In this proposed revision to the parent grant 5R01DK75549-3, entitled """"""""Molecular basis of host-microbiota signaling in the zebrafish gut"""""""", we propose to expand our analysis of intestinal alkaline phosphatase function to mice. Using a zebrafish model, we have demonstrated that IAP functions in an anti-inflammatory innate immune response to prevent excessive intestinal inflammation against the resident microbiota. We propose to test whether the function and regulation of IAP in microbial-host interactions is conserved among vertebrates. Our preliminary studies with IAP deficient mice indicate that this enzyme functions to prevent intestinal inflammatory as it does in zebrafish. We hypothesize that the host intestinal responses to LPS are balanced to provide protection against infection by Gram-negative pathogens while at the same time preventing damage from chronic inflammatory responses to resident gut bacteria. Therefore we predict that inhibition of IAP activity should increase host resistance to a well- characterized Gram-negative intestinal pathogen S. typhimurium (at the cost of increased inflammation). Whereas the normal set point of IAP activity may be selected to optimize the long-term health of the intestine, short-term inhibition of IAP may prove to be an effective prophylactic method to boost resistance to Gram-negative pathogens. We will validate or refute our model by specifically testing the following hypotheses: 1. IAP prevents excessive inflammation in response to bacteria in the mouse intestine. We will measure intestinal inflammation in IAP deficient mice reared in the presence or absence of microbes. 2. IAP is regulated by TLR signaling as part of a primary innate immune response to LPS. We will measure transcriptional regulation of IAP in the presence and absence of 1) bacterial LPS and 2) TLR4 mediate LPS sensing. 3. By detoxifying LPS, IAP reduces intestinal inflammatory responses to Salmonella. We will assay intestinal inflammation in response to S. typhimrium infection in wild type and IAP deficient mice. 4. By detoxifying LPS, IAP renders the intestinal epithelium more susceptible to Salmonella penetration. We will measure S. typhimuium translocation to mesenteric lymph nodes after oral infection of wild type and IAP deficient mice.
Project narrative We propose to study the function of Intestinal Alkaline Phosphatase (IAP) in host- bacterial interactions in the two model vertebrate hosts zebrafish and mouse. What we learn in these systems about how IAP promote tolerance to resident gut bacteria or alter susceptibility to pathogenic bacteria will be applicable to humans and will inform our understanding of infections with enteric pathogens and conditions such as Inflammatory Bowel Diseases.
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| Cheesman, Sarah E; Neal, James T; Mittge, Erika et al. (2011) Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88. Proc Natl Acad Sci U S A 108 Suppl 1:4570-7 |
| Bates, Jennifer M; Akerlund, Janie; Mittge, Erika et al. (2007) Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota. Cell Host Microbe 2:371-82 |
| Baden, Katrina N; Murray, James; Capaldi, Roderick A et al. (2007) Early developmental pathology due to cytochrome c oxidase deficiency is revealed by a new zebrafish model. J Biol Chem 282:34839-49 |
