NEW YORK (GenomeWeb News) – New research is illustrating the usefulness of RNA interference assays and comparative genomics for identifying genes involved in immunity across species.
In a paper appearing in the Proceedings of the National Academy of Sciences this week, researchers from the National Institutes of Health and Duke University used RNAi assays in a Caenorhabditis elegans and cell culture to identify dozens of genes suspected to be involved in innate immunity. They then pinpointed 11 genes that contribute to immunity in both systems, mapping their products onto a protein interaction network. The team further validated four of these in vivo in C. elegans mutants.
Because the genes are conserved in these systems, researchers say, they may also function in the human immune system, offering new therapeutic targets.
The innate immune system acts non-specifically to prevent infection, destroying viral and bacterial interlopers and damaged host cells. As such, it provides short-term defense, but not long-term, adaptive protection. Although it is crucial for host immunity, the innate immune system can become hyperactive, contributing to immunological diseases such as asthma, atherosclerosis, and sepsis.
In an effort to better understand this system, lead author Scott Alper, a geneticist affiliated with the National Heart, Lung, and Blood Institute and Duke University, and his colleagues used RNAi screening to identify genes that affect innate immunity in several different organisms — starting with the worm model C. elegans.
First, they went gene by gene, looking at the effect of knocking down each of the 2,416 genes on chromosome 1. The C. elegans model system is particularly well-suited for this sort of experiment, Alper explained, because it’s possible to feed the worms with Escherichia coli containing specific RNA molecules from a library.
Although C. elegans lack many of the components associated with immune systems in higher organisms such as humans, Alper said, they do express antimicrobial proteins in response to potential pathogens. The researchers exploited this property to gauge C. elegans immune responses, fusing a green fluorescent coding gene to the promoter of an antimicrobial protein called clec-85.
About 1.3 percent of the chromosome 1 genes tested — 32 of more than 2,400 — affected clec-85 expression.
Next, the researchers used a similar RNAi approach to individually knock down nearly 200 candidate genes related to immune function or stress response in various organisms in the published literature. Not surprisingly, Alper said, this approach provided a “much higher yield” of potential immune-related genes per gene tested: 25 of the 192 genes were required for complete expression of the clec-85-GFP.
When they mapped the gene products of the 32 genes identified with the chromosome 1 screen and the 25 genes from the subsequent candidate gene screen onto a C. elegans interactome database, the researchers found several different networks containing at least one of their proteins of interest.
One network contained four proteins identified in the screen — and each of these interacted with an enzyme called SIAH-1, which regulates the activity of the transcription factor NF-kappa-B. Consequently, the researchers suspected that other genes/gene products in this network might be part of the innate immune system too.
Again, they used RNAi to test this, inhibiting two genes — siah-1 and cri-3 — in the network. As expected, when either gene was inhibited, clec-85-GFP expression decreased.
Similarly, the researchers used siRNA in mouse macrophage cultures, individually knocking down 27 of the 59 immune genes identified in C. elegans and then assessing whether this affected cytokine production when the cells were exposed to E. coli lipopolysaccharides. Using this approach, they identified 11 genes that affect immune response in both C. elegans and murine macrophages.
They further validated four of these genes in vivo in mutant C. elegans strains that were exposed to E. coli or the pathogenic bacterium Pseudomonas aeruginosa.
Still, it’s too early to say which, if any, of the genes are involved in immune system signaling and which carry out downstream immune system processes. As well, Alper noted, there may be instances in which genes influence immune function by affecting overall viability.
In the future, Alper said, they plan to use available mouse models to investigate more of the candidate immune genes they detected. They also plan to look at whether any of the genes identified might play a role in human immunity. For example, he said, it may be possible to identify polymorphisms in their genes of interest that are associated with altered immune function.
The researchers also noted that the newly identified genes may be potential therapeutic targets, particularly for diseases related to aberrant innate immunity, such as asthma.
“Because the genes and pathways identified in this study exhibit evolutionarily conserved functions in several model systems, they are likely to be important mediators of the immune response and could be potential therapeutic targets for inflammatory disease,” they wrote.