Identification of innate immunity genes and pathways using a comparative genomics approach

Abstract

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.Caenorhabditis elegans ͉ macrophage

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

“…We identified several genes on chromosome I that were required for robust clec-85 expression in the presence of the nonpathogenic bacterium E. coli. Follow-up studies using RNAi in mouse macrophages, and knockouts in C. elegans and mice validated many of these genes as novel regulators of innate immunity (Alper et al 2008;De Arras et al 2012. This demonstrated the utility of this comparative genomics approach for innate immune regulator gene discovery.…”

“…elegans chromosome II-IV RNAi screen to identify regulators of clec-85::gfp production RNAi was performed in liquid culture in 96-well format as described previously (Alper et al 2007(Alper et al , 2008 using the Ahringer laboratory E. coli RNAi feeding library (Kamath and Ahringer 2003;. In brief, dsRNAproducing bacteria were inoculated from frozen stocks into LB, grown overnight at 37°, dsRNA synthesis was induced using IPTG, the bacteria were centrifuged and resuspended in nematode growth medium, nematode eggs isolated by bleaching (Wood 1988) were added to the wells, and the nematodes were allowed to grow at 25°i n the presence of the dsRNA producing bacteria for 3 days.…”

“…RNAi was performed largely as described previously (Alper et al 2008). Briefly, either pools of siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into the mouse macrophage cell line RAW264.7 using the Amaxa 96-well nucleofector shuttle.…”

“…clec-85 encodes a C-type lectin whose expression is induced by the bacterial pathogen Serratia marcescens (Mallo et al 2002). clec-85 expression is regulated by several known innate immunity signaling pathways in C. elegans including the p38 MAPK, insulin signaling, and TGFb signaling pathways in the presence of either pathogenic Pseudomonas aeruginosa or nonpathogenic Escherichia coli (Alper et al 2007;Alper et al 2008). We identified several genes on chromosome I that were required for robust clec-85 expression in the presence of the nonpathogenic bacterium E. coli.…”

“…We previously used an RNAi screen to target all of the genes on C. elegans chromosome I to identify genes that regulate expression of a clec-85::gfp fusion in transgenic nematodes (Alper et al 2008). clec-85 encodes a C-type lectin whose expression is induced by the bacterial pathogen Serratia marcescens (Mallo et al 2002).…”