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Identifier 000434953
Title Investigation of the molecular mechanisms that are involved in the anti-inflammatory function of CTLA-4-treated dendritic cells
Alternative Title Διερεύνηση των μοριακών μηχανισμών που εμπλέκονται στην ανοσοκατασταλτική δράση των δενδριτικών κυττάρων υπό την επίδραση του CTLA-4
Author Αξιώτης, Ισίδωρος
Thesis advisor Σιδηρόπουλος, Πρόδρομος
Reviewer Γουτακόλη, Παναγιώτα
Παπαδάκη, Γαρυφαλιά
Abstract DCs are the connecting step between innate and adaptive immunity and their major role is to present antigens to T cells. A mixture of instructions that reach naïve T cells affects their activation and leads them to acquire different effector phenotypes. These instructions are primarily derived by DCs which go through variable transcriptional and metabolic alterations. DCs produce three types of signals to stimulate T cell differentiation. Antigen presentation is the first signal (signal 1) but only in combination with co-activation signals (signal 2) they can induce T cell activation. One of the most essential secondary signals is promoted by the binding of CD80/86 on DC membrane with CD28 protein on T cell surface. CTLA-4, which is expressed by T cells, binds on CD80/86 to regulate T cell activation. A soluble form of CTLA-4 (CTLA-4–Ig) is used for treatment of autoimmune inflammatory diseases such as rheumatoid arthritis (RA). Also, the secretion of cytokines by DCs affects the polarization of T cells as well (signal 3). In parallel, DCs are also capable to preserve homeostatic conditions upon harmless or self-antigens by providing tolerogenic signals to T cells. Tolerogenic DCs are generated both in vivo and in vitro upon a variety of different agents, such as dexamethasone, and they are able to inhibit T cell responses via different mechanisms. Failure of self-tolerance drives overactivation of lymphocytes which leads to the development of autoimmunity. Drugs that promote anti-inflammatory properties of DCs are another therapeutic approach for autoimmune inflammation in diseases like RA. Recent studies, including ours, have noted that soluble CTLA-4 (CTLA-4 Ig is an approved treatment for RA) is able not only to block T cell stimulation but it induces reverse signaling in DCs and promotes anti-inflammatory properties. Hence, in order to further investigate the molecular mechanisms that drive the CTLA-4–mediated tolerogenic effects on DCs, we used an ex vivo setup where healthy human monocyte-derived DCs (moDCs) were cultured with CTLA-4–Ig. To address this, we analyzed the immunogenic profile of CTLA-4–Ig-treated moDCs. For the study of signal 1, we measured the levels of HLA-DR on moDCs surface which were elevated. T cell activation needs also signal 2 which is derived from co-stimulatory molecules. Among these, CD80 and CD86, through which CTLA-4 exerts its cell-extrinsic effects, were downregulated. The CD83 maturation marker was upregulated upon CTLA-4–Ig treatment. For the investigation of signal 3, we analyzed the mRNA of genes that encode important secreted factors. The expression of the pro-inflammatory IL-6 and IL-12b genes and of the anti-inflammatory IDO and IL-10 genes were inhibited in CTLA-4–Ig-treated moDCs. Interestingly, the IL-6 protein levels were found elevated. Moreover, tolerogenic characteristics of DCs are related to changes in cellular metabolism. To better characterize this, we performed RNAseq analysis of human moDCs treated with CTLA-4–Ig, which revealed 575 differentially expressed genes. Among them, the most deregulated pathways were those associated with mitochondria and oxidative phosphorylation. Also, a molecular pathway that is implicated in both metabolism and inflammatory responses is the Akt/mTOR-dependent pathway was found differentially regulated. It has been previously demonstrated that CTLA-4–Ig treatment on DCs activates the above pathway driving the induction of anti-inflammatory responses and the inhibition of autophagy, an important process for antigen presentation. Our results indicated an increase in mTOR protein expression while that of Akt was decreased. In summary, our findings support that CTLA-4–Ig is able to induce tolerogenic DCs via downregulation of co-stimulatory molecules and inhibition of pro-inflammatory cytokines and these changes are associated with deregulation of essential metabolic pathways.
Language English
Subject Abatacept
Soluble CTLA4
Δενδριτικά κύτταρα
Διαλυτό CTLA4
Issue date 2020-12-17
Collection   School/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
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