In a recent study published in the journal PNAS, researchers investigate the pronociceptive role of neutrophils in fibromyalgia using a murine model of chronic pain through hyperalgesic priming.

Study: Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia. Image Credit: Kaspars Grinvalds / Shutterstock.com

Background

Fibromyalgia is a debilitating condition with chronic pain known to originate from central nervous system (CNS) dysfunction; however, recent studies have reported a peripheral nervous system (PNS)-associated etiology of this condition. Immune dysregulation has been observed in fibromyalgia, including an increase in the number of neutrophils with enhanced microbicidal and chemotactic properties.

Elevated levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL-6), and interleukin (IL-8) have also been documented in fibromyalgia. Further research is needed to elucidate the etiological basis of fibromyalgia, guide treatment planning, and improve the standard of care for individuals with this disease.

About the study

In the present study, researchers performed hyperalgesic priming and used a back-translational adoptive transfer murine model to elucidate the immunological basis of mediating chronic pain by neutrophils among fibromyalgia patients.

The causal association between nociceptive activities underlying fibromyalgia-associated chronic pain and neutrophils was investigated using hyperalgesic mice and adoptive cell transfer from murine animals and individuals experiencing chronic-type pain to naïve murine animals. Pain behavior assessments, electrophysiological evaluations of nerve excitability, and PNS sensory ganglion imaging were also performed to examine pronociceptive neutrophilic activity.

The murine model received hyperalgesic priming to stimulate persistent and latent pain behavior, followed by two intramuscular carrageenan injections to stimulate acute inflammatory pain. Comparatively, phosphate-buffered saline (PBS) control mice were injected with carrageenan once.

Extracellular recordings from the murine dorsal horn were assessed to determine the influence of pain induction in mice on bilateral spinal cord neuronal excitability. Action potential firing by contralateral and ipsilateral dorsal horn neurons following a 10-second peripheral stimulation was assessed quantitatively.

The team investigated whether systemic circulating factors could underlie contralateral side hypersensitivity in the primed murine animals, for which serum and blood cells were isolated from control and primed mice and subsequently transferred to their naïve counterparts.

Immunological cells, including T lymphocytes, B lymphocytes, monocytes, and neutrophils, were administered intravenously to the naïve recipient murine animals. Furthermore, primed murine neutrophils were pharmacologically depleted with an intravenously administered anti-lymphocyte antigen 6 complex loci G6D (anti-Ly6G) antibody (clone 1A8) before the second intramuscular injection.

Neutrophil depletion was confirmed using fluorescence-activated cell sorting (FACS) of immune cells expressing Ly6-G, Ly6-C, and the cluster of differentiation 45 (CD45). Mechanical hypersensitivity was evaluated using the von Frey approach.

Antineutrophil antibodies and neutrophil sequestering antibodies were administered to primed mice to assess their influence on acute pain behavior and mechanical hypersensitivity, respectively.

The t-distributed stochastic neighbor embedding (tSNE) analysis characterized dorsal root ganglia (DRG)- and blood-derived neutrophils and compared immune cell subpopulations. Flow cytometry, immunohistochemistry, and whole-mount ex vivo multiphoton microscopy were also performed.

Study findings

Neutrophils invaded sensory ganglia and conferred mechanical hypersensitivity on recipient mice, whereas the adoptive transfer of serum, immunoglobulin, monocytes, or lymphocytes did not impact pain behavior.

The depletion of neutrophils prevented the establishment of widespread chronic pain in murine animals. Neutrophils from fibromyalgia patients also conferred pain in the mouse model.

These findings agree with the reported association between neutrophil-derived pain mediators and peripheral nerve sensitization. The study results also suggest targeting fibromyalgia through mechanisms that alter the activity of neutrophils and their interactions with sensory-type neurons.

Hyperalgesic priming in mice resulted in persistent pain behavior with prolonged spinal cord-level hypersensitivity to von Frey stimulation on the ipsilateral side and spatiotemporally spreading mechanical hypersensitivity to the opposite limb. These observations suggest a systemic effect of neutrophil migration on perceived nociception.

Neutrophil depletion can attenuate or delay pain development without impacting gross inflammatory responses. The neutrophils infiltrating sensory ganglia were both endogenous murine-origin neutrophils and exogenous human-origin neutrophils, thus indicating tissue-limited innate immunological pathways involved in recruiting exogenous and endogenous neutrophils into the nervous system in the state of chronic pain.

Transferring blood cells without serum from primed murine animals induced mechanical hypersensitivity for about two hours, which indicates that the cells comprised the algogenic etiological basis for chronic pain. Mice receiving neutrophils from their primed counterparts showed transient but robust mechanical hypersensitivity lasting for about one day.

Significant neutrophil infiltration was observed in the L4 segment of the primed murine DRG. The FACS analysis confirmed elevated Ly6G+/Ly6C+ CD45+ cell counts in the L3 to L5 segments of the DRG in primed mice.

The tSNE analysis showed enrichment of neutrophils with lowered levels of C-X-C motif chemokine receptor-2 (CXCR-2), L-selectin or leucocyte adhesion molecule 1 (LECAM-1), integrin αM, Ly6-G, and Ly6-C in the DRG, which is typical of an activated and migrated neutrophil phenotype.

Conclusions

Neutrophils appear to be significant contributing factors to peripheral pathological chronic pain in fibromyalgia. Neutrophils, derived either from mice with persistent pain for more than two weeks or from fibromyalgia patients enduring pain over several years, can undergo phenotypic changes that enable sustained sensitization of peripheral sensory neurons.

The current study suggests that neutrophils, particularly those that interact with sensory neurons, could be targeted to develop pain management therapeutics for fibromyalgia patients.