Dietary fatty acids modulate Th1 mediated immunity within the context of inflammatory disease

Abstract

Introduction: Polyunsaturated fatty acids (PUFA) are lipids with immunomodulatory effects. There are a wide variety of studies which have demonstrated the anti-inflammatory effects of fish derived n-3 PUFA, EPA and DHA and beef/dairy derived Conjugated linoleic acid (CLA). However, the exact mechanisms through which these PUFA exert there effects are not fully understood. Therefore the main objective of this thesis was to examine the molecular mechanisms through which PUFA exert there actions in relation to inflammatory bowel disease (IBD) and sepsis. Methods: A range of methods were used to examine the molecular effects associated with IBD and sepsis. Serum, colonic and supernatant cytokines were examined using ELISA. Protein levels and interactions were examined using Western blot and immunoprecipitation. Transcription factor binding assays were carried out to determine DNA binding affinity. mRNA levels were examined using RT-PCR. Results: Results demonstrate that in the intestinal epithelial cell line, Caco-2, the precursor fatty acid TVA does not exert the anti-inflammatory effects associated with CLA. While some anti-inflammatory effects were observed it is thought that this was due to the bioconversion of TVA to c9, t11-CLA. Results from Chapter 4 demonstrated that natural beef derived CLA has the potential to reduce systemic cytokine driven inflammation associated with LPS. In addition, DC isolated from animals fed beef-CLA demonstrated reduced expression of key molecules involved in the process of inflammation. Importantly, it was shown that CLA reduced production and mRNA expression of the LPS receptor, TLR4. Additionally results demonstrated an increase in PPARγ production, this is a transcription factor which is widely associated with anti-inflammatory effects in many disease states. Experiments carried out in Chapter 5 showed the effects of a low-CLA and high-CLA beef diet on mice challenged with DSS, an agent known to induce colitis. Both diet groups developed a mild form of colitis however clinical and biochemical analysis failed to find any differential effects between the two groups. Experiment detailed in Chapter 6 aimed to demonstrate the molecular mechanisms of the n-3 PUFA, EPA and DHA in DC. Results showed that both of these fatty acids had the ability to decrease both mRNA production and supernatant cytokine levels in DC. Key transcription factors known to influence the inflammatory process were analysed. Results demonstrated that the key pro-inflammatory transcription factor NF-κB was down regulated in both EPA and DHA however, further experiments demonstrated that only DHA had the ability to reduce NF-κB:DNA binding affinity. Analysis of PPARγ, a key transcription factor known to have anti-inflammatory effects showed increased production particularly in DHA treated cells. Furthermore immunoprecipitation experiments showed that these anit-inflammatory effects may be due to a physical interaction with NF-κB following activation by PUFA. Conclusion: Overall the studies described in this thesis demonstrate that naturally derived CLA and n-3 PUFA have potential for use as functional food. Importantly results show that CLA may be more applicable for certain diseases such as septic shock rather that IBD. This may reflect mechanisms of action which are more appropriate for different disease states.