Figure 1. Pathogen‐associated molecular patterns. Many microbes express molecules that are not expressed by mammals. These include unique carbohydrate and lipid structures, as well as nucleic acids such as single‐stranded (ss) or double‐stranded (ds) RNA, or DNA containing CpG motifs and a few proteins. Mammals have evolved PRRs that recognize broadly‐shared features of PAMPs. These receptors are encoded in the germline. A few examples of the type of molecule that can be a PAMP are indicated.
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Figure 2. Cellular locations of pattern recognition receptors. PRRs are located where they are most likely to be able to interact with different types of PAMPs and often where there is little possibility of their meeting host‐derived molecules that are cross‐reactive with PAMPs. Thus, TLRs may be expressed on the plasma membrane or in endosomes, while other types of PRRs are present within the cytoplasm. Some PRRs can also promote uptake of infectious agents.
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Figure 3. Functions of pattern recognition receptors. Most PRRs have signalling properties while some, such as the mannose receptor, are also endocytic receptors promoting uptake (e.g. phagocytosis). Agonist binding to PRRs activates intracellular signalling cascades of different types that modulate cellular functions. Some of their effects can be broadly divided into metabolic, cytoskeletal and changes in gene expression leading, for example, to cytokine secretion. Most of the cytokines secreted are pro‐inflammatory.
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Figure 4. General structure ofToll‐like receptors. TLRs have similar general structures, but the ways in which they interact with their agonists varies greatly. TLRs have a horseshoe‐shaped LRR domain connected to a globular TIR domain that is involved in signalling. In some cases, the agonists interact directly with the LRRs. In others cases, one or more accessory molecules may be involved in binding to LLRs and the triggering of signalling can involve several distinct molecular interactions. In general, the activation of signalling requires the dimerization of two TLR molecules.
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Figure 5. Signalling pathways from Toll‐like receptors. Following agonist binding and dimerization, different adapter molecules bind to TLRs. The key ones are Myd88, which is used by most TLRs, and TRIF, which is only used by one, TLR3; TLR4 can use either depending on its location. The canonical Myd88 pathway leads to NF‐κB activation, as well as activation of the MAP kinase pathways which activate the AP‐1 transcription factor; typically this results in activation of genes for pro‐inflammatory cytokines. In contrast, the TRIF pathway typically leads to activation of IRFs that act on “IFN‐responsive elements” in the DNA (not shown) and the activation of genes for Type I IFN production. Cross‐talk between these pathways can occur (e.g. stimulation of the Myd88 pathway can also lead to IFN production). Each pathway involves multiple signalling components that are not shown for simplicity.
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Figure 6. Western blotting. This technique permits identification of specific proteins in a cell lysate. Proteins are dissociated by typically treating with a detergent, sodium dodecylsulfate (SDS). The mix is then electrophoresed on a polyacrylamide gel in which the rate of a protein's migration when a charge is applied is proportional to its molecular size. This process is called SDS–polyacrylamide gel electrophoresis (PAGE). Antibodies cannot bind to proteins in the gel so typically a nitrocellulose membrane is apposed to the gel, and the proteins are made to migrate into the membrane, again by applying a current; this process is often called “blotting”. Once in the membrane, the proteins can be detected by using antibodies labelled with enzymatic or other labels.
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Figure 7. Inflammasomes. TLR agonists can lead to the transcription and translation of the IL‐1β precursor, pro‐IL‐1β. Inflammasomes are molecular complexes in the cytoplasm that contain multimerized NLRs, such as Nalp3, associated with a proteolytic enzyme, caspase‐1. In activated inflammasomes, caspase‐1 processes pro‐IL‐1 into the mature, functional form of IL‐1, which can then be secreted. Hence, particularly in DCs, two stages are needed for production of IL‐1 (IL‐18 is similarly processed), but how these pathways interact is not well understood; these pathways and requirements may differ in other cell types such as macrophages.
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Figure 8. Immunoprecipitation. This technique is used to isolate specific molecules from a complex mix in solution. Beads coated with antibodies specific for a particular molecule are added to the mix and will bind the specific molecule. Centrifugation can be used to isolate the beads that have bound the molecule; alternatively, magnetic beads can be used and can be captured in a magnetic field. By altering the ionic conditions, the specific molecule can be released from the beads and can be further characterized.
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Figure 9. Type I interferons. Type I IFNs (e.g. α and β) are potent anti‐viral molecules. They are secreted by cells containing viruses or other microbes for example, after recognition of viral nucleic acids by cytoplasmic PRRs. They are also secreted by cells of the innate immune system such as macrophages and plasmacytoid dendritic cells (pDCs) after PRR ligation. Type I IFNs act on other cells through a single plasma membrane receptor to induce synthesis of a variety of proteins that collectively act to inhibit viral replication.
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Figure 10. Analysis of cytokine secretion I: enzyme‐linked immunosorbent assays. Cytokines in solution can be assayed by using ELISAs, as can any other molecule for which antibodies recognizing different epitopes are available. In a multi‐stage procedure as illustrated for IL‐12, a specific antibody can be attached to a surface to capture the cytokine, and a second antibody to a different epitope on the cytokine can be added to detect the bound cytokine. Binding of the second antibody can be detected by adding a third, enzyme‐labelled, anti‐Ig antibody. Adding an enzyme substrate leads to production of a coloured reaction product than can be measured quantitatively. By comparing this with a standard preparation of the cytokine, the concentration can be measured. This particular type of ELISA is known as a sandwich ELISA (referring to the two different anti‐IL12 antibodies that are used); it is also called an antigen‐capture assay.
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Figure 11. Analysis of cytokine secretion II: intracellular flow cytometry. It can be important to know which individual cells in a population are secreting a particular molecule. To determine this, cells in suspension are permeabilized, allowing antibodies to enter. Antibody binding is detected by fluorescent labelling and the cells are analyzed in a flow cytometer. This permits the identification of specific cell types by their expression of particular cell surface molecules (phenotypic markers), and determining whether that cell has synthesized the cytokine in question. This technique is illustrated for a mixed population of macrophages in which only a subset is secreting IL‐12; analysis of phenotypic markers, which can be done simultaneously, is not shown.
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Figure 12. Tissue‐resident cells of innate immunity. Tissue‐resident cells can detect the presence of a microbe or its products. Many cells in peripheral tissues such as macrophages and mast cells in connective tissues, and epithelial cells in mucosal tissues, express PRRs that recognize PAMPs from infectious agents. PRR agonists trigger different cellular responses, often including cytokine secretion that triggers inflammation and initiates innate immune responses. DCs, responding to the PAMPs or the signals delivered by other resident cells, become activated and start to trigger adaptive immune responses. Other tissue‐resident cells, such as fibroblasts, are involved in repair of tissues damaged by the microbe.
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Figure 13. Anti‐microbial peptides. Cells such as mucosal epithelial cells and neutrophils can be stimulated by PAMPs to secrete peptides that have direct anti‐microbial properties. Molecules such as defensins and cathelicidins appear to act by several mechanisms, including bacterial membrane disruption and other actions on intracellular targets within the microbe.
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Figure 14. Mast cell functions. Mast cells are resident in connective tissues throughout the body. The can be activated by (1) mechanical damage, (2) by PAMPs acting on their PRRs, (3) through small molecules such as the C3a and C5a complement fragments, (4) and via cross‐linking of IgE bound to their specific FcRs. Activation results in very rapid degranulation with release of histamine and some other pre‐formed, stored mediators including some cytokines, and the later synthesis and release of lipid mediators such as leukotrienes and prostaglandins, and of many cytokines and chemokines.
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Figure 15. Macrophage activation. Blood monocytes are recruited into inflamed tissues where they can differentiate into macrophages. Stimulation via PRRs or other receptors leads to changes in their properties that include increased phagocytic ability, secretion of enzymes such as elastase and collagenase, and of angiogenic and fibroblast growth factors. These cells are now known as inflammatory macrophages. If these cells are primed by cytokines such as IFN‐γ (the most important) or TNF‐α, and stimulated via PRRs e.g. by LPS, they can acquire potent anti‐microbial properties. These fully activated macrophages produce many reactive oxygen and nitrogen species (ROIs, RNIs), and acquire specific FcRs (e.g. FCγR1) to target opsonized microbes into their microbicidal machinery. The most important early source of IFN‐γ is probably NK cells. Activated macrophages have increased expression of MHC class II molecules, which enables later recognition by activated CD4 T cells and delivery of IFN‐γ.
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Figure 16. Phagocytosis by macrophages. Particles can be internalized by macrophages in several ways. The figure illustrates two of the most important mechanisms. Antibody‐coated particles bind to FcRs and this stimulates actin‐dependent extension of cytoplasmic processes that engulf the particle by a zippering mechanism. In contrast, particles coated with complement C3b or iC3b appear to “sink” into the cell, but again this is actin‐dependent. The activation of the actin cytoskeleton involves signalling through the Rho family GTPases, examples of which are shown. In both cases the vacuole containing the particle, the phagosome, fuses with lysosomes, which discharge their contained enzymes into the phagosome.
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Figure 17. Assessment of apoptosis and necrosis. Apoptotic cells “flip” phosphatidylserine from the inner to the outer surface of the plasma membrane, where it can be detected by the binding of fluorescent Annexin V. Their plasma membranes are, however, still intact and this prevents the entry of molecules such as propidium iodide (PI), which binds DNA. In necrotic cells, however, PI can enter the cell and access and bind DNA. Using two‐colour flow cytometry, apoptotic cells (Annexin V‐positive) can be distinguished from necrotic cells (double‐positive for Annexin V and PI) and live cells (negative for both). The figure shows a dot‐plot as generated by two‐colour flow cytometry.
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Figure 18. Endothelial cells and leukocyte emigration. Leukocyte emigration from the blood into the tissues (extravasation) requires a highly regulated set of molecular interactions, shown here for neutrophils. (1) Inflammatory mediators secreted by alarm cells in response to PAMPs, and perhaps by direct action of PAMPs on endothelial cells, stimulate endothelial cells to express E‐ and P‐selectins on their luminal surface; the latter is stored in specialised granules (Webel‐Palade bodies) in endothelial cells, and is released on activation of the endothelial cell. Neutrophils have complementary ligands that make low‐affinity interactions with the selectins, allowing them to roll along the endothelium. (2) Chemokines, secreted by alarm cells, are translocated across the endothelium and bind to glycosylated molecules on the luminal surface. Chemokine receptors on the neutrophils recognize the corresponding chemokines and signal to integrin molecules, increasing their affinity for their ligands. (3) New expression of these corresponding ligands (e.g. stimulated by alarm signals) enables the neutrophils to adhere tightly and to flatten on the endothelium. (4) Other molecules such as CD31 then enable the neutrophils to migrate between, or through, the endothelial cells into the underlying connective tissue.
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Figure 19. Complement activation. The three pathways of complement activation converge with the formation of C3 convertases. In the lectin pathway, MBL or ficolin bind to bacterial carbohydrates and in association with the MASP proteases, activate C4 and C2 to form the C3 convertase C4b2a. In the classical pathway, C1q can bind to antibodies bound to a surface and in association with C1r and C1s, again activates C4 and C2 to form C4b2a. C1q may also bind to bacterial carbohydrates and initiate the cascade. In the alternative pathway, C3 is being continually broken down, but further activation is normally blocked by inhibitory factors. If C3 deposits on microbial surfaces however, inhibition is not effective and C3b combines with Factor B to form the C3 convertase C3bBb. C3b in combination with either C4a2a or C3bBb then creates a C5 convertase which cleaves C5 and activation continues to form the membrane attack complex (MAC). Each early stage generates a large (b) fragment, which is surface‐associated (e.g. on a microbe), and a small (a) fragment, which diffuses away. There is also enormous amplification of key components (e.g. C3b, C5b) as the cascade progresses.
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Figure 20. Anti‐microbial functions of neutrophils. Neutrophils can phagocytose opsonized microbes through their FcRs and complement receptors, and also express other receptors (e.g. PRRs) that promote phagocytosis. They possess several intracellular antimicrobial mechanisms including ROIs (for which NADPH oxidase is crucial), lysosomal enzymes such as acid proteases, and antimicrobial proteins and peptides, all of which can be released into the phagosome. Neutrophils can also form neutrophil extracellular traps (NETs) that contain chromatin and which may bind bacteria. These, perhaps together with secretion of other enzymes such as neutral proteases, may enable neutrophils to kill microbes extracellularly.
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Figure 21. Neutrophil chemotaxis. Neutrophils that have entered inflamed connective tissues from blood move towards the site of infection by migrating up a chemical concentration gradient, a process termed chemotaxis. Chemotactic factors are recognized by receptors on the neutrophil surface, that typically signal through G‐proteins and activate the cytoskeleton. The neutrophil can sense concentration differences over the length of the cell and this permits polarization of the cell, with pseudopodia being extended from the part of the cell in contact with the highest concentration. Important chemotactic factors for neutrophils include formylated bacterial peptides such as f‐MLP, specific types of chemokines and leukotrienes released from macrophages and mast cells, and complement component C5a.
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Figure 22. Phagosomal killing by neutrophils. Bacteria in neutrophil phagosomes are subject to several different killing mechanisms. ROIs such as hydrogen peroxide (H 2O 2), superoxide anion (O 2 −) and singlet oxygen (O •) can be directly toxic, but may also act to generate pH conditions appropriate for the actions of antimicrobial enzymes such as acid proteases. H 2O 2 can interact with chloride ions (Cl −) in the presence of myeloperoxidase to generate hypochlorous acid (HOCl). Proteins such as lactoferrin can sequestrate iron (Fe 3+)and make it unavailable for microbial metabolism. Lysosomal proteases break down dead bacteria but may also have more direct roles in killing. Anti‐bacterial peptides such as defensins cause membrane disruption and lysozyme can break down the cells of some Gram‐negative bacteria.
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Figure 23. Structure of an abscess. If pyogenic bacteria such as Staphylococcus aureus enter connective tissue they initiate acute inflammation, resulting in oedema and neutrophil recruitment. The bacteria can, however, resist and kill the neutrophils, and enzymes released by the bacteria and neutrophils cause liquefaction of the tissue, forming pus. Staphylococcus aureus also secretes coagulase, which precipitates fibrin, forming a barrier around the site of infection (not shown). At the same time, a healing reaction is starting to lay down collagen, secreted by fibroblasts, at the margins of the site of infection. The structure thus formed is an abscess. As the abscess develops, it erodes towards a surface, and will eventually rupture the surface and discharge its contents including live bacteria. This can be very serious if it discharges, for example, into the peritoneal cavity or a blood vessel, resulting in septicaemia and sometimes septic shock.
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Figure 24. Functions of inflammatory macrophages. Monocytes recruited into inflamed tissues can become macrophages that develop properties distinct from those of resident macrophages. These inflammatory macrophages are highly phagocytic. If acted on by PAMPs they are active secretors of catabolic enzymes that help to remodel tissues, release growth factors for blood vessels and fibroblasts, and secrete some plasma proteins such as complement components and coagulation factors. The latter help to increase the concentrations of those present at inflammatory sites, over and above the proteins that are recruited from the blood. The normal functions of inflammatory macrophages include assisting in healing and repair, but they can also be activated by IFN‐γ to become potent anti‐microbial cells.
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Figure 25. Natural killer cell life history. NK cells develop from CLP in the bone marrow. They are released into the blood as lymphocyte‐like cells, with conspicuous cytoplasmic granules (large granular lymphocytes). In the steady state a CD16 − subset of NK cells may enter the liver, lungs and spleen or return to the marrow. During inflammation another CD16 + subset may be recruited to inflamed tissues and their draining lymph nodes. NK cells are not long‐lived, most being replaced from precursors in days or weeks.
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Figure 26. Functions of natural killer cell subsets. NK cells comprise two main subsets based in part on expression of CD16, a specific FcR for IgG. CD16 – NK cells are active secretory cells. They themselves can be activated by cytokines secreted by different cells including macrophages (IL‐12, IL‐15, TNF‐α), DCs (IL‐12, IL‐15), activated T cells (IL‐2, not shown) and virally infected cells (Type I IFNs). These activated NK cells can the secrete cytokines including IFN‐γ and TNF‐α. CD16 +NK cells appear to be primarily cytotoxic cells. They may kill virally infected cells through granule‐dependent or death‐inducing receptor‐dependent mechanisms and they may also mediate ADCC when their FcRs are ligated. ADCC can be induced experimentally in culture, using antibody‐coated target cells, but its in vivo significance remains uncertain.
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Figure 27. Natural killer cell recognition and activation. NK cells possess granules that contain the pre‐formed apparatus (perforin and granzymes) needed to kill cells they recognize. They express several activating receptors which, on recognizing a ligand on the target cell, activate the killing mechanisms. NK cells, however, also express inhibitory receptors which, if they recognize MHC class I on the target cell, block the activation of the cytotoxic mechanisms. Thus, NK cells kill target cells on which MHC class I expression is decreased e.g. by viral infection or some tumour cells.
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Figure 28. Systemic effects of inflammation. Cytokines secreted by cells in inflamed tissues can enter the blood and affect distant tissues. These tissues include the liver where the acute‐phase response involves the increased synthesis of several proteins, some of which such as complement components have well‐defined roles in defence, whereas the functions of others are still obscure. Effects on the bone marrow include increased production and release of myeloid cells, particularly neutrophils and monocytes. Effects on the CNS include the induction of fever, loss of appetite and lethargy; on muscle and adipose these effects include increased catabolism to gererte energy.
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Figure 29. Septic shock. If LPS from Gram‐negative bacteria or lipoteichoic acid from Gram‐positive bacteria enters the bloodstream it can activate monocytes via TLR4 or TLR2/6 respectively. This can result in the release of pro‐inflammatory mediators such as IL‐1, IL‐6 and TNF‐α. In high concentration these cytokines can have potentially fatal effects. As well as causing fever, they can act on blood vessels to induce nitric oxide, and cause venous dilatation and increased permeability. This may lead to decreased venous return to the heart, resulting in severe hypotension (low blood pressure) and failure of organs such as the kidney and lungs.
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Figure 30. Cytokines in myelopoiesis. Bone marrow multi‐potential stem cells are acted on by SCF and Flt3, and probably other factors, to become CMPs. The CMP responds to other growth factors by dividing to form the different myeloid cell types. Some that are known or thought to be essential for production of monocytes and different types of granulocytes are shown. While it is clear that the production of the different myeloid cells is under feedback control, the details of the control mechanisms are still poorly understood.
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Figure 31. Possible mechanisms of alum adjuvanticity. Vaccines containing alum adjuvant stimulate very good, protective antibody responses against the antigen(s) they contain (e.g. tetanus toxoid). Alum may act as an adjuvant in several ways. (i) It may provide a “depot” of antigen at the site of injection, increasing the amount available to cells such as DCs. (ii) Through unknown routes it may lead to increased costimulatory molecule expression on DCs. Both would increase antigen presentation to, and activation of antigen‐specific T cells that could then help antibody responses to be triggered. (iii) It may be phagocytosed (e.g. by macrophages) and lead to damage or destabilization of endosomes. This may lead to activation of inflammasomes, perhaps through production of uric acid as a DAMP, and synthesis of pro‐inflammatory cytokines (e.g. IL‐1) to enhance innate immune responses and, in turn, adaptive immunity. We are gaining increased insights into these (and other) different mechanisms, but still have far to go, including understanding why alum does not efficiently stimulate other types of immunity (e.g. cytotoxic T lymphocytes) that may be more effective for vaccination against virally‐infected cells and tumours.
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