citations

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

Authors:

Alper S, Laws R, Lackford B, Boyd WA, Dunlap P, Freedman JH, Schwartz DA

In:

Source: Proc Natl Acad Sci USA
Publication Date: (2008)
Issue: 105(19): 7016-21

Research Area:

Immunotherapy / Hematology

Cells used in publication:

J774A.1: Species: mouse; Tissue Origin: ascites

Platform:

4D-Nucleofector® 96-well Systems

Experiment

To test the role of genes identified in th C. elegans screens on macrophage immune response, siRNAs (Dharmacon; pools of four siRNA duplexes per gene) were transfected into mouse macrophage cell line J774A.1 by using an Amaxa Nucleofector 96-well shuttle. Transfections were carried out with 2 µM siRNA and 100,000 cells per well. 24 to 36 h after transfection, ultrapure E. coli was added to a final concentration of 20 ng/ml. After 5 h, supernatant was collected, and cytokine production was assayed. Cell viability was monitored, and cell number was normalized by using fluorescin diacetate; cytokine production was normalized relative to a negative control siRNA. siRNAs were initially tested in triplicate; siRNAs that prevented IL-6 production were then assayed at least three more times.

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.

Open in PubMed