Dietary saturated fatty acids (FAs), such as palmitic acid (PA), play a major role in inflammation- associated disease. The mechanisms by which PA produces pro-inflammatory conditions through innate immunity are well-documented, but the pathways through which it acts on the adaptive immune system to produce disease are less clear. PA has been shown to act on dendritic cells (DCs), the immune cells that coordinate adaptive immune responses by regulating T cell activity. However, the molecular mechanisms by which PA activates DCs to influence downstream adaptive immune responses are unknown. This gap in knowledge prevents an understanding of immune cell biology that could provide new molecular targets for therapeutic drug discovery for diseases with inflammation-based etiology such as lupus and osteoarthritis. Thus, our long-term goal is to understand the molecular mechanisms underlying DC function for inflammatory diseases in order to identify therapeutic targets. The overall objective of this pre-doctoral research project is to determine the role of PA in regulating Toll-like receptor 4 (TLR4) signaling in DCs. Our central hypothesis is that PA stimulates DCs by inducing TLR4 signals and regulates these signals through ROS. This hypothesis was formulated based in part on the published work of others and on preliminary data and will be objectively tested through the following specific aims: (1) Determine whether PA binds TLR4 and its adapter protein MD-2;(2) Characterize PA induced DC activation;and (3) Evaluate PA induced ROS regulation of TLR4 signaling in DCs.
In Specific Aim 1, we will use isothermal titration calorimetry, fluorescence binding assays, and atomic force microscopy to determine the dissociation constant and thermodynamic parameters of PA interaction with TLR4/MD-2.
In Specific Aim 2, flow cytometry, RT-PCR, and siRNA knockdown will be used to characterize PA impact on co-stimulatory factor production, cytokine secretion, and T cell stimulation by DCs.
In Specific Aim 3, Immunofluorescence miscroscopy, and novel fluorescent H2O2 probes will be used to assess PA induced H2O2 production in DCs. Electrophoretic mobility shift assay (EMSA), western blot, and siRNA knockdown will be used to assess the roles that ROS play in TLR4 signaling in DCs. The approach is innovative, because it focuses on a new paradigm for the role H2O2 has in normal DC biology, and especially because it will employ application of recently developed fluorescent H2O2 probes and the development of a ternary binding model for TLR/MD-2 and a ligand. The proposed research is significant because, it will, for the first time, define a mechanistic role for H2O2 in the regulation of TLR4 signaling in DCs and define PA as a TLR4 ligand. Once H2O2 mechanisms are defined, targeted therapeutics such as ROS scavengers, anti-oxidants, and signaling inhibitors can be combined with dietary interventions to achieve maximum health benefits.
Dietary saturated fatty acids such as palmitic acid (PA) stimulate inflammation. The proposed research is relevant to public health because it will identify mechanisms underlying the impact of PA on dendritic cells, the immune cells that coordinate adaptive immune responses. Improved understanding of these mechanisms is highly likely to provide new therapeutic targets for many diseases by defining lipid induced-inflammation in disease etiology.