NF-��B in the Immune Response of Drosophila

The nuclear factor κB (NF-κB) pathways play a major role in Drosophila host defense. Two recognition and signaling cascades control this immune response. The Toll pathway is activated by Gram-positive bacteria and by fungi, whereas the immune deficiency (Imd) pathway responds to Gram-negative bacterial infection. The basic mechanisms of recognition of these various types of microbial infections by the adult fly are now globally understood. Even though some elements are missing in the intracellular pathways, numerous proteins and interactions have been identified. In this article, we present a general picture of the immune functions of NF-κB in Drosophila with all the partners involved in recognition and in the signaling cascades.The paramount roles of NF-κB family members in Drosophila development and host defense are now relatively well established and have been the subject of several in-depth reviews in recent years, including some from this laboratory (e.g., Hoffmann 2003; Minakhina and Steward 2006; Ferrandon et al. 2007; Lemaitre and Hoffmann 2007; Aggarwal and Silverman 2008). To avoid excessive duplication, we limit this text to the general picture that has evolved over nearly two decades—since the initial demonstration that the dorsal gene plays a role in dorsoventral patterning in embryogenesis of Drosophila and that it encodes a member of the NF-κB family of inducible transactivators (Nüsslein-Volhard et al. 1980; Steward 1987; Roth et al. 1989). In the early nineties, it became apparent that NF-κB also plays a role in the antimicrobial host defense of Drosophila (Engström et al. 1993; Ip et al. 1993; Kappler et al. 1993; Reichhart et al. 1993). We focus in this article on the immune functions of NF-κB and refer the reader to recent reviews for the roles of NF-κB in development (Roth 2003; Brennan and Anderson 2004; Moussian and Roth 2005; Minakhina and Steward 2006).The Drosophila genome codes for three NF-κB family members (Fig. 1). Dorsal and DIF (for dorsal-related immunity factor) are 70 kDa proteins, with a typical Rel homology domain, which is 45% identical to that of the mammalian counterparts c-Rel, Rel A, and Rel B. Dorsal and DIF lie some 10 kbp apart on the second chromosome and probably arose from a recent duplication (Meng et al. 1999). Both proteins are retained in the cytoplasm by binding to the same 54-kDa inhibitor protein Cactus, which is homologous to mammalian IκBs (Schüpbach and Wieshaus 1989; Geisler et al. 1992). The single Drosophila Cactus gene is closest to mammalian IκBα (Huguet et al. 1997). The third member of the family in Drosophila, Relish, is a 100-kDa protein with an amino-terminal Rel domain and a carboxy-terminal extension with typical ankyrin repeats, as found in Cactus and mammalian IκBs. Relish is similar to mammalian p100 and p105 and its activation requires proteolytic cleavage as in the case for these mammalian counterparts (reviewed in Hultmark 2003).Open in a separate windowFigure 1.The NF-κB and IκB proteins in Drosophila. The length in amino acids is indicated by numbers. REL, Rel-homology domain; NLS, nuclear localization sequence; PEST, proline, glutamic acid, serine, and threonine-rich segment; Ac, acidic domain.Put in simple terms, NF-κB family members function in the host defense of Drosophila to control the expression of genes encoding immune-responsive peptides and proteins. Prominent among the induced genes are those encoding peptides with direct antimicrobial activity. To exert this function, Dorsal and DIF are translocated to the nucleus following stimulus-induced degradation of the inhibitor Cactus, whereas Relish requires stimulus-induced proteolytic cleavage for nuclear translocation of its amino-terminal Rel domain. This paradigm is similar to that observed in mammalian immunity. Again, for the sake of simplicity, we may say that the stimulus-induced degradation of Cactus, and the concomitant release of Dorsal or DIF, is primarily observed during Gram-positive bacterial and fungal infections and mediated by the Toll signaling pathway. In contrast, stimulus-induced proteolytic cleavage of Relish, and concomitant nuclear translocation of its amino-terminal Rel domain, is the hallmark of the response to Gram-negative bacterial infection and mediated by the Imd signaling pathway. Whether these pathways are also involved in the multifaceted defense against viruses remains an open question (Zambon et al. 2005). The Toll pathway was further shown to be involved in hematopoiesis of flies (Qiu et al. 1998). Of note, the Cactus-NF-κB module also plays a central role in the elimination of Plasmodium parasites in infected mosquitoes (Frolet et al. 2006). In the following, we review our information of the two established signaling pathways, Toll and Imd, which lead to gene reprogramming through NF-κB in response to bacterial and fungal infections. We first consider the upstream mechanisms that mediate the recognition of infection and allow for a certain level of discrimination between invading microorganisms. Gene reprogramming in this context is best illustrated by the induction of the antimicrobial peptide genes, which serve as the most convenient readouts of the antimicrobial defense of Drosophila (see Samakovlis et al. 1990; Reichhart et al. 1992; Ferrandon et al. 1998). Flies produce at least seven families of mostly cationic, small-sized, membrane-active peptides, with spectra variously directed against Gram-positive (defensins) and Gram-negative (diptericins, attacins, and drosocin) bacteria, and against fungi (drosomycins and metchnikowins), or with overlapping spectra (cecropins) (reviewed in Bulet et al. 1999; Hetru et al. 2003). The primary site of biosynthesis of these peptides is the fat body, a functional equivalent of the mammalian liver. Blood cells also participate in the production of antimicrobial peptides. As a rule, these molecules are secreted into the hemolymph where they reach remarkably high concentrations to oppose invading microorganisms (Hetru et al. 2003). This facet of the antimicrobial host defense is generally referred to as systemic immune response. Of note, the gut and the tracheae also produce antimicrobial peptides in response to microbes (see Tzou et al. 2000; Onfelt Tingvall et al. 2001; Liehl et al. 2006; Nehme et al. 2007).During infection, the Toll and Imd pathways control the expression of hundreds of genes. In addition to the antimicrobial peptides, these genes encode proteases, putative cytokines, cytoskeletal proteins, and many peptides and proteins whose function in the host defense are still not understood (De Gregorio et al. 2001; Irving et al. 2001).     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Tumor Necrosis Factor �� Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-��B

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.     

The Nuclear Factor NF-��B Pathway in Inflammation

The nuclear factor NF-κB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-κB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway. NF-κB has long been considered the “holy grail” as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease. In this article, we discuss the role of NF-κB in inflammation in light of these recent studies.NF-κB has long been considered a prototypical proinflammatory signaling pathway, largely based on the activation of NF-κB by proinflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor α (TNFα), and the role of NF-κB in the expression of other proinflammatory genes including cytokines, chemokines, and adhesion molecules, which has been extensively reviewed elsewhere. But inflammation is a complex physiological process and the role of NF-κB in the inflammatory response cannot be extrapolated from in vitro studies. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB pathway in inflammation.