Understanding the innate immunity of the mucosal surfaces is crucial to protecting against infectious disease agents and inflammatory immune pathologies that can occur at these surfaces. One poorly understood innate immune mechanism is the generation and regulation of reactive oxygen species (ROS) production by dual oxidases in mucosal tissue. The objective of this application is to identify the components of ROS production and the mechanisms that prevent self-damage. C. elegans will be used because it is an accessible model organism with which we can address these questions. The central hypothesis is that at the site of infection, the intestinal cells generate extracellular ROS via Ce-Duox1 while simultaneously producing antioxidants and heat shock proteins to prevent self-damage. The rationale for the proposed research is that knowledge of the components and mechanisms involved in ROS production in C. elegans will likely be applicable to more complex animals and therefore further understanding of ROS's role in mucosal innate immunity.
Aim #1 will establish the localization of Ce-Duox1 and its ROS generating activity in response to pathogens. Based on the working hypothesis that Ce-Duox1 generates ROS in the intestine, the enzyme will be localized to this site of infection by immunofluorescence and GFP-tagging techniques. By using dyes sensitive to ROS, these species will also be localized to the site of infection.
Aim #2 will identify regulatory mechanisms and other co-factors involved in ROS production. We have established an assay in which we can detect ROS production from C. elegans in response to pathogens. Using RNAi and mutants to examine the loss of specific genes we will investigate the involvement of the p38 MAPK pathway, which has been implicated in our preliminary studies. Other established immune pathways will also be investigated. In addition to these targeted approaches, a forward-genetic screen will be carried out for mutants that exhibit changes in ROS production.
In Aim #3, we will investigate how the host minimizes damage caused by ROS. Antioxidant genes and heat shock proteins identified in preliminary studies by RNAi as having protective roles during infection will be further analyzed. Their putative protective roles will be confirmed by analyzing deletion mutants or transgenics that overexpress the gene-of-interest. They will be localized by GFP-tagging techniques to the site of infection. Finally, by examining lipofuscin accumulation and protein aggregation, these genes'effects on ROS-related damage will be assessed. In conclusion, C. elegans will be used as a model system to answer some important questions about ROS in mucosal immune response. As a result of the proposed investigations the components, regulators, and damage-controlling mechanisms of this response will be identified and localized. The research proposed is significant because knowledge of ROS production by the mucosa will potentially lead to new approaches for modulating this immune response in the treatment of infectious disease and inflammatory conditions in these tissues.
The research proposed in this application will lead to greater understanding of how an immune mechanism associated with mucosal surfaces, as found in the gastrointestinal and respiratory tracts, works. Specifically, using a tiny worm called C. elegans as a model, the mechanisms that generate, regulate and prevent self- damage from the response will be identified. Such knowledge is relevant to public health because it will lead to the potential manipulation of this immune response to the patient's advantage in the treatment of infections and autoimmune disorders associated with the mucosa.
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