Molecular genetics of Drosophila immunity

Molecular

genetics of Drosophila Y Tbny Ip and Michael

University

of California

San Diego,

immunity

Levine La Jolla, USA

insects resist bacterial infections through the induction of both cellular and humoral immune responses. The cellular response involves the mobilization of hemocytes, whereas the humoral response utilizes antibacterial peptides that are synthesized in the fat bodies and secreted into the circulating hemolymph. Recent studies suggest that the induction of the humoral response involves Rel-containing regulatory proteins, Dif and dorsal, which are related to mammalian NF-ICB. These regulatory proteins function as sequence-specific transcription factors that induce the expression of immunity genes, including cecropin and diptericin. In mammals, NF-KB has been implicated in both lymphocyte differentiation and the acute-phase response. The finding that insect and mammalian immunity involve related transcription factors offers the promise that genetic studies in Drosophila might lead to the identification of novel components mediating mammalian immunity. Current

Introduction

Opinion

in Genetics

and Development

.

1994, 4~672477

ent antibacterial peptides [16,17]. One of the proteins induced by infection in Hyalophora, hemolin, is a member of the immunoglobuhn superfamily [18]. It has been shown to bind to the surface of infecting bacteria, and a working model is that this binding of hemolin signals the activation of hemocytes and thereby initiates the cellular component of the immune response. Upon activation, the hemocytes are thought to release a ‘cytokine’ that triggers the induction of antibacterial proteins in the fat body [18].

Mammals initially respond to bacterial infection and injury through the induction of the acute-phase response. The first stfp in this process is the activation of macrophages to synthesize cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis Victor [l-3]. These molecules trigger hepatocytes to induce the production of a wide spectrum of ‘protective’ proteins that are secreted into the bloodstream [l-3]. The cytokines also function to mobilize other components of the immune system, including B and T cells [4-6]. IL-1 binds to a transmembrane receptor that triggers a signaling pathway causing the dissociation of nuclear factor (NF)-KB km the inhibitor IKB in the cytoplasm [7-lo]. NF-KB acts along with other transcription factors, such as C/EBP and NF-IL6, to induce the expression of acute-phase proteins in the liver, including the al-acid glycoprotein, C-reactive protein, haptoglobin, and serum amyloid A [1,9.11]. In this review, we consider those aspects of insect immunity that are most similar to the mammalian acute-phase response. Activated hemocytes are thought to participate in signaling the induction of antibacterial peptides in the fat body [12-141. A hallmark study by Boman and colleagues over 20 years ago [15] demonstrated a non-specific acquired immunity in Drosophila; adult flies that were injected with attenuated bacteria were protected against subsequent infections of normally lethal doses of the same or other bacterial strains. Ensuing studies in other insects identified similar phenomena, and the large size of the giant silkmoth Hyalophora cecropia &ilitated the characterization of a number of differ-

Several distinct groups of insect bactericidal peptides are known. Cecropins comprise a family of peptides of 35-39 amino acid residues [12,13,19’,20] that insert into the bacterial cell membrane and ultimately lyse the cells. Diptericins and attacins are characterized by the presence of a weakly conserved glycine-rich sequence motif called the G domain, although the function of this domain is not fully understood [12,13,19*,21]. The defensins are cystein-containing peptides that are structurally similar to charybdotoxin, a potassium channel blocking toxin in scorpions [13]. The cecropins, diptericins, defensins, and attacins all bind distinct components of the bacterial cell wall and membrane and thereby effect synergistic destruction of invading bacteria. Interestingly, a vertebrate cecropin has been identified in pig intestine [22], and vertebrate defensins have been isolated from neutrophils and macrophages [23,24]. Another component of the insect immunity is the activation of phenoloxidase and the clotting of the hemolymph. Phenoloxidase catalyzes the synthesis of melanin, resulting in the encapsulation of invading cells [25,26]. The ph enoloxidase proenzyme is activated by a

Abbreviations Ccc-Cecropin;

672

CIF-cecropia

immunoresponsive LPSlipopolysaccharide;

0. Current

factor;

Biology

D&dorsal-related NF-nuclear

immurtity factor.

Ltd ISSN 0959-437X

factor;

IL-interleukin;

Molecular genetics of Drosophila immunity Ip and Levine

serine protease cascade that is reminiscent of the complement and blood-clotting cascades of vertebrates. After the deposition of melanin, hemocytes form nodules around the surface of invading bacteria, possibly through interactions with hemolin [18]; these nodules are similar to those formed by macrophages in vertebrates. Hemocytes appear to fall into distinct classes, but these have been poorly characterized because of the lack of immunological and biochemical markers. In Drosophila, the best studied hemocytes are the crystal cells and the plasmatocytes. The latter cells differentiate into lamellocytes and podocytes upon stimulation [12,27]. Moledar

characterization

of immunity

genes

Several genes encoding antibacterial peptides have been cloned and characterized in Drosophila melanogaster. The Cecropins (Cecs)were cloned as a result of their homology with the Sanophaga sarcotoxin IA gene [28]. A total of five Ccc genes are present in the 99E region of chromosome 3. CerA 1 and A2 are strongly induced in response to infection or injury during larval and adult stages of the Drosophila life cycle [29], CecB and C are induced during pupal stages, and the fifth gene, Andropin, is structurally unrelated to Ccc and is constitutively expressed only in the ejaculatory duct of adult males [30]. Cross-hybridization was also used to isolate the diptericin gene, using a sequence fi-om the blowfly, Phonnia tarraenouae [21]. The single diptericin gene in Drosophila is induced in larval, pupal, and adult stages of the life cycle [31]. Recently, a Drosophila defensin gene was isolated using amino acid sequence information from the purified peptide [32]. This gene is induced in larval and adult stages, but to a substantially lower level than diptericin. The induction of antibacterial peptides is controlled at the level of transcriptional regulation [19]. Faye and colleagues [33] identified putative NF-KB recognition sequence motifs in the promoter regions of attacin genes in Hyalophora. Similar motifs were also identified in the promoter regions of other immunity genes, including the cecropin genes of Drosophila and Hyalophora [33-351. More recent studies have led to the identification of a cecropia immunoresponsive factor (CIF) that recognizes these tcB-like sequence motifs within crude nuclear extracts fi-om induced fat body preparations in Hyalophora [34,35]. On the basis of their inducibility and similar DNA-binding properties, it has been proposed that CIF might be related to mammalian NF-KB. Recent studies have also identified a t&like protein in the flesh fly Sarcophagaperegrina [36*]. Ccc and diptericin genes are the best characterized immunity genes in Drosophila. The closely linked Cede 7 and A2 genes encode exactly the same antibacterial peptide and display parallel patterns of expression in response to infection or injury [29]. The primary site of Ccc expression is the fat body, although expression is also detected in certain hemocytes [29]. One hemocyte cell line, mbn-2, can be induced to express Ccc upon addition of lipopolysaccharide (LPS) or microbial extracts

673

to the culture medium [37]. Similarly dipteridn is also induced in fat body, hemocyte, and mbn-2 cells [38-l. CetAl and diptericin promoter dissection studies have led to the identification of regulatory sequences that mediate tissue-specific induction in response to infection in transgenic larvae and adults [38**,3p”]. These promoter sequences include t&-like sequence motii; at least one is found in the CedlY promoter and two 17 bp repeats in the diptericin promoter. LPS treatment of mbn-2 cells results in the appearance of a DNAbinding activity, termed Drosophila immunoresponsive &tor (DIF) [39*], that recognizes these KB sequence motifs. UV cross-linking of mbn-2 extracts to the 17 bp KB mot& in the diptericin promoter reveals a DNA-binding protein of about 80 kDa in size [38”] Cloning

of Dif

Since the demonstration that the embryonic patterning gene dorsal is related to the avian oncogene rel [40], a number of r&containing genes have been identified. These include NF-tcB1 @50), NF-KBZ @52), RelA @65), and RelB [41-45]. The original NF-KB binding activity is a heteromeric complex containing the products of the NF-KBl and RelA genes p,8]. Recent studies, however, have shown that a number of different Rel-related proteins can form functional heteromeric complexes [46,47]. The fact that mammalian Rel-related proteins often function as heteromultimers prompted the search for additional rel-containing genes in Drosophila and led to the identification of a single new gene, called dorsal-related immunityfktor (01 [48*]. DiJ maps within 88 kb of the dorsal gene on chromosome 2. Among all the r&related genes, dorsal is most similar to D$ However, the two genes are related. only by virtue of the 300 amino acid rel homology region, and even there they share only about 50% amino acid identity. Dif and dorsal possesssimilar DNA-binding activities; both recognize the ‘oligoB’ sequence f?om the xen promoter with high affinity. Northern assaysindicate that DiJis expressed at very low levels in embryos, suggesting that DiGdorsal heteromeric complexes are not essential for gene regulation during early development. Df mRNA levels become substantial only during late periods of embryogenesis, and peak expression is observed during larval, pupal, and adult stages. The observation that the larval fat body corresponds to a major site of Dif expression has led to the suggestion that it might play a role in the Drosophila immune response.

Several lines of evidence indicate that the insect ht aody is analogous to the mammalian liver; for example, fimctionally related transcription f&tors regulate the expression of the alcohol dehydrogenase gene in the Drosophila fat body and mammalian liver [49-511. Moreover, NFKB has been implicated in the mammalian acute-phase response. Treatment of hepatocytes with the IL-l cytokine induces the nuclear translocation of NF-KFJ, and the promoter regions of a number of acute-phase genes possess t&like sequence motifs [9-111. Similarly, Dif is

.

674

Differentiation

and gene regulation

mainly localized in the cytoplasm of the larval fat body, but rapidly accumulates in nuclei upon infection or injury [48-l. Additional evidence that Dif might play a role in insect immunity is suggested by tissue culture studies. As mentioned earlier, LPS induces the appearance of a KB DNA-binding activity in mbn-2 hemocytes [37]. This binding activity is selectively blocked in crude extracts that are pre-incubated with anti-Dif antiserum [48’], suggesting that @encodes at least one component of the originally defined DIF activity in mbn-2 cells. It is currently unknown whether DIF is actually composed of multiple subunits; morevover, it should be noted that clarification of the role of Dyin immunity awaits genetic studies, including the identification and characterization of D$ mutants.

The role of

dorsalin immunity

Recent studies have implicated both Difand domrl in the immune response. The dorsal gene is maternally expressed and plays an essential role in the dorsoventral patterning of the early embryo [52-541. It is. however, also expressed at later periods of the life cycle, including larval stages [55*], and this later zygotic expression is not essential, in that donut animals derived from dorsal/+ heterozygotes are viable (although survival may be slightly reduced). Immunohistochemical studies have shown that the dorsal protein is present in the cytoplasm of&t bodies and rapidly accumulates in nuclei upon infection [55’]. Induction also results in enhanced levels of dorsal, particularly in adult males. Crude extracts of induced males contain a DNA-binding activity that recognizes the t&like sequence motif in the dipteriti’n promoter; these protein-DNA complexes form a ternary (‘supershift’) complex with anti-dorsal antibodies. Despite this compelling evidence that dorsal participates in immunity, the induction of diptericin expression appears normal in dorsul- mutants [55*]. Perhaps dorsal and Dif function redundantly and the removal of either activity is not sufficient to abolish an immune response. A reasonable, but unproven, view is that the optimal induction of immunity genes involves Dif-dorsal heteromeric complexes.

Signaling

pathways

The nuclear transport of dorsal during early Drosophila embryogenesis is regulated by 11 maternally expressed genes [53,54]. These include components of a cell signaling pathway which is triggered by the binding of a protein ligand, encoded by spiitxle [56], to a transmembrane receptor, Toll [571. An extracellular serine protease cascade is responsible for locally activating the spfzle ligand in ventral regions of early embryos. Interestingly, some of these serine proteases, particularly easter, are related to hemolymph-clotting enzymes in the horseshoe crab, fimulus [58,59]. The binding of the spatzle ligand to the Toll receptor triggers the induction of

pelle, a putative kinase, as welI as tube, a protein of no known homology [60,61]. This results in the dissociation of dorsal ti-om a cytoplasmic inhibitor, cactus [62,63]. Remarkably, a number of these signaling components are related to proteins implicated in the mammalian acute-phase response and lymphocyte differentiation. Not only is dorsal related to NF-KB, but cactus is related to the mammalian cytoplasmic inhibitor IKB (both share a series of related ankyrin repeats) [62,63]. In addition, the intracellular domain of the IL-l receptor is related to a corresponding region in the Toll receptor [64,65]. Current studies are addressing the issue of whether related Toll signaling pathways regulate dorsal activity during embryogenesis and Dif-dorsal activity in immunity. The first suggestion of joint regulation came horn the analysis of the Tolllob mutation, a dominant mutation resulting from a single amino acid substitution that causes the constitutive l&and-independent activation of the receptor [65]. Embryos derived from 7UOb/+ mothers constitutively express dorsal in all nuclei [66,67]. A similar situation is observed for Dif in the fat bodies of uninfected ‘IUOb/+ heterozygous larvae, although as much as half of the total Dif protein remains in the cytoplasm [48’]. Interestingly, such larvae also contain ‘melanotic tumors’ [48*,68,69]. The link between these tumors and constitutive nuclear expression of Dif in fat bodies is unclear. Perhaps hemocytes are somehow ‘activated’ in the absence of infection or injury in these mutants. The nuclear localization of Dif protein is, however, not sutficient to induce the expression of immunity genes such as CecAl and diptericin. Both genes are activated in TolllOb mutants only after infection (YT Ip, J Corbo, M Levine, unpublished data). Constitutive nuclear expression of Dif in TolllOb fit bodies is correlated with a fivefold increase in the steadystate levels of D$rnRNA and protein [48*]. In addition, infection of wild-type strains results in an increase in dorsal levels [55’]. Perhaps Dif and dorsal activate immunity genes as well as their own expression during the course of a normal immune response. Autoregulation has also been implicated in mammalian systems; for example, RelA appears to augment the expression of its inhibitor, IKB [70]. It has been proposed that autoregulation could explain why secondary bacterial infections in Drosophila result in a more robust response [ 151. The initial infection might result in augmented levels of Dif, dorsal, and perhaps other components of the regulatory pathway such as cactus. After this infection is cleared, higher levels of cytoplasm& Dif and dorsal should be found. When these animals are subjected to another infection, the augmented levels of Dif and dorsal might mediate a more efficient induction of immunity genes.

Conclusions

and future

perspectives

Striking parallels can be seen between insect immunity and the mammalian acute-phase response. It is reasonable to anticipate that the detailed characterization of

Molecular

the D&dorsal signaling pathway in Drosophila will be relevant to innate immunity in mammals. Conservation of these signaling pathways may also be found in lymphocyte differentiation. Current studies are aimed towards determining whether Dif and dorsal are subject to the same regulation in immunity that controls dorsal activity in early embryos. It should be noted, however, that it is equally likely that separate or redundant genetic regulatory networks control these disparate processes in Drosophila; for example, recent studies have identified a new transmembrane receptor protein, 18 wheeler [71], that is related to both Toll and the mammalian IL-l receptor. The demonstration that LPS induces the expression of immunity genes in both mbn-2 tissue culture cells and whole organisms (larvae and adults) offers the promise that similar receptors and signaling pathways mediate its activity in both insects and mammals. LPS has been linked to sepsis and various toxic shock syndromes in humans [72,73]. Ultimately, the combination of biochemical studies in mammalian systems and genetic studies in Drosophila should identie many new components in these conserved and important signaling processes.

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YT Ip and M Levine, Department of Biology, Center for Molecular Genetics, Bonner Hall, University of California San Diego, La Jolla, California 92093-0322, USA. YT Ip (present address), Program in Molecular Medicine, University of Massachusetts Medical Center, 373 Plantation Street, Worcester, Massachusetts 01605, USA.

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