Identification of lipid mediators using Drosophila to dissect function and role in inflammatory disease

Watson, Mark (2014). Identification of lipid mediators using Drosophila to dissect function and role in inflammatory disease. University of Birmingham. Ph.D.

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Abstract

Inflammation is widely accepted as an initiator, and involved in the exacerbation of many diseases. Key modulators of inflammatory responses are oxygenated metabolites of polyunsaturated fatty acids (PUFAs). There are two main classes of PUFAs, Omega-3 (n-3) and Omega-6 (n-6). Arachidonic acid, a derivative of omega-6 is known to produce pro-inflammatory mediators known as prostaglandins (C20, n-6) via cyclooxygenase (COX). COX (a member of the myeloperoxidase (MPO) family) is a major therapeutic target of intervention in inflammatory disease. We investigated whether Drosophila could be used to model and dissect new novel targets of inflammation. Using an inflammatory model based on a JAK temperature sensitive, gain-of-function mutation (hop\(^T\)\(^u\)\(^m\)), we have characterised lipid mediator-signaling pathways in Drosophila. Dietary supplementation with PUFAs was able to modulate inflammation, with omega-6 fatty acids increasing, and omega-3 fatty acids reducing, the severity of the hop\(^T\)\(^u\)\(^m\) inflammatory phenotype. While COX and MPO inhibitors were able to reduce inflammation, LC-MS analysis of Drosophila extracts failed to detect the existence of prostaglandins or their precursor, arachidonic acid (C20). Interestingly, however, we have been able to detect C18 oxygenated metabolites known as hydroxy-octadecadienoic acids (HODEs). HODEs are generated by the oxidation of omega-6 (C18, n-6) fatty acid, linoleic acid. Chiral analysis via LC-MS indicated a higher proportion of the enzymatic chiral form is generated of both 9- and 13-HODE in Drosophila samples. Members of the MPO family are able to produce HODEs. We have identified 3 candidate Drosophila COX/MPO homologues: Pxn, Pxt and Irc that may generate HODE inflammatory lipid mediators. We have shown them to have both COX and MPO activity in vitro. Deletions of these genes reduced the inflammatory phenotype in the hop\(^T\)\(^u\)\(^m\) assay. Functional characterization using deletions of these genes showed affects on longevity as well as survival upon infection. We show that Drosophila could be used to investigate 9- and 13-HODE inflammatory effects in diseases such as atherosclerosis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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