Hidroxiureia na doença falciforme: influência de polimorfismos nos genes MPO e SERPINA1 e implicações nas vias dos receptores tipo Toll e do Inflamassoma

Introduction: Sickle cell disease (SCD) is an inflammatory condition associated with vaso-occlusive and painful episodes intravascular hemolysis. SERPINA1 and myeloperoxidase (MPO)-463G>A gene polymorphisms are associated with vascular complications in sickle cell disease (SCD). SCD patients are treated with Hydroxyurea (HU), which increases levels of fetal hemoglobin (HbF) and decreases leukocytes. Toll-like receptors (TLR) play important role in the maintenance of the inflammatory status observed in these patients and the nod-like receptor protein 3 (NLRP3)-inflammasome platform could contribute this inflammation, since erythroid contents acts as danger signals (eDAMPs) for activating this pathway. Objectives: The aim of this study was to evaluate the possible influence of MPO and SERPINA1 genes polymorphisms in SCD patients in response to HU treatment and investigate whether this treatment can interfere with gene expression of TLR and NOD-induced red cells of patients with sickle cell anemia (SCA). The specific objectives were to evaluate the association between these polymorphisms and clinical and laboratory aspects in response to HU treatment; to evaluate the expression of TLR2, TLR4, TLR5 and TLR9 in peripheral blood mononuclear cells (PBMC) of SCA patients and the role that erythrocytes plays in the expression of these TLRs, and in vitro effect of Hydroxyurea; to investigate the expression of NLRP3, Caspase-1, interleukin (IL)-1β and IL-18 in PBMC of SCA patients and the role that erythrocytes plays in the expression of these molecules, and in vitro effect of Hydroxyurea. Methods: Blood samples from 356 SCD patients and 100 healthy volunteers were analyzed. Sixtynine patients were screened for -463G>A MPO and 129 for SERPINA1 polymorphisms by RT-PCR-based methods. PBMC and red blood cells of SCA patients (SS-RBC) and healthy volunteers (AA-RBC) were obtained. PBMC were challenged with SSor AA-RBC in presence of hydroxyurea (HU). TLRs and NODs gene expressions were performed by qPCR. Leukotriene-B4 (LTB4), IL-1β and IL-18 bind protein (BP) production were performed by Elisa. Nitrite production was measured by Griess reaction. Results: In the general SCD patients, independent of gene polymorphisms, HU-treated individuals exhibit increased levels of serum ferritin and HbF and reduction of leukocytes count. The MPO polymorphism was associated with reduction in platelet and leukocytes counts. Under HU treatment, patients with MPO polymorphism showed increased levels of ferritin and HbF, whereas these parameters were not changed after treatment of patients without polymorphism. In contrast, SCD patients with SERPINA1 polymorphism presented increased levels of ferritin and decreased levels of Alpha-1 antitrypsin (AAT). HU treatment significantly increased ferritin levels in both groups, whereas AAT were reduced only in the mutant group. HU-treated patients without SERPINA1 polymorphism exhibit reduction in total leukocytes count and AAT levels. TLR2, TLR4 and TLR5 are highly expressed in PBMC of SCA patients, comparing to healthy volunteers, whereas TLR9 expression was similar in both groups. Additionally, intact or lysed SS-RBC, but not AA-RBC, induces TLR9 expression, and lysed, both AAand SS-RBC, induces expression of TLR2, TLR4 and TLR5. Interestingly, HU treatment increases expression of TLR2 and does not interfere with expression of other TLRs. Moreover, although SS-RBC induces LTB4 and Nitrite, production, HU does not prevent LTB4 but reduces nitrite production. NLRP3 and IL-1β are highly expressed in PBMC of SCA patients when compared to healthy volunteers. In addition, we showed that intact or lysed SS-RBC, but not AA-RBC, induces Caspase-1 and IL-18 expression, and lysed AA-RBC induces expression of NLRP3 and IL-1β. Moreover, Intact SSRBC, but not AA-RBC, induces production of IL-1β. Treatment with hydroxyurea (HU) showed no interference with the expression of NLRP-3-inflammasome proteins, whereas induced production of IL-18BP. Conclusion: These results together point out that genetic change, such as mutations in MPO and SERPINA1 genes, interfere with the response to HU treatment. In addition, there are more evidence that RBCs, especially SS-RBCs, act as eDAMPs, stimulating TLR and NOD expression and contributing to inflammation. This study highlighted that HU does not prevent TLRand NLRP3-inflammasome-dependent inflammation. This knowledge could lead to the development of new therapeutic strategies, which act in different ways from those given by hydroxyurea.