Effect of lysed and non-lysed sickle red cells on the activation of NLRP3 inflammasome and LTB4 production by mononuclear cells.

OBJECTIVE AND DESIGN: This study tested the hypothesis that sickle red blood cell (SS-RBC) can induce inflammasome NLRP3 components gene expression in peripheral blood mononuclear cells (PBMCs) as well as interleukin-1ß (IL-1ß) and leukotriene B4 (LTB4) production. Additionally, we investigated the effect of hydroxyurea (HU) treatment in these inflammatory markers. METHODS: PBMCs from healthy donors (AA-PBMC) were challenged with intact and lysed RBCs from SCA patients (SS-RBC) and from healthy volunteers (AA-RBC). NLRP3, IL-1ß, IL-18 and Caspase-1 gene expression levels were assessed by quantitative PCR (qPCR). IL-1ß protein levels and LTB4 were measured by ELISA. RESULTS: We observed that lysed SS-RBC induced the expression of inflammasome NLRP3 components, but this increase was more prominent for CASP1 and IL18 expression levels. Moreover, we observed that intact SS-RBC induced higher production of IL-1ß and LTB4 than lysed SS-RBC. Although SCA patients treated with HU have a reduction in NLRP3 gene expression and LTB4 production, this treatment did not modulate the expression of other inflammasome components or IL-1ß production. CONCLUSIONS: Thus, our data suggest that caspase-1, IL-1ß and IL-18 may contribute to the inflammatory status observed in SCA and that HU treatment may not interfere in this inflammatory pathway.

Associations between TGF-ß1 Levels and Markers of Hemolysis, Inflammation, and Tissue Remodeling in Pediatric Sickle Cell Patients.

Transforming growth factor beta (TGF-ß) is a cytokine with important involvement in biological processes related to the pathogenesis of sickle cell disease (SCD), including endothelial and vascular dysfunction, inflammation, and hematopoietic homeostasis. This study is aimed at investigating associations between levels of TGF-ß1 and classical laboratory biomarkers and inflammatory mediators, as well as the tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), in pediatric patients (n = 123) with SCD in steady state: 84 with sickle cell anemia (HbSS) and 39 with hemoglobin SC disease (HbSC). A healthy control (HC) group of 59 individuals was also included. Hematological and biochemical analyses were carried out using electronic methods. TGF-ß1, TIMP-1, and MMP-9 plasma quantifications were performed by ELISA. TGF-ß1 plasma levels were higher in HbSS individuals than in HbSC and HC. In individuals with HbSS, TGF-ß1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets, and TIMP-1. In addition, HbSS individuals with TGF-ß1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-ß1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, and platelets, as well as levels of TIMP-1, VLDL-C, triglycerides, heme, and AST. Additionally, HbSC individuals with TGF-ß1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9, and TIMP-1, and decreased HDL-C. Our findings suggest that TGF-ß1 may play important roles in vascular remodeling, vasculopathy, angiogenesis, and inflammation in pediatric patients with SCD.

Transforming Growth Factor Beta Receptor 3 Haplotypes in Sickle Cell Disease Are Associated with Lipid Profile and Clinical Manifestations.

Individuals with sickle cell disease (SCD) present both chronic and acute inflammatory events. The TGF-ß pathway is known to play a role in immune response, angiogenesis, inflammation, hematopoiesis, vascular inflammation, and cell proliferation. Polymorphisms in the transforming growth factor-beta receptor 3 (TGFBR3) gene have been linked to several inflammatory diseases. This study investigated associations between two TGFBR3 haplotypes and classical laboratory parameters, as well as clinical manifestations, in SCD. We found that individuals with the GG haplotype presented higher levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides, non-HDL cholesterol, total proteins, and globulin than individuals with non-GG haplotypes. In addition, the GG haplotype was associated with a previous history of pneumonia. Individuals with the CGG haplotype presented increased plateletcrit, TC, LDL-C levels, and non-HDL cholesterol. The CCG haplotype was also associated with a previous history of pneumonia. Our findings suggest that individuals with the GG and CGG haplotypes of TGFBR3 present important alterations in lipid profile.

Hydroxyurea alters circulating monocyte subsets and dampens its inflammatory potential in sickle cell anemia patients.

Sickle cell anemia (SCA) is a hemolytic disease in which vaso-occlusion is an important pathophysiological mechanism. The treatment is based on hydroxyurea (HU), which decreases leukocyte counts and increases fetal hemoglobin synthesis. Different cell types are thought to contribute to vaso-occlusion. Nevertheless, the role of monocytes subsets remains unclear. We investigated frequencies of monocytes subsets in blood and their response to HU therapy, testing their ability to express pro-inflammatory molecules and tissue factor (TF). We identified major changes in monocyte subsets, with classical monocytes (CD14++CD16-) appearing highly frequent in who were not taking HU, whereas those with patrolling phenotype (CD14dimCD16+) were enriched in individuals undergoing therapy. Additionally, HU decreased the production of TNF-α, IL1-ß, IL-6, IL-8 as well as TF by the LPS-activated monocytes. Likewise, frequency of TF-expressing monocytes is increased in patients with previous vaso-occlusion. Moreover, activated monocytes expressing TF produced several pro-inflammatory cytokines simultaneously. Such polyfunctional capacity was dramatically dampened by HU therapy. The frequency of classical monocytes subset was positively correlated with percentage cytokine producing cells upon LPS stimulation. These findings suggest that classical monocytes are the subset responsible for multiple pro-inflammatory cytokine production and possibly drive inflammation and vaso-occlusion in SCA which is damped by HU.