Patients with Sickle Cell Disease Treated with Hidroxiurea Have Higher Expression of PD-L1 in Monocytes

Background: Inflammation plays a key role in pathophysiology of sickle cell disease (SCD). Monocytes are important mediator cells in the inflammation pathway. Despite some evidence that monocytes are more activated in SCD and can trigger vaso-occlusion, there is a remarkable lack of knowledge about how monocytes behave in patients with SCD and whether they are modulated by different treatments.

Objective: To assess possible changes in monocyte maturation and protein expression among sickle cell disease patients in different treatment modalities and compare them to healthy subjects.

Methods: 25 patients with SCD followed at the outpatient service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and 3 healthy controls were included. The inclusion criteria were: patients with sickle cell anemia (HbSS), over 18 years of age, without acute complications in the prior three months. Pregnancy was an exclusion criteria. Patients were divided in three groups: treatment with Hydroxyurea (HU) (n=10) using the same dose for at least three months; chronic exchange transfusion (n=9) (TP); and no specific treatment (NST) (n=6). We collected 3-5 ml of blood and performed peripheral blood mononuclear cells (PBMC) separation. Monocytes were analyzed by flow cytometry (Dx Flex, Beckman Coulter), using CD14 and CD16 as markers of subpopulations of monocytes as classical (CD14+/CD16-), intermediate (CD14+/CD16+) and non-classical (CD14-/CD16+). We also assessed the expression of CD 86 (a co-stimulation chemokine receptor), CD 163 (receptor for the conjugate hemoglobin-haptoglobin) and PD-L1 (an inhibitor receptor). Comparison between groups were made using ANOVA and Tukey test.

Results: Gender distribution yielded no significant difference. Age varied as shown: HU 30 ± 8y, TP 37 ± 9y, NST 35 ± 11y and 42 ± 19y for the controls. The mean of total leukocytes and monocytes were, respectively: 8,816 ± 2,133/mm³ and 900 ± 563/mm³ (HU), 11,487 ± 3,813/mm³ and 1,343 ± 754/mm³ (TP), 9,081 ± 1879/mm³ and 1,373 ± 432/mm³ (NST), 7,080 ± 642/mm³ and 700 ± 0/mm³ (control).Among the total nucleated cells, the percentage of classical monocytes was lower in HU compared with. Controls (3.8 ± 2.1% vs. 7.7 ± 1.3%, p 0.008), lower in TP vs NST (2.4 ± 1.8% vs. 5.6 ± 2.8%, p 0.04) and in TP vs Controls (2.4 ± 1.8% vs. 7.7 ± 1.3%, p 0.001). No differences were shown in the intermediate monocytes population. In turn, nonclassical monocytes were higher in HU compared with TP (0.44 ± 0.3% vs. 0.22 ± 0.2%, p 0.013) and in controls vs TP ( 0.35 ± 0.07% vs 0.22 ± 0.2%, p 0.017). The expression of CD 86 and CD 163 was not significantly different between any of the subsets of monocytes. In classical monocytes, expression of PD-L1 was higher in HU compared with NST (86.1 ± 17.4% vs. 43.9 ± 31.0%, p 0.015 ) and HU vs Controls ( 86.1 ± 17.4% vs. 34.9 ± 8.9%, p 0.019). Expression of PD-L1 in intermediate monocytes and in non classical monocytes were also higher in HU compared with controls (88.9 ± 16.3% vs. 58.6 ± 8.6%, p 0.02 and 79.6 ± 30.2% vs. 37.1 ± 4.6% , p 0.02 respectively). These results are depicted in table and figure 1.

Conclusion: Different treatments for sickle cell disease have affected the monocyte subsets distribution and PD-L1 expression in the population studied. HU and Transfusion Program induced a reduction in the Classical Monocyte subset, more pronounced in the latter. Classical monocytes are largely associated with a pro-inflammatory profile. The use of HU in SCD has been previously associated with less classical monocytes, and induction of monocyte maturation might help explain this finding; as for chronic transfusions, a migratory effect of monocytes to tissues has been proposed. The higher expression of PD-L1 in patients submitted to exchange transfusion and in use of HU indicate inhibition of inflammation, which is more pronounced in the HU group. Both treatments have immunomodulatory effects on SCD, however, to date, no inflammatory markers that assess response to treatment are available. Our findings might contribute to uncovering the role of PD-L1 in the chronic inflammation of SCD and to identify a potential biomarker. Nevertheless, further studies with larger cohorts are warranted in these settings.

No relevant conflicts of interest to declare.