Making connections in insect innate immunity.

I mmune response in the arthropod vector to virus infection is a critical determinant of transmission for arboviruses such as West Nile virus (WNV). The immune response modulates outcomes of infection such as viral load, incubation period required for transmission, and viral pathogenesis in the vector (1, 2). As insects appear to lack an adaptive immune response characteristic of vertebrates, this controlling response occurs through activation of apparently simple, linear innate immunity pathways. However, in PNAS, Paradkar et al. (3) describe a mechanism connecting two of these innate immunity pathways (the RNAi pathway and the Jak-STAT pathway) in mosquitoes through the action of a secreted signaling molecule, Vago, leading to an antiviral state in uninfected, responsive cells. This work provides evidence of an integrated, versatile insect immune system capable of communication between pathways and cells to give rise to an effective response. Innate immunity pathways have been well characterized in Drosophila, and orthologous pathways have been identified in a number of mosquito species (4, 5). These pathways are sufficient to mount protective responses to a variety of pathogens, including bacteria, fungi, and viruses. When considering the antiviral response, the RNAi, Toll, Imd, and JakSTAT pathways have been shown to contribute to innate immunity (reviewed in ref. 4). The RNAi pathway recognizes viral dsRNA that is produced as a result of viral RNA genome replication (6, 7). Through the RNAi pathway, viral dsRNA is cleaved by Dicer-2 and incorporated into the RNA-induced silencing complex, where it is used to bind viral RNA genomes, targeting them for degradation. The RNAi response is essential for controlling virus replication and limiting virusinduced pathology in insects (2, 6), and inherently provides response specificity. Signaling pathways such as the Toll, Imd, and Jak-STAT pathways have also been implicated in the insect antiviral response (8–11). These signaling pathways lead to the activation of transcription factors and the subsequent expression of antimicrobial peptides. Although these innate immune responses are typically described as linear pathways, it has long been assumed that the pathways must communicate to form an integrated and pathogen-specific immune response; however, the mechanisms and molecules that connect the innate immune pathways were to this point unknown. The Jak-STAT pathway has been well studied in mammals and is a complex system that uses multiple ligands, receptors, kinases, and transcription factors. There are four mammalian Jaks that associate with the cytoplasmic domains of a variety of membrane-bound receptors, including IFN and IL receptors (reviewed in ref. 12). The ligands (interferons and cytokines) bind to these receptors, inducing dimerization and bringing the Jaks into close proximity, allowing for transphosphorylation of the kinases as well as the cytoplasmic tails of the receptors. Phosphorylation recruits the STAT proteins; seven STATs are found in mammals (12). The STATs are phosphorylated by Jak and dimerize as homoor heterodimers. Following dimerization, the STATs translocate to the nucleus and activate transcription of antimicrobial peptide genes, among others (12, 13). Combinations of various ligands, receptors, Jaks, and STATs increase the versatility and specificity of the immune response in mammals. In contrast, the insect Jak-STAT pathway is relatively simple (Fig. 1A), possessing three ligands (Upd, Upd2, Upd3), one receptor (Domeless), one Jak (Hopscotch), and one STAT (STAT92E), as characterized in Drosophila (4). The ligands differentially activate the pathway; Upd and Upd2 are involved in developmental processes, whereas Upd3 responds to septic injury and bacterial challenge (14–16). Several genes have been reported to be STAT-responsive following pathogen-induced activation of the pathway. Some of these target genes are associated with phagocytosis (tep1), whereas others have been implicated in the general stress response and hemocyte proliferation (totA and raf, respectively) (14). When specifically considering viral infection, the Jak-STAT pathway has been shown to be activated by and produce an antiviral response to Drosophila C virus, Sindbis virus, WNV, and Dengue virus, among others (10, 17, 18). Virus infection induces a specific STAT-responsive transcription profile, characterized by increased transcription of previously unidentified genes and negligible change in standard pathway targets identified following bacterial infection in Drosophila (10). Specifically, the transcription of the gene vir-1 is highly up-regulated during Drosophila C virus infection of Drosophila, Fig. 1. (A) Characterized pathway for activation of STAT. The Upd3 ligand binds Domeless, leading to recruitment of hopscotch (Jak) and STAT. Phosphorylation of STAT allows nuclear translocation, promoter binding, and activation of transcription. (B) Dicer-2–dependent activation of STAT transcription via Vago signaling. Dicer-2 binds viral dsRNA and, through an unknown mechanism, activates expression of Vago. Processed and secreted Vago binds to an unknown cellular receptor, resulting in Jak-STAT activation and expression of vir-1 and yet-uncharacterized antiviral genes.