Dissecting Signaling Networks that Control Innate Immune Responses in Drosophila
Zusammenfassung der Projektergebnisse
The innate immune system is the predominant system of host defense in most organisms. It needs to be tightly regulated, as over-activation can result in chronic inflammatory diseases and cancer. Due to the evolutionary conservation of most central signaling networks, well-established model organisms like Drosophila play a key role in dissecting cellular processes involved in innate immune responses. Genetic screens have identified two signaling pathways that regulate NF-kB-dependent immune gene transcription after microbial challenge in Drosophila: the Toll and immune deficiency (Imd) pathways. Nevertheless, significant gaps remained in our understanding of how the activation of intracellular signaling elicits specific transcriptional programs. Here, we developed and optimized cell-based assays targeting different branches of immune signaling. RNAi screens identified several novel candidates of the Toll and Imd signaling pathways. We studied selected candidates in depth in vitro as well as in vivo using genetic and molecular approaches, epistasis analyses and infection models. For example, GTPase-activating protein 1 (GAP1) was identified as a negative regulator of the Imd pathway. GAP1 is a regulator of the RAS/MAPK pathway, and we identified 8 further components of this pathway in a large-scale RNAi screen. Based on these results, we established a model, where RAS/MAPK signaling limits the immune response in the presence of microbial challenge, but also prevents spurious immune induction in the absence of infection by controlling the transcription of an Imd inhibitor. We also identified the well-conserved gene Akirin as being a component of the Imd pathway. Epistasis analyses placed it downstream of Imd acting in parallel with the NF-kB transcription factor. Other candidates whose function were characterized in detail, were for example Inhibitor of Apoptosis Protein 2 (IAP2), which we placed downstream of Imd, but upstream of NF-kB and Deformed Epidermal Autoregulatory Factor 1 (Deaf1), which we identified as a component of the Toll pathway that acts downstream of the NF-kB factors. The systematic RNAi screening approaches resulted in new insights into the function of the innate immune system and into the crosstalk between different signaling pathways. Because of the evolutionary conservation of these signaling pathways, our research also contributes to a better understanding of mammalian immune responses.
Projektbezogene Publikationen (Auswahl)
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(2005). An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Rep. 6:979-84
Gesellchen V, Kuttenkeuler D, Steckel M, Pelte N, Boutros M
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(2005). E-RNAi: a web application to design optimized RNAi constructs. Nucleic Acids Res. 33:W582-8
Arziman Z, Horn T, Boutros M
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(2008). Akirins are highly conserved nuclear proteins required for NF-kappaB-dependent gene expression in drosophila and mice. Nat Immunol. 9:97-104
Goto A, Matsushita K, Gesellchen V, El Chamy L, Kuttenkeuler D, Takeuchi O, Hoffmann JA, Akira S, Boutros M, Reichhart JM
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(2008). RNAi screening in cultured Drosophila cells. Methods Mol Biol. 420:139-53
Steinbrink S, Boutros M
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(2008). The art and design of genetic screens: RNA interference. Nat Rev Genet. 9:554-66
Boutros M, Ahringer J
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(2010). A large-scale RNAi screen identifies Deaf1 as a regulator of innate immune responses in Drosophila. J Innate Immun. 2:181-94
Kuttenkeuler D, Pelte N, Ragab A, Gesellchen V, Schneider L, Blass C, Axelsson E, Huber W, Boutros M
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(2010). E-RNAi: a web application for the multi-species design of RNAi reagents--2010 update. Nucleic Acids Res. 38:W332-9
Horn T, Boutros M
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(2011). Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells. EMBO J. 30:1123-36
Ragab A, Buechling T, Gesellchen V, Spirohn K, Boettcher AL, Boutros M
