Immunopharmacological mechanisms underlying SARS-CoV-2 spike-induced persistent pain in mice.

SARS-CoV-2 infection is frequently associated with persistent pain, yet the underlying mechanisms remain poorly understood. Given that viral structural proteins can act as pathogen-associated molecular patterns, we investigated whether the Spike protein is sufficient to induce long-lasting nociceptive alterations and the immune pathways involved. Intraplantar administration of recombinant Spike protein in mice elicited robust and persistent mechanical and thermal hypersensitivity, accompanied by transient local inflammation, sustained systemic cytokine alterations, and prolonged spinal neuroinflammation. Spike-induced paw inflammation and persistent mechanical hypersensitivity were dependent on Toll-like receptor 4 (TLR4), as both pharmacological inhibition and genetic deficiency of TLR4 abolished these responses. Mechanistically, depletion of mononuclear phagocytes or blockade of T-cell co-stimulation prevented the development of persistent mechanical hypersensitivity, supporting a role for coordinated innate and adaptive immune mechanisms in the establishment of long-lasting nociceptive sensitization. In parallel, Spike induced increases in lymphocyte-associated cytokines, including IFN-γ, IL-4, and IL-17, with modest and transient changes in peripheral compartments but pronounced and sustained elevations in the spinal cord. Among these, IFN-γ emerged as a critical contributor to these responses, as its genetic deletion abolished both inflammation and persistent hypersensitivity. Collectively, these findings demonstrate that the SARS-CoV-2 Spike protein is sufficient to trigger persistent pain-like states through a TLR4-driven neuroimmune cascade involving phagocytes, T cells, and IFN-γ signaling. This study provides mechanistic insight into neuroimmune pathways that may contribute to persistent pain following SARS-CoV-2 infection and establishes a tractable mechanistic experimental model for investigating Spike-induced neuroimmune mechanisms of chronic pain.