Epithelial immunity the local immune response

The systemic immune response has been studied through the direct introduction of pathogens into the body cavity. This approach has limited the study of the immune response to the steps involved in recognition and antimicrobial response in the fat body. While these studies have revealed insights on the molecular basis of the Drosophila immune response, they may not reflect the most common mode of host interaction with potential pathogens. In metazoans, the epithelia of the digestive, respiratory, and genital tracts are constantly exposed to microbes of both indigenous and environmental origin. Thus, these routes have the potential to be the major routes of infection for a host. The next section of this chapter will focus on the immune epithelial response.

2.3.1 A gut-associated immune system

The systemic mode of infection bypasses the layered steps of a given host-microbe interaction by directly activating the immune response. The mere presence of a microbial elicitor is sufficient to induce an immune response. However, the circulatory system of animals is generally sterile, which is not the case for epithelia, especially that of the digestive tract that is frequently associated with an indigenous microbiota. In contrast to the systemic response, epithelia must tolerate the presence of some microbes while responding to potential pathogens. This implies a tight and specific regulation of the immune response in epi-thelia, balancing between immune activation and bacterial tolerance. Physical attributes of the host and host epithelial environment are one factor that can prevent either colonization or immune activation, thereby reducing the number of microbes that truly interact with the host. For this reason, only a handful of bacteria have been described as being infectious to Drosophila via oral ingestion (Serratia marcescens, Enterococcus faecalis, Vibrio cholerae, Erwinia carotovora, Pseudomonas entomophila) (Basset et al, 2000; Vodovar et al, 2005; Nehme et al, 2007; reviewed in Vallet-Gely et al., 2008). These bacteria are capable of persisting in the gut and/or inducing a local immune response (Figure 2.1). Among them, E. carotovora is able to induce a strong local and systemic immune response, but differs in that infection does not kill the host (Basset et al., 2000). The use of these bacteria has demonstrated that two complementary effector mechanisms are key in controlling bacterial persistence and infection in the gut: generation of ROS and local production of AMPs. ROS production

In Drosophila, oral ingestion of bacteria induces rapid ROS synthesis in the gut by a NADPH oxidase enzyme, called dDuox. Adult flies in which dDuox expression is silenced by RNA interference (RNAi) show a marked increase in mortality following ingestion of microbe-contaminated food (Ha et al., 2005a). Ingested bacteria were shown to persist and proliferate throughout the intestinal tract of dDuox RNAi flies. To maintain the homeo-static redox balance perturbed by the ingestion of microbes, wild-type flies also express an antioxi-dant system composed of an extracellular immune-regulated catalase (IRC) (Ha et al, 2005a, 2005b). This ROS-dependent gut immunity is not affected by the Imd pathway and provides an initial barrier against ingested microbes. However, it has been shown that ROS-resistant bacteria are still controlled by local AMP expression (Ryu et al., 2006). Local AMP expression

As observed in systemic infection, several AMP genes under the control of the Imd pathway (e.g. Diptericin and Attacin) are expressed in the digestive tract upon oral infection by Gram-negative bacteria (Tzou et al., 2000). This local AMP production is critical in the host response to control oral infection by Gram-negative bacteria; flies lacking a functional Imd pathway in the gut are more susceptible upon oral infection with P. entomophila and S. marcescens (Liehl et al, 2006; Nehme et al, 2007). The relevance of the local production of AMP is also revealed by the strategy of some entomopathogens that produce abundant extracellular metalloproteases that are capable of degrading AMPs (Liehl et al, 2006).

Similarly, as in the systemic response, the local immune response is inducible and triggered by the recognition of Gram-negative peptidoglycan by PGRP-LC activating the Imd pathway (Zaidman-Remy et al., 2006). AMP genes are also expressed in specific domains along the digestive tract (Figure 2.1), revealing that the gut is a complex and compartmentalized organ with distinct immuno-reactive domains (Senger et al, 2006; Buchon et al, 2009). Additionally, transcription factors involved in defining cell identity, like the homeobox gene caudal, have been identified in controlling AMP expression in the gut. Caudal represses the expression of AMP in the posterior part of the midgut despite the presence of a functional Imd pathway in this location (Ryu et al, 2008). It was proposed that the suppression of AMP by Caudal is essential for the establishment of a beneficial microbiota in this segment of the gut (see below).

2.3.2 Gut microbiota and bacterial tolerance

Host ingestion of microbes typically results in benign interactions. Examination of gut-associated microbes of animals has demonstrated tolerance to a diverse and complex consortium of bacteria. Molecular analysis of the Drosophila-associated microbiota has revealed a relatively low species diversity, dominated by Acetobacter, Gluconobacter, and Lactobacillus spp. (Corby-Harris et al., 2007; Cox and Gilmore, 2007; Ren et al., 2007; Ryu et al., 2008). The constant presence of this microbiota raises the question as to why they do not generate a state of permanent immune activation in the gut and why this microbiota is not eliminated by the gut immune defence. This topic is also discussed in Chapter 7 in this volume. Host factors that scavenge peptidoglycan and promote microbiota tolerance

A central role in bacterial tolerance of the gut has been attributed to amidase PGRPs, as they are proposed to scavenge peptidoglycan released by gut microbes (Bischoff et al, 2006; Zaidman-Remy et al., 2006). Resident bacteria may have a low rate of growth in the gut and therefore would release only low amounts of peptidoglycan that can be readily hydrolysed by amidase PGRPs, whereas infectious bacteria release large amounts of peptidoglycan while proliferating (Zaidman-Remy et al., 2006). This implies a threshold response for local immune activation to differentiate between indigenous micro-organisms and invading pathogens. This is supported by experiments showing that RNAi extinction of amidase-encoding genes PGRP-SC1/2 or PGRP-LB, induces higher Diptericin expression after oral bacterial infection compared to wild-type flies (Bischoff et al, 2006; Zaidman-Remy et al., 2006).

Thus, amidase PGRPs downregulate the immune response and modulate the immune reactivity of the fly. Reduction of the immune response by amidase PGRPs may also prevent damage to host tissues from prolonged immune activity as demonstrated by increased lethality and developmental defects of PGRP-SC1/2 RNAi larvae orally infected with bacteria (Bischoff et al, 2006). In addition to amidase PGRPs, negative regulators of the Imd pathway, such as Pirk, also prevent activation of the Imd pathway by the gut microbiota, thus promoting tolerance (Lhocine et al, 2008). Host transcription factors that promote the establishment of the gut microbiota However, despite this role of amidase PGRPs, the microbiota still activates the Imd pathway as reflected by the permanent nuclear translocation of Relish along the gut. The homeobox transcription factor Caudal has been shown to downregulate AMP expression at the transcriptional level in the posterior part of the midgut. This appears to be critical for the establishment of a normal gut microbiota. RNAi inhibition of caudal leads to increased expression of antimicrobial peptides in the posterior part of the gut and long-term mortality. This increased mortality and loss of gut immune regulation is associated with an imbalance of the gut microbiota (Ryu et al., 2008). Both the lack of Caudal or artificial over-expression of AMPs increased the representation of an AMP-resistant minor constituent of the microbiota, resulting in the observed pathology. This study identified one mode of interaction between the microbiota and host immunity and suggests a complex interplay between the host, gut micro-biota, and innate immune response.

2.3.3 Other epithelial responses

Use of Drosophila green fluorescent protein (GFP) reporter genes has revealed that all epithelia in communication with the external environment (e.g. gut) express a subset of AMPs (Ferrandon et al, 1998; Tzou et al, 2000). Expression of AMPs in epithelia is inducible and regulated by the Imd pathway. In some tissues, AMPs are constitu-tively expressed and in this case their expression is thought to be governed by developmental genes such as Caudal (Ryu et al., 2008; reviewed by Uvell and Engstrom, 2007).

In Drosophila, tracheae are formed by invaginations of the ectoderm and thus, are lined by a cuticular intima that is continuous with the external cuticle. Tracheae are likely to be exposed constantly to microbes, although little is known about pathogens that can infect this tissue or the specifics of tracheal immune defense (Wagner et al., 2008). Natural infection with Gram-negative bacteria such as Erwinia carotovora induces the expression of AMPs in the trachea (Tzou et al., 2000). Interestingly, the antifungal peptide gene Drosomycin is induced in the trachea by the Imd pathway in contrast to the regulation observed during the systemic response. Additionally, infection induces local activation of the phenoloxidase cascade leading to melanization that may confine the spread of infection (Tang et al, 2008).

Another epithelium that risks injury and infection is the genital epithelia of females following copulation. In order to prevent infection, many AMPs are expressed in this tissue. This is the case for Drosomycin which is constitutively expressed in spermathecae, receptacles that store spermatozo-ids (Ferrandon et al, 1998). Interestingly, it has been shown that sex peptide, a short peptide present in the sperm of males, induces local AMP expression in females (Peng et al., 2005). This induction appears to be independent of any microbial elicit-ors and may limit the entry of potential infectious agents just after copulation (see also Chapter 15 in this volume).

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