Neuroimmune signaling through spinal IL-17–TNF-α/p38 MAPK drives acute pain in sickle cell disease
Yugal Goel, Donovan A Argueta, Kendall O’Daniel, Carolina Mireles, Reina A Lomeli, Richard Prince, Joel Friedman, Kalpna GuptaAbstract
Background
Acute pain episodes in sickle cell disease (SCD) are unpredictable, frequently require hospitalization, and are associated with reduced survival. While vasoocclusion and hypoxia are major triggers, the neuroimmune mechanisms that amplify nociception during these events remain incompletely defined. Hemolysis and vasoocclusion release damage-associated signals, including heme, that promote oxidative stress and inflammatory activation in the periphery and central nervous system (CNS). Increasing evidence supports a cytokine-driven axis in which interleukin-17A (IL-17A) amplifies tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) signaling and engages stress kinases such as p38 mitogen-activated protein kinase (p38 MAPK), thereby sustaining microglial activation, neuroinflammation, and neuronal dysfunction. Defining how this IL-17A–TNF-α/IL-6–p38 MAPK cascade links systemic inflammation to spinal mechanisms of pain during hypoxic stress is essential for developing preventive, non-opioid strategies for acute SCD pain.
Methods
Acute vasoocclusive stress was modeled using hypoxia/reoxygenation (H/R; 8% O2 for 3 h followed by reoxygenation at room air) in homozygous HbSS-BERK sickle mice with HbAA-BERK controls. Mechanical sensitivity, cold sensitivity, and grip force were assessed at baseline, immediately after H/R-1, 24 h post-H/R-1, immediately after H/R-2, and 24 h post-H/R-2 (H/R-1 and H/R-2 separated by 48 h). To test the role of IL-17A signaling, anti–IL-17A antibody (0.25 µg/g body weight) was administered intrathecally 4 h prior to each H/R exposure. To evaluate therapeutic modulation, mice received daily transdermal curcumin (TDC; VAS-101, Vascarta Inc.) or vehicle for 2 weeks prior to H/R, which was continued until the end of the study. Plasma cytokines and spinal neuroinflammatory signaling were analyzed by immunoassays and immunofluorescence. Mechanistic studies were performed in primary dorsal root ganglion (DRG) neurons, primary microglia, and HT22 hippocampal neurons exposed to a hypoxic sickle microenvironment (TNF-α + hemin ± CoCl2-induced hypoxia), with or without the p38 MAPK inhibitor neflamapimod.
Results
Vehicle-treated HbSS-BERK mice displayed increased mechanical and cold hypersensitivity at all post-H/R time points, as predicted, compared with baseline and HbAA controls (P < 0.0001). Grip force declined after H/R-1 (P < 0.05) and further after H/R-2 (P < 0.0001), indicating progressive musculoskeletal hyperalgesia. H/R increased spinal phospho-p38 MAPK and elevated IL-17, TNF-α, and IL-6 compared with normoxia (P < 0.05). Under normoxia, HbSS-BERK mice exhibited higher basal microglial activation, indicated by increased ionized calcium-binding adapter molecule 1 (Iba1) immunoreactivity, and higher phospho-p38 MAPK immunoreactivity in the spinal dorsal horn compared with controls; both were amplified after H/R. Plasma IL-17A was significantly increased in H/R treated compared with normoxic sickle mice (P < 0.05), consistent with a systemic inflammatory response to HR. Intrathecal anti–IL-17A antibody significantly reduced mechanical (P < 0.0001), cold (P < 0.0001), and musculoskeletal hyperalgesia (P < 0.05), attenuated spinal microglial activation, and decreased spinal IL-17 and TNF-α following H/R-2. In primary sickle microglia, TNF-α plus hemin under hypoxic conditions markedly increased IL-17A production, and this response was attenuated by TDC or neflamapimod, supporting microglia as a p38 MAPK-regulated source of IL-17A. Pre-treatment with TDC significantly attenuated H/R-evoked mechanical and cold hypersensitivity at 24 h post-H/R-1, H/R-2, and 24 h post-H/R-2 (P < 0.0001), improved grip force (P < 0.05), suppressed spinal microglial and p38 MAPK activation, reduced systemic and spinal cytokines, and decreased oxidative stress while preserving mitochondrial membrane potential in DRG and HT22 neurons.
Conclusions
Acute hypoxia reoxygenation in sickle mice engages an IL-17A–TNF-α/p38 MAPK axis that links systemic inflammation to spinal microglial activation and downstream neuronal oxidative stress, culminating in mechanical, cold, and musculoskeletal hyperalgesia. TDC disrupts this multi-level inflammatory cascade and mitigates acute pain behaviors, supporting its potential as a preventive, non-opioid drug candidate for acute pain episodes in SCD.