SARS-CoV-2 Spike Peptides Trigger Nociceptive Responses Through Spinal TLR4 Pathways

COVID19 has affected over 700 million people worldwide, with a significant portion of the population experiencing severe pulmonary and circulatory complications, often accompanied by symptoms such as prostration and pain. Moreover, the manifestation of these symptoms and other COVID19-related complications varies depending on viral mutations, particularly those occurring in the spike (S) protein of SARS CoV2. Therefore, the present study aimed to investigate the effects of three different S proteins on nociceptive threshold, as well as the spinal involvement of TLR4 and microglia in this process. Male C57BL/6 mice received intrathecal administration of three synthetic peptides (PSPD2001, PSPD2002 and PSPD2003) derived from the SARS CoV2 S protein or saline. Nociceptive threshold was assessed using the von Frey filament test before and after peptide administration. The spinal involvement of Toll-like receptor 4 (TLR4), microglia, p38 MAPK, and NF kappaB was evaluated using specific antagonists and inhibitors. mRNA expression of TLR4 was assessed by RT PCR, pro-inflammatory cytokine levels by ELISA, and microglial activation in the dorsal horn of the spinal cord was analyzed by immunofluorescence in wild-type, CX3CR1 GFP positive and TLR4 KO mice. In addition, molecular dynamics analysis was performed to assess the temporal stability of the PSPD2003 TLR4 complex. Pharmacological data demonstrated that the peptides induced nociception involving TLR4, microglia, p38 MAPK, and NF kappaB. Notably, PSPD2003 increased TLR4 mRNA expression and elevated TNFalpha; and IL6 levels in the spinal cord. PSPD2003 also enhanced microglial activation in the spinal cord, which was abolished in TLR4 KO; mice. Molecular dynamics analysis results robustly demonstrate that PSPD2003 forms a stable and functionally relevant complex with TLR4. These findings suggest that SARS CoV 2 S protein derived peptides contribute to pain during COVID-19 infection, with spinal TLR4 and microglia playing key roles in this process.