Innate cellular responses frontline defenders

The immune system includes a complex array of cells and biomolecules, which interact to provide protection from challenge by pathogenic microbes, and impaired immune function leads to increased risk of infection by foodborne pathogens. Antigens - substances that induce an immune response - are often components of invading microbes. The immune system can be divided into two branches - the innate (or nonspecific) and the adaptive (or specific) immune response. The innate response (the focus of this chapter) includes certain cell types and molecules able to react to the presence of invading microorganisms and their components, but without a high degree of specificity. Innate immunity is characterized by the speed of its response, and by a lack of memory. Rapid response speed is essential for initial host protection, and reflects the ability of cells in the innate immune system to react rapidly to contact with pathogens, a property enhanced by their pre-positioning at the luminal interface. Lack of memory of such contact, however, means that the efficiency and effectiveness of the innate immune response does not improve with repeated exposure, unlike the adaptive immune response.

Cells of the innate immune system include neutrophils, monocytes, and macrophages, all of which are phagocytes. Phagocytic cells ingest invading pathogens, with the goal ofdigesting and destroying the invader, using an array of enzymes, reactive oxygen intermediates, and nitric oxide. Macrophages are the key phagocytic cell type in tissues. Monocytes, their precursors, are present in the blood, as are neutrophils. Macrophages recognize invading microorganisms through receptors that detect non-self components, including carbohydrates such as mannose (Delves and Roitt, 2000). Macrophages and neutrophils both interact with complement (a protein component of the innate immune response) and antibodies (components of the adaptive immune response) to improve their rates of phagocytosis of invading microbes through the process of opsonization. Once foodborne pathogens are phagocytosed, they are exposed to an impressive intracellular array of defenses, including lysozyme, antimicrobial peptides, and nitric oxide, and the respiratory burst-derived mediators superoxide anion, hypochlorous acid and hydroxyl radicals. Neutrophil killing of pathogens is now believed to be mainly due to the activity of destructive enzymes within intracellular vacuoles, rather than to the direct actions of reactive oxygen species (Segal, 2005).

Neutrophils play an essential role in defense at the intestinal epithelial layer. IEC respond to foodborne pathogens, such as Salmonella Typhimurium, by producing IL-8, a chemoattractant cytokine, which is then secreted from the basolateral side of the epithelial layer, stimulating neutrophil migration to sites of infection (Gewirtz et al., 1999). S. Typhimurium challenge leads to release of a second chemoattractant (pathogen-elicited epithelial chemoattractant, or PEEC) from IECs in the apical direction. PEEC release stimulates neutrophil translocation across the epithelial lining to the lumen. From this vantage point, neutrophils can actively phagocytose and destroy bacteria (reviewed in Sansonetti, 2004).

Some foodborne pathogens succeed in invading the host and causing disease owing to their ability to counteract macrophage activity. Shigella flexneri initially infect IEC and multiply inside them, inducing actin nucleation into comet-like tails that propel the bacteria through the cytoplasm. The resulting cellular extensions can penetrate into neighbouring IEC and other cell types, and allow Shigella to infect adjacent cells without being seen by cells of the immune system. Shigella flexneri is then able to defeat macrophages by binding to caspase 1 and inducing programmed cell death (apoptosis) following macrophage infection (Zychlinsky et al., 1992). It has been suggested that entero-invasive E. coli (EIEC) use this host evasion strategy as well (Kaper et al., 2004), allowing both Shigella and EIEC to evade macrophages and cause serious illness. Listeria monocytogenes also induces actin polymerization and 'comet tail' formation, causing it to be projected through the IEC and directly into the membranes of adjacent cells, thus escaping detection by phagocytes (Daniels et al., 2000). Enteropathogenic E. coli use their ability to signal host cell cyto-skeletal rearrangements to inhibit phagocytosis through a process involving tyrosine dephosphorylation of infected macrophage proteins (Goosney et al., 1999b). Yersinia enterocolitica is also able to inhibit phagocytosis, using certain of its Yersinia outer-protein (Yop) components (such as YopJ), to effectively paralyze macrophages. Y. enterocolitica inhibits host inflammatory responses by both macrophages and IEC through inhibition of MAP kinases and NFkB signaling (reviewed in Boyer and Lemichez, 2004).

Natural killer (NK) cells are also participants in the innate immune response. NK cells are closely related to T cells, and use a range of cell surface receptors to recognize their targets and regulate their cytolytic activity (reviewed in Lanier, 2005). Their key role is to respond to virus-infected cells in the early stages of infection by killing infected target cells. NK cells are capable of detecting and killing certain types of malignant cells. They can recognize targets through antibody-dependent cellular cytotoxicity, or through unique NK receptors that respond to certain molecules present on all cell types. The killing ability of NK cells is kept in check by killer-inhibitory receptors (KIRs), which recognize (major histocompatibility) MHC class I molecules. Down-regulation of Class I MHC molecules following certain types of virus infection or on malignant cells releases NK cells from KIR-mediated inhibition, allowing them to kill infected or transformed target cells (reviewed in Hamerman et al., 2005).

Target cell killing involves the insertion of perforin (a pore-forming protein) into the target cell membrane, and injection of granzymes, cytotoxic molecules that trigger target cell apoptosis. Owing to their mode of action and target recognition, NK cells are involved in innate defense against intracellular bacteria such as Listeria monocytogenes and Salmonella (Chin et al., 2002; Wick, 2004). NK cells also provide a source of interferon 7, which then acts to activate the adaptive immune response and promote the activation of T helper 1 cells (Chin et al, 2002).

NKT cells are a recently identified cell type expressing a semi-invariant TCR a chain, which allows them to recognize glycolipid antigens from Gramnegative bacteria (Kronenberg, 2005). This ability suggests NKT cells also play a role in responses to foodborne pathogens. For example, NKT cells have been shown to respond to Salmonella Typhimurium by producing interferon 7, a cytokine that promotes antimicrobial host defenses (Kinjo et al., 2005). Recent studies suggest that NKT cells may be most important as a defense against bacteria that lack such TLR stimuli as lipopolysaccharide, and their precise role in defense against other foodborne pathogens remains to be determined (Mattner et al, 2005).

Dendritic cells (DCs) provide a 'bridge' or interface between the innate and adaptive immune systems. Like other cells in the innate immune system, DCs react to the presence of pathogens using relatively nonspecific receptors. DCs are able to process and present antigens to the central participants in the adaptive immune response: T cells. Macrophages and monocytes can act as 'antigen-presenting cells' (APCs) and carry out a bridging role between innate and adaptive responses. Since T cells do not respond to 'free' antigen - only to antigen that is presented by APCs - this ability of DCs, monocytes and macrophages is crucial in bridging the transition from innate to adaptive immunity. DCs, macrophages and monocytes also exert their influence over the adaptive immune response through cytokine production, discussed in the preceding section. Interdigitating dendritic cells continually endocytose antigen, becoming activated when certain of their cell surface receptors recognize pathogen-associated molecular patterns (PAMPs). PAMPs include such microbial components as lipopolysaccharide, mannans, teichoic acids, and CpG motifs in DNA (reviewed in Akira and Takeda, 2004). Receptors for PAMPs, often referred to as pathogen recognition receptors (PRRs), include the toll-like receptors (TLRs), the LPS receptor CD14, and the Nod receptors.

TLRs are an evolutionarily conserved family of cell surface receptors that play a central and essential role in innate immunity through recognition of certain key microbial determinants (reviewed in Vasselon and Detmers, 2002). TLR-2 is involved in recognition of components of Gram-positive bacteria, including lipoteichoic acid (LTA), peptidoglycan and lipoproteins. TLR-4 recognizes LPS from Gram-negative bacteria, TLR-5 recognizes bacterial flagellin, TLR-9 recognizes unmethylated CpG dimers from bacterial DNA and TLR-3 recognizes viral double-stranded RNA (Akira and Takeda, 2004). Nod receptors are nucleotide-binding oligomerization proteins located in the cell cytoplasm rather than on the cell surface, and are also able to recognize peptidoglycan (Mumy and McCormick, 2005). Nod 1 recognizes D-Glu-meso-DAP, a degradation product of peptidoglycan that is naturally released by Gramnegative bacteria. Nod 2 recognizes muramyl dipeptide, which is essentially the minimal peptidoglycan unit, giving Nod 2 the ability to detect both Gramnegative and Gram-positive bacteria (Carneiro et al., 2004). Their intracellular location allows the Nod proteins to play a key role in detection of invasive enteric pathogens, such as Salmonella and Shigella. Overall, the recognition array composed of PRRs allows cells of the innate immune system to detect a broad range of invaders such as foodborne pathogens without requiring a high degree of specificity.

8.4.1 How do cells participating in the innate immune response recognize pathogens?

IECs and other cell types involved in the innate response react to foodborne and other pathogens detected through the TLR and Nod systems by activating the pro-inflammatory pathway controlled by nuclear factor kB (NFkB) (Mumy and McCormick, 2005). NFkB is a DNA-binding protein that acts as a central control point for expression of several genes encoding pro-inflammatory cytokine production following TLR activation (Medzhitov et al., 1997). NFkB is present in the IEC cytoplasm in an inactive form, in a complex with a member of the inhibitory IkB family, IkBa. Pro-inflammatory signals, including those sent by many PAMPs through TLR binding, lead to phosphorylation, ubiquitination and subsequent proteolysis of IkBa, releasing NFkB. In this free state, NFkB moves into the nucleus, and binds to regulatory control sequences in DNA, activating transcription of genes encoding pro-inflammatory cytokines (Thanos and Maniatis, 1995). Several enteric pathogens such as enteroinvasive E. coli, enteropathogenic E. coli, Salmonella Dublin and Yersinia enterocolitica stimulate the production of pro-inflammatory cytokines by IEC, including IL-8 and TNFa, through NFkB activation (Elewaut et al., 1999; Savkovic et al., 1997). In this way, the IEC response to infection with enteroinvasive foodborne pathogens is coordinated through activation of a common signaling pathway, leading to a pro-inflammatory response.

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