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

Pain is a common neurological manifestation of COVID-19, yet the mechanisms by which SARS-CoV-2 spike protein fragments contribute to nociceptive processing remain poorly understood. We investigated whether spike-derived peptides directly activate spinal neuroimmune pathways involved in pain signalling.

Male C57BL/6 mice received intrathecal administration of three synthetic SARS-CoV-2 spike-derived peptides (PSPD2001, PSPD2002 or PSPD2003) or saline. Mechanical nociception was assessed using the von Frey test. The involvement of spinal Toll-like receptor 4 (TLR4), microglia and p38 MAPK/NF-κB signalling was investigated using pharmacological antagonists, TLR4 knockout mice, RT-qPCR, ELISA, immunofluorescence, CX3CR1^GFP/+ mice, human C20 microglial cells and molecular dynamics simulations.

All spike-derived peptides induced mechanical nociception, with PSPD2003 producing the most pronounced response. PSPD2003 increased spinal TLR4 expression, elevated TNF-α and IL-6 levels and promoted activation of dorsal horn microglia, demonstrated by increased TMEM119- and CX3CR1-positive cells. These nociceptive and neuroinflammatory effects were abolished by pharmacological inhibition of TLR4, microglia, p38 MAPK and NF-κB signalling, as well as in TLR4^-/- mice, demonstrating that TLR4 signalling is essential for PSPD2003-induced pain. PSPD2003 also induced a hypertrophic phenotype in human microglial cells, while molecular dynamics simulations supported a stable interaction with the TLR4/MD-2 complex.

These findings identify a previously unrecognized neuroimmune mechanism whereby a SARS-CoV-2 spike-derived peptide triggers spinal nociception through TLR4-dependent microglial activation and downstream p38 MAPK/NF-κB signalling, highlighting the spinal TLR4-microglia axis as a potential therapeutic target for COVID-19-associated and post-viral pain.

This study provides the first evidence that SARS-CoV-2 spike-derived peptides directly activate a spinal TLR4-dependent neuroimmune pathway to induce nociception. By integrating behavioural, pharmacological, genetic, cellular and computational approaches, it identifies microglial activation and p38 MAPK/NF-κB signalling as key mechanisms linking viral peptides to pain, providing a mechanistic framework for COVID-19- and post-viral pain and supporting TLR4 as a potential therapeutic target.