Modulation of protein expression in Mycobacterium tuberculosis-stimulated TB- IRIS patient-derived PBMCs by omega n-3 polyunsaturated fatty acids

Master Thesis


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University of Cape Town

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory disorder which affects up to 54% of TB-HIV co-infected patients. Its pathogenesis remains unclear, and although treatment with the corticosteroid prednisone has shown some benefits, there is no specific treatment currently available for TB-IRIS. N-3 polyunsaturated fatty acids (PUFA) have safely and successfully been used in the treatment of other inflammatory diseases, and we hypothesized that they will have beneficial anti-inflammatory effects on TB-IRIS patient-derived immune cells. To investigate this hypothesis, we used mass spectrometry (MS)-based proteomic methods to probe the secretome of peripheral blood mononuclear cells (PBMCs) re-stimulated ex vivo with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL), and treated with n-3 PUFA. Optimization experiments were performed on cells obtained from eight healthy donors to assess the secretome changes induced by re-stimulation with Mtb WCL and treatment with n-3 PUFA. In addition, experiments were repeated using PBMCs obtained from five non-IRIS and five TB-IRIS patients. The secretome was prepared via chloroform/methanol precipitation and overnight Trypsin digestion, and investigated via MS-based shotgun proteomics. MaxQuant was used for protein identification and Perseus for statistical analysis. Stimulation with Mtb WCL shifted the secretome of healthy PBMCs towards an inflammatory state, and this was altered by treatment with EPA/DHA via changes in the regulation of several proteins. Preliminary results from TB-IRIS patient-derived PBMCs show the same trend. These promising early results suggest the potential benefits of n-3 PUFA dietary supplementation for patients with TB-IRIS, and warrant further studies with increased sample size to confirm findings, and the possible inclusion of n-3 PUFA in a future clinical trial on patients with TBIRIS.