Project Summary: Pain is the most common clinical complication of sickle cell disease (SCD) from infancy through adulthood. Individuals with SCD experience severe, recurrent acute pain during a vaso-occlusive episode (VOE) that can last for days. Patients also develop lifelong chronic pain at steady state that occurs in addition to ongoing acute pain episodes. The acute and chronic pain in SCD is currently treated almost entirely with opioids, which have serious adverse side effects, addiction risk, and in some cases fail to completely alleviate pain or even exacerbate it further. The mechanistic causes of the intense, episodic pain that drives patients to seek emergency health care have been minimally investigated and present a major gap in understanding and treating acute SCD pain. Moreover, the causes of chronic SCD pain at steady state are insufficiently understood. Our proposal will tackle these critical gaps by using data derived from patients with SCD to drive our hypotheses that will be directly tested in a preclinical SCD mouse model. Metabolomic analysis of patient plasma showed that free heme was elevated in patients with SCD at steady state compared to healthy controls. Consistent with this, our preliminary data in SCD mice showed that scavenging heme with the heme binding protein haptoglobin alleviated steady state hypersensitivity. Furthermore, plasma taken from patients with SCD seeking acute pain care showed that TNFα gene transcription was highly increased in a reporter cell line. Consistent with this, our preliminary data in SCD mice showed that inhibition of TNFα using the FDA- approved TNFα antibody infliximab completely alleviated VOE-associated mechanical hypersensitivity. Taken together, these compelling data support our hypotheses that free heme instigates chronic, steady state pain in SCD through direct access to the dorsal root ganglia (DRG), while TNFα drives acute VOE-associated SCD pain. Furthermore, we hypothesize that both free heme and TNFα also mediate persistent and acute pain, respectively, by initiating immune cell infiltration into DRG sensory neurons that contribute to their sensitization. By exploring these mechanisms in the context of SCD pain, we may uncover novel therapeutic targets that provide more effective analgesia and avoid the harmful effects of prominently prescribed opioid therapy used for SCD pain. These studies may also reveal more about pain mechanisms present in other pain conditions where heme and TNFα are also dysregulated, including fibromyalgia, spinal disc herniation, arthritis, inflammatory bowel disease, ankylosing spondylitis, and psoriasis.