tetano
Editor, Senior Moderator
Eur J Pain
. 2026 Aug;30(7):e70343.
doi: 10.1002/ejp.70343.
SARS-CoV-2 Spike Peptides Trigger Nociceptive Responses Through Spinal TLR4 Pathways
Bruno Eduardo Silva[SUP] 1 [/SUP], Rayner Ribeiro Cardoso[SUP] 1 [/SUP], Lívia Maria Ribeiro Rosário[SUP] 1 [/SUP], João Paulo Prado[SUP] 1 [/SUP], Rafaela Silva Dos Santos[SUP] 1 [/SUP], Flávio Protasio Veras[SUP] 1 [/SUP], Mylena de Souza[SUP] 1 [/SUP], Eduardo Maffud Cilli[SUP] 2 [/SUP], Danilo Olivier[SUP] 3 [/SUP], Marco Antonio de Andrade Belo[SUP] 4 [/SUP], Ives Charlie-Silva[SUP] 2 [/SUP], Ester Siqueira Caixeta[SUP] 5 [/SUP], Angel Roberto Barchuk[SUP] 6 [/SUP], Albená Nunes-Silva[SUP] 7 [/SUP], Thiago Roberto Lima Romero[SUP] 8 [/SUP], Giovane Galdino[SUP] 1 [/SUP]
Affiliations
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[SUP]GFP/+[/SUP] 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[SUP]-/-[/SUP] 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.
Keywords: cytokines; microglia; pain; spike protein; toll‐like 4 receptors.
. 2026 Aug;30(7):e70343.
doi: 10.1002/ejp.70343.
SARS-CoV-2 Spike Peptides Trigger Nociceptive Responses Through Spinal TLR4 Pathways
Bruno Eduardo Silva[SUP] 1 [/SUP], Rayner Ribeiro Cardoso[SUP] 1 [/SUP], Lívia Maria Ribeiro Rosário[SUP] 1 [/SUP], João Paulo Prado[SUP] 1 [/SUP], Rafaela Silva Dos Santos[SUP] 1 [/SUP], Flávio Protasio Veras[SUP] 1 [/SUP], Mylena de Souza[SUP] 1 [/SUP], Eduardo Maffud Cilli[SUP] 2 [/SUP], Danilo Olivier[SUP] 3 [/SUP], Marco Antonio de Andrade Belo[SUP] 4 [/SUP], Ives Charlie-Silva[SUP] 2 [/SUP], Ester Siqueira Caixeta[SUP] 5 [/SUP], Angel Roberto Barchuk[SUP] 6 [/SUP], Albená Nunes-Silva[SUP] 7 [/SUP], Thiago Roberto Lima Romero[SUP] 8 [/SUP], Giovane Galdino[SUP] 1 [/SUP]
Affiliations
- PMID: 42549977
- PMCID: PMC13435826
- DOI: 10.1002/ejp.70343
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[SUP]GFP/+[/SUP] 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[SUP]-/-[/SUP] 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.
Keywords: cytokines; microglia; pain; spike protein; toll‐like 4 receptors.