Toll-Like Receptor 4 Knockout Attenuates Neurogenic Inflammation and Hyperalgesia In Sickle Mice

Pain in sickle cell anemia (SCA) is accompanied by inflammation, vascular dysfunction and ischemia/reperfusion (IR) injury. We found that activated cutaneous mast cells in HbSS-BERK sickle mice release cytokines and neuropeptides and stimulate Evans blue leakage from the vasculature resulting in neurogenic inflammation and hyperalgesia (Vincent et al., 2013, Blood). Toll-like receptor 4 (TLR4) signaling stimulates mast cell activation and plays a causative role in inflammatory and neuropathic pain. Skin mast cells and spinal cords from HbSS-BERK sickle mice showed a several-fold increase in gene expression of TLR4 transcripts as compared to control mice expressing normal human hemoglobin (p<0.001 & 0.01, respectively). We hypothesized that TLR4 mediates mast cell activation-induced neurogenic inflammation and hyperalgesia in SCA. In TLR4 knockout (KO) mice, response to lipopolysaccharide and thermal and mechanical stimuli is attenuated; and spinal glial activation and release of inflammatory cytokines are reduced with accompanying resistance to IR injury. We backcrossed HbSS-BERK mice expressing human sickle hemoglobin and HbAA-BERK control mice expressing normal human hemoglobin with TLR4-KO mice to obtain HbSS-BERK with TLR4-KO (TLR4-KO-SS), and littermate TLR4-KO, HbSS-BERK and HbAA-BERK to examine the contribution of TLR4 to chronic pain and IR-induced acute pain in SCA. Pain behaviors included grip force measurement for deep tissue pain, mechanical sensitivity to von Frey filaments for cutaneous hyperalgesia and sensitivity to heat and cold for thermal hyperalgesia, as described by us for sickle mice (Kohli et al., Blood 2010). The measure of mechanical threshold and suprathreshold to a 1.0 g von Frey fiber showed a significant reduction in mechanical sensitivity in TLR4-KO-SS as compared to HbSS-BERK (p<0.001 for both measures). Similarly, deep pain and thermal sensitivity were significantly reduced in TLR4-KO-SS as compared to HbSS-BERK (p<0.01 for each measure). All pain profiles in TLR4-KO-SS were similar to HbAA-BERK and TLR4-KO, suggestive of a contribution of TLR4 in chronic pain in sickle mice. We next examined pain evoked by hypoxia/reoxygenation (HR) simulating acute pain following vasoocclusive crisis (VOC). HR evoked a significant increase in mechanical and heat sensitivity and in deep tissue pain in HbSS-BERK mice, which was sustained for 7 days, last period of observation (p<0.05 Vs baseline at normoxia). In contrast, TLR4-KO-SS did not show a significant increase in any of the pain measures following HR, suggesting that TLR4 mediates HR-induced injury in SCA. To analyze neurogenic inflammation we quantified the leakage of Evans blue dye in response to PBS, substance P (SP) and capsaicin in the skin, 7 days after the incitement of HR. PBS-treated skin showed significantly increased leakage of Evans blue following HR in HbSS-BERK, as compared to HbSS-BERK under normoxia (p<0.01). In contrast, Evans blue leakage following HR in TLR4-KO-SS was significantly reduced as compared to HbSS-BERK under HR as well as under normoxia (p<0.01 and 0.05, respectively). In TLR4-KO-SS, SP- and capsaicin-induced Evans blue leakage was approximately 50% that of HbSS-BERK, following HR, demonstrating that TLR4 contributes to neurogenic inflammation in sickle mice. TLR4 is also expressed on endothelial cells of the vasculature, which may contribute to HR-evoked vascular dysfunction directly and also via neurogenic inflammation caused by mast cells and peripheral nerve fibers. We observed that mast cell degranulation was reduced by ∼50% in TLR4-KO-SS as compared to HbSS-BERK (p<0.001) and the number of mast cells were reduced by ∼90% in TLR4-KO-SS as compared to HbSS-BERK (p<0.001) under HR. It is likely that TLR4 also mediates the recruitment, and/or proliferation of mast cells in addition to activating the existent mast cells. Together, these data suggest that TLR4 contributes to mast cell degranulation, neurogenic inflammation and hyperalgesia in sickle mice. Therefore, targeting TLR4 with novel pharmacological antagonists/agents may reduce inflammation and pain and prevent IR injury in SCA.