The mononuclear phagocyte system (MPS) exemplifies cells differentiation generating functional and phenotypic diversity. Cells of the MPS origin from bone marrow hematopoietic stem cells differentiating through progenitor intermediates to monocytes, which enter circulation, migrate into tissue and terminally differentiate to replenish resident dendritic cells and macrophages. Macrophages differ from their monocytic precursors morphologically and functionally. Monocytes and macrophages are both critical effectors and regulators of inflammation and the innate immune response e.g., by producing growth factors and cytokines. The phenotypic and functional diversity of MPS cells depends on differentiating programs being strictly related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. PLCs are strictly tissue specific and the expression panel, as well as the subcellular localization differs in quiescent cells compared to the pathological counterpart. Our previous work demonstrated the involvement of PLC enzymes during differentiation of macrophages from unpolarized (M0) into M1 and M2. Human leukemia model cell lines with monocytic characteristics such as the human cell line THP-1, frequently used as a model system with monocytic properties. THP-1 cells can be differentiated into macrophage-like cells that resemble properties of mature macrophages by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA), resulting in cells with increased adherence and loss of proliferative activity. In the present project we aim to investigate the differentiation of THP-1 cells. Once differentiated, we want to investigate: 1) the involvement of PI signaling, 2) the production of cytokines and 3) the effects of the tumour environment.
Our group analyzed the variation of the panel of expression and localization of PLC enzymes in a number of normal and pathological tissues or cells during last years (2004-2017). Our group demonstrated that differently stimulated M0 macrophages can differentiate in M1 and M2 showing different panel of PLC expression and different localization. Thus we suggested that PLC enzymes can act upon macrophage differentiation. The role of each MPS cell¿s state is relatively well known, as well as the stimuli inducing differentiation were described. However, the signal transduction pathways and the molecules leading to differentiation in monocytes with respect to stimulated monocytes was not analyzed. As PLC enzymes were described to be modified under inflammation stimuli (such as LPS in astrocytes and HUVEC, in endometriosis ¿ Lo Vasco et al 2011a and b, 2012, 2014, 2016 and 2017), these enzymes might be modified in macrophages. At now, no data are available about the PLC isoforms expressed in THP cells (unstimulated or quiescent). Our results will improve literature data listing the PLC isoforms expressed in the MPS system. Moreover, we want to localize the expressed PLC isoforms within the cells. In fact, it is well known that changes in selected PLC isoforms¿ cellular localization might modify the activities of the cell.
Notably, recent studies were addressed to evaluate the environment the cells come into contact. In fact, it was suggested that the molecules (especially chemokines, but also others) present in the environment might act upon the recruitment or the differentiation of cells, with special regard to immune cells. Many research efforts were addressed to investigate the tumoral microenvironment and its components. We aim to investigate the effects of osteosarcoma microenvironment upon survival, morphology and immune-related genes¿ expression in THP. We will use the media culture of two different lines of in vitro cultured human osteosarcoma cells, such as 143B (highly metastasizing cells) and MG-63 cells (considered as osteoblast-like cells).
MPS cells can produce chemokines or cytokines. In the present project we aim investigate THP ability to produce cytokines after stimulation with different stimuli.
Our results will improve knowledge about the signal transduction of PI in MPS system, allowing further studies about the role of each enzyme. Furthermore, our results might represent the baseline to modify the expression of PLC (using RNA silencing and/or PLC inhibitors) in order to manipulate the MPS system.