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Identifier 000442092
Title Delineating molecular mechanisms of Dendritic cell/monocyte activation mediated by neutrophil extracellular traps underlying Rheumatoid Arthritis pathogenesis
Alternative Title Διερεύνηση των μοριακών μηχανισμών της ενεργοποίησης των μονοκυττάρων/δενδριτικών κυττάρων που διαμεσολαβούνται από εξωκυτταρικές παγίδες ουδετερόφιλων και συμβάλουν στην παθογένεια της Ρευματοειδούς Αρθρίτιδας
Author Γωνιανάκη, Ειρήνη
Thesis advisor Παπαδάκη, Γαρυφαλιά
Reviewer Σιδηρόπουλος, Πρόδρομος
Μπερτσιάς, Γεώργιος
Βεργίνης, Παναγιώτης
Abstract Rheumatoid arthritis (RA) is a chronic autoimmune disease that involves multiple immune cell types and biological processes. Complexity of RA in combination with inadequate treatment responses and frequent patient relapses, underscore the necessity to delineate the molecular mechanisms underlying the disease. Neutrophils are the most abundant white blood cell type and the first cells that reach the inflamed synovium. Besides releasing several inflammatory cytokines, neutrophils also externalize chromatin fibers called extracellular traps. RA patients present increased spontaneous formation of NETs, which indicates an important role in pathogenesis of the disease. Moreover, our previous work revealed that RA-derived NETs show an increased potential to induce the maturation of dendritic cells (DCs) from healthy individuals. Mechanistically, we showed that exposure of DCs to Collagen induced Arthritis (CIA)-derived NETs induced DC maturation characterized by significant up-regulation of CD80 and CD86 costimulatory molecules as well as elevated secretion of the inflammatory cytokine IL-6. In agreement, CIA-NET-treated DCs promoted the induction of antigen-specific Th1 cells in vitro. In the present study, our main purpose was to delineate the mechanisms implicated in NET-induced activation of DCs. For this purpose, we focused on monocytes as they represent the progenitor cells of monocyte derived dendritic cells (moDCs) and exhibit similar properties with DCs. To address our hypothesis, first, we provoked NET formation with IgG antibodies isolated from sera of active RA patients and observed increased NET release on healthy neutrophils as compared with neutrophils treated with IgG isolated from healthy volunteers. Next, we focused on the effect of RA IgG-induced NETs on the activation of monocytes. We measured the secreted protein levels of cytokines in monocytes treated with NETs and observed increased expression of Tumor Necrosis Factor alpha (TNF-α) indicative of monocytes activation. According to recent evidence, induction of autophagy is highly linked with the activation of monocytes. For that reason, we focused on the autophagic machinery of monocytes. Interestingly, monocytes treated with RA IgG-induced NETs showed decreased expression of the autophagic marker LC3 and increased expression of p62 in confocal microscopy. In Western blotting, p62 levels also tend to increase. The above evidence suggests impaired autophagy in monocytes treated with RA IgG NETs. Next, we sought to determine the intracellular signaling events mediating the NET-dependent downregulation of monocyte autophagic machinery. To address this, we performed flow cytometry on monocytes treated with NETs for the phosphorylated intracellular markers of the mTOR pathway, pmTOR and pAkt and the downstream molecules of the mTORC1 pathway, pS6 and p4EBP1 and observed increased expression of the PI3K/Akt/mTOR axis. 6 To extend our findings in vivo, we used the collagen induced arthritis mouse model (CIA). CIA bone marrow neutrophils (PMNs) present increased spontaneous NET formation as compared with control BM PMNs. Treatment of bone marrow derived dendritic cells (BMDCs) with CIA NETs revealed increased antigen presenting capacity as compared with the control. Collectively, our findings indicate that RA IgG induced NETs may promote monocyte activation through decreased autophagy and increased PI3K/Akt/mTOR signaling pathway.
Language English
Subject Autophagy
Issue date 2021
Collection   School/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work
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