Janeway

Immunology_Janeway. B. Fibrinogen-like domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver C. C-type lectin domain, affinity for oligosaccharides containing acetylated sugars, synthesized in the liver D. Fibrinogen-like domain, affinity for carbohydrates such as fucose and N-acetylglucosamine (GlcNAc), synthetized in the liver and lungs 2.4 Fill-in-the-Blanks: For each of the following sentences, fill in the blanks with the best word selected from the list below. Not all words will be used; each word should be used only once. Like MBLs, ficolins form oligomers with ________ and ________. Such interaction allows the oligomer to cleave the complement components ________ and ________. Once these are cleaved, they form ________, a C3 convertase, which cleaves ________ and permits the formation of the membrane-attack complex.


Immunology_Janeway

Immunology_Janeway. 2.5 Short Answer: One way in which the alternative pathway activates is by spontaneous hydrolysis of the C3 thioester bond that is normally used to covalently attach to the pathogen’s surface. How can the alternative pathway proceed to form a membrane-attack complex if the C3 convertase that initiates this process is soluble? 2.6 Fill-in-the-Blanks: Paroxysmal nocturnal hemoglobinuria, a disease characterized by episodes of intravascular red blood cell lysis, is the result of red blood cells losing the expression of ________ and ________, which renders them susceptible to lysis by the ________ pathway of the complement system. 2.7 Matching: Match each of the following complement-regulatory proteins with the pathological manifestation that would develop if this factor were defective: A. C1INH 1. Atypical hemolytic uremic syndrome B. Factor H & factor I 2. Hereditary angioedema


Immunology_Janeway

Immunology_Janeway. A. C1INH 1. Atypical hemolytic uremic syndrome B. Factor H & factor I 2. Hereditary angioedema C. DAF 3. Paroxysmal nocturnal hemoglobinuria 2.8 Multiple Choice: Diseases such as cryoglobulinemia and systemic lupus erythematosus usually present with low C3 and C4 levels in blood due to the activation of the classical complement pathway. In contrast, diseases such as dense deposit disease or C3 glomerulonephritis generally have low C3 due to activation of the alternative complement pathway. What would be the expected levels of C2 and C4 in a patient suffering from dense deposit disease or C3 glomerulonephritis? A. Normal B. High C. Low D. High C4 and low C3 2.9 True or False: Mucins secreted at a mucosal surface exhibit direct microbicidal activities. 2.10 Short Answer: Neisseria meningitidis and Staphylococcus aureus each prevent complement activation in different ways. Explain how each does so.


Immunology_Janeway

Immunology_Janeway. 2.10 Short Answer: Neisseria meningitidis and Staphylococcus aureus each prevent complement activation in different ways. Explain how each does so. 2.11 True or False: Both neutrophils and Paneth cells of the gut secrete antimicrobial peptides, such as defensins, only upon stimulation. 2.12 Short Answer: What are two products of the C3 convertase? Name three downstream events that can result from the formation of these products and lead to clearance of the microbe. 2.13 True or False: CD21 (CR1) is a complement receptor expressed on B cells that binds C3dg (a C3b breakdown product) and serves as a co-receptor to augment signaling and trigger a stronger antibody response. References. Section references. 2-1 Infectious diseases are caused by diverse living agents that replicate in their hosts. Kauffmann, S.H.E., Sher, A., and Ahmed, R.: Immunology of Infectious Diseases. Washington, DC: ASM Press, 2002.


Immunology_Janeway

Immunology_Janeway. Kauffmann, S.H.E., Sher, A., and Ahmed, R.: Immunology of Infectious Diseases. Washington, DC: ASM Press, 2002. Mandell, G.L., Bennett, J.E., and Dolin, R. (eds): Principles and Practice of Infectious Diseases, 4th ed. New York: Churchill Livingstone, 1995. 2-2 Epithelial surfaces of the body provide the first line of defense against infection. Gallo, R.L., and Hooper, L.V.: Epithelial antimicrobial defense of the skin and intestine. Nat. Rev. Immunol. 2012, 12:503–516. 2-3 Infectious agents must overcome innate host defenses to establish a focus of infection. Gorbach, S.L., Bartlett, J.G., and Blacklow, N.R. (eds): Infectious Diseases, 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2003. Hornef, M.W., Wick, M.J., Rhen, M., and Normark, S.: Bacterial strategies for overcoming host innate and adaptive immune responses. Nat. Immunol. 2002, 3:1033–1040. 2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins.


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Immunology_Janeway. 2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins. Cash, H.L., Whitham, C.V., Behrendt, C.L., and Hooper, L.H.: Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 2006, 313:1126–1130. De Smet, K., and Contreras, R.: Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol. Lett. 2005, 27:1337–1347. Ganz, T.: Defensins: antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 2003, 3:710–720. Mukherjee, S., Zheng, H., Derebe, M.G., Callenberg, K.M., Partch, C.L., Rollins, D., Propheter, D.C., Rizo, J., Grabe, M., Jiang, Q.X., and Hooper, L.V.: Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 2014, 505:103–107. Zanetti, M.: The role of cathelicidins in the innate host defense of mammals. Curr. Issues Mol. Biol. 2005, 7:179–196. 2-5 The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b.


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Immunology_Janeway. 2-5 The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b. Gros, P., Milder, F.J., and Janssen, B.J.: Complement driven by conformational changes. Nat. Rev. Immunol. 2008, 8:48–58. Janssen, B.J., Christodoulidou, A., McCarthy, A., Lambris, J.D., and Gros, P.: Structure of C3b reveals conformational changes that underlie complement activity. Nature 2006, 444:213–216. Janssen, B.J., Huizinga, E.G., Raaijmakers, H.C., Roos, A., Daha, M.R., Nilsson-Ekdahl, K., Nilsson, B., and Gros, P.: Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 2005, 437:505–511. 2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade.


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Immunology_Janeway. 2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade. Bohlson, S.S., Fraser, D.A., and Tenner, A.J.: Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol. Immunol. 2007, 44:33–43. Fujita, T.: Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2002, 2:346–353. Gál, P., Harmat, V., Kocsis, A., Bián, T., Barna, L., Ambrus, G., Végh, B., Balczer, J., Sim, R.B., Náray-Szabó, G., et al: A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations. J. Biol. Chem. 2005, 280:33435–33444.


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Immunology_Janeway. Héja, D., Kocsis, A., Dobó, J., Szilágyi, K., Szász, R., Závodszky, P., Pál, G., Gál, P.: Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2. Proc. Natl Acad. Sci. USA 2012, 109:10498–10503. Wright, J.R.: Immunoregulatory functions of surfactant proteins. Nat. Rev. Immunol. 2005, 5:58–68. 2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway. McGrath, F.D., Brouwer, M.C., Arlaud, G.J., Daha, M.R., Hack, C.E., and Roos, A.: Evidence that complement protein C1q interacts with C-reactive protein through its globular head region. J. Immunol. 2006, 176:2950–2957. 2-8 Complement activation is largely confined to the surface on which it is initiated.


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Immunology_Janeway. 2-8 Complement activation is largely confined to the surface on which it is initiated. Cicardi, M., Bergamaschini, L., Cugno, M., Beretta, A., Zingale, L.C., Colombo, M., and Agostoni, A.: Pathogenetic and clinical aspects of C1 inhibitor deficiency. Immunobiology 1998, 199:366–376. 2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin in the presence of pathogens. Fijen, C.A., van den Bogaard, R., Schipper, M., Mannens, M., Schlesinger, M., Nordin, F.G., Dankert, J., Daha, M.R., Sjoholm, A.G., Truedsson, L., et al.: Properdin deficiency: molecular basis and disease association. Mol. Immunol. 1999, 36:863–867. Kemper, C., and Hourcade, D.E.: Properdin: new roles in pattern recognition and target clearance. Mol. Immunol. 2008, 45:4048–4056.


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Immunology_Janeway. Kemper, C., and Hourcade, D.E.: Properdin: new roles in pattern recognition and target clearance. Mol. Immunol. 2008, 45:4048–4056. Spitzer, D., Mitchell, L.M., Atkinson, J.P., and Hourcade, D.E.: Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly. J. Immunol. 2007, 179:2600–2608. Xu, Y., Narayana, S.V., and Volanakis, J.E.: Structural biology of the alternative pathway convertase. Immunol. Rev. 2001, 180:123–135. 2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation. Golay, J., Zaffaroni, L., Vaccari, T., Lazzari, M., Borleri, G.M., Bernasconi, S., Tedesco, F., Rambaldi, A., and Introna, M.: Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000, 95:3900–3908.


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Immunology_Janeway. Spiller, O.B., Criado-Garcia, O., Rodriguez De Cordoba, S., and Morgan, B.P.: Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack. Clin. Exp. Immunol. 2000, 121:234–241. Varsano, S., Frolkis, I., Rashkovsky, L., Ophir, D., and Fishelson, Z.: Protection of human nasal respiratory epithelium from complement-mediated lysis by cell-membrane regulators of complement activation. Am. J. Respir. Cell Mol. Biol. 1996, 15:731–737. 2-11 Complement developed early in the evolution of multicellular organisms. Fujita, T.: Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2002, 2:346–353. Zhang, H., Song, L., Li, C., Zhao, J., Wang, H., Gao, Q., and Xu, W.: Molecular 2-16 Complement control proteins regulate all three pathways of cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri. Mol. Immunol. 2007, 44:3492–3500.


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Immunology_Janeway. 2-12 Surface-bound C3 convertase deposits large numbers of C3b activity. Rawal, N., and Pangburn, M.K.: Structure/function of C5 convertases of complement. Int. Immunopharmacol. 2001, 1:415–422. 2-13 Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for the bound complement proteins. Gasque, P.: Complement: a unique innate immune sensor for danger signals. Mol. Immunol. 2004, 41:1089–1098. Helmy, K.Y., Katschke, K.J., Jr., Gorgani, N.N., Kljavin, N.M., Elliott, J.M., Diehl, L., Scales, S.J., Ghilardi, N., and van Lookeren Campagne, M.: CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens. Cell 2006, 124:915–927. 2-14 The small fragments of some complement proteins initiate a local inflammatory response. Barnum, S.R.: C4a: an anaphylatoxin in name only. J. Innate Immun. 2015, 7:333-9. Kohl, J.: Anaphylatoxins and infectious and noninfectious inflammatory diseases. Mol. Immunol. 2001, 38:175–187.


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Immunology_Janeway. Barnum, S.R.: C4a: an anaphylatoxin in name only. J. Innate Immun. 2015, 7:333-9. Kohl, J.: Anaphylatoxins and infectious and noninfectious inflammatory diseases. Mol. Immunol. 2001, 38:175–187. Schraufstatter, I.U., Trieu, K., Sikora, L., Sriramarao, P., and DiScipio, R.: Complement C3a and C5a induce different signal transduction cascades in endothelial cells. J. Immunol. 2002, 169:2102–2110. 2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens. Hadders, M.A., Beringer, D.X., and Gros, P.: Structure of C8α-MACPF reveals mechanism of membrane attack in complement immune defense. Science 2007, 317:1552–1554. Parker, C.L., and Sodetz, J.M.: Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8γ is not essential. Mol. Immunol. 2002, 39:453–458.


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Immunology_Janeway. Parker, C.L., and Sodetz, J.M.: Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8γ is not essential. Mol. Immunol. 2002, 39:453–458. Scibek, J.J., Plumb, M.E., and Sodetz, J.M.: Binding of human complement C8 to C9: role of the N-terminal modules in the C8α subunit. Biochemistry 2002, 41:14546–14551. complement activation and protect the host from their destructive effects. Ambati, J., Atkinson, J.P., and Gelfand, B.D.: Immunology of age-related macular degeneration. Nat. Rev. Immunol. 2013, 13:438–451. Atkinson, J.P., and Goodship, T.H.: Complement factor H and the hemolytic uremic syndrome. J. Exp. Med. 2007, 204:1245–1248. Jiang, H., Wagner, E., Zhang, H., and Frank, M.M.: Complement 1 inhibitor is a regulator of the alternative complement pathway. J. Exp. Med. 2001, 194:1609–1616.


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Immunology_Janeway. Jiang, H., Wagner, E., Zhang, H., and Frank, M.M.: Complement 1 inhibitor is a regulator of the alternative complement pathway. J. Exp. Med. 2001, 194:1609–1616. Miwa, T., Zhou, L., Hilliard, B., Molina, H., and Song, W.C.: Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack. Blood 2002, 99:3707–3716. Singhrao, S.K., Neal, J.W., Rushmere, N.K., Morgan, B.P., and Gasque, P.: Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis. Am. J. Pathol. 2000, 157:905–918. Smith, G.P., and Smith, R.A.: Membrane-targeted complement inhibitors. Mol. Immunol. 2001, 38:249–255.


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Immunology_Janeway. Smith, G.P., and Smith, R.A.: Membrane-targeted complement inhibitors. Mol. Immunol. 2001, 38:249–255. Spencer, K.L., Hauser, M.A., Olson, L.M., Schmidt, S., Scott, W.K., Gallins, P., Agarwal, A., Postel, E.A., Pericak-Vance, M.A., and Haines, J.L.: Protective effect of complement factor B and complement component 2 variants in age-related macular degeneration. Hum. Mol. Genet. 2007, 16:1986–1992. Spencer, K.L., Olson, L.M., Anderson, B.M., Schnetz-Boutaud, N., Scott, W.K., Gallins, P., Agarwal, A., Postel, E.A., Pericak-Vance, M.A., and Haines, J.L.: C3 R102G polymorphism increases risk of age-related macular degeneration. Hum. Mol. Genet. 2008, 17:1821–1824. 2-17 Pathogens produce several types of proteins that can inhibit complement activation.


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Immunology_Janeway. 2-17 Pathogens produce several types of proteins that can inhibit complement activation. Blom, A.M., Rytkonen, A., Vasquez, P., Lindahl, G., Dahlback, B., and Jonsson, A.B.: A novel interaction between type IV pili of Neisseria gonorrhoeae and the human complement regulator C4B-binding protein. J. Immunol. 2001, 166:6764–6770. Serruto, D., Rappuoli, R., Scarselli, M., Gros, P., and van Strijp, J.A.: Molecular mechanisms of complement evasion: learning from staphylococci and meningococci. Nat. Rev. Microbiol. 2010, 8:393–399. The Induced Responses of Innate Immunity


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Immunology_Janeway. In Chapter 2 we introduced the innate defenses—such as epithelial barriers, secreted antimicrobial proteins, and the complement system—that act immediately upon encounter with microbes to protect the body from infection. We also introduced the phagocytic cells that lie beneath the epithelial barriers and stand ready to engulf and digest invading microorganisms that have been flagged for destruction by complement. These phagocytes also initiate the next phase of the innate immune response, inducing an inflammatory response that recruits new phagocytic cells and circulating effector molecules to the site of infection. In this chapter we describe how phagocytic cells of the innate immune system detect microbes or the cellular damage they cause, how they destroy these pathogens, and how they orchestrate downstream inflammatory responses through production of cytokines and chemokines (chemoattractant cytokines). We also introduce other cells of the innate immune system—a diverse array of


Immunology_Janeway

Immunology_Janeway. orchestrate downstream inflammatory responses through production of cytokines and chemokines (chemoattractant cytokines). We also introduce other cells of the innate immune system—a diverse array of specialized innate lymphoid cells (ILCs) including the natural killer cells (NK cells)—that contribute to innate host defenses against viruses and other intracellular pathogens. Also in this stage of infection dendritic cells initiate adaptive immune responses, so that if the infection is not cleared by innate immunity, a full immune response will ensue.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. The basis of the adaptive immune system’s enormous capacity for antigen recognition has long been appreciated. In contrast, the basis of recognition of microbial products by innate immune sensors was discovered only in the late 1990s. Initially, innate recognition was considered to be restricted to relatively few pathogen-associated molecular patterns, or PAMPs, and we have already seen examples of such recognition of microbial surfaces by complement (see Chapter 2). In the last several years, with the discovery of an increasing number of innate receptors that are capable of discriminating among a number of closely related molecules, we have come to realize that a much greater flexibility in innate recognition exists than had been previously thought.


Immunology_Janeway

Immunology_Janeway. The first part of this chapter examines the cellular receptors that recognize pathogens and signal for a cellular innate immune response. Regular patterns of molecular structure are present on many microorganisms but do not occur on the host body’s own cells. Receptors that recognize such features are expressed on macrophages, neutrophils, and dendritic cells, and they are similar to the secreted molecules, such as ficolins and histatins, described in Chapter 2. The general characteristics of these pattern recognition receptors (PRRs) are contrasted with those of the antigen-specific receptors of adaptive immunity in Fig. 3.1. A new insight is that self-derived host molecules can be induced that indicate cellular infection, damage, stress, or transformation, and that some innate receptors recognize such proteins to mediate responses by innate immune cells. Such indicator molecules have been termed ‘damageassociated molecular patterns,’ or DAMPs, and some of the molecules in this class


Immunology_Janeway

Immunology_Janeway. recognize such proteins to mediate responses by innate immune cells. Such indicator molecules have been termed ‘damageassociated molecular patterns,’ or DAMPs, and some of the molecules in this class can be recognized by receptors also involved in pathogen recognition, such as the Toll-like receptors (TLRs).


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. Induced innate responses to infection. Fig. 3.1 Comparison of the characteristics of recognition molecules of the innate and adaptive immune systems. The innate immune system uses germline-encoded receptors while the adaptive immune system uses antigen receptors of unique specificity assembled from incomplete gene segments during lymphocyte development. Antigen receptors of the adaptive immune system are clonally distributed on individual lymphocytes and their progeny. Typically, receptors of the innate immune system are expressed non-clonally, that is, they are expressed on all the cells of a given cell type. However, NK cells express various combinations of NK receptors from several families, making individual NK cells different from one another. A particular NK receptor may not be expressed on all NK cells.


Immunology_Janeway

Immunology_Janeway. Coordination of the innate immune response relies on the information provided by many types of receptors. Pattern recognition receptors can be classified into four main groups on the basis of their cellular localization and their function: free receptors in the serum, such as ficolins and histatins (discussed in Chapter 2); membrane-bound phagocytic receptors; membrane-bound signaling receptors; and cytoplasmic signaling receptors. Phagocytic receptors primarily signal for phagocytosis of the microbes they recognize. A diverse group of receptors, including chemotactic receptors, help to guide cells to sites of infection, and other receptors, including PRRs and cytokine receptors, can control the activity of effector molecules at those sites.


Immunology_Janeway

Immunology_Janeway. In this part of the chapter we first look at the recognition properties of phagocytic receptors and of signaling receptors that activate phagocytic microbial killing mechanisms. Next we describe an evolutionarily ancient pathogen system of recognition and signaling, the Toll-like receptors (TLRs), the first of the innate sensor systems to be discovered, and several recently discovered systems that detect intracellular infections by sensing cytoplasmic microbial cell-wall components, foreign RNA, or foreign DNA. 3-1 After entering tissues, many microbes are recognized, ingested, and killed by phagocytes.


Immunology_Janeway

Immunology_Janeway. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, in most cases, it is immediately recognized by resident phagocytic cells. The main classes of phagocytic cells in the innate immune system are macrophages and monocytes, granulocytes, and dendritic cells. Macrophages are the major phagocyte population resident in most normal tissues at homeostasis. They can arise either from progenitor cells that enter the tissues during embryonic development, and then self-renew at steady state during life, or from circulating monocytes. Studies suggest that the embryonic progenitors arise from either the fetal liver, the yolk sac, or an embryonic region near the dorsal aorta called the aorta–gonad–mesonephros (AGM), although the relative contribution of these origins is still debated. Macrophages are found in especially large numbers in connective tissue: for example, in the submucosal layer of the gastrointestinal tract; in the submucosal layer of


Immunology_Janeway

Immunology_Janeway. these origins is still debated. Macrophages are found in especially large numbers in connective tissue: for example, in the submucosal layer of the gastrointestinal tract; in the submucosal layer of the bronchi, and in the lung interstitium—the tissue and intercellular spaces around the air sacs (alveoli)—and in the alveoli themselves; along some blood


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. vessels in the liver; and throughout the spleen, where they remove senescent blood cells. Macrophages in different tissues were historically given different names, for example, microglial cells in neural tissue and Kupffer cells in the liver. The self-renewal of these two types of cells is dependent on a cytokine called interleukin 34 (IL-34) that is produced in these tissues and acts on the same receptor as macrophage-colony stimulating factor (M-CSF).


Immunology_Janeway

Immunology_Janeway. During infection or inflammation, macrophages can also arise from monocytes that leave from the circulation to enter into tissues. Monocytes in both mouse and human develop in the bone marrow and circulate in the blood as two main populations. In humans, 90% of circulating monocytes are the ‘classical’ monocyte that expresses CD14, a co-receptor for a PRR described later, and function during infection by entering tissues and differentiating into activated inflammatory monocytes or macrophages. In mice, this monocyte population expresses high levels of the surface marker Ly6C. A smaller population are the ‘patrolling monocytes’ that roll along the endothelium rather than circulating freely in the blood. In humans, they express CD14 and CD16, a type of Fc receptor (FcγRIII; see Section 10-21), and are thought to survey for injury to the endothelium but do not differentiate into tissue macrophages. In mice, they express low levels of Ly6C.


Immunology_Janeway

Immunology_Janeway. The second major family of phagocytes comprises the granulocytes, which include neutrophils, eosinophils, and basophils. Of these, neutrophils have the greatest phagocytic activity and are the cells most immediately involved in innate immunity against infectious agents. Also called polymorphonuclear neutrophilic leukocytes (PMNs, or polys), they are short-lived cells that are abundant in the blood but are not present in healthy tissues. Macrophages and granulocytes have an important role in innate immunity because they can recognize, ingest, and destroy many pathogens without the aid of an adaptive immune response. Phagocytic cells that scavenge incoming pathogens represent an ancient mechanism of innate immunity, as they are found in both invertebrates and vertebrates.


Immunology_Janeway

Immunology_Janeway. The third class of phagocytes in the immune system is the immature dendritic cells that reside in lymphoid organs and in peripheral tissues. There are two main functional types of dendritic cells: conventional (or classical) dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). Both types of cells arise from progenitors within the bone marrow that primarily branch from cells of myeloid potential, and they migrate via the blood to tissues throughout the body and to peripheral lymphoid organs. Dendritic cells ingest and break down microbes, but, unlike macrophages and neutrophils, their primary role in immune defense is not the front-line, large-scale direct killing of microbes. A major role of cDCs is to process ingested microbes in order to generate peptide antigens that can activate T cells and induce an adaptive immune response. They also produce cytokines in response to microbial recognition that activate other types of cells against infection. cDCs are thus considered to


Immunology_Janeway

Immunology_Janeway. T cells and induce an adaptive immune response. They also produce cytokines in response to microbial recognition that activate other types of cells against infection. cDCs are thus considered to act as a bridge between innate and adaptive immune responses. pDCs are major producers of a class of cytokines known as type I interferons, or antiviral interferons, and are considered to be part of innate immunity; they are discussed in detail later in the chapter.


Immunology_Janeway

Immunology_Janeway. Because most microorganisms enter the body through the mucosa of the gut and respiratory system, skin, or urogenital tract, macrophages in the submucosal tissues are the first cells to encounter most pathogens, but they are soon reinforced by the recruitment of large numbers of neutrophils to sites of infection. Macrophages and neutrophils recognize pathogens by means of cell-surface receptors that can discriminate between the surface molecules of pathogens and those of the host. Although they are both phagocytic, macrophages and neutrophils have distinct properties and functions in innate immunity. MOVIE 3.1


Immunology_Janeway

Immunology_Janeway. MOVIE 3.1 MOVIE 3.2 lipid receptor (CD36) scavenger receptors (SR-A I/II, MARCO) mannose receptor CTLD Dectin-1 Bound material is internalized in phagosomes and broken down in phagolysosomes Macrophages have phagocytic receptors that bind microbes and their components mannose receptor Dectin-1 (˜-glucan receptor) scavenger receptor complement receptor Fc receptor complement receptor lipid receptor


Immunology_Janeway

Immunology_Janeway. The process of phagocytosis is initiated when certain receptors on the surface of the cell—typically a macrophage, neutrophil, or dendritic cell—interacts with the microbial surface. The bound pathogen is first surrounded by the phagocyte plasma membrane and then internalized in a large membrane-enclosed endocytic vesicle known as a phagosome. The phagosome fuses with one or more lysosomes to generate a phagolysosome, in which the lysosomal contents are released. The phagolysosome also becomes acidified, acquires antimicrobial peptides and enzymes, and undergoes enzymatic processes that produce highly reactive superoxide and nitric oxide radicals, which together kill the microbe (Fig. 3.2). Neutrophils are highly specialized for the intracellular killing of microbes, and contain different types of cytoplasmic granules—the primary granules and secondary granules described in Section 2-4. These granules fuse with phagosomes, releasing additional enzymes and antimicrobial peptides that


Immunology_Janeway

Immunology_Janeway. types of cytoplasmic granules—the primary granules and secondary granules described in Section 2-4. These granules fuse with phagosomes, releasing additional enzymes and antimicrobial peptides that attack the microbe. Another pathway by which extracellular material, including microbial material, can be taken up into the endosomal compartment of cells and degraded is receptor-mediated endocytosis, which is not restricted to phagocytes. Dendritic cells and other phagocytes can also take up pathogens by a nonspecific process called macropinocytosis, in which large amounts of extracellular fluid and its contents are ingested.


Immunology_Janeway

Immunology_Janeway. Macrophages and neutrophils constitutively express a number of cell-surface receptors that stimulate the phagocytosis and intracellular killing of microbes bound to them, although some also signal through other pathways to trigger responses such as cytokine production. These phagocytic receptors include several members of the C-type lectin-like family (see Fig. 3.2). For example, Dectin-1 is strongly expressed by macrophages and neutrophils and recognizes β-1,3-linked glucans (polymers of glucose), which are common components of fungal cell walls in particular. Dendritic cells also express Dectin-1, as well as several other C-type lectin-like phagocytic receptors, which will be discussed in relation to pathogen uptake for antigen processing and presentation in Chapter 9. Another C-type lectin, the mannose receptor (MR) expressed by macrophages and dendritic cells, recognizes various mannosylated ligands, including some present on fungi, bacteria, and viruses; it was once suspected to


Immunology_Janeway

Immunology_Janeway. the mannose receptor (MR) expressed by macrophages and dendritic cells, recognizes various mannosylated ligands, including some present on fungi, bacteria, and viruses; it was once suspected to have an important role in resistance to microbes. However, experiments with mice that lack this receptor do not support this idea. The macrophage mannose receptor is now thought to function mainly as a clearance receptor for host glycoproteins such as β-glucuronidase and lysosomal hydrolases, which have mannose-containing carbohydrate side chains and whose extracellular concentrations are raised during inflammation.


Immunology_Janeway

Immunology_Janeway. A second set of phagocytic receptors on macrophages, called scavenger receptors, recognize various anionic polymers and acetylated low-density lipoproteins. These receptors are structurally heterogeneous, consisting of at least six different molecular families. Class A scavenger receptors are membrane proteins composed of trimers of collagen domains (see Fig. 3.2).


Immunology_Janeway

Immunology_Janeway. Fig. 3.2 Macrophages express receptors that enable them to take up microbes by phagocytosis. First panel: macrophages residing in tissues throughout the body are among the first cells to encounter and respond to pathogens. They carry cell-surface receptors that bind to various molecules on microbes, in particular carbohydrates and lipids, and induce phagocytosis of the bound material. Second panel: Dectin-1 is a member of the C-type lectin-like family built around a single C-type lectin-like domain (CTLD). Lectins in general are based on a carbohydrate-recognition domain (CRD). The macrophage mannose receptor contains many CTLDs, with a fibronectin-like domain and a cysteine-rich region at its amino terminus. Class A scavenger receptors such as MARCO are built from collagen-like domains and form trimers. The receptor protein CD36 is a class B scavenger receptor that recognizes and internalizes lipids. Various complement receptors bind and internalize complement-coated bacteria. Third


Immunology_Janeway

Immunology_Janeway. form trimers. The receptor protein CD36 is a class B scavenger receptor that recognizes and internalizes lipids. Various complement receptors bind and internalize complement-coated bacteria. Third panel: phagocytosis of receptor-bound material is taken into intracellular phagosomes, which fuse with lysosomes to form an acidified phagolysosome in which the ingested material is broken down by lysosomal hydrolases.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. They include SR-A I, SR-A II, and MARCO (macrophage receptor with a collagenous structure), which all bind various bacterial cell-wall components and help to internalize bacteria, although the basis of their specificity is poorly understood. Class B scavenger receptors bind high-density lipoproteins, and they internalize lipids. One of these receptors is CD36, which binds many ligands, including long-chain fatty acids. A third set of receptors of crucial importance in macrophage and neutrophil phagocytosis is the complement receptors and Fc receptors introduced in Chapters 1 and 2. These receptors bind to complement-coated microbes or to antibodies that have bound to the surface of microbes and facilitate the phagocytosis of a wide range of microorganisms. 3-2 G-protein-coupled receptors on phagocytes link microbe recognition with increased efficiency of intracellular killing.


Immunology_Janeway

Immunology_Janeway. 3-2 G-protein-coupled receptors on phagocytes link microbe recognition with increased efficiency of intracellular killing. Phagocytosis of microbes by macrophages and neutrophils is generally followed by the death of the microbe inside the phagocyte. As well as the phagocytic receptors, macrophages and neutrophils have other receptors that signal to stimulate antimicrobial killing. These receptors belong to the evolutionarily ancient family of G-protein-coupled receptors (GPCRs), which are characterized by seven membrane-spanning segments. Members of this family are crucial to immune system function because they also direct responses to anaphylatoxins such as the complement fragment C5a (see Section 2-14) and to many chemokines, recruiting phagocytes to sites of infection and promoting inflammation.


Immunology_Janeway

Immunology_Janeway. The fMet-Leu-Phe (fMLF) receptor is a G-protein-coupled receptor that senses the presence of bacteria by recognizing a unique feature of bacterial polypeptides. Protein synthesis in bacteria is typically initiated with an N-formylmethionine (fMet) residue, an amino acid present in prokaryotes but not in eukaryotes. The fMLF receptor is named after a tripeptide, formylmethionyl-leucyl phenylalanine, for which it has a high affinity, although it also binds other peptide motifs. Bacterial polypeptides binding to this receptor activate intracellular signaling pathways that direct the cell to move toward the most concentrated source of the ligand. Signaling through the fMLF receptor also induces the production of microbicidal reactive oxygen species (ROS) in the phagolysosome. The C5a receptor recognizes the small fragment of C5 generated when the classical or lectin pathways of complement are activated, usually by the presence of microbes (see Section 2-14), and signals by a similar


Immunology_Janeway

Immunology_Janeway. recognizes the small fragment of C5 generated when the classical or lectin pathways of complement are activated, usually by the presence of microbes (see Section 2-14), and signals by a similar pathway as the fMLF receptor. Thus, stimulation of these receptors both guides monocytes and neutrophils toward a site of infection and leads to increased antimicrobial activity; these cell responses can be activated by directly sensing unique bacterial products or by messengers such as C5a that indicate previous recognition of a microbe.


Immunology_Janeway

Immunology_Janeway. The G-protein-coupled receptors are so named because ligand binding activates a member of a class of intracellular GTP-binding proteins called G proteins, sometimes referred to as heterotrimeric G proteins to distinguish them from the family of ‘small’ GTPases typified by Ras. Heterotrimeric G proteins are composed of three subunits: Gα, Gβ, and Gγ, of which the α subunit is similar to the small GTPases (Fig. 3.3). In the resting state, the G protein is inactive, not associated with the receptor, and a molecule of GDP is bound to the α subunit. Ligand binding induces conformational changes in the receptor that allow it to bind the G protein, which results in the displacement of the GDP from the G protein and its replacement with GTP. The active G protein dissociates into two components, the Gα subunit and a complex consisting of a Gβ and a Gγ subunit. Each of these components can interact with other intracellular signaling molecules to transmit and amplify the signal. G proteins can


Immunology_Janeway

Immunology_Janeway. the Gα subunit and a complex consisting of a Gβ and a Gγ subunit. Each of these components can interact with other intracellular signaling molecules to transmit and amplify the signal. G proteins can activate a wide variety of downstream enzymatic


Immunology_Janeway

Immunology_Janeway. MOVIE 3.3 GPCR chemokine or fMet-Leu-Phe Before ligand binding a GPCR is not associated with a G protein Ligand binding causes a conformational change in the receptor which enables it to associate with the G protein G protein dissociates into ˜ and °˛ subunits, both of which can activate other proteins The ˜ subunit cleaves GTP to GDP, allowing the ˜ and °˛ subunits to reassociate Inactive G protein has GDP bound G protein releases GDP and binds GTP Activation of the GTPases Rac, Rho, and Cdc42 stimulates chemotaxis or the respiratory burst Signaling terminates ˜°˛GTP GTP GDP GDP heterotrimeric G protein Rac/Rho Cdc42 Chemotaxis Respiratory burst


Immunology_Janeway

Immunology_Janeway. Fig. 3.3 G-protein-coupled receptors signal by coupling with intracellular heterotrimeric G proteins. First panel: G-protein-coupled receptors (GPCRs) such as the fMet-Leu-Phe (fMLF) and chemokine receptors signal through GTP-binding proteins known as heterotrimeric G proteins. In the inactive state, the α subunit of the G protein binds GDP and is associated with the β and γ subunits. Second panel: the binding of a ligand to the receptor induces a conformational change that allows the receptor to interact with the G protein, which results in the displacement of GDP and binding of GTP by the α subunit. Third panel: GTP binding triggers the dissociation of the G protein into the α subunit and the βγ subunit, each of which can activate other proteins at the inner face of the cell membrane. In the case of fMLF signaling in macrophages and neutrophils, the α subunit of the activated G protein indirectly activates the GTPases Rac and Rho, whereas the βγ subunit indirectly activates the


Immunology_Janeway

Immunology_Janeway. In the case of fMLF signaling in macrophages and neutrophils, the α subunit of the activated G protein indirectly activates the GTPases Rac and Rho, whereas the βγ subunit indirectly activates the GTPase Cdc42. The actions of these proteins result in the assembly of the NADPH oxidase, resulting in a respiratory burst. Chemokine signaling acts by a similar pathway and activates chemotaxis. Fourth panel: The activated response ceases when the intrinsic GTPase activity of the α subunit hydrolyzes GTP to GDP, and the α and βγ subunits reassociate. The intrinsic rate of GTP hydrolysis by α subunits is relatively slow, and signaling is regulated by additional GTPase-activating proteins (not shown), which accelerate the rate of GTP hydrolysis.


Immunology_Janeway

Immunology_Janeway. targets, such as adenylate cyclase, which produces the second messenger cyclic AMP; and phospholipase C, whose activation gives rise to the second messenger inositol 1,3,5-trisphosphate (IP3) and the release of free Ca2+.


Immunology_Janeway

Immunology_Janeway. Signaling by fMLF and C5a receptors influences cell motility, metabolism, gene expression, and cell division through activation of several Rho family small GTPase proteins. The α subunit of the activated G protein indirectly activates Rac and Rho, while the βγsubunit indirectly activates the small GTPase Cdc42 (see Fig. 3.3). Activation of these GTPases is controlled by guanine nucleotide exchange factors (GEFs) (see Fig. 7.4, which exchange GTP for GDP bound to the GTPase. The G proteins activated by fMLF activate the GEF protein PREX1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein), which can directly activate Rac. Other GEFs, including members of the Vav family that are controlled by other types of receptors (see Section 7-19), can also activate Rac activity, and their activity synergizes with the actions of fMLF and C5a.


Immunology_Janeway

Immunology_Janeway. The activation of Rac and Rho helps to increase the microbicidal capacity of macrophages and neutrophils that have ingested pathogens. Upon phagocytosing microbes, macrophages and neutrophils produce a variety of toxic products that help to kill the engulfed microorganism (Fig. 3.4). The most important of these are the antimicrobial peptides described in Section 2-4, reactive nitrogen species such as nitric oxide (NO), and ROS, such as the super-oxide anion (O2–) and hydrogen peroxide (H2O2). Nitric oxide is produced by a high-output form of nitric oxide synthase, inducible NOS2 (iNOS2), whose expression is induced by a variety of stimuli, including fMLF.


Immunology_Janeway

Immunology_Janeway. Activation of the fMLF and C5a receptors is directly involved in generating ROS. Superoxide is generated by a multicomponent, membrane-associated NADPH oxidase, also called phagocyte oxidase. In unstimulated phagocytes, this enzyme is inactive because it is not fully assembled. One set of subunits, the cytochrome b558 complex (composed of p22 and gp91), is localized in the plasma membranes of resting macrophages and neutrophils, and it appears in lysosomes after the maturation of phagolysosomes. The other components, p40, p47, and p67, are in the cytosol. Activation of phagocytes induces the cytosolic subunits to join with the membrane-associated cytochrome b558 to form a complete, functional NADPH oxidase in the phagolysosome membrane (Fig. 3.5). The fMLF and C5a receptors participate in the process by activating Rac, which functions to promote the movement of the cytosolic components to the membrane to assemble the active NADPH oxidase.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. ˜-Defensins (HNP1–4), °-defensin Cathelicidin, macrophage HBD4, cathelicidin, azurocidin, Antimicrobial peptides bacterial permeability inducing protein (BPI), lactoferricin Acidifcation Toxic nitrogen oxides Toxic oxygen-derived products pH°3.5–4.0, bacteriostatic or bactericidal Nitric oxide NO Superoxide O2 – , hydrogen peroxide H2O2, singlet oxygen 1O2 •, hydroxyl radical •OH, hypohalite OCl– Lysozyme: digests cell walls of some Gram-positive bacteria Acid hydrolases (e.g., elastase and other proteases): break down ingested microbes Lactoferrin (sequesters Fe2+), vitamin B12-binding protein


Immunology_Janeway

Immunology_Janeway. The NADPH oxidase reaction results in a transient increase in oxygen consumption by the cell, which is known as the respiratory burst. It generates superoxide anion within the lumen of the phagolysosome, and this is converted by the enzyme superoxide dismutase (SOD) into H2O2. Further chemical and enzymatic reactions produce a range of toxic ROS from H2O2, including the hydroxyl radical (•OH), hypochlorite (OCl–), and hypobromite (OBr–). In this way, the direct recognition of bacterially derived polypeptides or previous pathogen recognition by the complement system activates a potent killing mechanism within macrophages and neutrophils that have ingested microbes via their phagocytic receptors. However, phagocyte activation can also cause extensive tissue damage because hydrolytic enzymes, membrane-disrupting peptides, and reactive oxygen species can be released into the extracellular environment and are toxic to host cells.


Immunology_Janeway

Immunology_Janeway. Neutrophils use the respiratory burst described above in their role as an early responder to infection. Neutrophils are not tissue-resident cells, and they need to be recruited to a site of infection from the bloodstream. Their sole function is to ingest and kill microorganisms. Although neutrophils are eventually present in much larger numbers than macrophages in some types of acute infection, they are short-lived, dying soon after they have accomplished a round of phagocytosis and used up their primary and secondary granules. Dead and dying neutrophils are a major component of the pus that forms in abscesses and in wounds infected by certain extracellular capsulated bacteria such as streptococci and staphylococci, which are thus known as pus-forming, or pyogenic, bacteria. Macrophages, in contrast, are long-lived cells and continue to generate new lysosomes.


Immunology_Janeway

Immunology_Janeway. Patients with a disease called chronic granulomatous disease (CGD) have a genetic deficiency of the NADPH oxidase, which means that their phagocytes do not produce the toxic oxygen derivatives characteristic of the respiratory burst and so are less able to kill ingested microorganisms and clear an infection. The most common form of CGD is an X-linked heritable disease that arises from inactivating mutations in the gene encoding the gp91 subunit of cytochrome b558. People with this defect are unusually susceptible to bacterial and fungal infections, especially in infancy, though they remain susceptible for life. One autosomal recessive form of NADPH oxidase deficiency, p47phox deficiency, has very low but detectable activity and causes a milder form of CGD.


Immunology_Janeway

Immunology_Janeway. Fig. 3.4 Bactericidal agents produced or released by phagocytes after uptake of microorganisms. Most of the agents listed are directly toxic to microbes and can act directly in the phagolysosome. They can also be secreted into the extracellular environment, and many of these substances are toxic to host cells. Other phagocyte products sequester essential nutrients in the extracellular environment, rendering them inaccessible to microbes and hindering microbial growth. Besides being directly bacteriostatic or bactericidal, acidification of lysosomes also activates the many acid hydrolases that degrade the contents of the vacuole.


Immunology_Janeway

Immunology_Janeway. O2–OCl–H2O2phagosomeprimarygranuleNADPHoxidasesecondarygranulegp91p22Rac2fMLFlysosomeBacterial fMet-Leu-Phe peptides activate Rac2, and bacteria are taken up into phagosomes Phagosomes fuse with primary and secondary granules. Rac2 induces assembly of a functional NADPH oxidase in the phagolysosome membrane, leading to generation of O2 – . Acidifcation as a result of ion infux releases granule proteases from granule matrix p67p47p40Neutrophils engulf and kill the microbes to which they bind primarygranulesecondarygranulemicrobelysosomeK+K+K+SOD


Immunology_Janeway

Immunology_Janeway. Fig. 3.5 The microbicidal respiratory burst in phagocytes is initiated by activation-induced assembly of the phagocyte NADPH oxidase. First panel: neutrophils are highly specialized for the uptake and killing of pathogens, and contain several different kinds of cytoplasmic granules, such as the primary and secondary granules shown in the first panel. These granules contain antimicrobial peptides and enzymes. Second panel: in resting neutrophils, the cytochrome b558 subunits (gp91 and p22) of the NADPH oxidase are localized in the plasma membrane; the other oxidase components (p40, p47, and p67) are located in the cytosol. Signaling by phagocytic receptors and by fMLF or C5a receptors synergizes to activate Rac2 and induce the assembly of the complete, active NADPH oxidase in the membrane of the phagolysosome, which has formed by the fusion of the phagosome with lysosomes and primary and secondary granules. Third panel: active NADPH oxidase transfers an electron from its FAD cofactor


Immunology_Janeway

Immunology_Janeway. of the phagolysosome, which has formed by the fusion of the phagosome with lysosomes and primary and secondary granules. Third panel: active NADPH oxidase transfers an electron from its FAD cofactor to molecular oxygen, forming the superoxide ion O2– (blue) and other free oxygen radicals in the lumen of the phagolysosome. Potassium and hydrogen ions are then drawn into the phagolysosome to neutralize the charged superoxide ion, increasing acidification of the vesicle. Acidification dissociates granule enzymes such as cathepsin G and elastase (yellow) from their proteoglycan matrix, leading to their cleavage and activation by lysosomal proteases. O2– is converted by superoxide dismutase (SOD) to hydrogen peroxide (H2O2), which can kill microorganisms, and can be converted by myeloperoxidase, a heme-containing enzyme, to microbicidal hypochlorite (OCl–) and by chemical reaction with ferrous (Fe2+) ions to the hydroxyl (•OH) radical.


Immunology_Janeway

Immunology_Janeway. In addition to killing microbes engulfed by phagocytosis, neutrophils use another rather novel mechanism of destruction that is directed at extracellular pathogens. During infection, some activated neutrophils undergo a unique form of cell death in which the nuclear chromatin, rather than being degraded as occurs during apoptosis, is released into the extracellular space and forms a MOVIE 3.4 fibril matrix known as neutrophil extracellular traps, or NETs (Fig. 3.6). NETs act to capture microorganisms, which may then be more efficiently phagocytosed by other neutrophils or macrophages. NET formation requires the generation of ROS, and patients with CGD have reduced NET formation, which may contribute to their susceptibility to microorganisms.


Immunology_Janeway

Immunology_Janeway. Macrophages can phagocytose pathogens and produce the respiratory burst immediately upon encountering an infecting microorganism, and this can be sufficient to prevent an infection from becoming established. In the nineteenth century, the immunologist Élie Metchnikoff believed that the innate response Pattern recognition by cells of the innate immune system. of macrophages encompassed all host defenses; indeed, invertebrates such as the sea star that he was studying rely entirely on innate immunity to overcome infection. Although this is not the case in humans and other vertebrates, the innate response of macrophages still provides an important front line of defense that must be overcome if a microorganism is to establish an infection that can be passed on to a new host.


Immunology_Janeway

Immunology_Janeway. Pathogens have, however, developed a variety of strategies to avoid immediate destruction by macrophages and neutrophils. Many extracellular pathogenic bacteria coat themselves with a thick polysaccharide capsule that is not recognized by any phagocytic receptor. In such cases, however, the complement system can recognize microbial surfaces and coat them with C3b, thereby flagging them for phagocytosis via complement receptors, as described in Chapter 2. Other pathogens, for example, mycobacteria, have evolved ways to grow inside macrophage phagosomes by inhibiting their acidification and fusion with lysosomes. Without such devices, a microorganism must enter the body in sufficient numbers to overwhelm the immediate innate host defenses and to establish a focus of infection. 3-3 Microbial recognition and tissue damage initiate an inflammatory response.


Immunology_Janeway

Immunology_Janeway. 3-3 Microbial recognition and tissue damage initiate an inflammatory response. An important effect of the interaction between microbes and tissue macrophages is the activation of macrophages and other immune cells to release small proteins called cytokines and chemokines, and other chemical mediators. Collectively, these proteins induce a state of inflammation in the tissue, attract monocytes and neutrophils to the infection, and allow plasma proteins to enter the tissue from the blood. An inflammatory response is usually initiated within hours of infection or wounding. Macrophages are stimulated to secrete pro-inflammatory cytokines, such as TNF-α, and chemokines by interactions between microbes and microbial products and specific receptors expressed by the macrophage. We will examine how the cytokines interact with pathogens later in the chapter, but first we describe some general aspects of inflammation and how it contributes to host defense.


Immunology_Janeway

Immunology_Janeway. Inflammation has three essential roles in combating infection. The first is to deliver additional effector molecules and cells from the blood into sites of infection, and so increase the destruction of invading microorganisms. The second is to induce local blood clotting, which provides a physical barrier to the spread of the infection in the bloodstream. The third is to promote the repair of injured tissue.


Immunology_Janeway

Immunology_Janeway. Inflammatory responses are characterized by pain, redness, heat, and swelling at the site of an infection, reflecting four types of change in the local blood vessels, as shown in Fig. 3.7. The first is an increase in vascular diameter, leading to increased local blood flow—hence the heat and redness—and a reduction in the velocity of blood flow, especially along the inner walls of small blood vessels. The second change is the activation of endothelial cells lining the blood vessel to express cell-adhesion molecules that promote the binding of circulating leukocytes. The combination of slowed blood flow and adhesion molecules allows leukocytes to attach to the endothelium and migrate into the tissues, a process known as extravasation. All these changes are initiated by the pro-inflammatory cytokines and chemokines produced by activated macrophages and parenchymal cells.


Immunology_Janeway

Immunology_Janeway. Once inflammation has begun, the first white blood cells attracted to the site are neutrophils. These are followed by monocytes (Fig. 3.8), which upon activation are called inflammatory monocytes and can produce various pro-inflammatory cytokines, but are distinguishable from macrophages by their lack of expression of the adhesion G-protein-coupled receptor E1, commonly Fig. 3.6 Neutrophil extracellular traps (NETs) can trap bacteria and fungi. This scanning electron micrograph of activated human neutrophils infected with a virulent strain of Shigella flexneri (pink rods) shows the stimulated neutrophils forming NETs (blue, indicated by arrows). Bacteria trapped within NETs are visible (lower arrow). Photo courtesy of Arturo Zychlinsky.


Immunology_Janeway

Immunology_Janeway. Fig. 3.7 Infection stimulates macrophages to release cytokines and chemokines that initiate an inflammatory response. Cytokines produced by tissue macrophages at the site of infection cause the dilation of local small blood vessels and changes in the endothelial cells of their walls. These changes lead to the movement of leukocytes, such as neutrophils and monocytes, out of the blood vessel (extravasation) and into the infected tissue; this movement is guided by chemokines produced by the activated macrophages. The blood vessels also become more permeable, allowing plasma proteins and fluid to leak into the tissues. Together, these changes cause the characteristic inflammatory signs of heat, pain, redness, and swelling at the site of infection.


Immunology_Janeway

Immunology_Janeway. Fig. 3.8 Monocytes circulating in the blood migrate into infected and inflamed tissues. Adhesion molecules on the endothelial cells of the blood vessel wall capture the monocyte and cause it to adhere to the vascular endothelium. Chemokines bound to the vascular endothelium then signal the monocyte to migrate across the endothelium into the underlying tissue. The monocyte, now differentiating into an inflammatory monocyte, continues to migrate, under the influence of chemokines released during inflammatory responses, toward the site of infection. Monocytes leaving the blood are also able to differentiate into dendritic cells (not shown), depending on the signals that they receive from their environment. called F4/80. Monocytes are also able to give rise to dendritic cells in the tissues, depending on signals that they receive from their environment. In the later stages of inflammation, other leukocytes such as eosinophils and lymphocytes also enter the infected site.


Immunology_Janeway

Immunology_Janeway. The third major change in local blood vessels is an increase in vascular permeability. Thus, instead of being tightly joined together, the endothelial cells lining the blood vessel walls become separated, leading to an exit of fluid and proteins from the blood and their local accumulation in the tissue. This accounts for the swelling, or edema, and pain—as well as the accumulation in tissues of plasma proteins such as complement and MBL that aid in host defense. The changes that occur in endothelium as a result of inflammation are known generally as endothelial activation. The fourth change, clotting in microvessels in the site of infection, prevents the spread of the pathogen via the blood.


Immunology_Janeway

Immunology_Janeway. These changes are induced by a variety of inflammatory mediators released as a consequence of the recognition of pathogens by macrophages, and later by neutrophils and other white blood cells. Macrophages and neutrophils secrete lipid mediators of inflammation—prostaglandins, leukotrienes, and platelet-activating factor (PAF)—which are rapidly produced by enzymatic pathways that degrade membrane phospholipids. Their actions are followed by those of the chemokines and cytokines that are synthesized and secreted Pattern recognition by cells of the innate immune system. by macrophages and inflammatory monocytes in response to pathogens. The cytokine tumor necrosis factor-α (TNF-α, also known simply as TNF), for example, is a potent activator of endothelial cells. We describe TNF-αand related cytokines in more detail in Section 3-15.


Immunology_Janeway

Immunology_Janeway. Besides stimulating the respiratory burst in phagocytes and acting as a chemoattractant for neutrophils and monocytes, C5a also promotes inflammation by increasing vascular permeability and inducing the expression of certain adhesion molecules on endothelium. C5a also activates local mast cells (see Section 1-4), which are stimulated to release their granules containing the small inflammatory molecule histamine as well as TNF-α and cathelicidins.


Immunology_Janeway

Immunology_Janeway. If wounding has occurred, the injury to blood vessels immediately triggers two protective enzyme cascades. One is the kinin system of plasma proteases that is triggered by tissue damage to generate several polypeptides that regulate blood pressure, coagulation, and pain. Although we will not fully describe its components here, one inflammatory mediator produced is the vasoactive peptide bradykinin, which increases vascular permeability to promote the influx of plasma proteins to the site of tissue injury. It also causes pain. Although unpleasant to the victim, pain draws attention to the problem and leads to immobilization of the affected part of the body, which helps to limit the spread of the infection.


Immunology_Janeway

Immunology_Janeway. The coagulation system is another protease cascade that is triggered in the blood after damage to blood vessels, although its full description is also outside our present scope. Its activation leads to the formation of a fibrin clot, whose normal role is to prevent blood loss. With regard to innate immunity, however, the clot physically encases the infectious microorganisms and prevents their entry into the bloodstream. The kinin and the coagulation cascades are also triggered by activated endothelial cells, and so they can have important roles in the inflammatory response to pathogens even if wounding or gross tissue injury has not occurred. Thus, within minutes of the penetration of tissues by a pathogen, the inflammatory response causes an influx of proteins and cells that may control the infection. Coagulation also forms a physical barrier in the form of blood clots to limit the spread of infection. Damage to tissues can occur in the absence of infection by microbes, such as


Immunology_Janeway

Immunology_Janeway. the infection. Coagulation also forms a physical barrier in the form of blood clots to limit the spread of infection. Damage to tissues can occur in the absence of infection by microbes, such as physical trauma, ischemia, and metabolic or autoimmune disorders. In such sterile injury, many of the changes associated with infection, such as neutrophil recruitment, can also occur, in addition to activation of the kinin system and clot formation.


Immunology_Janeway

Immunology_Janeway. 3-4 Toll-like receptors represent an ancient pathogen-recognition system.


Immunology_Janeway

Immunology_Janeway. Section 1-5 introduced pattern recognition receptors (PRRs), which function as sensors for pathogen-associated molecular patterns (PAMPs). Cytokine and chemokine production by macrophages is the result of signaling by these PRRs that is induced by a wide variety of pathogen components. The existence of these receptors was predicted by Charles Janeway, Jr., before mechanisms of innate recognition were known, based on the requirement for adjuvants in driving immune responses to purified antigens. Jules Hoffmann discovered the first example of such a receptor, for which he was awarded part of the 2011 Nobel Prize in Physiology or Medicine. The receptor protein Toll was identified earlier as a gene controlling the correct dorso-ventral patterning embryo of the fruitfly Drosophila melanogaster. But in 1996, Hoffmann discovered that in the adult fly, Toll signaling induces the expression of several host-defense mechanisms, including antimicrobial peptides such as drosomycin, and is critical


Immunology_Janeway

Immunology_Janeway. But in 1996, Hoffmann discovered that in the adult fly, Toll signaling induces the expression of several host-defense mechanisms, including antimicrobial peptides such as drosomycin, and is critical for defense against Gram-positive bacteria and fungal pathogens.


Immunology_Janeway

Immunology_Janeway. Mutations in Drosophila Toll or in signaling proteins activated by Toll decreased the production of antimicrobial peptides and led to susceptibility of the adult fly Fig. 3.9 Toll is required for antifungal responses in Drosophila melanogaster. Flies that are deficient in the Toll receptor are dramatically more susceptible than wild-type flies to fungal infection. This is illustrated here by the uncontrolled hyphal growth (arrow) of the normally weak pathogen Aspergillus fumigatus in a Toll-deficient fly. Photo courtesy of J.A. Hoffmann.


Immunology_Janeway

Immunology_Janeway. MOVIE 3.5 to fungal infections (Fig. 3.9). Subsequently, homologs of Toll, called Toll-like receptors (TLRs), were found in other animals, including mammals, in which they are associated with resistance to viral, bacterial, and fungal infection. In plants, proteins with domains resembling the ligand-binding regions of TLR proteins are involved in the production of antimicrobial peptides, indicating the ancient association of these domains with this means of host defense. 3-5 Mammalian Toll-like receptors are activated by many different pathogen-associated molecular patterns.


Immunology_Janeway

Immunology_Janeway. There are 10 expressed TLR genes in humans and 12 in mice. Each TLR is devoted to recognizing a distinct set of molecular patterns that are essentially not found in healthy vertebrate cells. Initially called pathogen-associated molecular patterns (PAMPs), these molecules are general components of both pathogenic and nonpathogenic microorganisms, and so are sometimes called microbial-associated molecular patterns, or MAMPs. Between them, the mammalian TLRs recognize molecules characteristic of Gram-negative and Gram-positive bacteria, fungi, and viruses. Among these, the lipoteichoic acids of Gram-positive bacterial cell walls and the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria (see Fig. 2.9) are particularly important in the recognition of bacteria by the innate immune system, and are recognized by TLRs. Other microbial components also have a repetitive structure. Bacterial flagella are made of a repeated flagellin subunit, and bacterial DNA has abundant


Immunology_Janeway

Immunology_Janeway. immune system, and are recognized by TLRs. Other microbial components also have a repetitive structure. Bacterial flagella are made of a repeated flagellin subunit, and bacterial DNA has abundant repeats of unmethylated CpG dinucleotides (which are often methylated in mammalian DNA). In many viral infections, a double-stranded RNA intermediate is part of the viral life cycle, and frequently the viral RNA contains modifications that can be used to distinguish it from normal host RNA species.


Immunology_Janeway

Immunology_Janeway. The mammalian TLRs and their known microbial ligands are listed in Fig. 3.10. Because there are relatively few TLR genes, the TLRs have limited specificity compared with the antigen receptors of the adaptive immune system. However, they can recognize elements of most pathogenic microbes and are expressed by many types of cells, including macrophages, dendritic cells, B cells, stromal cells, and certain epithelial cells, enabling the initiation of antimicrobial responses in many tissues.


Immunology_Janeway

Immunology_Janeway. TLRs are sensors for microbes present in extracellular spaces. Some mammalian TLRs are cell-surface receptors similar to Drosophila Toll, but others are located intracellularly in the membranes of endosomes, where they detect pathogens or their components that have been taken into cells by phagocytosis, receptor-mediated endocytosis, or macropinocytosis (Fig. 3.11). TLRs are single-pass transmembrane proteins with an extracellular region composed of 18–25 copies of a leucine-rich repeat (LRR). Each LLR of a TLR protein is composed of around 20–25 amino acids, and multiple LRRs create a horseshoe-shaped protein scaffold that is adaptable for ligand binding and recognition on both the outer (convex) and inner (concave) surfaces. Signaling by mammalian TLRs is activated when binding of a ligand induces formation of a dimer, or induces conformational changes in a preformed TLR dimer. All mammalian TLR proteins have in their cytoplasmic tail a TIR (for Toll–IL-1 receptor) domain, which


Immunology_Janeway

Immunology_Janeway. ligand induces formation of a dimer, or induces conformational changes in a preformed TLR dimer. All mammalian TLR proteins have in their cytoplasmic tail a TIR (for Toll–IL-1 receptor) domain, which interacts with other TIR-type domains, usually in other signaling molecules, and is also found in the cytoplasmic tail of the receptor for the cytokine interleukin-1β(IL-1β). For years after the discovery of the mammalian TLRs it was not known whether they made direct contact with microbial products or whether they sensed the presence of microbes by some indirect means. Drosophila Toll, for example, does not recognize pathogen products directly, but instead it is activated when it binds a cleaved version of a self protein, Spätzle. Drosophila has other direct pathogen-recognition molecules, and these trigger the proteolytic cascade that ends in the cleavage


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. Fig. 3.10 Innate immune recognition by Toll-like receptors. Each of the human or mouse TLRs whose specificity is known recognizes one or more microbial molecular patterns, generally by direct interaction with molecules on the pathogen surface. Some Toll-like receptor proteins form heterodimers (e.g., TLR-1:TLR-2 and TLR-6:TLR-2). LPS, lipopolysaccharide. of Spätzle. In this sense Toll is not a classical pattern recognition receptor. However, X-ray crystal structures of several mammalian dimeric TLRs bound to their ligands show that at least some mammalian TLRs make direct contact with microbial ligands. Fig. 3.11 The cellular locations of the mammalian Toll-like receptors.


Immunology_Janeway

Immunology_Janeway. Fig. 3.11 The cellular locations of the mammalian Toll-like receptors. TLRs are transmembrane proteins whose extracellular region contains 18–25 copies of the leucine-rich repeat (LRR), but these cartoons depict only 9 LRRs for simplicity. Some TLRs are located on the cell surface of dendritic cells, macrophages, and other cells, where they are able to detect extracellular pathogen molecules. TLRs are thought to act as dimers. Only those that form heterodimers are shown in dimeric form here; the rest act as homodimers. TLRs located intracellularly, in the walls of endosomes, can recognize microbial components, such as DNA, that are accessible only after the microbe has been broken down. The diacyl and triacyl lipopeptides recognized by the heterodimeric receptors TLR-6:TLR-2 and TLR-1:TLR-2, respectively, are derived from the lipoteichoic acid of Gram-positive bacterial cell walls and the lipoproteins of Gram-negative bacterial surfaces.


Immunology_Janeway

Immunology_Janeway. Mammalian TLR-1, TLR-2, and TLR-6 are cell-surface receptors that are activated by various ligands, including lipoteichoic acid and the diacyl and triacyl lipoproteins of Gram-negative bacteria. They are found on macrophages, dendritic cells, eosinophils, basophils, and mast cells. Ligand binding induces the formation of heterodimers of TLR-2 and TLR-1, or of TLR-2 and TLR-6. The X-ray crystal structure of a synthetic triacyl lipopeptide ligand bound to TLR-1 and TLR-2 shows exactly how it induces dimerization (Fig. 3.12). Two of the three lipid chains bind to the convex surface of TLR-2, while the third binds to the convex surface of TLR-1. Dimerization brings the cytoplasmic TIR domains of the TLR chains into close proximity with each other to initiate signaling. Similar interactions are presumed to occur with the diacyl lipopeptide ligands that induce the dimerization of TLR-2 and TLR-6. The scavenger receptor CD36, which binds long-chain fatty acids, and Dectin-1, which binds


Immunology_Janeway

Immunology_Janeway. are presumed to occur with the diacyl lipopeptide ligands that induce the dimerization of TLR-2 and TLR-6. The scavenger receptor CD36, which binds long-chain fatty acids, and Dectin-1, which binds β-glucans (see Section 3-1), both cooperate with TLR-2 in ligand recognition.


Immunology_Janeway

Immunology_Janeway. TLR-5 is expressed on the cell surface of macrophages, dendritic cells, and intestinal epithelial cells; it recognizes flagellin, a protein subunit of bacterial flagella. TLR-5 recognizes a highly conserved site on flagellin that is buried and inaccessible in the assembled flagellar filament. This means that the receptor is activated only by monomeric flagellin, which is produced by the breakdown of flagellated bacteria in the extracellular space. Mice, but not humans, express TLR-11 and TLR-12, which share with TLR-5 the ability to recognize an intact protein. TLR-11 is expressed by macrophages and dendritic cells, and also by liver, kidney, and bladder epithelial cells.


Immunology_Janeway

Immunology_Janeway. TLR-12 is also expressed in macrophages and dendritic cells, and is more broadly expressed in hematopoietic cells than TLR-11, but is not expressed by the epithelial tissues where TLR-11 is expressed. TLR-11-deficient mice develop urinary infections caused by uropathogenic strains of Escherichia coli, although the bacterial ligand for TLR-11 has not yet been identified. TLR-11 and TLR-12 have an overlapping function in that both recognize protozoan parasites such as Toxoplasma gondii and Plasmodium falciparum. They bind to protein motifs that are present in the protozoan actin-binding protein profilin but absent in mammalian profilins. TLR-11 and TLR-12 are both required in macrophages and conventional dendritic cells for activation by T. gondii profilin, but TLR-12 plays a more dominant role. Mice lacking TLR-11 develop more severe tissue injury than normal mice on infection with


Immunology_Janeway

Immunology_Janeway. Fig. 3.12 Direct recognition of pathogen-associated molecular can activate TLR1:TLR2 dimers; it has three fatty-acid chains bound patterns by TLR-1 and TLR-2 induces dimerization of the TLRs to a polypeptide backbone. Two fatty-acid chains bind to a pocket and signaling. TLR-1 and TLR-2 are located on cell surfaces (left on the convex surface of the TLR-2 ectodomain, and the third chain panel), where they can directly recognize bacterial triacyl lipoproteins associates with a hydrophobic channel in the convex binding surface (middle panel). The convex surfaces of their extracellular domains of TLR-1, inducing dimerization of the two TLR subunits and bringing have binding sites for the lipid side chains of triacyl lipopeptides. In their cytoplasmic Toll–IL-1 receptor (TIR) domains together to initiate the crystal structure (right panel), the ligand is a synthetic lipid that signaling. Structure courtesy of Jie-Oh Lee. Pattern recognition by cells of the innate immune system.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. Toxoplasma, whereas mice lacking TLR-12 die rapidly after infection. TLR-10 is expressed in humans, but TLR-10 is a pseudogene in mice. Its ligand and function are currently not known.


Immunology_Janeway

Immunology_Janeway. Not all mammalian TLRs are cell-surface receptors. The TLRs that recognize nucleic acids are located in the membranes of endosomes, to which they are transported via the endoplasmic reticulum. TLR-3 is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells; it recognizes double-stranded RNA (dsRNA), which is a replicative intermediate of many types of viruses, not only those with RNA genomes. dsRNA is internalized either by the direct endocytosis of viruses with double-stranded RNA genomes, such as rotavirus, or by the phagocytosis of dying cells in which viruses are replicating, and it encounters the TLRs when the incoming endocytic vesicle or phagosome fuses with the TLR-containing endosome. Crystallographic analysis shows that TLR-3 binds directly to dsRNA. The TLR-3 ectodomain (the ligand-binding domain) has two contact sites for dsRNA: one on the amino terminus and a second near the membrane-proximal carboxy terminus. The twofold symmetry of dsRNA


Immunology_Janeway

Immunology_Janeway. The TLR-3 ectodomain (the ligand-binding domain) has two contact sites for dsRNA: one on the amino terminus and a second near the membrane-proximal carboxy terminus. The twofold symmetry of dsRNA allows it to bind simultaneously to two TLR-3 ectodomains, inducing a dimerization that brings the TIR domains of TLR-3 together and activates intracellular signaling. This can be verified by using poly I:C to artificially induce signaling. A synthetic polymer composed of inosinic and cytidylic acid, poly I:C binds to TLR-3 and functions as an analog of dsRNA; poly I:C is often used experimentally to activate this pathway. Mutations in the ectodomain of human TLR-3, which produce a dominantly acting loss-of-function mutant receptor, have been associated with encephalitis that is caused by a failure to control the herpes simplex virus.


Immunology_Janeway

Immunology_Janeway. TLR-7, TLR-8, and TLR-9, like TLR-3, are endosomal nucleotide sensors involved in the recognition of viruses. TLR-7 and TLR-9 are expressed by plasmacytoid dendritic cells, B cells, and eosinophils; TLR-8 is expressed primarily by monocytes and macrophages. TLR-7 and TLR-8 are activated by single-stranded RNA (ssRNA), which is a component of healthy mammalian cells, but it is normally confined to the nucleus and cytoplasm and is not present in endosomes. Many virus genomes, for example, those of orthomyxoviruses (such as influenza) and flaviviruses (such as West Nile virus), consist of ssRNA. When extracellular particles of these viruses are endocytosed by macrophages or dendritic cells, they are uncoated in the acidic environment of endosomes and lysosomes, exposing the ssRNA genome for recognition by TLR-7. Mice lacking TLR-7 have impaired immune responses to viruses such as influenza. In abnormal settings, TLR-7 may be activated by self-derived ssRNA. Normally, extracellular RNases


Immunology_Janeway

Immunology_Janeway. by TLR-7. Mice lacking TLR-7 have impaired immune responses to viruses such as influenza. In abnormal settings, TLR-7 may be activated by self-derived ssRNA. Normally, extracellular RNases degrade the ssRNA released from apoptotic cells during tissue injury. But in a mouse model of lupus nephritis, an inflammatory condition of the kidney, TLR-7 recognition of self ssRNA was observed to contribute to disease. Several studies have identified polymorphisms in the human TLR-7 gene that are associated with increased risk of the autoimmune disease systemic lupus erythematosus, suggesting a potential role in this disease. The role for TLR-8 has not been established as clearly from mouse model systems as for TLR-7. TLR-9 recognizes unmethylated CpG dinucleotides. In mammalian genomes, CpG dinucleotides in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. But in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another


Immunology_Janeway

Immunology_Janeway. in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. But in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another pathogen-associated molecular pattern.


Immunology_Janeway

Immunology_Janeway. The delivery of TLR-3, TLR-7, and TLR-9 from the endoplasmic reticulum to the endosome relies on their interaction with a specific protein, UNC93B1, which is composed of 12 transmembrane domains. Mice lacking this protein have defects in signaling by these endosomal TLRs. Rare human mutations in UNC93B1 have been identified as causing susceptibility to herpes simplex encephalitis, similarly to TLR-3 deficiency, but do not impair immunity to many other viral pathogens, presumably because of the existence of other viral sensors, which are discussed later in this chapter. 3-6 TLR-4 recognizes bacterial lipopolysaccharide in association with the host accessory proteins MD-2 and CD14.


Immunology_Janeway

Immunology_Janeway. Not all mammalian TLRs bind their ligands so directly. TLR-4 is expressed by several types of immune-system cells, including dendritic cells and macrophages, and is important in sensing and responding to numerous bacterial infections. TLR-4 recognizes the LPS of Gram-negative bacteria by a mechanism that is partly direct and partly indirect. The systemic injection of LPS causes a collapse of the circulatory and respiratory systems, a condition known as shock. These dramatic effects of LPS are seen in humans as septic shock, which results from an uncontrolled systemic bacterial infection, or sepsis. In this case, LPS induces an overwhelming secretion of cytokines, particularly TNF-α (see Section 3-15), causing systemic vascular permeability, an undesirable effect of its normal role in containing local infections. Mutant mice lacking TLR-4 function are resistant to LPS-induced septic shock but are highly sensitive to LPS-bearing pathogens such as Salmonella typhimurium, a natural


Immunology_Janeway

Immunology_Janeway. containing local infections. Mutant mice lacking TLR-4 function are resistant to LPS-induced septic shock but are highly sensitive to LPS-bearing pathogens such as Salmonella typhimurium, a natural pathogen of mice. In fact, TLR-4 was identified as the receptor for LPS by positional cloning of its gene from the LPS-resistant C3H/HeJ mouse strain, which harbors a naturally occurring mutation in the cytoplasmic tail of TLR-4 that interferes with the receptor’s ability to signal. For this discovery, the 2011 Nobel Prize in Physiology or Medicine was partly awarded to Bruce Buetler.


Immunology_Janeway

Immunology_Janeway. LPS varies in composition among different bacteria but essentially consists of a polysaccharide core attached to an amphipathic lipid, lipid A, with a variable number of fatty-acid chains per molecule. To recognize LPS, the ectodomain of TLR-4 uses an accessory protein, MD-2. MD-2 initially binds to TLR-4 within the cell and is necessary both for the correct trafficking of TLR-4 to the cell surface and for the recognition of LPS. MD-2 associates with the central section of the curved ectodomain of TLR-4, binding off to one side as shown in Fig. 3.13. When the TLR4–MD-2 complex encounters LPS, five lipid chains of LPS bind to a deep hydrophobic pocket of MD-2, but not directly to TLR-4, while a sixth lipid chain remains exposed on the surface of MD-2. This last lipid chain and parts of the LPS polysaccharide backbone can then bind to the convex side of a second TLR-4 ectodomain, inducing TLR-4 dimerization that activates intracellular signaling pathways.


Immunology_Janeway

Immunology_Janeway. TLR-4 activation by LPS involves two other accessory proteins besides MD-2. While LPS is normally an integral component of the outer membrane of Gram-negative bacteria, during infections it can become detached from the membrane and be picked up by the host LPS-binding protein present in the blood and in extracellular fluid in tissues. LPS is transferred from LPS-binding protein to a second protein, CD14, which is present on the surface of macrophages, neutrophils, and dendritic cells. On its own, CD14 can act as a phagocytic receptor, but on macrophages and dendritic cells it also acts as an accessory protein for TLR-4. 3-7 TLRs activate NFκB, AP-1, and IRF transcription factors to induce the expression of inflammatory cytokines and type I interferons.


Immunology_Janeway

Immunology_Janeway. 3-7 TLRs activate NFκB, AP-1, and IRF transcription factors to induce the expression of inflammatory cytokines and type I interferons. Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses that together result in the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and the antiviral cytokines interferon-α and -β (IFN-α and IFN-β), the type I interferons. TLR signaling achieves this by activating several different signaling pathways that each activate different transcription factors. As mentioned earlier, ligand-induced dimerization of two TLR ectodomains brings the cytoplasmic TIR domains together, allowing them to interact with the TIR domains of cytoplasmic adaptor molecules that initiate intracellular signaling. There are Pattern recognition by cells of the innate immune system.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. four such adaptors used by mammalian TLRs: MyD88, MAL (also known as TIRAP), TRIF, and TRAM. It is significant that the TIR domains of the different TLRs interact with different combinations of these adaptors (Fig. 3.14). Most TLRs interact only with MyD88, which is required for their signaling.


Immunology_Janeway

Immunology_Janeway. Fig. 3.13 TLR-4 recognizes LPS in association with the accessory protein MD-2. Panel a: a side view of the symmetrical complex of TLR-4, MD-2, and LPS. TLR-4 polypeptide backbones are shown in green and dark blue. The structure shows the entire extracellular region of TLR-4, composed of the LRR region (shown in green and dark blue), but lacks the intracellular signaling domain. The MD-2 protein is shown in light blue. Five of the LPS acyl chains (shown in red) are inserted into a hydrophobic pocket within MD-2. The remainder of the LPS glycan and one lipid chain (orange) make contact with the convex surface of a TLR-4 monomer. Panel b: the top view of the structure shows that an LPS molecule makes contact with one TLR-4 subunit on its convex (outer) surface, while binding to an MD-2 molecule that is attached to the other TLR-4 subunit. The MD-2 protein binds off to one side of the TLR-4 LRR region. Panel c: schematic illustration of relative orientation of LPS binding to MD-2 and


Immunology_Janeway

Immunology_Janeway. that is attached to the other TLR-4 subunit. The MD-2 protein binds off to one side of the TLR-4 LRR region. Panel c: schematic illustration of relative orientation of LPS binding to MD-2 and TLR-4. Structures courtesy of Jie-Oh Lee.


Immunology_Janeway

Immunology_Janeway. Fig. 3.14 Mammalian TLRs interact with different TIR-domain adaptor molecules to activate downstream signaling pathways. The four signaling adaptor molecules used by mammalian TLRs are MyD88 (myeloid differentiation factor 88), MAL (MyD88 adaptor-like, also known as TIRAP, for TIR-containing adaptor protein), TRIF (TIR domain-containing adaptor-inducing IFN-β), and TRAM (TRIF-related adaptor molecule). All TLRs interact with MyD88, except TLR-3, which interacts only with TRIF. The table indicates the known pattern of adaptor interactions for the known TLRs. TLR-3 interacts only with TRIF. Other TLRs use either MyD88 paired with MAL, or TRIF paired with TRAM. Signaling by the TLR-2 heterodimers (TLR-2/1 and TLR-2/6) requires MyD88/MAL. TLR-4 signaling uses both of these adaptor pairs, MyD88/MAL and TRIF/TRAM, which is used during endosomal signaling by TLR-4. Importantly, the choice of adaptor influences which of the several downstream signals will be activated by the TLR.


Immunology_Janeway

Immunology_Janeway. Signaling by most TLRs activates the transcription factor NFκB (Fig. 3.15), which is related to DIF, the factor activated by Drosophila Toll. Mammalian TLRs also activate several members of the interferon regulatory factor (IRF) transcription factor family through a second pathway, and they activate members of the activator protein 1 (AP-1) family, such as c-Jun, through yet another signaling pathway involving mitogen-activated protein kinases (MAPKs). NFκB and AP-1 act primarily to induce the expression of pro-inflammatory cytokines and chemotactic factors. The IRF factors IRF3 and IRF7 are particularly important for inducing antiviral type I interferons, whereas a related factor, IRF5, is involved in the production of pro-inflammatory cytokines. Here we will describe how TLR signaling induces the transcription of various cytokine genes; later in the chapter, we will explain how those cytokines exert their various actions.


Immunology_Janeway

Immunology_Janeway. We consider first the signaling pathway triggered by TLRs that use MyD88. Two protein domains of MyD88 are responsible for its function as an adaptor. MyD88 has a TIR domain at its carboxy terminus that associates with the TIR domains in the TLR cytoplasmic tails. At its amino terminus, MyD88 has a death domain, so named because it was first identified in signaling proteins involved in apoptosis, a type of programmed cell death. The MyD88 death domain associates with a similar death domain present in other intracellular signaling proteins. Both MyD88 domains are required for signaling, since rare mutations in either domain are associated with immunodeficiency characterized by recurrent bacterial infections in humans. The MyD88 death domain recruits and activates two serine–threonine protein kinases—IRAK4 (IL-1receptor associated kinase 4) and IRAK1—via their death domains. This IRAK complex performs two functions: it recruits enzymes that produce a signaling scaffold, and uses this


Immunology_Janeway

Immunology_Janeway. kinases—IRAK4 (IL-1receptor associated kinase 4) and IRAK1—via their death domains. This IRAK complex performs two functions: it recruits enzymes that produce a signaling scaffold, and uses this scaffold to recruit other molecules that are then phosphorylated by the IRAKs.


Immunology_Janeway

Immunology_Janeway. To form a signaling scaffold, the IRAK complex recruits the enzyme TRAF6 (tumor necrosis factor receptor-associated factor 6), which is an E3 ubiquitin ligase that acts in cooperation with UBC13, an E2 ubiquitin ligase, and its cofactor Uve1A (together called TRIKA1) (see Fig. 3.15). The combined activity of TRAF-6 and UBC13 is to ligate (unite with a chemical bond) one ubiquitin molecule to another protein, which can be another ubiquitin molecule, and thereby generate protein polymers. The polyubiquitin involved in signaling contains linkages between the lysine 63 on one ubiquitin and the carboxy terminus of the next, forming K63 linkages. This polyubiquitin polymer can be initiated on other proteins, including TRAF-6 itself, or produced as free linear ubiquitin polymers, and can be extended to produce polyubiquitin chains that act as a platform—or scaffold—that bind to other signaling molecules. Next, the scaffold recruits a signaling complex consisting of the polyubiquitin-binding


Immunology_Janeway

Immunology_Janeway. to produce polyubiquitin chains that act as a platform—or scaffold—that bind to other signaling molecules. Next, the scaffold recruits a signaling complex consisting of the polyubiquitin-binding adaptor proteins TAB1, TAB2, and the serine–threonine kinase TAK1 (see Fig. 3.15). By being brought onto the scaffold, TAK1 is phosphorylated by the IRAK complex, and activated TAK1 propagates signaling by activating certain MAPKs, such as c-Jun terminal kinase (JNK) and MAPK14 (p38 MAPK). These then activate AP-1-family transcription factors that transcribe cytokine genes.


Immunology_Janeway

Immunology_Janeway. TAK1 also phosphorylates and activates the IκB kinase (IKK) complex, which is composed of three proteins: IKKα, IKKβ, and IKKγ (also known as NEMO, for NFκB essential modifier). NEMO functions by binding to polyubiquitin chains, which brings the IKK complex into proximity with TAK1. TAK1 Pattern recognition by cells of the innate immune system. NF˜B NF˜B p50 p65 I˜B I˜B I˜B degradation cytokine genes TRAF-6 UBC13, Uve1A MyD88 MAL nucleus TAK1 TAK1 TAB1/2 TAB1/2 IRAK4IRAK1 (E3 ligase) (E2 ligase) ubiquitin polyubiquitin chain IKK° IKK˛IKK˝ (NEMO) IKK˝ IKK°/˛ IKK I˜B is degraded, releasing NF˜B into the nucleus to induce expression of cytokine genes TAK1 associates with IKK and phosphorylates IKK°, which phosphorylates I˜B TRAF-6 is polyubiquitinated, creating a scaffold for activation of TAK1 Dimerized TLRs recruit IRAK1 and IRAK4, activating the E3 ubiquitin ligase TRAF-6


Immunology_Janeway

Immunology_Janeway. Fig. 3.15 TLR signaling can activate the transcription factor NFκB, which induces the expression of pro-inflammatory cytokines. First panel: TLRs signal via their cytoplasmic TIR domains, which are brought into proximity to each other by ligand-induced dimerization of their ectodomains. Some TLRs use the adaptor protein MyD88, and others use the MyD88/MAL pair to initiate signaling. The MyD88 death domain recruits the serine–threonine kinases IRAK1 and IRAK4, in association with the ubiquitin E3 ligase TRAF-6. IRAK undergoes autoactivation and phosphorylates TRAF-6, activating its E3 ligase activity. Second panel: TRAF-6 cooperates with an E2 ligase (UBC13) and a cofactor (Uve1A) to generate polyubiquitin scaffolds (yellow triangles) by attachment of ubiquitin through its lysine 63 (K63). This scaffold recruits a complex of proteins composed of the kinase TAK1 (transforming growth factor-β-activated kinase 1) and two adaptor proteins, TAB1 (TAK1-binding protein 1) and TAB2. TAB1 and


Immunology_Janeway

Immunology_Janeway. This scaffold recruits a complex of proteins composed of the kinase TAK1 (transforming growth factor-β-activated kinase 1) and two adaptor proteins, TAB1 (TAK1-binding protein 1) and TAB2. TAB1 and TAB2 function to bind to polyubiquitin, bringing TAK1 into proximity with IRAK to become phosphorylated (red dot). Third panel: activated TAK1 activates IKK, the IκB kinase complex. First, the IKKγ subunit (NEMO) binds to the polyubiquitin scaffold and brings the IKK complex into proximity to TAK1. TAK1 then phosphorylates and activates IKKβ. IKKβ then phosphorylates IκB, the cytoplasmic inhibitor of NFκB. Fourth panel: phosphorylated IκB is targeted by a process of ubiquitination (not shown) that leads to its degradation. This releases NFκB, which is composed of two subunits, p50 and p65, into the nucleus, driving the transcription of many genes including those encoding inflammatory cytokines. TAK1 also stimulates activation of the mitogen-activated protein kinases (MAPKs) JNK and p38,


Immunology_Janeway

Immunology_Janeway. the nucleus, driving the transcription of many genes including those encoding inflammatory cytokines. TAK1 also stimulates activation of the mitogen-activated protein kinases (MAPKs) JNK and p38, which phosphorylate and activate AP-1 transcription factors (not shown).


Immunology_Janeway

Immunology_Janeway. phosphorylates and activates IKKβ. IKKβ then phosphorylates IκB (inhibitor of κB), which is a distinct molecule whose name should not be confused with IKKβ. IκB is a cytoplasmic protein that constitutively binds to the transcription factor NFκB, which is composed of two subunits, p50 and p65. The binding of IκB traps the NFκB proteins in the cytoplasm. Phosphorylation by IKK induces the degradation of IκB, and this releases NFκB into the nucleus, where it can drive transcription of genes for pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. The actions of these cytokines in the innate immune response are described in the second half of this chapter. The outcome of TLR activation can also vary depending on the cell type in which it occurs. For example, activation of TLR-4 via MyD88 in specialized epithelial cells such as the Paneth cells of the intestine (see Section 2-4) results in the production of antimicrobial peptides, a mammalian example of the ancient function of


Immunology_Janeway

Immunology_Janeway. MyD88 in specialized epithelial cells such as the Paneth cells of the intestine (see Section 2-4) results in the production of antimicrobial peptides, a mammalian example of the ancient function of Toll-like proteins.


Immunology_Janeway

Immunology_Janeway. The ability of TLRs to activate NFκB is crucial to their role of alerting the immune system to the presence of bacterial pathogens. Rare instances of inactivating mutations in IRAK4 in humans cause an immunodeficiency, IRAK4 deficiency, which, like MyD88 deficiency, is characterized by recurrent bacterial infections. Mutations in human NEMO produce a syndrome known as X-linked hypohidrotic ectodermal dysplasia and immunodeficiency or NEMO deficiency, which is characterized by both immunodeficiency and developmental defects. The nucleic-acid-sensing TLRs—TLR-3, TLR-7, TLR-8, and TLR-9—activate members of the IRF family. IRF proteins reside in the cytoplasm and are inactive until they become phosphorylated on serine and threonine residues in