Background & Aims
We have recently shown a key role for neutrophils in mediating the spatiotemporal spread of hypersensitivity in a model of chronic widespread pain [1]. Our data shows a fundamental pro-nociceptive action of neutrophils derived from patients or mice with chronic widespread pain when administered to naïve recipient mice. Neutrophils release extracellular vesicles (NDEVs) in response to numerous immunological stimuli, acting on and directly modulating target immune cells. This function separates NDEVs from most other EVs that are primarily involved in intercellular communication, thereby substantiating a role for NDEVs as mobile functional units of neutrophils themselves. We hypothesise that NDEV release and consequent actions of cargo release enable neutrophils to sensitise sensory neurons and trigger nociceptive signalling.
Methods
To identify the pro-nociceptive mechanism through which neutrophils sensitise neurons, we have optimised a protocol to isolate NDEVs. We have utilised flow cytometry, transmission electron scanning microscopy and western blot analysis to characterise NDEV phenotypes, including size distribution, concentration and protein content. To identify a specific pro-nociceptive capacity of NDEVs, we have isolated NDEVs from patients with fibromyalgia and pain free controls to examine phenotypic, proteomic and functional differences between cohorts. Functional differences were quantified using calcium imaging of primary afferent neurons following application of NDEVs. We have also used a back translational model of adoptive transfer of neutrophils into naïve recipient mice to examine neutrophil infiltration into cervical ganglia within the body.
Results
We have successfully optimised a protocol of NDEV isolation from stimulated and unstimulated neutrophils where we see increased NDEV release following PMA stimulation. Similarly, we see increased NDEV release with longer stimulation periods. Our NDEV size profile and the presence of a phospholipid bilayer has been confirmed using TEM and western blot, identifying the transmembrane tetraspanin CD63. Phenotypic characterisation of NDEVs derived from patients with fibromyalgia reveals increased release when compared to controls. Functional studies demonstrate neuronal hypersensitivity following the application of NDEVs. NDEV-mediated neuronal hypersensitivity may reflect a mechanism employed by neutrophils to trigger chronic widespread pain in fibromyalgia. Pilot data also demonstrates neutrophil infiltration into trigeminal ganglion following adoptive transfer of patient neutrophils into naïve recipient mice, housing primary afferent nociceptors that innervate supracervical regions.
Conclusions
We have identified specific phenotypic and functional differences between NDEVs derived from patients with fibromyalgia compared to pain-free controls. Our data demonstrating this altered phenotypic profile reflect novel mechanisms through which neutrophils sensitise peripheral sensory neurons to produce chronic widespread pain in FM.
References
[1] Caxaria S, Bharde S, Fuller AM, Evans R, Thomas B, Celik P, Dell’Accio F, Yona S, Gilroy D, Voisin MB, Wood JN, Sikandar S. Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia. Proc Natl Acad Sci U S A. 2023 Apr 25;120(17):e2211631120. doi: 10.1073/pnas.2211631120. Epub 2023 Apr 18. PMID: 37071676; PMCID: PMC10151464.
Presenting Author
Romy Evans
Poster Authors
Topics
- Specific Pain Conditions/Pain in Specific Populations: Fibromyalgia