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Sarah Steele (left), Alejandro Aballay (right).
To test whether the worms' tendency to seek low oxygen concentrations played a role in their sensitivity, Styer visited Bargmann's lab to use an apparatus developed there: a low-oxygen growth chamber. In a low-oxygen environment, the mutant worms did slightly better, but they still succumbed to P. aeruginosa in greater numbers than did normal worms.
To study the role of the pathogen-avoiding behavior as a factor, the researchers grew both normal and mutant worms on two kinds of culture dishes: dishes completely coated with P. aeruginosa—the worms had nowhere to hide—and dishes that allowed the worms to avoid the pathogen.
When they had nowhere to hide, the mutant worms died in droves, but normal worms persisted. However, normal worms cultured on plates where they couldn't hide from the deadly bacteria died at a higher rate than normal worms cultured on plates that allowed them to avoid P. aeruginosa. This result indicated, according to Aballay, that pathogen avoidance is part of the C. elegans defense response but cannot account for all the differences between normal and mutant worms.
Then the team cultured the worms with the human pathogen Salmonella enterica, which kills worms but does not elicit an avoidance behavior. Mutant worms died in far greater numbers than normal worms. The normal worms weren't avoiding the Salmonella, and they still weren't dying like the mutant worms. “This tells you it's an immune response—with no room for ambiguity,” says Aballay.
With a crippled immune system as one culprit in the mutant worms' weakness, Styer and Steele set out to use the npr-1 mutants to map the biological pathways connecting the worm's nervous and immune systems. The two students measured the activity of thousands of genes in the mutant to detect which ones the npr-1 mutation affected. They confirmed that the NPR-1 protein regulates the expression of several genes that are markers of the innate immune response. Many of those genes are active in intestinal cells, which are in direct contact with pathogens and play a direct role in immunity.
“The results of the genetic studies really surprised me,” says Aballay. “I wasn't expecting that the neural circuit was going to control innate immune response in such a specific way.”
The NPR-1 protein functions as a “brake on a brake” for the immune system. The normal protein inhibits neurons that inhibit the immune system, so it is free to attack invading microbes. But when a mutation wrecks the npr-1 gene, those neurons set the biological equivalent of a parking brake on the immune system; the worm can't protect itself against pathogens. The researchers published their findings October 17, 2008, in Science.
“We have conclusively demonstrated that neurons in C. elegans regulate innate immunity,” says Aballay. Significantly, he says, the genes in the worms' nervous system–immune pathway have direct counterparts in humans, lending broader significance to the findings.
Photos: Steele: Ashley Taufen; Aballay: Jon Gardiner - Duke Photography.