To combat infection, the fruit fly Drosophila melanogaster relies on multiple innate defense reactions, many of which are shared with higher organisms. These reactions include the use of physical barriers together with local and systemic immune responses. First, epithelia, such as those beneath the cuticle, in the alimentary tract, and in tracheae, act both as a physical barrier and local defense against pathogens by producing antimicrobial peptides and reactive oxygen species. Second, specialized hemocytes participate in phagocytosis and encapsulation of foreign intruders in the hemolymph. Finally, the fat body, a functional equivalent of the mammalian liver, produces humoral response molecules including antimicrobial peptides. Here we review our current knowledge of the molecular mechanisms underlying Drosophila defense reactions together with strategies evolved by pathogens to evade them.

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The Host Defense of Drosophila melanogaster

The mammalian Rel/NF-κB family of transcription factors, including RelA, c-Rel, RelB, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100), plays a central role in the immune system by regulating several processes ranging from the development and survival of lymphocytes and lymphoid organs to the control of immune responses and malignant transformation. The five members of the NF-κB family are normally kept inactive in the cytoplasm by interaction with inhibitors called IκBs or the unprocessed forms of NF-κB1 and NF-κB2. A wide variety of signals emanating from antigen receptors, pattern-recognition receptors, receptors for the members of TNF and IL-1 cytokine families, and others induce differential activation of NF-κB heterodimers. Although work over the past two decades has shed significant light on the regulation of NF-κB transcription factors and their functions, much progress has been made in the past two years revealing new insights into the regulation and functions of NF-κB...

Regulation and Function of NF-κB Transcription Factors in the Immune System

Reactive Oxygen Species Promote TNFα-Induced Death and Sustained JNK Activation by Inhibiting MAP Kinase Phosphatases

Osteoimmunology is an interdisciplinary research field focused on the molecular understanding of the interplay between the immune and skeletal systems. Although osteoimmunology started with the study of the immune regulation of osteoclasts, its scope has been extended to encompass a wide range of molecular and cellular interactions, including those between osteoblasts and osteoclasts, lymphocytes and osteoclasts, and osteoblasts and haematopoietic cells. Therefore, the two systems should be understood to be integrated and operating in the context of the 'osteoimmune' system, a heuristic concept that provides not only a framework for obtaining new insights by basic research, but also a scientific basis for the discovery of novel treatments for diseases related to both systems.

Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems

The molecular understanding of osteoclast differentiation.

Transcription factor, nuclear factor kappaB (NF-kappaB), is required for osteoclast formation in vivo and mice lacking both of the NF-kappaB p50 and p52 proteins are osteopetrotic. Here we address the relative roles of the two catalytic subunits of the IkappaB kinase (IKK) complex that mediate NF-kappaB activation, IKKalpha and IKKbeta, in osteoclast formation and inflammation-induced bone loss. Our findings point out the importance of the IKKbeta subunit as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Although IKKalpha is required for RANK ligand-induced osteoclast formation in vitro, it is not needed in vivo. However, IKKbeta is required for osteoclastogenesis in vitro and in vivo. IKKbeta also protects osteoclasts and their progenitors from tumor necrosis factor alpha-induced apoptosis, and its loss in hematopoietic cells prevents inflammation-induced bone loss.

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IκB kinase (IKK)β, but not IKKα, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss

Multicellular organisms defend themselves from microbial infections by mounting an innate immune response that serves to neutralize pathogenic invaders (Kimbrell and Beutler 2001; Janeway and Medzhitov 2002). Receptors that recognize pathogen-associated molecular patterns (PAMPs) trigger signaling cascades that culminate in expression of immune effector genes encoding antimicrobial peptides, cytokines, chemokines, and various inflammatory mediators. In both vertebrates and invertebrates, the NF-κB family of transcription factors serves a pivotal role in signal-induced gene activation during the innate immune response (Silverman and Maniatis 2001).

Owing to the broad evolutionary conservation of innate immunity, studies in genetically tractable invertebrates, especially Drosophila melanogaster, have contributed greatly to our understanding of the mechanisms underlying innate immunity. Drosophila, like other invertebrates, lacks an adaptive immune system, being entirely dependent on innate immunity for its resistance to microbial infection. Genetic screens for Drosophila mutants susceptible to pathogenic challenge have identified two distinct signaling cascades—the Toll and Imd pathways—that govern expression of antimicrobial peptides (Hoffmann and Reichhart 2002; Tzou et al. 2002; Hultmark 2003).

The Toll pathway, originally characterized for its role in establishing embryonic dorsoventral polarity, mediates resistance to fungal and Gram-positive infections by activating two NF-κB proteins: Dorsal and Dif (Wasserman 2000). The Imd pathway induces expression of a distinct set of genes coding for antimicrobial peptides, such as Diptericin and Attacin, that provide immunity to Gram-negative bacteria. Components of the Imd pathway include a peptidoglycan recognition protein (PGRP-LC; Choe et al. 2002; Gottar et al. 2002; Ramet et al. 2002), as well as several counterparts to signal transducers involved in NF-κB activation in mammalian cells. These include IMD itself, a homolog of the TNF signaling pathway kinase RIP (Georgel et al. 2001), as well as TAK1, a MAP kinase kinase kinase (MAPKKK), and two IκB kinase (IKK) complex components, IKKβ (ird5), and IKKγ (Kenny; Rutschmann et al. 2000; Silverman et al. 2000, 2003; Lu et al. 2001; Vidal et al. 2001). In conjunction with the IKK complex, the Drosophila caspase Dredd functions in proteolytic activation of a third NF-κB family protein, Relish, that directly activates antibacterial peptide genes (Leulier et al. 2000; Stoven et al. 2000, 2003). There is also evidence that the Drosophila FADD homolog acts downstream of IMD, independently of TAK1 and IKK, to control Dredd activation (Leulier et al. 2002; Naitza et al. 2002).

Although Relish, Dif, and Dorsal are critical for transcriptional induction of antimicrobial peptides, they are unlikely to be the sole mediators of PAMP-triggered signal transduction. In mammalian macrophages and dendritic cells, Toll-like receptors (TLRs) specific to particular PAMP ligands induce activation of ERK, JNK, and p38 MAP kinases (MAPKs) in addition to NF-κB (Akira et al. 2001). Furthermore, the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana encode no orthologs of NF-κB, but rely instead on MAPK signaling cascades to mediate innate immune responses (Asai et al. 2002; Kim et al. 2002; Mallo et al. 2002).

JNK signaling is required for a variety of biological processes in flies and mammals, including developmental morphogenesis, apoptosis, and immunity (Stronach and Perrimon 1999; Chang and Karin 2001). In mammals, JNK activation is required for inflammation-induced apoptosis (Deng et al. 2003; Maeda et al. 2003). Curiously, JNK activation is negatively regulated by the IKK-NF-κB pathway (De Smaele et al. 2001; Tang et al. 2001). Although this cross-talk appears to be an important mediator of the antiapoptotic function of NF-κB (Karin and Lin 2002), the mechanism by which NF-κB negatively regulates JNK activation in mammals is not well understood (Kyriakis 2001). In Drosophila, lipopolysaccharide (LPS) has been reported to activate JNK (Sluss et al. 1996), which is required for induction of a subset of immune response genes (Boutros et al. 2002), as well as the NF-κB family member Relish, which directs transcription of a distinct group of immune response loci (Boutros et al. 2002). However, it has been difficult to assess the biological role of the JNK module in the innate immune response because loss-of-function mutations in this pathway are usually embryonic lethal (Glise et al. 1995; Riesgo-Escovar et al. 1996; Sluss et al. 1996).

We have found that activation of the Relish module in Drosophila results in attenuation of JNK activity after challenge with a commercial LPS preparation. Moreover, we demonstrate that Relish activation is mechanistically linked to switching off the JNK signaling module after LPS stimulation. In the absence of Relish activation, LPS treatment causes a sustained activation of JNK, which, in turn, leads to prolonged expression of JNK target genes. Biochemical experiments reveal that the negative regulation of JNK signaling involves proteasomal degradation of TAK1. Hence, it appears that the Relish-dependent genes not only serve their own immune effector roles, but also participate in shaping the temporal pattern of the JNK-mediated immune response. These findings reveal an unanticipated cross-talk between Drosophila innate immune pathways and hold promise for mechanistic insight into the mammalian counterpart of this regulatory interaction.

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Targeting of TAK1 by the NF-κB protein Relish regulates the JNK-mediated immune response in Drosophila

Transcription factor NF-kappaB has been well recognized as a pivotal player in osteclastogenesis and inflammation-induced bone loss. Here, we discuss our recent results obtained using a genetic approach in mice that indicate the importance of IKKbeta, and not IKKalpha, as a transducer of signals from receptor activator of NF-kappaB (RANK) to NF-kappaB. Ablation of IKKbeta results in lack of osteoclastogenesis and unresponsiveness of IKKbeta-deficient mice to inflammation-induced bone loss. In the need of a more effective therapy for the treatment of inflammatory diseases causing bone resorption, specific inhibition of IKKbeta represents a logical alternative strategy to the current therapies.

Maria Grazia Ruocco

Papers

IκB kinase (IKK)β, but not IKKα, is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss

Targeting of TAK1 by the NF-κB protein Relish regulates the JNK-mediated immune response in Drosophila

Control of Osteoclast Activity and Bone Loss by IKK Subunits: New Targets for Therapy