Pharmacological Inhibition of TLR4 Reduces Mast Cell Activation, Neuroinflammation and Hyperalgesia in Sickle Mice
Pharmacological Inhibition of TLR4 Reduces Mast Cell Activation, Neuroinflammation and Hyperalgesia in Sickle Mice
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TLR4 的药理抑制可减少镰状小鼠肥大细胞激活、神经炎症和痛觉过敏
DOI：
10.1182/blood.v126.23.278.278
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发表时间：
2015
期刊：
影响因子：
20.3
通讯作者：
K. Gupta
中科院分区：
文献类型：
--
作者：
Jianxun Lei;Ying Wang;J. Paul;Susan T. Thompson;Ritu Jha;K. Gupta
镰状细胞病（SCD）的特征是慢性溶血、炎症、血管功能障碍和疼痛。早些时候，我们表明肥大细胞活化有助于神经炎症和疼痛，并且伴随着肥大细胞上toll样受体4（TLR4）表达的增加（Vincent et al.，Blood 2013），并且TLR4的遗传缺失改善HbSS-BERK镰状小鼠中的神经源性炎症和痛觉过敏。其他一些研究表明，外周系统中TLR4表达增加，并参与镰状病理生物学。我们认为，游离血红素，由于溶血，激活TLR4在中枢神经系统除了外周激活，这进一步加剧了神经炎症和痛觉过敏。与HbAA-BERK对照小鼠的脊髓相比，HbSS-BERK镰状小鼠的脊髓显示TLR4的3倍mRNA转录和氯化血红素的2倍增加。因此，用药理学抑制剂靶向TLR4可以提供减轻外周和中枢炎症和痛觉过敏的治疗方法。在本研究中，我们研究了TLR4抑制剂TAK242对HbSS-BERK镰状小鼠肥大细胞活化、神经炎症和痛觉过敏的药理学抑制潜力。对镰状小鼠静脉内施用TLR4抑制剂TAK242（lmg/kg体重/天）5天。在招募时的基线和5天治疗期间定期进行感觉测试，并在治疗结束后再进行8天，以评价von Frey细丝的机械痛觉过敏、热/冷反应的热痛觉过敏和肌肉骨骼/深部组织痛觉过敏的握力。在用TAK242处理5天后，通过ELISA分析从皮肤活组织检查和脊髓释放的细胞因子、类胰蛋白酶（肥大细胞活化的标志物）和P物质的释放。TAK242显著降低类胰蛋白酶的释放（TAK 242：5.178 ± 0.7613 pg/ml vs溶剂：8.801 ± 0.9403 pg/ml，p = 0.0181），P物质（TAK 242：11.56 ± 1.945 pg/ml vs溶媒：25.51 ± 4.283 pg/ml，p = 0.018）和IL-6（TAK242：15.59 ± 0.4541 pg/ml vs溶媒：29.74 ± 0.8249 pg/ml，p = 0.0045），表明TAK242减少SCD诱导的肥大细胞活化和炎症。TAK242还显著降低P物质（TAK242：0.7198 ± 0.0587 pg/mg vs溶媒：0.931 ± 0.0676 pg/mg，p = 0.0462）和p38/MAPK磷酸化（p = 0.0184），以及背侧皮肤血流量（TAK242：6.392 ± 0.3857 PU vs溶媒：12.32 ± 0.5575 PU，p <0.0001），表明TAK242改善了SCD诱发的炎症和伤害感受机制的中枢和外周激活。此外，TAK 242给药逐渐减少了机械、深层组织和热痛觉过敏，直至5天治疗（p <0.01，vs溶媒HbSS）。然而，停止治疗导致治疗后第8天观察到的痛觉过敏逐渐增加。TAK 242还显著降低缺氧/复氧引起的急性疼痛，并加速缺氧/复氧损伤的恢复。这些数据揭示了药理学抑制TLR4对镰状小鼠的炎症和痛觉过敏的显著治疗效果。靶向TLR4抑制的疗法可能在改善镰状病理学和疼痛方面是潜在有益的。
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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.
Disclosures No relevant conflicts of interest to declare.