IMMUNITY AGAINST BACTERIAL INFECTIONS

1. IMMUNITY AGAINST BACTERIAL INFECTIONS

2. The immune defense mechanisms elicited against pathogenic bacteria are determined by their: • surface chemistry; • mechanism of pathogenicity; and • whether they are predominantly extracellular or also have the ability to survive inside mammalian cells.

3. There are four main types of bacterial cell wall The four main types of bacterial cell wallbelong to the following groups. • Gram-positive bacteria; • Gram-negative bacteria; • mycobacteria; • spirochetes.

4. Fig. no. 01 The components indicated with an asterisk (*) are recognized by the innate immune system as a non-specific ‘danger’ signal that selectively boosts some aspects of immune activity.

5. Different immunological mechanisms have evolved to destroy the cell wall structure of the different groups of bacteria. • All types have an inner cell membrane and a peptidoglycan wall. • Gram-negative bacteria also have an outer lipid bilayer in which lipopolysaccharide (LPS) is embedded. • Lysosomal enzymes and lysozyme are active against the peptidoglycan layer, whereas cationic proteins and complement are effective against the outer lipid bilayer of the Gram-negative bacteria.

6. The compound cell wall of mycobacteria is extremely resistant to breakdown, and it is likely that this can be achieved only with the assistance of the bacterial enzymes working from within. • Some bacteria also have fimbriae or flagellae, which can provide targets for the antibody response, others have an outer capsule. These can impede the functions of phagocytes or complement, but they also act as targets for the antibody response.

7. Pathogenicity varies between two extreme patterns The two extreme patterns of pathogenicity are: • toxicity without invasiveness; • invasiveness without toxicity However, most bacteria are intermediate between these extremes, having some invasiveness assisted by some locally acting toxins and spreading factors (tissue-degrading enzymes). • Corynebacteriumdiphtheriaeand Vibriocholeraeare examples of organisms that are toxic, but not invasive. Because their pathogenicity depends almost entirely on toxin production, neutralizing antibody to the toxin is probably sufficient for immunity, though antibody binding to the bacteria and so blocking their adhesion to the epithelium could also be important. • In contrast, the pathogenicity of most invasive organisms does not rely so heavily on a single toxin, so immunity requires killing of the organisms themselves.

8. Fig. no. 02

9. Innate immune responses are the first line of immune defense • The innate immune system provides the first line of immune defense by detecting the immediate presence and nature of infection. • Many different cells are involved in generating innate responses including phagocytic cells and NK cells. • It is also becoming clear that early recognition of bacteria by antigen-presenting cells (APCs), for example dendritic cells, determines the phenotype of the adaptive response. • Innate immune recognition relies on a growing number of receptors, termed pattern recognition receptors(PRRs)that have evolved to recognize pathogen associated molecular patterns (PAMPs).

10. The first lines of defense (do not depend on antigen recognition) • The body’s first line of defense against pathogenic bacteria consists of simple barriers to the entry or establishment of the infection. • The skin and exposed epithelial surfaces have non-specific or innate protective systems, which limit the entry of potentially invasive organisms. • Intact skin is impenetrable to most bacteria. Additionally, fatty acids produced by the skin are toxic to many organisms. Indeed, the pathogenicity of some strains correlates with their ability to survive on the skin. • Epithelial surfaces are cleansed, for example, by ciliary action in the trachea or by flushing of the urinary tract. • Many bacteria are destroyed by pH changes in the stomach and vagina, both of which provide an acidic environment. In the vagina, the epithelium secretes glycogen, which is metabolized by particular species of commensal bacteria, producing lactic acid.

11. Commensals can limit pathogen invasion • Commensals can limit pathogen invasion through the production of antibacterial proteins termed colicins. • When the normal flora are disturbed by antibiotics, infections by Candida spp. or Clostridium difficile can occur,and the latter is a major cause of antibiotic-induced colitis and diarrhea.

12. The second line of defense is mediated by recognition of bacterial components • If organisms do enter the tissues, they can be combated initially by further elements of the innate immune system. • Numerous bacterial components are recognized in ways that do not rely on the antigen-specific receptors of either B cells or T cells. These types of recognition are phylogenetically ancient ‘broad-spectrum’ mechanisms that evolved before antigen-specific T cells and immunoglobulins, allowing protective responses to be triggered by common microbial components bearing so-called ‘pathogen-associated molecular patterns’ (PAMPs).

13. The host molecules that recognize these microbial components are referred to as the ‘pattern recognitionmolecules’ of the innate immune system. • Collectins and ficolins , the Toll-like receptors, the mannose receptor, and theNOD proteins all recognize PAMPs. • The immune system (PRR) has selected PAMPs for recognition because they are not only characteristic of microbes, but are essential for their growth and cannot be easily mutated to evade discovery.

14. Bacterial PAMPs activate cells via Toll-like receptors (PRR) • Many bacterial PAMPs activate cells via Toll-like receptors (TLRs). These are homologs of a receptor mediating antimicrobial immune responses in the fruit fly (Drosophila spp.). • The TLR family is made up of at least ten different TLR molecules which differ in the microbial structures they recognize. • The most prominent TLRs involved in recognition of bacterial components are TLR1, 2, 4, 5, 6, and 9.

15. TLRs are preferentially expressed on phagocytes, dendritic cells, and epithelial cells at sites of bacterial entry to the host. • Each cell type can express a different combination of receptors and this repertoire can be altered by inflammatory stimuli, allowing the greatest possible recognition coverage to a diverse range of pathogens.

16. Many organs contain cells belonging to the mononuclear phagocyte lineage. These cells are derived from blood monocytes and ultimately from stem cells in the bone marrow. Fig. no. 03

17. Other important pattern recognition receptors include: • the mannose receptor; and • scavenger receptors. Of course, recognition of bacteria also occurs in the absence of cells via: • complement; • C-reactive protein (CRP); • mannose-binding lectin in the blood; • surfactant protein A in the lungs.

18. Fig. no. 04 Several common bacterial PAMPs are recognized by molecules present in serum and by receptors on cells. These recognition pathways result in activation of the alternative complement pathway (factors C3, B, D, P), with consequent release of C3a and C5a; activation of neutrophils, macrophages, and NK cells; triggering of cytokine and chemokine release; mast cell degranulation, leading to increased blood flow in the local capillary network; and increased adhesion of cells and fibrin to endothelial cells. These mechanisms, plus tissue injury caused by the bacteria, may activate the clotting system and fibrin formation, which limit bacterial spread.

19. LPS is the dominant activator of innate immunity in Gram-negative bacteria • Injection of pure LPS into mice or even humans is sufficient to mimic most of the features of acute Gram negative infection, including massive production of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF), leading to severe shock.

20. Recognition of LPS is a complex process involving molecules that bind LPS and pass it on to cell membrane associated receptors on leukocytes, and endothelial and other cells, which initiate this proinflammatory cascade; these events are illustrated in following Figure. • Binding of LPS to TLR4 is a critical event in immune activation. TLR4 knockout mice are resistant to LPS induced shock and there is some evidence that polymorphisms in human TLR4 may influence the course of infection with these bacteria.

21. Fig. 05 LPS released from Gram-negative bacteria becomes bound to LPS-binding protein (LBP) which promotes transfer of LPS to either soluble CD14 (sCD14) or to a GPI-linked membrane form of the protein (mCD14) expressed on neutrophils and macrophages. LBP then dissociates and the mCD14–LPS complex, in association with Toll-like receptor 4 (TLR4) and MD2, transduces signals that cause increased expression of integrins (adhesion molecules) and increased release of many proinflammatory cytokines including TNFá and IL-1. These in turn activate endothelial cells and drive the acute phase response in the liver. One product of the acute phase response is further LBP.

22. Other PAMPs • Most capsular polysaccharides are not potent activators of inflammation (though some can activate macrophages) and instead attempt to shield the bacterium from host immune defenses.  • Components of the cell wall, including peptidoglycans and lipoteichoic acids, are the dominant activators of innate immunity, and TLR2, often in cooperation with TLR1 or TLR6, is the major TLR involved. • The LBP and CD14, which bind LPS, are also involved in recognition of lipid-containing bacterial components from mycoplasmas, mycobacteria, and spirochetes.  • Other molecules that trigger innate immunity include mycoplasma lipoproteins (via TLR 2/6), flagellin (via TLR5), and DNA (due to its distinct CpG motifs) via TLR9.

23. Most pattern recognition receptors are expressed on the plasma membrane of cells, making contact with microbes during the process of binding and/or phagocytosis. • However, others are designed to detect intracellular pathogens and their products inside phagosomes (such as TLR9) or in the cytosol. • NOD-1 and NOD-2 proteins recognize peptidoglycans of both Gram-positive and Gram-negative bacteria (Fig. 6).

24. NOD-1 is an intracellular pattern recognition molecule. Peptidoglycan from, for example, Shigella flexneri and some strains of Escherichia coli binds to the protein. A domain (RICK) then activates IKK leading to the transcription of proteins dependent on NF-κB. Fig. no. 06

25. Epithelial cells of the gut and lung have few TLRs on their luminal surface, but can be triggered by pathogens that: • actively invade the cell (such as Listeria spp.); • inject their components (such as Helicobacter pylori); or • actively reach the basolateral surface (e.g. Salmonella spp.). • This may explain why constant exposure to non-pathogenic microbes in the intestine and airways does not induce a chronic state of inflammation – the host waits until they move beyond the lumen, signifying the presence of a real pathogenic threat.

26. LYMPHOCYTE-INDEPENDENT BACTERIAL RECOGNITION PATHWAYS HAVE SEVERALCONSEQUENCES Complement is activated via the alternative pathway Complement activation can result in the killing of some bacteria, particularly those with an outer lipid bilayer susceptible to the lytic complex (C5b–9). • Perhaps more importantly, complement activation releases C5a, which attracts and activates neutrophils and causes degranulation of mast cells. • The consequent release of histamine and leukotriene (LTB4) contributes to further increases in vascular permeability (Fig. no.04). • Opsonization of the bacteria, by attachment of cleaved derivatives of C3, is also critically important in subsequentinteractions with phagocytes.

27. Release of proinflammatory cytokines increases the adhesive properties of the vascular endothelium • The rapid release of cytokines such as TNF and IL-1 (Fig. no. 05) from macrophages increases the adhesive properties of the vascular endothelium and facilitates the passage of more phagocytes into inflamed tissue. • Combined with the release of chemokines such as CCL2, CCL3, and CXCL8, this directs the recruitment of different leukocyte populations. Epithelial cells, neutrophils, and mast cells are also important sources of proinflammatory cytokines.

28. Some of the chemokine receptors found on particular leukocytes and the chemokines they respond to are listed. The cells are grouped according the principal types of effector response. Note that TH1 cells and mononuclear phagocytes both express chemokine receptor CCR5, which allows them to respond to chemokine CCL3, while TH2 cells, eosinophils, and basophils express CCR3, which allows them to respond to CCL11. This allows selective recruitment of sets of leukocytes into areas with particular types of immune/inflammatory response. Both groups of cells express chemokine receptors CCR1 and CCR2, which allow responses to macrophage chemotactic proteins (CCL2, CCL7, CCL8, and CCL13). Neutrophils express chemokine receptors CXCR1 and CXCR2, which allow them to respond to CXCL8 (IL-8) and CXCL1 and CXCL2. Bracketed entries indicate that only a subset of cells express that receptor. Fig. no. 07

29. IL-1, TNF, and IL-6 also initiate the acute phase response, increasing the production of complement componentsas well as other proteins involved in scavengingmaterial released by tissue damage and, In the case of CRP,an opsonin for improving phagocytosis of bacteria. • When NK cells are stimulated by the phagocyte derived cytokines IL-12 and IL-18 they rapidly releaselarge quantities of interferon-γ (IFNγ). This response happens within the first day of infection, well before the clonal expansion of antigen-specific T cells, and provides a rapid source of IFNγ to activate macrophages. • This Tcell-independent pathway helps to explain the considerableresistance of mice with SCID (severe combinedimmune deficiency, a defect in lymphocyte maturation)to infections such as with Listeria monocytogenes.

30. Pathogen recognition generates signals that regulate the lymphocyte-mediated response • The signals generated following the recognition of pathogens not only generate a cascade of innate immune events, but also regulate the development of the appropriate lymphocyte-mediated response. •  Dendritic cells (DCs) are crucial for the initial priming of naive T cells specific for bacterial antigens. • Contact with bacteria in the periphery induces immature DCs to migrate to the draining lymph nodes and augments their antigen-presenting ability by increasing their: • display of MHC molecule–peptide complexes; • expression of co-stimulatory molecules (such as CD40, CD80, and CD86); and • secretion of T cell differentiating cytokines.

31. Some of this DC activation occurs secondary to their production of cytokines such as type I IFN. • Activated macrophages also act as antigen-presenting cells (APCs), but probably function more at the site of infection, providing further activation of effector rather than naive T cells. • Binding of bacterial components to pattern recognition receptors such as TLRs induces a local environment rich in cytokines such as IFNγ, IL-12, and IL-18, which promote T cell differentiation down the TH1 rather than TH2 pathway.

32. ANTIBODY PROVIDES AN ANTIGENSPECIFIC PROTECTIVE MECHANISM • The relevance to protection of interactions of bacteria with antibody depends on the mechanism of pathogenicity. • Antibody clearly plays a crucial role in dealing with bacterial toxins: • it neutralizes diphtheria toxin by blocking the attachment of the binding portion of the molecule to its target cells; • similarly it may block locally acting toxins or extracellular matrix-degrading enzymes, which act as spreading factors; • Antibody can also interfere with motility by binding to flagellae.

33. An important function on external and mucosal surfaces, often performed by secretory IgA , is to stop bacteria binding to epithelial cells – for instance, antibody to the M proteins of group A streptococci gives type-specific immunity to streptococcal sore throats. • It is likely that some antibodies to the bacterial surface can block functional requirements of the organism such as binding of iron-chelating compounds or intake of nutrients (Fig. 08). • An important role of antibody in immunity to nontoxigenic bacteria is the more efficient targeting of complement.

34. This diagram lists the stages of bacterial invasion (blue) and indicates the antibacterial effects of antibody (yellow) that operate at the different stages. Antibodies to fimbriae, lipoteichoic acid, and some capsules block attachment of the bacterium to the host cell membrane. Antibody triggers complement-mediated damage to Gramnegative outer lipid bilayers. Antibody directly blocks bacterial surface proteins that pick up useful molecules from the environment and transport them across the membrane. Antibody to M proteins and capsules opsonizes the bacteria via Fc and C3 receptors for phagocytosis. Bacterial factors that interfere with normal chemotaxis or phagocytosis are neutralized. Bacterial toxins may be neutralized by antibody, as may bacterial spreading factors that facilitate invasion (e.g. by the destruction of connective tissue or fibrin). Fig. no. 08

35. Naturally occurring IgM antibodies, which bind to common bacterial structures such as phosphorylcholine, are important for protection against some bacteria (particularly streptococci) via their complement fixing activity. • Specific, high-affinity IgG antibodies elicited in response to infection are most important – children with primary immune deficiencies in B cell development or in T cell help have increased susceptibility to extracellular rather than intracellular bacteria.

36. With the aid of antibodies, even organisms that resist the alternative (i.e. innate) complement pathway (Fig. 09) are damaged by complement or become coated with C3 products, which then enhance the binding and uptake by phagocytes. • The most efficient complement-fixing antibodies in humans are IgM, then IgG3 and to a lesser extent IgG1, whereas IgG1 and IgG3 are the subclasses with the highest affinity for Fc receptors.

37. A variety of molecules facilitate the binding of the organisms to the phagocyte membrane. These are in addition to the TLR system (e.g. TLR4 for LPS, TLR5 for flagellin, and TLR2 [plus TLR1/TLR6] for bacterial lipoproteins and peptidoglycans). The precise nature of the interaction will determine whether uptake occurs and whether cytokine secretion and appropriate killing mechanisms are triggered. Recognition invariably involves combinations of different receptor families. Note that apart from complement, antibody, and mannose-binding lectin (MBL), which bind to the bacterial surface, the other components are constitutive bacterial molecules. Fig. no. 09

38. Pathogenic bacteria may avoid the effects of antibody • Neisseria gonorrhoeae is an example of a pathogenic bacterium that uses several immune evasion strategies • (Fig. 10) and humans can be repeatedly infected with N. gonorrhoeae with no evidence of protective immunity. • Antibodies may also be important for effective immunity against some intracellular bacteria such asLegionella and Salmonella spp.

39. N. gonorrhoeae is an example of a bacterium that uses several strategies to avoid the damaging effects of antibody. First, it fails to evoke a large antibody response, and the antibody that does form tends to block the function of damaging antibodies. Second, the organism secretes an IgA protease to destroy antibody. Third, blebs of membrane are released, and these appear to adsorb and so deplete local antibody levels. Finally, the organism uses three strategies to alter its antigenic composition: (i) the LPS may be sialylated, so that it more closely resembles mammalian oligosaccharides and promotes rapid removal of complement; (ii) the organism can undergo phase variation, so that it expresses an alternative set of surface molecules; (iii) the gene encoding pilin, the subunits of the pilus, undergoes homologous recombination to generate variants. N. gonorrhoeae also impairs T cell activation by engaging a co-inhibitory receptor CEACAM-1 on the lymphocyte surface by one of its OPA proteins. Fig. 10.

40. Pathogenic bacteria can avoid the detrimental effects of complement • Some bacterial capsules are very poor activators of the alternative pathway (Fig. 11). •  For other bacteria, long side chains (O antigens) on their LPS may fix C3b at a distance from the otherwise vulnerable lipid bilayer. •   Similarly, smooth-surfaced Gram negative organisms (Escherichia coli, Salmonella spp., Pseudomonas spp.) may fix but then rapidly shed the C5b–C9 membrane lytic complex. •  Other organisms exploit the physiological mechanisms that block destruction of host cells by complement. • When C3b has attached to a surface it can interact with factor B leading to further C3b amplification or it can become inactivated by factors H and I. Capsules rich in sialic acid (as host cell membranes are) seem to promote the interaction with factors H and I. Neisseria meningitidis, E. coli K1, and group B streptococci all resist complement attachment in this way. • The M protein of group A streptococci acts as an acceptor for factor H, thus potentiating C3bB dissociation. • These bacteria also have a gene for a C5a protease.

41. Bacteria avoid complement-mediated damage by a variety of strategies. (1) An outer capsule or coat prevents complement activation. (2) An outer surface can be configured so that complement receptors on phagocytes cannot obtain access to fixed C3b. (3) Surface structures can be expressed that divert attachment of the lytic complex (MAC) from the cell membrane. (4) Membrane-bound enzyme can degrade fixed complement or cause it to be shed. (5) The outer membrane can resist the insertion of the lytic complex. (6) Secreted decoy proteins can cause complement to be deposited on them and not on the bacterium itself. Fig. 11.

42. ULTIMATELY MOST BACTERIA ARE KILLED BY PHAGOCYTES • A few, mostly Gram-negative, bacteria are directly killed by complement. • However, immunity to most bacteria, whether considered as extracellular or intracellular pathogens, ultimately needs the killing activity of phagocytes. This process involves several steps. • Bacterial components attract phagocytes by chemotaxis • Unlike neutrophils, which in the uninfected host are found almost entirely in the blood, resident macrophages are constitutively present in tissues where exposure to pathogens first occurs (such as alveolar macrophages in the lung and Kupffer cells in the liver).

43. These macrophages have some killing activity, but invariably need to be supplemented by recruitment of neutrophils and/or monocytes across the blood vessel wall. • Phagocytes are attracted by: • bacterial components such as f-Met-Leu-Phe (which is chemotactic for leukocytes); • complement products such as C5a; and • locally released chemokines and cytokines derived from resident macrophages and epithelial cells.

44. The cellular composition of this inflammatory response varies according to the pathogen and the time since infection. For instance: • acute infection with encapsulated bacteria such as Streptococcus pyogenes give rise to tissue lesions rich in neutrophils (typical of so-called pyogenic or pus forming infections); • at the other extreme, chronic infections with M. tuberculosis result in granulomas rich in macrophages,macrophage-derived multinucleated giant cells, andT cells; • other organisms, such as Listeria and Salmonella spp., result in lesions of more mixed composition.

45. The choice of receptors is critical • The choice of receptors used for attachment of the phagocyte to the organism is critical and will determine: • the efficiency of uptake; • whether killing mechanisms are triggered; • whether the process favors the pathogen by subverting immunity. • The binding can be mediated by lectins on the organism (e.g. on the fimbriae of E. coli), but receptors on the phagocyte are the most important. • These either bind directly to the bacterium or indirectly via host complement and antibody deposited on the bacterial surface (opsonization).

46. • direct binding is mediated by pattern recognition molecules including Toll-like receptors and scavenger receptors (such as SRA, MARCO), mannose receptor, and dectin-1b; • opsonization is mediated through complement receptors such as CR1, CR3, and CR4, which recognize complement fragments deposited on the organism via the alternative or classic complement pathways. • Complement can also be fixed by MBL present in serum, which can itself bind to C1q receptors and CR1.  • Additionally, Fc receptors on the phagocyte (FcγRI, FcγRII, and FcγRIII, bind antibody that has coated bacteria ( Fig. 09), whereas various integrins can bind fibronectin and vitronectin opsonized particles.

47. Uptake can be enhanced by macrophage activating cytokines • The binding of an organism to a receptor on the macrophage membrane does not always lead to its uptake. • For example, zymosan particles (derived from yeast) bind via the glucan-recognizing lectin-like site on the CR3 of the macrophage and are taken up, whereas erythrocytes coated with iC3b are not, even though the iC3b also binds to CR3. • This can, however, be enhanced by macrophage activating cytokines such as granulocyte–macrophagecolony stimulating factor (GM-CSF).

48. Different membrane receptors vary in theirefficiency at inducing a microbicidal response • Just as the binding of an organism to membrane receptors does not guarantee uptake, so different membrane receptors vary in their efficiency at inducing a microbicidal response – for example, mannose receptors and Fc receptors are particularly efficient at inducing the respiratory burst, but complement receptors are not, providing an evasion strategy for some organisms.

49. Phagocytic cells have many killing methods • The killing pathways of phagocytic cells can be: • oxygen dependent; or • oxygen independent. • In summary, one oxygen-dependent pathway involves the reduction of oxygen to superoxide anion (which is molecular oxygen to which a single unpaired electron has been added). • This then interacts with numerous other molecules to give rise to a series of free radicals and other toxic derivatives, which can kill bacteria and fungi. • Recent studies in neutrophils suggests that the oxidative burst may also act indirectly, by promoting the flux of K+ ions into the phagosome and activating microbicidal proteases.

50. A second oxygen-dependent pathway involves the creation of nitric oxide (NO•) from the guanidino nitrogen of L-arginine. This in turn leads to further toxic substances such as the peroxynitrites, which result from interactions of NO• with the products of the oxygen reduction pathway. • Cytokine activation by IFNγ and TNFγ leads to production of inducible NO synthase, which generates NO from L-arginine.