Heme Induced Spinal Microglial Cell Activation By TLR4 and Endoplasmic Reticulum Stress in Sickle Mice

Sickle cell disease (SCD) is associated with pain, which remains a major challenge to treat. Earlier, we showed that peripheral mechanisms including mast cell activation in the skin contribute to pain in sickle mice (Vincent et al., Blood 2013). Mast cell activation in sickle mice was accompanied by a significant increase in toll-like receptor 4 (TLR4) as compared to mast cells from control mice. Since peripheral as well as central mechanisms are involved in nociception, we examined the central mechanisms underlying pain in SCD. TLR4 signaling is involved in inflammatory and neuropathic pain (Wang et al., FASEB 2013 and Hutchinson et al., Eur J Neurosci 2008). Microglial cells, the “macrophages” of the central nervous system in the spinal cord are critically involved in the development and maintenance of pain. Binding of an endogenous ligand to TLR4 is an important step in the regulation of microglial activity in pain facilitation. We hypothesized that heme, released during hemolysis in SCD, is a ligand for TLR4 expressed on spinal microglia.

Methods. We isolated microglial cells from the spinal cords of HbSS-BERK (sickle) and HbAA-BERK (control) mice. To assess mitochondrial activity, we analyzed reactive oxygen species (ROS) and ATP, since increased ROS and decreased ATP are suggestive of mitochondrial dysfunction, which in turn is influenced by endoplasmic reticulum (ER) stress. ROS in the microglial cells was determined by utilizing the cell permeable reagent 2’,7’-dichlorofluorescein, which is oxidized by ROS to form a fluorescent compound, with the max excitation and emission spectra of 495 nm and 529 nm, respectively. ATP production was measured by a luminescence based assay from PerkinElmer (ATPlite).

Results. Stimulation of microglia from control and sickle mice with hemin in vitro led to a several-fold increase in TLR4 gene transcripts in a time-dependent manner. Additionally, hemin induced the production of proinflammatory cytokines, TNF-α and IL-6, and ROS compared to vehicle-treated microglial cells from both sickle and control mice (p<0.01 for both). TAK-242 and LPS-RS, inhibitors of TLR4, ameliorated hemin-induced ROS production in microglial cells (p<0.01 and p<0.001 vs. hemin, respectively). Microglial cells treated with hemin showed a significant reduction in ATP content (p<0.01 vs. vehicle). Furthermore, hemin treatment increased expression of the ER stress protein, XBP1, in sickle and control microglial cells (40% increase in the expression of XBP1 compared to unstimulated), which was attenuated by the TLR4 inhibitor, LPS-RS (30% decrease compared to hemin stimulated), suggesting that hemin-induced TLR4 activation leads to ER stress. The ER stress inhibitor, salubrinal, attenuated hemin-induced ROS production from microglial cells (p<0.01 vs. vehicle). Moreover, hemin significantly stimulated the phosphorylation of p38MAPK, Stat3, Akt and MAPK/ERK in a time-dependent manner in both control and sickle glial cells. Whole spinal cord lysates from sickle mice showed significantly higher density of protein bands for phosphorylated p38MAPK, Stat3, Akt and MAPK/ERK, as compared to those from control mice, indicative of ongoing heme-induced glial activation and nociceptive signaling in spinal cords of sickle mice. Complementary to nociceptive signaling, ROS was significantly higher in sickle as compared to control mice spinal cords (p<0.05). Since hemin activates glial cells from control mice, it is a likely cause of microglial activation in sickle mice and because it further augments activation of glial cells from sickle mice, it may lead to a sustained activation of spinal glia. Therefore, hemin induces ER stress via activation of TLR4 resulting in the generation of ROS, oxidative stress and inflammation leading to the activation of microglial cells, which in turn release mediators that excite and sensitize spinal nociceptive neurons, thus maintaining chronic pain. These data suggest that inhibitors of TLR4 and ER stress may be of therapeutic benefit in treating pain in SCD.