Heme Causes Pain in Sickle Mice <i>via</i> Toll-Like Receptor 4-Mediated Reactive Oxygen Species- and Endoplasmic Reticulum Stress-Induced Glial Activation

<b><i>Aims:</i></b> 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 (<i>TLR4</i>), 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. <b><i>Results:</i></b> Spinal heme levels, TLR4 transcripts, oxidative stress, and ER stress were significantly higher in sickle mice than controls. <i>In vitro</i>, 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. <b><i>Innovation:</i></b> 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. <b><i>Conclusion:</i></b> Heme-induced microglial activation <i>via</i> TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain <i>via</i> ER stress in SCD. <i>Antioxid. Redox Signal</i>. 34, 279-293.