Tese de Doutoramento Medicina, Investigação Clínica 2022 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

High-density lipoproteins (HDL) are the densest plasma lipoproteinsand theircomplex protein and lipid constitution interfereswith several physiological mechanisms. The most well characterized HDL functionis reverse cholesterol transport,but other functions have also been attributed to HDL, namelyanti-oxidative, vasodilatoryand anti-inflammatory properties. However, the HDL immunomodulatoryfunction is poorly understood. HDL mediation of cholesterol efflux and consequent lipid raft disruption is one of the main mechanisms by which HDL modulatestheimmune response, but other mechanisms are also involved.Most ofthe existent knowledge comes from animal studies, withlimitations related to the animal model lipid metabolism. Thefew human studiesare heterogeneous and demonstrate both anti-inflammatory and pro-inflammatory effects.Paradoxically, HDL can also suffer transformation to dysfunctional particles, by many different mechanisms. One of these mechanisms is related with the production of antibodies to HDL particles, with themain target beingapolipoprotein A-I (ApoA-I).In this thesis, I questioned the role ofHDL in immune functionwitha specialfocus on the HDL effects on T celllipid metabolism and T cell response, using cells from patients with systemic lupus erythematosus and healthy controls.This work studied: 1)the conditions in which HDL induces cholesterol depletion from CD4+T cells in vitro; 2)the HDL influence on ABCA1 and lipid raft organization inthe plasma membrane (PM)of cultured CD4+T cells; 3) the HDL effect on immune conjugate formation; 4) CD4+T cell lipid metabolismin relation toanti-HDL antibodies; 5)the presence of anti-ABCA1 antibodies; 6) the HDL modulation of T cell response in vitro.The main methodologies used in this thesiswere peripheral blood mononuclear cells (PBMCs)isolation by density gradient separation, immune-based assays such as ELISA, cell culture experimentsandflow cytometry.The results give importantclues to the importance of HDL to T cell metabolism and response: 1)HDL depletes cholesterol from the PM of CD4+T cellsin 24hourcultures, suggesting that this is the ideal time-lapse for in vitrostudies; 2)cholesterol content in the PM of healthy CD4+T cells varies between different T cell subsets, with effector memory (EM)T cells showing the highestlevels of PM cholesteroland less abundant glycosphingolipids (lipid raft constituents); 3) anti-HDL antibodies associate with an increase in the prevalence of EM T cells and a decrease in naïve and regulatory T cells (Treg). The presence of anti-HDL antibodies is also associated withanincreased expression of lipid rafts in CD4+T cells from SLE patients and withaderegulated membrane cholesterol and lipid transporter ABCA1; 4)anti-ABCA1 antibodies are presentin some patients with SLE; 5)24 hour culture with HDL induces the expression of TGF-β1 in CD4+T cellswithout affecting inflammatory cytokines; 6)HDL inhibited the proliferation of CD4+T cells from patients with SLE but did not affect the proliferation in healthy T cells; 7)in the presence of HDL,TCRzeta phosphorylation is reduced. In conclusion, this worksupports the conceptthat HDL has essentially regulatory functions in the immune system, with a notorious effect in the production of TGF-β1 in CD4+T cells. HDL seems toreduceexaggerated inflammation and maintain normal immune function, withimmune modulatory effects that are context dependent. Modifications in lipid metabolism occur in lymphocytes from patients with SLE, reinforcing the importance ofthelipid metabolism for the immune response. These discoveries add new information to the current knowledge on HDL immune functionin humansthat will hopefully be further studied in the research of atherosclerosis, autoimmune diseases and other pathologiesin which lipid metabolism anomalies concurwith immune dysfunction.