Aims: Lifelong pain is a hallmark feature of sickle cell disease (SCD). How sickle pathobiology evokes pain remains unknown. We hypothesize that increased cell-free heme due to ongoing hemolysis activates toll-like receptor 4 (TLR4), leading to the formation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Together, these processes lead to spinal microglial activation and neuroinflammation, culminating in acute and chronic pain. Results: Spinal heme levels, TLR4 transcripts, oxidative stress, and ER stress were significantly higher in sickle mice than controls. In vitro, TLR4 inhibition in spinal cord microglial cells attenuated heme-induced ROS and ER stress. Heme treatment led to a time-dependent increase in the characteristic features of sickle pain (mechanical and thermal hyperalgesia) in both sickle and control mice; this effect was absent in TLR4-knockout sickle and control mice. TLR4 deletion in sickle mice attenuated chronic and hypoxia/reoxygenation (H/R)-evoked acute hyperalgesia. Sickle mice treated with the TLR4 inhibitor resatorvid; selective small-molecule inhibitor of TLR4 (TAK242) had significantly reduced chronic hyperalgesia and had less severe H/R-evoked acute pain with quicker recovery. Notably, reducing ER stress with salubrinal ameliorated chronic hyperalgesia in sickle mice. Innovation: Our findings demonstrate the causal role of free heme in the genesis of acute and chronic sickle pain and suggest that TLR4 and/or ER stress are novel therapeutic targets for treating pain in SCD. Conclusion: Heme-induced microglial activation via TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain via ER stress in SCD. Antioxid. Redox Signal. 34, 279-293.

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