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Identification of new regulators of proinflammatory signaling pathways Helena Dráberová Protein 4.1R has been described in immune system as regulator of migration and cell adhesion, but was also shown to play a role in activation of T lymphocytes. Polymorphism in gene ORMDL-3 is associated with asthma risk in children and correlates with increased ORMDL-3 expression. This disertation thesis describes the function of proteins 4.1R and ORMDL-3 in activation of mast cells after stimulation of FcεRI receptor. IL-17 is a proinflammatory cytokine that plays a role in immune response against fungal and yeast infections. IL-17 however also plays a role in the pathology of autoimmune diseases such as reumatoid arthritis, psoriasis and multiple sclerosis. IL-17 signaling is tightly regulated, however the exact mechanism has not been described. This disertation thesis describes the IL-17R complex by mass spectrometry and analyze the function of its known and newly discovered components in cells deficient in individual proteins by method CRISPR-Cas9. Last part focuses on the discovery of new subunit of IL-17RC protein CMTM4, which role in IL-17 signaling has not been described so far. CMTM4 stabilizes IL-17RC and is required for its surface expression. In vitro data are supported by data from autoimmune model of...

The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity

Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...

Maintaining energy homeostasis at reduced temperatures is essential for the survival of the organisms. In this diploma thesis, we determined the impact of cold stress and cold adaptation on the rat immune system. A number of different factors participate at the process of thermoregulation, but the adrenergic signalling plays a crucial role. The binding of norepinephrine to β-adrenergic receptors leads to the formation of brown adipose tissue, which is necessary for non-shivering thermogenesis, as well as for energy balance. Bioactive products of adipocytes subsequently modulate the immune system, this process is significantly influenced by signalling of nerve cells. In order to understand neuro-immune interaction during the cold adaptation, we monitored changes in immune cell populations and the production of soluble products in rats treated with specific inhibitors of β-adrenergic receptors. Relationship between the immune and nervous system seems to be very important in many biological processes. Deciphering basic mechanisms of the influence of cold adaptation on immune cells can therefore explain other clinically relevant topics, such as treatment of obesity. Key words: immune system, cold adaptation, norepinephrine, adrenergic receptor, cytokines, brown adipose tissue, non-shivering thermogenesis

The system of mononuclear phagocytes includes macrophages that modulate their phenotype based on microenvironmental signals. Their properties vary considerably in a differentiated stage. M1 macrophages, which are classically activated (typically by IFN-γ), are involved in phagocytosis and produce some pro-inflammatory cytokines, that can stimulate other immune cells. A phenotypically different cell population are M2 macrophages, which are alternatively activated by exposure by Th2 cytokines. M2 macrophages produce preferentially anti-inflammatory cytokines IL-10, TGF-β and participate in repair and tissue healing. The main aim of this study was to standardize a model of differentiation of THP-1 cells and human monocytes towards the M2 phenotyp using an in vitro model. This is represented in particular by increased expression of CD163 and CD206 molecules. The second aim was to assess the dynamics of expression (and co-expression) of CD163 and CD206 molecules in monocytes of patients after kidney transplantation. Expression of surface markers was determined by flow cytometry. Both THP-1 cells and human monocytes, isolated from buffy coat fraction, were stimulated by IL-4, TNF-α, TGF-β and IL-10. Changes in CD163 and CD206 expression were measured after day 1, day 3 and day 6 of stimulation. The most...

Leishmania major is an intracellular parasite which often successfully multiplies and disseminates in a body of the host thanks to strategies that help it to escape the components of immune system of the host organism. Thus, the parasites evoke an impairment of regulatory pathways that in physiological conditions lead to an expression of genes involved in a response to L. major and its efficient elimination. Gene targeted deletion, also called gene knock-out, can result in phenotypic alteration and associated enhanced susceptibility or resistance of the host. Although such detected genes do not have to signify their variability in population and hence they may not be responsible for the worsened outcome of leishmaniasis of some people necessarily, studies in which they are analysed and general knowledge being also a subject of this thesis help us together with techniques of forward genetics to reveal the biochemical pathways during the infection and their elements that influence the outcome of the disease and might be useful for researches of new medicine drugs or gene therapy.

Monocytes and macrophages are important components of the innate immune response. These mononuclear phagocytes form a heterogeneous cell population, of which phenotype and functions can be modified under the influence of different signals coming from the surrounding microenvironment. The aim of this work was to modulate the phenotype of these cells by a variety of stimulants and to compare the changes induced on the model of THP-1 monocytic cell line and on the human peripheral blood monocytes. Surface marker expression was analyzed by flow cytometry. Further on, IL-8 production was evaluated by Luminex assay and the concentration of soluble calprotectin was assessed by ELISA. The most significant changes in surface marker expression were induced by exposure to IFNγ. This cytokine increased the expression of CD54, CD14 and HLA-DR on the surface of THP-1 cell line. Higher concentrations of IFNγ promoted higher apoptotic rate and augmented calprotectin expression and production in THP-1 cell line. On the surface of monocytes, IFNγ stimulation resulted only in the upregulation of CD54 expression. IL-4 increased the expression of CD36 by THP-1 cell line and inhibited the expression of CD163 by human monocytes. LPS stimulation caused the suppression of HLA-DR activation in monocytes and enhanced IL-8...

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...