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| Author(s): |
Rafaela Mendonça Baggio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2019-03-08 |
| Examining board members: |
Nicola Amanda Conran Zorzetto;
Fabíola Traina;
Erich Vinicius De Paula;
Alessandra Gambeiro;
Vanessa Moreira
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| Advisor: | Nicola Amanda Conran Zorzetto |
| Abstract | |
Sickle cell anemia (SCA) has a complex pathophysiology that results in a chronic inflammatory state, which plays a fundamental role in the initiation and propagation of the vaso-occlusive processes that are associated with the disease, as well as many of its variable manifestations, such as acute chest syndrome, pulmonary hypertension, stroke and nephropathy. It is probable that the chronic inflammatory state in SCA occurs as the result of intravascular hemolysis and ischemia/reperfusion events. Consequently, the vaso-occlusive process is initiated by the adhesion of red blood cells and activated leukocytes to the activated endothelium of the microcirculation, forming a physical barrier to blood flow. Currently, the treatment of SCA is limited to blood transfusion, hematopoietic stem cell transplant and some pharmacotherapeutics, such as hydroxyurea. We postulate that the formation of the inflammasome complex in the leukocytes of patients with SCA may potentially contribute significantly to the inflammatory state in this disease. The inflamasome is a multimeric protein complex, formed in inflammatory cells, which often consists of a sensor molecule (such as NLRP3), an adapter protein called ASC (Apoptosis associated speck like protein containing a CARD) and caspase-1. The formation of the inflammassome can be stimulated by a number of molecules released during an infectious process or during tissue damage / metabolic imbalances; these molecules are known as pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs), respectively. The aim of this project was to demonstrate the existence of inflammation in the neutrophils and monocytes of individuals with SCA, as well as to identify the components of this complex and the possible DAMPs that may contribute to inflammasome formation in this disease. The techniques used for this study were: ELISA, flow cytometry, immunofluorescence and confocal microscopy, imaging flow cytometry, quantitative PCR (PCRq), Western blotting, co-immunoprecipitation, culture of primary and lineage cells, plasmid cloning, transfection assays, and Bimolecular Fluorescence complementation. Circulating concentrations of both IL-1 ? and IL-18 were significantly elevated in the population of patients with SCA studied. Furthermore, novel findings demonstrate that neutrophils from the SCA individuals (SCA neutrophils) studied release IL-1? in increased amounts, when compared to neutrophils from healthy individuals. However, interestingly, while the processing of this cytokine in SCA neutrophils was dependent on increased caspase-1 activity, it was independent of the participation of NLRP3 and the ASC molecule. Therefore, we suggest that the processing of IL-1? by neutrophils in SCA individuals is not mediated by the classic formation of the inflammatory molecule, NLRP3. In monocytes from SCA individuals, we observed the processing and release of IL-1? and IL-18, but we did not find significant differences in their release to the extracellular medium, as compared to monocytes from healthy individuals; however, given the increased numbers of these leukocytes in the SCA circulation, we cannot exclude the participation of the monocytes in the elevated concentrations of IL-1? and IL-18 observed in SCA. The inflammassome observed in SCA monocytes appears to be mediated by the classical NLRP3 inflammasome with recruitment of the ASC molecule for activation of caspase-1. Circulating DAMPs are significantly augmented in SCA individuals, and we also showed that the plasma of SCA individuals is able to stimulate the formation of inflammassome in neutrophils and, importantly, is able to activate the inflammassome machinery in transfected non-immune cells. These findings are of importance for the improvement of our understanding of the pathophysiology of SCA, since mechanisms that induce IL-1? processing amplify inflammatory responses and may constitute important therapeutic targets for the disease (AU) | |
| FAPESP's process: | 16/08729-8 - Investigation of the inflammasome formation in patients with sickle cell anemia |
| Grantee: | Rafaela Mendonça Baggio |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |