Levels of inflammatory markers are differentially expressed in sickle cell anemia and sickle cell trait

Abstract Although sickle cell anemia (SCA) is related to inflammation, the profile of inflammatory markers in sickle cell trait (SCT) is poorly studied. This is a cross‐sectional study of inflammatory biomarkers carried out involving adults with SCA in steady state, SCT and controls. The SCA group had higher levels of lactato dehydrogenase, IL‐1β, IL‐6, IL‐10, and tumor necrosis factor alpha than the others, while the SCT group had similar levels to control group. In addition, SCA group had lower IL‐8/IL‐10 and soluble triggering receptor expressed on myeloid cells‐1/IL‐10 ratios. These findings indicate that individuals with SCT do not have a chronic inflammatory profile and reinforce that cytokines are involved in the maintenance of the inflammatory state in SCA.


INTRODUCTION
Sickle cell anemia (SCA) is the most prevalent hereditary hemoglobinopathy in the world and is characterized by the production of a variant form of hemoglobin, the S hemoglobin (HbS) [1]. Individuals with SCA are homozygous for HbS, and their erythrocytes assume a sickle shape. Recurrent vaso-occlusion, the ischemia-reperfusion process and consequent activation and injury of vascular endothelial cells induce a chronic inflammatory response in the individual with SCA, which is propagated by high levels of inflammatory cytokines, decreased nitric oxide bioavailability and oxidative stress [2]. On the other hand, the heterozygous genotype (Hb-AS) is sickle cell trait (SCT), considered a benign condition, which morbidity and mortality rates are comparable to the general population's [3]. Even though most individuals with SCT are asymptomatic, studies point out possible complications for this condition, such as hematuria, renal papillary necrosis, hyposthenuria, splenic infarction, exertional rhabdomyolysis, and sudden exercise-related death [4].
Although much is already known about the involvement of inflammatory mediators in SCA pathophysiology, there is no clear information regarding inflammatory markers in SCT. Therefore, the present study assessed the levels of the inflammatory markers, which were most previously studied in SCA and are associated with inflammatory i (IL-1β, IL-6, IL-8, and tumor necrosis factor alpha [TNF-α]) and modulator (IL-10) patterns in non-infectious inflammatory diseases, in adults with SCT and SCA in steady state. Inflammatory markers were measured in the serum, which was obtained from the peripheral blood of participants and stored at was measured using the optimized ultraviolet kinetic method.

Statistical analysis
The data normality analysis was performed using the D'Agostino-Pearson test. For variation comparisons between groups, the Kruskal-Wallis test was used, followed by the Dunn test for specific differences between groups. GraphPad Prism 5.0 was used to assess the cytokine values. Differences were considered significant if p < 0.05.  (Table 1).

Fifty-five individuals in the
Differences between Hb-SS, Hb-AS and Hb-AA groups were noted for most of the inflammatory markers, except for sTREM-1. The levels of LDH ( Figure 1A), IL-1β ( Figure 1B), IL-6 ( Figure 1C), IL-10 ( Figure 1E), and TNF-α ( Figure 1F) were higher in the Hb-SS group than the others. The Hb-SS group had lower IL-8 levels than the Hb-AS group ( Figure 1D). Comparing the Hb-AA and Hb-AS groups, the latter had lower levels of LDH and IL-1β ( Figure 1A and 1B).

DISCUSSION
Several inflammatory biomarkers are involved in the pathophysiology of SCA, but as far as we know, this is the first study that comparatively assessed these markers in adults with SCA and SCT. This study confirmed the high levels of these markers in patients with SCA in a steady state and its importance is to know the inflammatory immune profile of individuals with SCT, since they are candidates for bone marrow donors from relatives with SCA.
Although SCT is considered a benign condition, study found that it contributes to inflammatory markers and worsen vascular dysfunction in diabetics [5], what could suggest a pro-inflammatory profile in findings regarding the levels of these markers in this context, however, conclude that regular physical activity reduces endothelial activation and vascular adhesion responses in individuals with SCT [6,7].
The present study confirmed that Hb-SS individuals have higher levels of LDH, which reflects the pathophysiology of the disease. The Hb-AS individuals had the lowest levels of LDH among all that corroborates their asymptomatic condition, in addition to ruling out the occurrence of tissue damage linked to it.
The Hb-SS group also showed higher levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and anti-inflammatory IL-10 than the Hb-AA group, which is pointed out in the scientific literature. Previous study observed that IL-1β followed the same pattern as TNF-α, with higher levels in Hb-SS than Hb-AA individuals, as well as in stable Hb-SS individuals compared to those in crisis [8]. The evidence of higher pro-inflammatory cytokines in stable Hb-SS individuals than in those in crisis [9] corroborates both the results of the current study, as it TREM-1 is a receptor constitutively expressed on neutrophils and monocyte subsets, linked to inflammatory response in infections or non-infectious inflammatory pathologies, such as sepsis and rheumatoid arthritis [13,14], and it can also be produced in its soluble form (sTREM-1). Activation of TREM-1 in neutrophils is related to increased production of pro-inflammatory cytokines and reduction of antiinflammatory cytokines [15], which highlights its pro-inflammatory role. The levels of sTREM-1 were similar between the groups, perhaps because the patients were in steady state. There are no previous reports on sTREM-1 in SCA. It would be valuable to study sTREM-1 levels in sickle cell disease patients in vaso-occlusive crisis, and whether this correlates with other measures of severity.
IL-10 levels were higher in the Hb-SS group than the others, as previously reported, both in Hb-SS individuals in crisis or in steady state when compared to healthy individuals [8], that could indicate compensating mechanism. IL-10 levels in Hb-AS individuals were lower than in Hb-SS ones, what, along with similar levels in the Hb-AA group, corroborates the idea that Hb-AS individuals do not have a chronic inflammatory state. Based on these results, an analysis of the ratio between inflammatory cytokines and IL-10 was performed in order to estimate a suppression profile. It was seen that, in Hb-SS individuals in steady state, the ratios of IL-1β, IL-6 and TNF-α followed the same pattern as the levels of these cytokines in the groups, showing a minor influence of IL-10 on these cytokines, while the ratios of IL-8 and sTREM-1, which are related, respectively, to the recruitment and activation of neutrophils, showed a profile more suppressed by IL-10. Thus, we could hypothesize that in Hb-SS individuals in a steady state the role of neutrophils in the chronic inflammatory process is less intense and that this inflammatory state is mediated especially by IL-1 β, IL-6 and TNF-α.

CONCLUSION
The

FUNDING INFORMATION
This publication was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Supeperior (CAPES).

CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The project was approved by the Ethics Committee in Research Involving Human Beings of the Federal University of Sergipe (CEP HU/UFS), through serial number 2.897.835. Individuals who expressed their consent by appreciating and signing the Free and Informed Consent Term participated in the study.