Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the model nematode C. elegans. We previously identified an immunostimulatory xenobiotic, called RPW-24, that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the toxic effects of the compound. We identified seven toxicity suppressors from a screen of approximately 100,000 mutagenized haploid genomes, and found that five of these strains contain loss-of-function mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1 and pmk-1). These data indicate that the toxicity of RPW-24 is directly related to its stimulatory effects on the p38 MAPK pathway. We therefore predicted that a genetic screen aimed at identifying endogenous activators of the p38 MAP kinase pathway would uncover mutants that phenocopy the addition of RPW-24 to wild-type animals. We screened approximately 170,000 haploid genomes for dominant mutations that activated the p38 MAPK-dependent pathogen response gene (F08G5.6::GFP) and identified a single allele, which had a gain-of-function (gof) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK PMK-1. We found that the nsy-1(gof) allele had greater levels of phosphorylated p38 MAPK, caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. As was the case in wild-type worms that were exposed to RPW-24, a nsy-1(gof) mutation was toxic to developing animals, a phenotype that could be suppressed through RNAi-mediated knockdown of the nsy-1 gene. Thus, a gain-of-function mutation in nsy-1 and xenobiotic-induced hyperactivation of the p38 MAPK pathway each drive immune responses that are protective during infection, but are toxic to nematodes under normal growth conditions. These data suggest that there are physiological mechanisms that regulate the p38 MAPK pathway to ensure cellular homeostasis in C. elegans.
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