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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 1 , Rayner Ribeiro Cardoso 1 , Lívia Maria Ribeiro Rosário 1 , João Paulo Prado 1 , Rafaela Silva Dos Santos 1 , Flávio Protasio Veras 1 , Mylena de Souza 1 , Eduardo Maffud Cilli 2 , Danilo Olivier 3 , Marco Antonio de Andrade Belo 4 , Ives Charlie-Silva 2 , Ester Siqueira Caixeta 5 , Angel Roberto Barchuk 6 , Albená Nunes-Silva 7 , Thiago Roberto Lima Romero 8 , Giovane Galdino 1
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, CX3CR1GFP/+ 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.
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 1 , Rayner Ribeiro Cardoso 1 , Lívia Maria Ribeiro Rosário 1 , João Paulo Prado 1 , Rafaela Silva Dos Santos 1 , Flávio Protasio Veras 1 , Mylena de Souza 1 , Eduardo Maffud Cilli 2 , Danilo Olivier 3 , Marco Antonio de Andrade Belo 4 , Ives Charlie-Silva 2 , Ester Siqueira Caixeta 5 , Angel Roberto Barchuk 6 , Albená Nunes-Silva 7 , Thiago Roberto Lima Romero 8 , Giovane Galdino 1
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, CX3CR1GFP/+ 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.
Keywords: cytokines; microglia; pain; spike protein; toll‐like 4 receptors.