L. PNEUMOPHILA AND LEGIONNAIRES' DISEASE

Legionella pneumophila is a Gram-negative facultative intracellular pathogen capable of growing in both protozoan and mammalian host cells. L. pneumophila is found in natural and artificial water reservoirs and less often in soil and organic matter (Fields, 1996; Szymanska et al., 2004). Optimal proliferation conditions for Legionella are those in which water temperatures are between 25°C and 42°C, calcium and magnesium salt-containing sediments are present, and are further enhanced by the presence of algae and protozoa (Szymanska et al., 2004). In hostile conditions, Legionella and other organisms become attached to surfaces in an aquatic environment, forming a biofilm (Langmark et al., 2005). L. pneumophila can be isolated from such natural water sources as lakes, ponds and streams; however, artificial reservoirs such as plumbing fixtures, hot water tanks, whirlpool spas and cooling towers, all possess excellent conditions for Legionella proliferation inside protozoan hosts and are the source of most outbreaks (Fliermans et al., 1981; Yee and Wadowsky, 1982).

The first recognized outbreak of L. pneumophila occurred in Philadelphia in 1976 during a state convention of the American Legion (Fraser et al., 1977). During this outbreak a total of 221 people contracted the disease, 34 of whom subsequently died. A new Gram-negative bacterium was isolated from both patients and the air-conditioning system of the hotel that was the source of the outbreak (McDade et al., 1977). This isolated organism was named Legionella pneumophila (Brenner et al., 1979). There are 48 different species of Legionella found in nature.

INTRODUCTION

Pathogenic bacteria have evolved several strategies to gain access across epithelial surfaces particularly those lining the mucosae. After their epithelial transcytosis bacteria find a first line of immune defense represented by professional phagocytes, including macrophages and dendritic cells. These cells are particularly apt at bacterial uptake, killing and processing for the initiation/maintenance of adaptive immune responses. Furthermore, intracellular bacteria can induce by epithelial cells the release of inflammatory mediators and cytokines that will recruit other immune cells, particularly neutrophils. Dendritic cells are not simply passive players waiting for possible invaders, they can actively participate to bacterial sampling by intercalating between epithelial cells. This mechanism is not restricted to pathogenic bacteria. Since gut dendritic cells have been thoroughly studied, in this chapter we will focus on dendritic cells located in the intestinal mucosa and on their role in the uptake and handling of luminal bacteria.

THE ANATOMY OF THE INTESTINAL MUCOSAL EPITHELIUM AND THE GUT ASSOCIATED LYMPHOID TISSUE (GALT)

The intestinal epithelium is the first line of defense toward dangerous microorganisms. It opposes a physical, electric and chemical barrier against luminal bacteria. The permeability of the barrier is regulated by the presence of both tight junctions (TJ) between epithelial cells (ECs) and a negatively charged mucous glycocalix. TJ seal adjacent ECs to one another and regulate solute and ion flux between cells. The glycocalix sets the size of macromolecules that can reach the apical membrane of ECs and opposes an electric barrier to bacteria.

DENDRITIC CELLS PRIME ANTI-BACTERIAL CD4+ AND CD8+ T CELLS IN VIVO

It is now widely accepted that dendritic cells (DCs) are crucially required for the priming of T cell responses. Major histo-compatibility complex (MHC) class I and class II presentation pathways ensure the priming of CD8+ and CD4+ T cell, respectively. They thus represent major checkpoints for the induction of adaptative protective immunity toward intracellular bacteria. In this chapter, we will focus on the basic cell biology and physiological regulation of these pathways in the context of bacterial infection. In accordance with the literature, we will refer to “cross presentation” for MHC class I pathways involved in the presentation of non cytosolic antigens.

Listeria, Mycobacteria and Salmonella were shown to actually infect DCs in situ. Some studies have addressed the capacity of DCs purified from infected animals to activate in vitro T cells specific for bacteria-encoded antigens. Intravenous infection of mice with Salmonella and with Mycobacterium bovis BCG (bacillus Calmette-Guérin) leads to the infection of both spleen DC subsets (CD8α− CD11c+ and CD8α+ CD11c+). Both subsets display some MHC class I and II complexes formed after the processing of bacteria-encoded antigens. Bacterial infection may also promote apoptosis, resulting in the delivery of bacterial antigens to DCs upon the phagocytosis of infected apoptotic bodies (see Section 3.5).

Whatever may be the mechanism (infection or dead cells cross presentation), the absolute requirement of DCs to induce anti-bacterial T cell priming was elegantly demonstrated in the Listeria model.

INTRODUCTION

Toll-like receptors (TLRs) play essential roles in innate immune responses. The name TLR is derived from a Drosophila protein, Toll, which detects fungal infection in the fruit fly. The immune system in Drosophila is entirely dependent on a limited number of germline-encoded receptors for pathogen recognition. In contrast, the vertebrate immune system is characterized by the evolution of acquired immunity in addition to innate immunity. Acquired immunity is mediated by T and B cells, which utilize rearranged receptors. This system is advantageous for detecting pathogens with high specificity, eradicating infection in the late stages and establishing an immunological memory. However, the mammalian innate immune system plays critical roles in the initial defense against invading pathogens and subsequent activation of the acquired immune system. Innate immune cells, such as macrophages and dendritic cells (DCs), sense pathogens through TLRs, phagocytose them and evoke immune responses.

To date, 12 different TLRs have been reported in either humans or mice. The innate immunity system targets a set of molecular structures that are unique to microorganisms and shared by various pathogens, but absent from host cells. By recognizing these “pathogen-specific” patterns, the innate immunity system is able to prevent autoimmune responses. Members of the TLR family of proteins are characterized by extracellular leucine-rich repeat (LRR) motifs responsible for ligand recognition, a transmembrane region and a cytoplasmic tail containing a Toll/IL-1 receptor homology (TIR) domain.

The gut represents the largest lymphoid tissue of the whole body. The delicate task of the intestinal immune system is the discrimination of harmless food antigens and the commensal bacterial flora from harmful pathogens. Under normal physiologic conditions, immune tolerance is induced to non-pathogenic stimuli while effective immune responses are generated toward dangerous pathogens. Thus “decision making” is an important feature of the intestinal immune system. If inappropriate responses are generated, serious inflammation of the small and large intestine may develop. Crohn's disease (CD) and ulcerative colitis are the two prototypes of such inflammatory bowel disease that are believed to develop as a consequence of a disregulated immune response toward harmless antigens. Despite our limited knowledge on the mechanisms of such “decision making” in the gut, recent evidence suggest an important role of intestinal dendritic cells. Dendritic cells (DCs) can be found in large numbers throughout the gastrointestinal tract where they usually build a tight network underlying the epithelium. This chapter will discuss their contribution to the induction of tolerance and immunity in the intestinal immune system as well as a possible role of these DCs in localized immune responses predisposing the terminal ileum for the development of inflammatory bowel disease (IBD).

DENDRITIC CELLS IN THE INTESTINAL IMMUNE SYSTEM: AN OVERVIEW

The intestinal immune system can be functionally separated into an inductive site and an effector site. The prototypic inductive site in the small intestine is the Peyer's patch, a localized lymphoid structure placed within the bowel wall.

INTRODUCTION

Immunity to a bacterial pathogen requires the generation of bacteria-specific T cells with appropriate effector function. Eliciting T cells during infection requires internalization of the bacteria and processing of bacterial proteins to generate peptides for presentation on major histocompatibility complex (MHC) class I (MHC-I) and/or MHC class II (MHC-II) molecules, depending on the pathogen. As not all host cells have the capacity to phagocytose bacteria, and not all bacterial pathogens have the capacity to actively invade non-phagocytic cells, phagocytic antigen presenting cells, macrophages and immature dendritic cells (DCs), are the key players in generating adaptive immunity to bacteria.

Both macrophages and immature dendritic cells can present antigens from the bacteria they internalize on their own MHC-I and MHC-II molecules and thus carry out so-called direct presentation of bacterial antigens (Sundquist et al., 2004; Harding et al., 2003). Direct presentation of bacterial antigens on MHC-II is the expected outcome following phagocytosis of bacteria and is the event necessary to elicit CD4+ T cells. However, both macrophages and DCs can also present antigens from internalized bacteria on MHC-I, molecules most renowned for their presentation of peptides derived from endogenously synthesized proteins (Rock and Goldberg, 1999), and generate CD8+ T cells (Sundquist et al., 2004; Harding et al., 2003).

Given the capacity of both macrophages and DCs to directly present bacterial antigens on MHC-I and MHC-II, these cells in principle could initiate adaptive immunity during primary infection. However, it is only DCs that have this ability (Banchereau and Steinman, 1998).

Natural killer (NK) cells represent a distinct lymphoid population characterized by unique phenotypic and functional features. NK cells were originally identified on a functional basis as this denomination was assigned to lymphoid cells capable of lysing tumor cell lines in the absence of prior stimulation in vivo or in vitro. Both their origin and the mechanism(s) mediating their function remained mysterious until recently. Regarding their origin, it has been shown that NK cells derive from a precursor common to T cells and expressing the CD34+CD7+ phenotype. In addition, functional NK cells can be obtained in vitro and in vivo from (CD34+) haematopoietic precursors isolated from several different sources. The cell maturation in vitro has been shown to require appropriate feeder cells and/or IL-15. The molecular mechanisms underlying the ability of NK cells to discriminate between normal and tumor cells, predicted by the “missing self hypothesis”. have been clarified only during the past decade. It has been shown that NK cells recognize MHC-class I molecules through surface receptors delivering inhibitory, rather than activating, signals. Accordingly, NK cells lyse target cells that have lost (or express low amounts of) MHC class I molecules. This event occurs frequently in tumors or in cells infected by some viruses such as certain herpesviruses or adenoviruses. In addition to providing a first line of defence against viruses, NK cells release various cytokines and chemokines.

INTRODUCTION

Innate immunity is an ancient and highly conserved system that provides the first line of defense upon encounter with pathogenic organisms. Activation of innate immune responses is a complex process involving multiple components and distinct steps. The cellular components of innate immunity include neutrophils, monocytes, macrophages and dendritic cells (DCs). These cells are capable of direct microbicidal activity that partially depends on inducible nitric synthase (iNOS) and NADPH oxidase complex that catalyze production of toxic anti-microbial compounds. Additionally, they secrete a vast array of pro-inflammatory mediators such as cytokines and chemokines and can recruit and activate other inflammatory cells, thus amplifying the immune cascade. Apart from their role in restricting microbial growth, innate immune responses also provide the inflammatory context in which adaptive T- and B-cell immune responses develop.

Dendritic cells are derived from hematopoietic progenitor cells in the bone marrow and are found in the peripheral circulation as well as in the lymphoid and non-lymphoid tissues. Dendritic cells can be subdivided into several subsets based on the expression of the cell surface markers and different subsets have been ascribed distinct functions during the immune response. Since their discovery, dendritic cells have been studied extensively with regard to their role as antigen-presenting cells. However, it is becoming increasingly clear that dendritic cells also play an important role during the innate immune responses to microbial pathogens.

INTRODUCTION

Antigen-presenting cells (APC), such as dendritic cells (DCs) and macrophages, are located throughout the body to sense and capture invading pathogens and to trigger immune responses to fight such invaders. In addition, in the absence of danger signals, DCs have an active role in the induction of T cell tolerance and the maintenance of homeostasis. The recognition and internalization of pathogens is mediated by so-called pathogen-recognition receptors, germ-line encoded cell surface receptors that include toll-like receptors (TLR) and C-type lectins (CLR). It is becoming increasingly clear that during the long co-evolution with their hosts, pathogens have evolved mechanisms to misuse pathogen-recognition receptors to suppress or evade immune responses and thus to escape clearance. In this chapter, we will review recent examples of how pathogens evade immune activation by targeting recognition receptors on APC and subverting their function.

BACTERIAL RECEPTORS ON ANTIGEN-PRESENTING CELLS

APC interact with invading pathogens via pathogen-recognition receptors that bind conserved patterns of carbohydrates, lipids, proteins and nucleic acids in classes of microbes. This variety of receptors and conserved ligands recognized ensures that most, if not all, microbes can be detected by the immune system, either by a single or by combinations of receptors. Pathogen-recognition receptors include TLR and CLR (Figure 9.1). To date, 11 TLR have been identified (see Chapter 2) that each targets specific pathogenic structures, such as lipopolysaccharide (TLR4), viral dsRNA (TLR3) and bacterial peptidoglycans (TLR2/TLR6).

DENDRITIC CELL SUBPOPULATIONS

Dendritic cells (DCs) have an essential function in the immune system by participating in primitive defense responses that constitute the innate immunity, as well as in the induction and regulation of antigen-specific immune responses. This allows DCs to control infections caused by parasitic and microbial pathogens, to block tumour growth and to exert a precise regulation of T cell, B cell and NK cell immune responses. In addition, DCs also fulfill a pivotal role in the induction and maintenance of T cell tolerance. The functional diversity characterizing the DC system relies essentially on the remarkable plasticity of the DC differentiation process, which dictates the acquisition of DC functional specialization through the generation of a large collection of DC subpopulations (reviewed by Shortman and Liu, 2002). Dendritic cells are located both in the lymphoid organs (such as the spleen or the lymph nodes), and in non-lymphoid tissues (such as the skin or the liver), and can be classified in two major categories: conventional DCs (cDCs), and plasmacytoid DCs (pDCs). Whereas in turn cDCs comprise multiple DC subpopulations endowed with specific functions, little is known about the functional heterogeneity of pDCs. A summary of the most relevant phenotypic and functional characteristics of the main DC subpopulations present in mice is shown in Table 1.1.

A first group of cDCs includes those that are common, and largely restricted, to the majority of organized lymphoid organs of the immune system, and perform their specific functions, as immature or mature DCs, within these organs.

INTRODUCTION

Infection with pathogenic bacteria can result in acute or chronic disease, which can be life threatening, especially in young, elderly or other immunocompromised individuals. Humans are also infected with a wide range of commensal bacteria, as part of our normal gut flora, and the immune system must be capable of controlling immune responses against these beneficial bacteria, while at the same time generating effector immune responses against pathogenic micro-organisms. In addition, pathogenic bacteria have evolved strategies for delaying or preventing their elimination by evading or subverting protective immune responses of the host.

Innate immunity to bacteria

The initial inflammatory response to pathogenic bacteria involves the release of cytokines and chemokines and the recruitment of neutrophils, monocytes, dendritic cells (DCs) and lymphocytes to the site of infection. Tissue macrophages and neutrophils quickly phagocytose and attempt to kill the bacteria. Macrophages and DCs are activated through binding of conserved, secreted or cell surface bacterial products to pathogen recognition receptors (PRR). This leads to activation of immune response genes, including those coding for inflammatory cytokines, chemokines and co-stimulatory molecules expressed on the surface of DCs and macrophages, that are involved in antigen presentation (Janeway and Medzhitov, 2002).

Bacteria are phagocytosed by neutrophils and macrophages and this is facilitated through activation of the alternative complement pathway by bacterial cell wall components, resulting in the production of C3b, which together with antibodies help to opsonize the bacteria.

Dendritic cells (DCs) comprise a family of professional antigen presenting cells that are unique in their ability to activate T lymphocytes. Dendritic cells patrol all the tissues at the interface with the external world, including skin and mucosal surfaces, for the presence of invaders. The DC system is characterized by a remarkable plasticity that allows the induction both of immunity and tolerance toward the encountered antigens. This is achieved through the combination of a number of different factors, including the subsets of DCs, their activation state and environmental cells that can regulate DC function. DCs are present in the periphery in an immature form that is particularly apt at capturing antigens and at deciphering the messages associated therein. After an activation stimulus that is delivered by some antigens (including bacteria) or by inflammatory cytokines released during inflammation, activated DCs acquire a migratory phenotype and reach the draining lymph node. Here, DCs present the antigens captured in the periphery and initiate T cell adaptive immune responses.

This book describes how the intimate interplay between dendritic cells, bacteria and the environment dictates the induction of immunity or tolerance to bacteria and how pathogenic bacteria have evolved strategies to escape DC patrolling. The first section introduces the complexity of the DC system describing the different subpopulations of DCs and their role in the induction of immune responses.

Introduction

The Plasmodiophoromycota are a group of obligate (i.e. biotrophic) parasites. The best-known examples attack higher plants, causing economically significant diseases such as club-root of brassicas (Plasmodiophora brassicae), powdery scab of potato (Spongospora subterranea; formerly S. subterranea f. sp. subterranea) and crook-root disease of watercress (S. nasturtii; formerly S. subterranea f. sp. nasturtii). In addition to damaging crops directly, some species (S. subterranea, Polymyxa betae, P. graminis) also act as vectors for important plant viruses (Adams, 1991; Campbell, 1996). Other species infect roots and shoots of non-cultivated plants, especially aquatic plants. Algae, diatoms and Oomycota are also attacked. If the nine species of Haptoglossa, which parasitize nematodes and rotifers, are included in the Plasmodiophoromycota, the phylum currently comprises 12 genera and 51 species (Dick, 2001a). Genera are separated from each other largely by the arrangement of resting spores in the host cell (Waterhouse, 1973). This feature has also been used for naming most genera; for instance, in Polymyxa, numerous resting spores are contained within each sorus, whereas in Spongospora the resting spores are grouped loosely in a sponge-like sorus (Fig. 3.6). Accounts of the Plasmodiophoromycota have been given by Sparrow (1960), Karling (1968), Dylewski (1990) and Braselton (1995, 2001).

Taxonomic considerations

Plasmodiophoromycota have traditionally been studied by mycologists and plant pathologists. Many general features of their biology and epidemiology are similar to those of certain members of the Chytridiomycota such as Olpidium (see p. 145).