SARS
‐
CoV
‐2 Spike Peptides Trigger Nociceptive Responses Through Spinal
TLR4
Pathways
Bruno Eduardo Silva, Rayner Ribeiro Cardoso, Lívia Maria Ribeiro Rosário, João Paulo Prado, Rafaela Silva dos Santos, Flávio Protasio Veras, Mylena de Souza, Eduardo Maffud Cilli, Danilo Olivier, Marco Antonio de Andrade Belo, Ives Charlie‐Silva, Ester Siqueira Caixeta, Angel Roberto Barchuk, Albená Nunes‐Silva, Thiago Roberto Lima Romero, Giovane Galdino ABSTRACT
Background
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.
Methods
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.
Results
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.
Conclusions
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.
Significance Statement
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.