Sickle Cell Inflammatory Environment Is Associated with Products of the Eicosanoid Synthesis Pathways

Background: Sickle cell disease (SCD) is a group of inherited anemia characterized by heterogeneous clinical outcome, including hemolysis, chronic inflammation, and vaso-occlusive/painful crisis.

Aims: We evaluated inflammatory and anti-inflammatory mediators, such as TNFα, IL-10, IL-12, IL-1β, IL-6, and IL-8, TGF-beta, tissue inhibitor of metalloproteinase (TIMP1), matrix metalloproteinase 9 (MMP9), heme, and leukotriene B4 (LTB₄), and prostaglandin E2 (PGE₂), the last two are products of the eicosanoid synthesis pathways, in SCD patients (in steady-state and in crisis-state) and healthy controls. For the same groups, in order to establish biomarkers of crisis and steady-state, we also investigated association among inflammatory/anti-inflammatory mediators and markers of hemolysis, inflammation, hepatic dysfunction, renal and lipid metabolism.

Methods. We assessed 129 SCD steady-state patients (SP), 23 SCD in crisis patients (CP), and 67 healthy individuals (HC) age- and sex-matched with patients groups. Hematological analyzes were performed by automatic cell counter and hemoglobin profile by HPLC. Biochemistry analyses of inflammation and infection markers, as well as lipid, hepatic, and kidney metabolism markers were investigated by immunochemistry assays. Plasma levels of TNFα, IL-10, IL-12, IL-1β, IL-6 and IL-8 were measured using Cytometric Bead Array (CBA) according to the manufacturer's protocol. Plasma concentration of total heme was measured by QuantiChrom Heme Assay Kit. PGE₂, LTB₄, TGF-beta, TIMP1 and MMP9 levels were estimated in plasma samples and supernatants by ELISA, according to the manufacturer's instructions.

Results. Steady-state SCD patients had the highest values of LTB4, PGE2, TIMP1, MMP9, IL-8, and IL-12 concentration compared with CP and HC groups (p < 0.0001). However, crisis-state SCD patients had the highest values of IL-1β, IL-6, IL-10, TNFα compared to HC and SP groups, and had a the lowest levels of LTB₄, PGE₂,TGF-β, MMP9, IL-8 and IL-12 levels (p < 0.0001 for both analysis). Significant difference of heme levels was not finding among the three groups. The ROC curve showed that LTB₄, PGE₂ and TGF- β are important markers related to steady state and IL-1β, IL-6, and TNFα are markers of crisis in SCD.

Conclusions: The finding of difference in inflammatory markers level in steady state and crisis SCD patients suggest that these markers can be used to monitoring patients and to predict crisis events. The finding of similar heme concentration between steady-state and crisis-state SCD patients may be explained by the hyperhemolysis phenomenon, showing that even in steady-state, these patients continuous to have hemolysis and, consequently, continuous to generate heme and reactive oxygen species.