The Toll pathway is important for an antiviral response in <em>Drosophila</em>

Abstract

The innate immune system plays an important role in immune responses against multiple pathogens in various species. In mammalian systems, the innate immune system provides the first line of defense against pathogens before activation of acquired immune responses. In insects, the entire immune system is innate and has been shown to respond to bacteria, fungi, parasites, and, as our results show, viruses. Because of the striking homology between the Drosophila and mammalian innate immune systems, one example being the Toll pathway, Drosophila has become the model system of choice for the study of innate immune responses.

Because of the nonvariable nature of innate immune responses, activation primarily occurs by recognition of distinct pathogen-associated molecular patterns (PAMPs), which are shared by multiple pathogens (1). In mammalian systems, multiple Toll-like receptors (TLRs) have been found that activate the immune response via detection of a range of PAMPs including: lipopoly-saccharide (TLR4), lipoproteins (TLR2), dsRNA (TLR3), flagellin (TLR5), CpG DNA (TLR9), and various antiviral compounds (TLR7) (2, 3). In addition to these PAMPs, TLRs can also be activated by recognition of “self” patterns normally present inside of cells such as heat shock proteins and uric acid (4, 5). When activated, these TLRs are involved in the expression of inflammatory cytokines and costimulatory molecules that activate the adaptive immune system (3). In contrast, of the 10 TLRs identified in Drosophila melanogaster, only one has been definitively identified as playing a role in innate immunity. Additionally, unlike the limited PAMP sets recognized by each mammalian TLR, this one Drosophila Toll is able to respond to bacterial, fungal, and viral infections (6). Drosophila mounts an immune response against these pathogens through the use of both humoral and cellular responses. The identified humoral response in Drosophila consists primarily of the Toll pathway and the IMD pathway, which regulate antimicrobial peptide (AMP) expression in the fat body, a flattened tissue in the fly abdomen that is functionally equivalent to the mammalian liver (7, 8).

The Toll pathway is activated by Gram ^{bacterial and fungal infections via binding of PAMPs to peptidoglycan receptor proteins (PGRPs) (-SA,-SD) and Gram binding proteins (}1, 3, 9, 10). This binding initiates a serine protease cascade that cleaves Spätzle, the ligand of the Toll transmembrane receptor protein (10, 11). Once this cleaved form of Spätzle is bound, Toll signaling directs the phosphorylation and degradation of Cactus, an IκB-like protein that inhibits the NF-κB like transcription factors Dorsal and Dif (12). Destruction of Cactus allows translocation of these transcription factors to the nucleus, causing a rapid increase in expression of multiple AMPs (11-13). The Toll pathway also plays an important role in both maternal effect embryonic patterning and larval hematopoiesis (14, 15). This hematopoietic developmental effect may be significant for immune responses as hemocytes mediate nearly all cellular immune responses, including phagocytosis, melanization, and encapsulation, and also signal the fat body to initiate AMP production during infection.

The IMD pathway is activated in a similar fashion to the Toll pathway. It is believed that the IMD pathway is triggered by interaction between the Gram ^{bacteria PAMP diaminopimelic acid peptidoglycan and the PGRP-LC transmembrane receptor (}10, 16). Imd, a death domain adaptor protein with significant similarities to the mammalian Receptor Interaction Protein, is then recruited by and binds to dFadd (17, 18). dFadd interacts with the caspase Dredd, which in turn associates with and is thought to cleave phosphorylated Relish, a bipartite NF-κB-type transcription factor (18-20). Relish is phosphorylated by the Drosophila IκB kinase complex, which is activated by the mitogen-activated protein kinase kinase kinase Tak1 in an Imd-dependent manner (21-24). The cleaved N-terminal domain of Relish then translocates to the nucleus, where it regulates the transcription of various immune response-related genes (20).

Inactivation of either these pathways results in increased susceptibility to select microorganisms. Inactivation of the Toll pathway, for example, eliminates induction of the antifungal peptide Drosomycin and increases susceptibility to fungal and Gram ^{infections. However, these flies are able to induce the antibacterial peptide} Diptericin normally and can resist Gram ^{infections (}12, 25-27).

In addition to bacterial and fungal pathogens, multiple viruses that infect Drosophila have been identified. Drosophila C virus, for instance, has been studied, and its pathogenesis has been examined in depth (28). Another virus, Drosophila X virus (DXV), is a member of the Birnavirus family and has an icosahedral nucleocapsid and bisegmented dsRNA genome (Fig. 5, which is published as supporting information on the PNAS web site). Despite extensive research into DXV's genome, many of its pathological effects in Drosophila have yet to be thoroughly defined (29, 30). Infection was shown to induce anoxia sensitivity and eventual death, but the specific cause was unknown (30).

In our studies, we have developed several assays to identify mutant lines with altered sensitivity to DXV infection. Additionally, we find that the Toll pathway is an essential component of viral resistance in flies. Dif¹ mutants, which do not have a functional Toll pathway, develop higher DXV titers and succumb to death by anoxia more rapidly then WT flies. Tl^10b, a Toll gain-of-function mutant, succumbs to similar early onset death but has a reduced DXV titer. These results provide an example of an identified Drosophila innate immune related pathway playing a role in viral susceptibility. Our results suggest that the Toll pathway is able to reduce replication of DXV and possibly other viral pathogens. Further characterization of this pathway in relation to viral resistance should yield insight into this branch of the innate immune response in Drosophila.

Acknowledgments

Notes

Author contributions: R.A.Z. and L.P.W. designed research; R.A.Z. performed research; R.A.Z. and V.N.V. contributed new reagents/analytic tools; R.A.Z. analyzed data; R.A.Z. wrote the paper; and M.N. screened flies and collected data.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PAMP, pathogen-associated molecular patterns; AMP, antimicrobial peptide; DXV, Drosophila X virus; dpi, days postinfection.