Sphingosine-1-Phosphate (S1P) Signaling through S1P Receptor 1 (S1PR1) in Macrophage Contributes to Pain in Sickle Cell Disease

Sickle cell disease (SCD) is the most common autosomal recessive hemolytic disorder affecting more than 100,000 African-Americans and millions around the world. The severe health complication of patients with SCD is chronic pain, which is the leading cause of SCD patient morbidity and hospitalization. Our proposed research builds on the results from our nonbiased high-throughput metabolomics screen, which revealed erythrocyte sphingosine-1-phosphate (S1P), a bioactive lipid, is significantly elevated in the circulation of humans and mice with SCD. We identified that erythrocyte-sphingosine kinase 1 (Sphk1), the enzyme that generates S1P, promote erythrocyte sickling and the genetic deletion of Sphk1 significantly reduce pain symptoms in SCD mice. S1P is a pleiotropic molecule that signals via five g-protein coupled receptors (GPCRs) known as S1P receptors (S1PR1-5). S1PR1 is widely expressed in many cell types, including neurons and immune cells. We treated SCD mice with FDA approved drug, FTY720, to specifically target S1PR1. We demonstrated that mice had improved mechanical and thermal pain sensitivity. Notably, using flow cytometry, we found that the percentage of macrophages infiltrated to skin and percentage of S1PR1 expression in macrophages in the skin were significantly induced in SCD mice compared to the controls. This implies that elevated S1PR1 in macrophage contributes to chronic pain in SCD mice. To test this possibility, we treated SCD mice with clodronate liposomes to deplete tissue macrophages. We identified that mice had reduced sensitivity to mechanical and thermal stimuli assessments. Altogether, we provide both genetic and pharmacological evidence that elevated plasma S1P signaling via S1PR1 likely on macrophages contributes to chronic pain in SCD. Additionally, S1PR1 is known to mediate pro-inflammatory cytokine secretion in immune cells. We identified that SCD mice have elevated levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, and is likely involved in SCD pain. To study the role of IL-6 in SCD pain, we generated SCD-IL-6^-/- mice and observed improved mechanical and thermal pain sensitivity in these mice. From these evidences, we hypothesize that Sphk1 mediate S1P-S1PR1 activation in macrophages and IL-6 induction to promote SCD pain. Our work addresses a knowledge gap regarding an inflammatory mediated mechanism involved in SCD pain and novel therapies to counteract pain in SCD.