The enrichment of T-regulatory cells as a potential therapeutic approach for cerebral vasculopathy in sickle cell disease

Abstract Cerebral vasculopathy is a major contributor to morbidity and mortality in sickle cell disease (SCD). The complex interactions between altered hematologic and vascular factors lead to cerebrovascular injury. In SCD, chronic anemia and presence of hemoglobin S (HbS) cause an increase in cerebral blood flow (CBF), impair cerebrovascular reserve and increase the risk of ischemic injuries. These events in association with intimal hyperplasia, progressive stenosis of large arteries such as the internal carotid, and intraluminal thrombosis are responsible for overt and silent strokes. Moreover, intravascular hemolysis and autooxidation of HbS release heme, which promotes endothelial dysfunction, inflammation, and thrombogenesis. These processes may worsen cerebral perfusion and induce vessel remodeling, resulting in acute and chronic cerebrovascular complications. To investigate whether intravascular elevation of heme alters cerebrovascular architecture and CBF, we infused ferric heme (20 mmoles/kg bw) in SCD (SS) mice. Magnetic resonance angiography (MRA) revealed both vasoconstriction and aneurysmal lesions following heme challenge. Two-photon laser scanning microscopy of cerebral blood vessels exhibited alterations in cerebral microvascular caliber, characterized by sparse large pial vessels. These events were associated with substantial reduction in CBF and increased thrombogenesis in isolated cerebral microvessels from SS mice. Cerebrovascular injury is accompanied by accumulation of regulatory T cells (Tregs), a specialized subset of T lymphocytes (CD3+CD4+CD25+Foxp3+). Tregs play critical roles preserving cerebrovascular integrity, and their depletion impairs functional recovery after cerebral injury. In SCD, studies have reported altered Treg frequency and function, but it remains unclear whether reduced Treg numbers directly contribute to increased susceptibility to cerebrovascular injury. We hypothesized that adequate accumulation of Tregs may promote vasodilation and augment CBF following acute hemolytic events in SCD, potentially mitigating ischemic complications. We found that 24h after acute heme challenge, Tregs were substantially reduced in the SS mice brain (n=5; p<0.01). This reduction was accompanied by acute vasoconstriction and a decrease in CBF (n=5; p<0.01). Interestingly, Tregs started accumulating in the SS mice brain 48 hours following heme challenge, which was accompanied by a partial restoration of the CBF (n=4). Treatment with IL-2 protein and IL-2 antibody complex (IL2/IL-2 Ab; 2:1 molar ratio) is an established approach to boost the number and function of Tregs. Thus, we injected SS mice with IL2/IL-2 Ab complexes intravenously for three consecutive days followed by intravenous heme injection. In comparison to IgG-injected control SS mice, the IL2/IL-2 Ab complex-treated SS mice exhibited a higher accumulation of regulatory Tregs (n=6; p<0.001) in their brains after 24 hours. Notably, the CBF was preserved in IL2/IL-2 Ab complex-treated SS mice, which was associated with reduction in vasoconstriction and microvascular thrombin accumulation. Overall, our findings suggest that the increased accumulation of Tregs in the brain augments CBF and improves the morphological homeostasis of the cerebral microvasculature in SCD. Collectively, our results support the therapeutic potential of Treg-based immunomodulation to mitigate cerebral vasculopathy in SCD. Future studies are warranted to define the mechanisms by which Tregs confer vascular protection and to evaluate the long-term efficacy and safety of this approach in preclinical and clinical settings.