The pathophysiology of sickle cell disease (SCD) is mainly driven by intravascular hemolysis and recurrent vaso-occlusion, both leading to a cascade of complex interconnected pathophysiological processes 1. Several studies have demonstrated elevated inflammatory proteins in SCD, with interleukin-1β (IL-1β) and interleukin-6 (IL-6) recognized as important mediators of immune activation 2. Hemolysis-induced free heme results in NLRP3 inflammasome activation through TLR4 on macrophages and contributes to the pro-inflammatory profile in SCD 3. IL-18, another pro-inflammatory cytokine upregulated by the NLRP3 inflammasome, is involved in neutrophil recruitment and vaso-occlusion, and has been associated with SCD-related cardiomyopathy 4, 5. Furthermore, pro-angiogenic markers such as angiopoietins and VEGF are often elevated in SCD patients, reflecting neovascularization in response to tissue hypoxia 6. An increased ANGPT-2/ANGPT-1 ratio results in vascular destabilization and increased endothelial permeability and has been associated with SCD complications such as retinopathy, vaso-occlusive episodes (VOE) and acute chest syndrome 6, 7. Although allogeneic hematopoietic cell transplantation (HCT) eliminates the chronic hemolysis, already established vascular pathology could perpetuate inflammatory and angiogenic cascades despite normal hemoglobin production. Moreover, HCT itself can activate inflammatory responses through recurrent infections and graft-versus-host disease (GvHD). In this study, we assessed the effects of HCT on pro-inflammatory proteins, anti-inflammatory/immunoregulatory proteins, proteins involved in angiogenesis and hypoxic regulators. Furthermore, we examined associations of these protein values with pre-transplantation disease characteristics and treatment, and post-transplantation complications, treatments, and donor-related factors. Adults with SCD and an indication for allogeneic HCT were eligible for this longitudinal cohort study. Candidates for matched sibling donor (MSD) transplantation received preconditioning with azathioprine and hydroxyurea for 3 months, followed by a non-myeloablative conditioning with alemtuzumab and 3 Gy total body irradiation (TBI) 8. This approach results in mixed chimerism. Recipients of haploidentical transplantations received a reduced-intensity conditioning with thiotepa and post-transplantation cyclophosphamide 9. This approach generally results in full donor chimerism. All patients received sirolimus for approximately 1 year post-transplantation. EDTA plasma samples were collected at baseline (pre-transplantation) and at > 12 months post-transplantation. A panel of 24 proteins was selected from the Olink Flex library. An overview of these proteins and their functions is summarized in Table S1. All proteins were analyzed using Olink's proximity extension assay (PEA) technology as described in the supplementary methods. Olink provided reference values of 15 unmatched, healthy adult donors. Changes in protein values after transplantation as compared to baseline were assessed, using a paired t-test or Wilcoxon signed-rank test as appropriate. To identify potential effect modifiers, we compared changes in values between subgroups (e.g., follow-up timepoint, donor type) using Mann–Whitney U tests. Benjamini-Hochberg's false discovery rate (FDR) correction was used for multiple testing (R version 2024.12.1). Thirty four patients who underwent HCT for SCD at Amsterdam UMC were included (50% female, median age 27 (range 18–49), Table 1). Most patients (n = 25, 74%) had follow-up samples collected at > 24 months post-transplant, while 9 patients (26%) were sampled at 12 months. A comparison of baseline values in SCD patients with reference values is shown in Table S2. Compared to reference medians, SCD patients had elevated values of pro-inflammatory (IL-1β, IL-18, IL-8, PTX3), immunoregulatory (TACI, BAFF-R, IL-10, CD200, CD200R1, IL-1RN), angiogenesis-related markers (ANGPT-1, ANGPT-2, VEGF-A, CXCL12) and hypoxia-related (EPO and EGLN1) proteins. Associations between the inflammatory and angiogenic proteins with routine laboratory markers of hemolysis and inflammation are shown in Figure S1 and detailed in the Supplementary Results. No potential associations between protein values and baseline characteristics (hydroxyurea use, chronic transfusion, azathioprine, severe genotype) remained significant after multiple testing correction (Supplementary Results, Figure S2). At > 12 months post-transplantation, a significant decline was observed in pro-inflammatory proteins IL-18 (p 24 months) only affected changes in IL-6 (p 24 months. Protein values at baseline were not predictive of GvHD occurrence and no association was found between protein values and chimerism levels within the MSD group (data not shown). In this study, we observed a profound pro-inflammatory and pro-angiogenic state in SCD patients, with significant decreases in protein values involved in inflammation, angiogenesis and hypoxic responses > 12 months post-transplantation. The important role of hemolysis as the driving force behind the pro-inflammatory state in SCD is supported by the association between total bilirubin levels and IL-18, a cytokine that promotes T helper cell differentiation and interferon-γ production. Importantly, our data show that cure by HCT not only reversed hemolysis but also successfully reversed the chronic inflammatory cascade, as illustrated by the significant decline in IL-18 values despite potential transplantation-related inflammation. This decline in IL-18 is particularly encouraging since high values are associated with VOE and organ complications, underscoring its potential as a biomarker for disease activity and successful transplantation 4, 5. While elevated IL-18 values at baseline and shortly after transplantation have been associated with an increased risk of GvHD, graft failure, and non-relapse mortality in other studies, our study population demonstrated favorable transplantation outcomes despite universally elevated baseline IL-18 values 10-12. Interestingly, the six patients who experienced declining donor chimerism warranting intervention to prevent graft rejection exhibited significantly elevated baseline IL-18 values compared to the patients without imminent graft rejection. Although the pro-inflammatory state dominates in SCD, there were also signs of compensatory anti-inflammatory mechanisms at baseline. While increased IL-10 values have been described previously, we additionally observed elevated IL-1RN values, a natural inhibitor of pro-inflammatory cytokines IL-1α and IL-1β. IL-1RN values correlated with leukocyte counts and bilirubin levels, suggesting a compensatory anti-inflammatory response proportional to the rate of hemolysis and ensuing inflammation. We also observed elevated CD200 and CD200R1 at baseline, an axis that is involved in reduction of inflammatory cytokine production, and has been described to protect against atherosclerosis 13. This might contribute to the relatively lower prevalence of atherosclerosis and coronary artery disease in SCD patients. After transplantation, CD200R1 values decreased significantly, potentially reflecting a normalization of compensatory anti-inflammatory responses due to reduced inflammatory burden. Disrupted angiogenic pathways in SCD are demonstrated by increased VEGF-A and CXCL12 values and an increased ANGPT-2/ANGPT-1 ratio. High ANGPT-2 values have been associated with pulmonary hypertension and other adverse clinical outcomes 7, 14. In the present study, HCT resulted in decreased ANGPT-2 values, restoring the ANGPT-2/ANGPT-1 ratio, and thereby potentially improving vascular stability. The restored angiopoietin ratio could contribute to a stabilizing effect of HCT on sickle cell retinopathy and to improvement of markers of pulmonary hypertension and cerebral blood flow 15-17. Importantly, CXCL12 values also declined significantly after transplantation. This pro-angiogenic and inflammatory chemokine is involved in revascularization of ischemic tissue and has also been associated with pulmonary hypertension in patients with SCD 18. Hypoxic response was also evident at baseline, as shown by elevated EPO values. The strong positive association between EPO values and HbF% has also been described in beta-thalassemia patients and is likely the result of the higher oxygen affinity of HbF compared to HbA 19. Resolution of hemolysis and concomitant decrease in HbF% following HCT illustrate the reversal of the hypoxic state characterizing SCD. Our study was limited by a lack of ethnicity-matched controls. Additionally, baseline samples were collected during azathioprine/hydroxyurea preconditioning in the majority of MSD recipients, which might have affected baseline values, potentially masking HCT-mediated improvements in pre-inflammatory proteins. Moreover, four baseline blood samples were collected shortly after exchange transfusion, potentially also masking HCT-mediated improvements, as we have recently shown that transfusion ameliorates inflammatory and angiogenic processes of SCD 20. Furthermore, the relatively small sample size and stringent multiple testing correction prevented robust subgroup analyses. To conclude, this study confirms that SCD patients have a pronounced pro-inflammatory and pro-angiogenic profile, with significant reduction of key inflammatory and angiogenic proteins following allogeneic HCT. Resolution of hemolysis and ischemia–reperfusion injury is likely the main driver of amelioration of chronic inflammation and angiogenesis post-transplantation. Future larger studies are needed to assess the role of pre-transplant pro-inflammatory proteins on post-transplant outcomes. Additionally, prospective studies should investigate whether the observed normalization of inflammatory and angiogenic markers translates to improvement in SCD-related organ damage. E.N. conceptualized the study. E.N. and E.D. enrolled and cared for patients. M.Z. organized measurements, gathered data, and performed the statistical analyses. M.Z. and E.N. drafted the manuscript. All authors critically reviewed the manuscript. The authors extend their gratitude to all the patients who participated in the study. This study was funded by the Dutch Sickle Cell Foundation (Het Sikkelcelfonds). This study was carried out in accordance with the principles of the Declaration of Helsinki (seventh revision, 2013). Written informed consent was provided by all participants. Bart J. Biemond: Novartis, BMS, Pfizer, Novo Nordisk (research funding); Pfizer, BMS/Celgene (advisory board consultancy); Novo Nordisk, Sanofi (honoraria for lectures and podcasts). Erfan Nur: Novartis (research funding), Vertex (speakers bureau), Roche (steering Committee) and other authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1: Supporting information. Table S1: Plasma proteins and function summary. Table S2: Quantified plasma protein values. Figure S1: Heatmap depicting associations between circulating plasma proteins and routine laboratory parameters at baseline. Red tiles represent positive associations, while blue tiles indicate negative associations. Color intensity is proportional to correlation coefficient magnitude (Spearman). Statistical significance following false discovery rate (FDR) correction is denoted by asterisk: * p < 0.05, ** p < 0.01, *** p < 0.001. Figure S2: Heatmap depicting associations between circulating plasma proteins and clinical parameters at baseline. Red tiles represent positive associations, while blue tiles indicate negative associations. Color intensity is proportional to the rank-biserial correlation (Mann–Whitney U test). No associations were significant after false discovery rate (FDR) correction. RBC, red blood cell. Figure S3: Longitudinal individual routine laboratory parameters at baseline and after cure by allogeneic hematopoietic cell transplantation (n = 34). Black lines show mean (normally distributed delta) or median (non-normally distributed delta) as appropriate. Red trajectories indicate significant changes after false discovery rate (FDR) correction. Light blue shaded areas represent validated reference ranges. Y-axis shows appropriate units: HbS %; Bilirubin total μmol/L; LDH U/L; Hemoglobin g/dL; Neutrophils, Reticulocytes, and Leukocytes ×109/L; Figure S4: Heatmap depicting associations between circulating plasma proteins and clinical parameters at follow-up. Red tiles represent positive associations, while blue tiles indicate negative associations. Color intensity is proportional to the rank-biserial correlation (Mann–Whitney U test). No associations were significant after false discovery rate (FDR) correction. GvHD, graft versus host disease. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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Zwolsman et al. (Wed,) studied this question.