Pharmacological Inhibition of TLR4 Reduces Mast Cell Activation, Neuroinflammation and Hyperalgesia in Sickle Mice

Sickle cell disease (SCD) is characterized by chronic hemolysis, inflammation, vascular dysfunction, and pain. Earlier we showed that mast cell activation contributes to neuroinflammation and pain and is accompanied by increased toll-like receptor 4 (TLR4) expression on mast cells (Vincent et al., Blood 2013), and that genetic deletion of TLR4 ameliorates neurogenic inflammation and hyperalgesia in HbSS-BERK sickle mice. Several other studies have shown increased TLR4 expression in peripheral system and its involvement in sickle pathobiology. We propose that free heme, due to hemolysis, activates TLR4 in the central nervous system in addition to peripheral activation, which further exacerbates neuroinflammation and hyperalgesia. Spinal cords of HbSS-BERK sickle mice show 3-fold mRNA transcripts for TLR4 and a 2-fold increase in hemin as compared to the spinal cords of HbAA-BERK control mice. Therefore, targeting TLR4 with pharmacological inhibitors may provide a therapeutic approach to attenuate peripheral and central inflammation and hyperalgesia. In the present study we examined the potential of pharmacological inhibition of mast cell activation, neuroinflammation and hyperalgesia in HbSS-BERK sickle mice with TLR4 inhibitor, TAK242. Sickle mice were administrated intravenously with TLR4 inhibitor TAK242 (1 mg/kg body weight/day) for 5 days. Sensory testing was performed at baseline at recruitment and periodically during the 5-day treatment and for another 8 days after concluding the treatment to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the 5-day treatment with TAK242, release of cytokines, tryptase (marker of mast cell activation) and substance P released from skin biopsies and spinal cords were analyzed by ELISA. TAK242 significantly decreased the release of tryptase (TAK242: 5.178 ± 0.7613 pg/ml vs vehicle: 8.801 ± 0.9403 pg/ml, p = 0.0181), substance P (TAK242: 11.56 ± 1.945 pg/ml vs vehicle: 25.51 ± 4.283 pg/ml, p = 0.018), and IL-6 (TAK242: 15.59 ± 0.4541 pg/ml vs vehicle: 29.74 ± 0.8249 pg/ml, p = 0.0045) from skin biopsies, suggesting that TAK242 reduced SCD-induced mast cell activation and inflammation. TAK242 also significantly decreased substance P (TAK242: 0.7198 ± 0.0587 pg/mg vs vehicle: 0.931 ± 0.0676 pg/mg, p = 0.0462) and phosphorylation of p38/MAPK (p = 0.0184) in the spinal cord, as well as dorsal cutaneous blood flow (TAK242: 6.392 ± 0.3857 PU vs vehicle: 12.32 ± 0.5575 PU, p < 0.0001), indicating that TAK242 ameliorated SCD-evoked central and peripheral activation of inflammation and nociceptive mechanisms. Furthermore, TAK242 administration gradually reduced the mechanical, deep tissue, and thermal hyperalgesia upto 5-day treatment (p < 0.01, vs vehicle HbSS). However, discontinuation of treatment led to a gradual increase in hyperalgesia observed upto day-8 post-treatment. TAK242 also significantly decreased acute pain induced by hypoxia/reoxygenation and accelerated recovery from injury of hypoxia/reoxygenation. These data reveal the significant therapeutic effect of pharmacological inhibition of TLR4 on inflammation and hyperalgesia in sickle mice. Therapies targeting TLR4 inhibition may be potentially beneficial in ameliorating sickle pathobiology and pain.