Establishing a proteomics-based monocyte assay to assess differential innate immune responses

Sammanfattning: The innate immune system represents the first line of organism defense. Monocytes and their derivatives, the macrophages, play a central role in this response. The monocyte response to various stimuli is complex and includes simultaneous activation in a variety of ways. The common methods currently used to estimate this response usually focus on one of the modes of activation. The aim of this thesis was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the monocyte response to various challenges. The underlying hypothesis of the project was that the cell proteome response to different activating molecules would reflect the immunogenicity of these molecules. The assay is based on comparison between the proteomes of human monocytic THP-1 cells treated with a stimulus of interest and the ‘reference’ proteomes obtained from THP-1 cells treated with positive and negative controls. The study described in Paper I (Project 1) revealed two main types of monocyte responses and features of monocyte separation into adherent and non-adherent subpopulations. The first subpopulation differentiated into macrophages, while the second one was activated by cytokines according to the changes in their proteomes compared to the control cells. The Pam3CSK4 and PMA stimuli were concluded to be the best positive controls for pro-inflammatory activation and differentiation, respectively, among the tested nine treatments (Paper II, Project 2). Anti- cancer drugs with known mechanisms of action were chosen as positive controls for cell death (Paper III). The workflow of the assay was optimized and validated in Paper II (Project 2). The established assay was applied to identify the response of monocytes to peptide containing isoaspartate (Paper II, Project 3) and nanoparticles (Paper III, Project 3). The epidermal growth factor (EGF) peptide containing isoaspartate primed monocytes for differentiation but not for pro-inflammatory activation. The three studied nanoparticles induced distinct proteome signatures despite equivalent overall toxicity effect (induction of 50±10% cell death). The CdTe-NH2 nanoparticles induced the strongest response characterized by down-regulation of topoisomerases and similar to the effect of Camptothecin. The response to CuO nanoparticles included up-regulation of proteins involved in heat response. The gold nanoparticles showed the weakest response among the studied three nanoparticles and induced up-regulation of NF-κB and a number of proteins related to energy metabolism consistent with pro-inflammatory activation. The established and validated assay has several possibilities for extension as well as optimization for particular aims of the experiment. For instance, the identification of induced post- translational modifications upon stimulation can be performed (Paper IV, Project 4). Because of its versatility, robustness and specificity, this new assay is likely to find a niche among the more established immunological methods.