Abstract
In atherosclerosis disease, macrophages present intra-plaque antigens in the form of peptides on major histocompatibility (MHC) class II molecules to T lymphocytes. Macrophages stimulated by low density lipoprotein (LDL) may present differential peptide profiles on MHC complex molecules resulting in an altered immune response. The main aim of this study was to stimulate macrophages with LDL in vitro, to induce expression of novel patterns of MHC-associated peptides to be subsequently identified by LC-MS analysis. Then, to complete the study, the aim was to elucidate the affect of these identified peptides on macrophage function. To achieve this, J774. 2 mouse macrophages were treated with human native and oxidized LDL. Peptides were extracted from macrophage MHC complexes using citrate-phosphate buffer (pH 3, 4 and 5). Samples were fractionated and then analyzed by LC/MS/MS. MASCOT analysis, IEDB and NetMHCII 1.0 bioinformatics platforms were used to predict peptide-binding affinity to MHC complexes. We also aimed to evaluate the effect of novel identified peptides (Pep4 & Pep5) on macrophages and ox-LDL functions using flow cytometry analysis. In addition, the metabolite profiles derived from macrophage pre-treated with ox-LDL and n-LDL were assessed. Results showed that 135 peptides from 100 proteins with 27 overlapping proteins and 40 overlapping peptides among the different groups were identified. Based on predictive MHC binding analysis algorithms, 22 of these peptides were found to be MHC class I peptides and 24 were MHC class II peptides. Importantly, 66.1% of these peptides had high promiscuity values. Flow cytometry analysis showed a clear inhibition in the expression of TLR-2, TLR-4 and CD 14 by macrophages pre-treated with ox-LDL and then with Pep3, Pep4 and Pep5. Moreover, different metabolite profiles were induced by each condition, and different types of triglyceride were identified in ox-LDL which were missing in n-LDL conditions. These findings may contribute to future identification of disease-associated markers and may also facilitate the potential development of therapeutic tools to treat atherosclerosis.