Sickle cell disease (SCD) is one of the most prevalent monogenic disease in the world caused by a one point mutation at position 6 of the β-globin gene, which results in abnormal production of hemoglobin S (1–3). SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to diverse clinical complications, such as chronic hemolytic anemia, splenic sequestration crises, pain, acute chest syndrome, strokes, leg ulcers, retinopathy, dactylitis, priapism, pulmonary hypertension, heart failure, and increased susceptibility to infections (1). The hallmarks of the disease are the vaso-occlusion crises (VOC), severe pain episodes characterized by tissue ischemia and infarction (1, 4), associated with increased levels of soluble VCAM-1, ICAM-1, endothelin-1, IL-6, IL-8, and TNF-α (5–9).

The pathogenesis of SCD is a multifactorial process that comprehends recurrent vaso-occlusions, ischemia-reperfusion injury and oxidative stress (10). In SCD patients, hemoglobin S polymerizes at low oxygen levels, resulting in altered cellular architecture of red blood cells (RBC) and exposure of adhesion molecules on the cell surface, including CD36, CD47, CD49d, and BCAM/Lu (11–14). Sickle red blood cells have a reduced lifespan, leading to accelerated red cell turnover. Circulating reticulocytes, increased in number, express high concentrations of adhesion molecules, contributing to increased adhesiveness of the sickle cells and the occurrence of VOC (11, 15). In addition, sickle cells are more susceptible to lysis and release of free hemoglobin, lactate dehydrogenase (LDH) and arginase in the plasma (16, 17). Free hemoglobin and circulating arginase consume nitric oxide (NO), an important vasodilator (18), further contributing to occlusive crises. Free hemoglobin also releases heme molecules, which in turn activate the immune system and thereby intensify VOC episodes (19, 20).

The vascular endothelium contributes to the inflammatory process in SCD patients. During VOC, damaged endothelial cells trigger the coagulation cascade and induce expression of VCAM-1 (cell-vascular adhesion molecule 1), ICAM-1 (intercellular adhesion molecule 1), P-selectin and E-selectin, which facilitate the adherence of blood cells to the endothelium (17). Endothelial cells produce endothelin-1, not only a potent vasoconstrictor, but also a pro-inflammatory molecule that increases levels of soluble VCAM-1 and ICAM-1 (21, 22), stimulates monocytes to secrete inflammatory cytokines in vitro (23) and induces production of superoxide by neutrophils in vitro (24, 25).

The management of SCD is a challenge (3, 26, 27) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment (28–31). To date, hundreds of SCD patients have already been successfully transplanted worldwide (29, 31). Patients with HLA-identical sibling donors have high overall and event-free survival rates, ranging from 90% to 100% (29, 32). The graft rejection rate (graft failure) varies extensively among different studies (31) and addition of anti-thymocyte globulin (ATG) to the transplant regimen is associated with a decrease in the incidence of graft rejection from 22.6% to 3% (33).

To our knowledge, the effects of allo-HSCT on the pathophysiological processes of SCD have not yet been fully investigated. We would like to understand if systemic inflammation and endothelial dysfunction subside after transplantation and how these events correlate with clinical complications, such as secondary graft failure and graft versus host disease (GVHD). Therefore, in this work we assessed red blood cell adhesiveness, intensity of hemolysis, vascular tone markers and systemic inflammation, in SCD patients treated with allo-HSCT.

This study included 32 SCD patients who were treated with allogeneic HSCT. Patients were followed-up for a mean (standard deviation) time of 30 (13) months, and clustered according to post-transplant chimerism determined by VNTR. Two patients from the graft failure group died at 7 and 49 months after transplantation, both due to infectious complications.

Hematological parameters normalize in SCD patients successfully engrafted following allogeneic HSCT (29, 33, 38–43). Indeed, here we show that HSCT modifies key elements associated with SCD pathogenesis.

Sickle-cell patients have increased numbers of reticulocytes, with high expression of adhesion molecules such as CD49d, CD36 and Lu-BCAM (11). Accordingly, we detected increased percentages of reticulocytes expressing CD49d in our SCD patients at baseline, when compared to controls. After HSCT, patients with successful engraftment had lower frequencies of reticulocytes expressing CD49d and CD36 than patients with graft failure, suggesting that HSCT substitutes highly adherent abnormal cells by normal adherent cells. A similar mechanism has been described in sickle cell patients under treatment with hydroxyurea, with decreased expression of adhesion molecules, such as CD36 and CD49d, on reticulocytes (11, 44).

Our SCD patients also had higher percentage of reticulocytes expressing CD47 at baseline when compared to the healthy donors. These percentages were reduced after transplantation in patients with good engraftment. Surprisingly, however, the expression of CD47 was increased in mature RBC of SCD after transplantation. CD47 is involved with increased cell adhesion in SCD patients as part of disease mechanism (45, 46). CD47 expressed on SCD reticulocytes is able to bind to thrombospondin, resulting in VLA-4 activation, thereby increasing their adhesion properties (45, 46). CD47 also plays an important role in regulating the clearance of senescent RBC by macrophages, as it binds to macrophage inhibitory receptors (SIRPα) and prevents phagocytosis (47, 48). This may explain the greater expression of this molecule in mature RBC after transplantation.

Plasma concentrations of P-selectin, VCAM-1 and ICAM-1 are increased in SCD patients, a phenomenon associated with severe clinical symptoms and tissue damage (6, 7, 9, 49, 50). In our SCD patients, however, baseline concentrations of P-selectin were similar to those from healthy controls, whereas concentrations of ICAM-1 were lower than controls. VCAM-1 plasma concentrations, conversely, were higher than controls at baseline and decreased significantly after HSCT, especially in patients with good engraftment. The divergences between ICAM-1 and P-selectin levels measured in our patients and those reported by the literature may be due to number of evaluated patients and should be further evaluated in future studies.

Lactate dehydrogenase (LDH) levels were reduced up to 5 years after HSCT in patients with good engraftment, which is in accordance with the successful hematological recovery observed in our patients. Although LDH has been established as a biomarker of hemolysis and is associated with severe clinical symptoms of SCD, such as priapism, leg ulcers, pulmonary hypertension and death (16), it also reflects tissue damage, which is involved in the pathogenesis of the disease (51). Thus, the decreasing levels of LDH may reflect not only an improvement of hemolysis, but also from reduction in tissue damage and ischemia.

In our patients, IL-15 and IL-18 levels remained elevated in SCD patients who experienced graft failure, in contrast with those with sustained engraftment. It is not clear if the quantitative changes of these cytokines are cause or consequence of graft failure since autologous recovery of abnormal hematopoiesis after graft failure may also resume the pre-transplantation inflammatory status. Organ transplantation studies suggest that IL-15 mediates allograft rejection mainly by NK activation (52) and IL-18 has been associated with poor allograft function (53). Conversely, IL-18 is associated with hemolysis and endothelial activation in SCD patients, suggesting that this cytokine may contribute to vaso-occlusion pathophysiology (54). Interestingly, Cerqueira and collaborators (54) demonstrated a positive correlation between IL-18 and uric acid plasma levels in SCD patients. Uric acid is a damage-associated molecular pattern (DAMP) and may trigger the NLRP3 inflammasome pathway, resulting in secretion of IL-1β and IL-18 (54). In our study, however, we were not able to find an association between uric acid levels and clinical outcomes, possibly because of the administration of cyclosporine A to the patients, a known urate retention agent (data not shown). Nevertheless, we believe that NLRP3 inflammasome activation could be a possible underlying mechanism of IL-18 production in SCD patients that underwent graft failure after HSCT. The participation of NLRP3 inflammasome and possible triggers in graft failure should be more specifically investigated in future studies.

Heme levels in our SCD patients were not higher than control levels at baseline, possibly because patients were clinically stable immediately before transplantation, not presenting sickle cell crises or severe anemia. Shortly after HSCT, procedure-related events may have contributed to transient increase in heme levels, since toxicity from the conditioning regimen, infections, blood component transfusions with high levels of free heme, ABO incompatibility, adverse effects from drugs and graft versus host disease (GVHD) may activate the endothelium and the immune system (55–57). Later on follow-up, heme levels declined and resumed normal levels, which can be considered another indication of favorable outcomes after HSCT. Heme is critical for endothelial and inflammatory activation in SCD (17, 19, 58, 59). Indeed, in patients who presented graft failure, heme levels increased after 2 years, due to recurrence of hemolytic anemia and, probably, of the endothelial dysfunction, as supported by the VCAM-1 levels. Furthermore, heme has also been described as an activator molecule of NLRP3 inflammasome (60), which may explain the increased levels of IL-18 observed in SCD patients at pre-transplantation and in those who underwent graft failure.

Recent reports have associated high levels of pre-transplant IL-18 with non-relapse and overall mortalities (61) and delayed platelet recovery in transplanted patients (62). We speculate that the mixed chimerism achieved after allogeneic HSCT, marked by 20-40% HbS in recipient sickle red blood cells, may lead to release of endogenous ligands that trigger NLRP3 inflammasome activation (such as heme, uric acid). This activation may initiate the secretion of mature forms of IL-18 from endothelial cells to promote further inflammatory processes and oxidative stress in the endothelium. Administration of immunotherapeutic agents (such as drugs targeting NLRP3 inflammasome pathway) may diminish endothelial activation and, as consequence, decrease systemic inflammation in SCD patients after HSCT. Future studies addressing the role of NLRP3 inflammasome activation in graft failure may shed light on HSCT protocol improvements for SCD patients.

Finally, considering the pivotal role of endothelial function in clinical events associated with SCD, we evaluated mediators of vascular tonus associated with VOC in SCD patients (12, 63, 64). Endothelin-1 levels were increased at baseline, when compared to healthy controls, and decreased after HSCT, especially in patients with good engraftment. Baseline endothelin-1 plasma levels in patients who later developed graft failure were already higher than in patients with good engraftment after HSCT. However, ROC curve analyses failed to demonstrate associations between levels of endothelin-1 at baseline and transplant outcomes, probably due to reduced number of patients analyzed. A potential predictive role of endothelin-1 should be tested in a larger cohort of SCD patients treated with HSCT.

Notably, the presence of acute GVHD in our cohort of patients seem not to affect the assessment of systemic inflammatory parameters, except for the levels of HGF and TGF-β. The levels of HGF were increased early post-transplant in SCD patients with aGVHD, which is in accordance with the literature that have described HGF as an important biomarker for aGVHD occurrence (65, 66).

We acknowledge here some limitations of this work. First, this study lacked a treatment control and a non-SCD transplanted control group of patients for comparisons. We would need an age-matched group of patients with other hematological reconstitution, such as aplastic anemia, also treated with allo-HSCT, for demonstrating whether the sustained systemic inflammation after is specific of SCD. Second, the number of overall patients evaluated in this study is small for biomarkers and multivariate analyses. Thereby, statistical analyses are difficulted and there is a potential risk of bias, which should be acknowledged. Furthermore, these data need to be validated in other carefully designed controlled clinical studies and/or in other cohorts of patients.

Here, we showed that systemic inflammation persists in SCD patients long-term after allo-HSCT, evidenced by altered levels of plasma pro-inflammatory cytokines (mainly TNF-α) adhesion molecules (P-selectin and ICAM-1), indicating that transplantation does not equally affect all aspects of SCD pathogenesis. At 24 months or later after transplantation, TNF-α levels were persistently high. In SCD patients who underwent graft failure, IL-18 and IL-15 levels were significantly increased at 24 months after allo-HSCT. Following transplantation, we observed increased production of healthy mature erythrocytes, decreased circulating reticulocyte counts and adhesiveness, decreased hemolysis, and improved vascular tone markers in SCD patients with successful engraftment.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics Committee of the Ribeirão Preto Medical School, University of São Paulo. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Study concept and design: KM, BS, and MO. Study supervision: KM, BS, and MO. Acquisition of clinical data: TC, LD-J, CS, AS, JE, FP, RG-C, AP, GS, BS, and MO. Acquisition of laboratorial data: JA, TM, KL, and PP. Analysis and interpretation of data: JA, TC, KM, and MO. Administrative, technical, or material support: DC and OH. Obtained funding: KM, BS, MO, DC, and OH. Drafting of the manuscript: JA, TC, KM, and MO. Critical revision of the manuscript: JA, TC, TM, KL, PP, TC, LD-J, CS, AS, JE, FP, RG-C, AP, GS, DC, OH, BS, and MO. All authors contributed to the article and approved the submitted version.

This work was supported by two Brazilian foundations: São Paulo Research Foundation (Center for Cell-Based Research, CTC-CEPID-FAPESP, grant #2013/08135-2; grant #2014/00088-8, grant #2016/22330-0) and the Coordination for the Improvement of Higher Education Personnel (CAPES; Finance Code 001). This work was supported by State funding from the French National Research Agency under “Investissements d’Avenir” program (ANR-10-IAHU-01) and ANR-16-CE14-0024-03 «Tiebet ». JTC de Azevedo was supported by a labex GR-Ex fellowship. The labex GR-Ex, reference ANR-11-LABX-0051, is funded by the program “Investissements d’avenir” of the French National Research Agency, reference ANR-11-IDEX-0005-02.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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