Background & Aims

Chronic pain activates the peripheral nervous and immune systems to both generate a nociceptive signal, and to regulate inflammation1. Immune cells, particularly macrophages, regulate neuroimmune interactions including modulating long-lasting pain behaviors in rodents2,3 and patient4. In addition, macrophages can be “trained” by prior insult to respond with greater inflammation to a subsequent insult5. This effect may be retained in bone marrow, where new monocytes/macrophages are generated and is also susceptible to injury6,7. However, the effect of chronic pain in regards to bone marrow alterations, and resulting differential neuroimmune interactions is unknown. We address this gap in knowledge by evaluating human donor and rodent pain, bone marrow derived macrophages (BMDMs), macrophage signals to dorsal root ganglia (DRG) sensory neurons. We tested the hypothesis that chronic pain increases macrophage-derived inflammation, which sensitizes sensory neurons in mouse and human.

Methods

Rodent: Four weeks after a spared nerve injury, vertebral bone marrow was seeded at 100,000 cells and differentiated into macrophages. Cells were left untreated or stimulated with LPS and interferon-gamma to promote inflammation8. 24 hours later the conditioned media (CM) was removed and 1) was used for content analyses by cytokine arrays, 2) was applied to DRG sensory neurons (1:1 by volume) overnight for patch clamp electrophysiology, and 3) injected into the hind paw of naïve animals (10 µL) for pain-like behavior monitoring. In addition, BMDMs were scraped and processed for quantitative PCR.
Human: Vertebral bone marrow from organ donors with and without pain history was isolated and differentiated as in mouse. Immunohistochemistry confirmed differentiation. CM analyses for content and cultured donor DRG neuron9 incubation with CM were completed as in mouse.

Results

Vertebral bone marrow differentiation was confirmed by an increased Iba-1 positive area. Transcript analyses of mouse and donor BMDM indicate expected inflammatory profiles with increased pro-inflammatory expression following stimulation, suggesting healthy macrophages. In pain-reporting patients, preliminary data indicate that there is a basal pro-inflammatory profile of bone marrow compared to those that did not report pain. Similarly, in rodents BMDMs from animals with a history of pain had increased inflammatory RNA profiles compared to controls. CM applied to sensory neurons in mouse and human increased the number of neurons that had spontaneous activity. History of pain appears to decrease neurons’ rheobase and increase action potential kinetics in mouse and human neurons. Finally, CM injected into hind paw of naïve animals led to robust guarding and mechanical hypersensitivity with a possible difference between SNI-associated CM and controls.

Conclusions

Preliminary data indicate that both mouse and human bone marrow are “trained” because of chronic pain. The extent to which this occurs is currently unclear, however, the result is increased DRG sensitization which is a likely contributing factor in the reported persistent pain. Further investigations into how bone marrow is modified by chronic pain and how macrophages from “trained” bone marrow alter neuronal sensitivity is currently being investigated. Our data suggest both unique and overlapping mechanisms between species in which marrow may be modified by a history of pain, and that this alters DRG neuron excitability and pain-like behaviors. Results from this study will be relevant in identifying critical neuroimmune factors for sensitization that can be targeted for pain relief.

References

1Yang, J. X. et al. Potential Neuroimmune Interaction in Chronic Pain: A Review on Immune Cells in Peripheral and Central Sensitization. Front Pain Res (Lausanne) 3, 946846 (2022). https://doi.org/10.3389/fpain.2022.946846
2Shepherd, A. J. et al. Macrophage angiotensin II type 2 receptor triggers neuropathic pain. Proceedings of the National Academy of Sciences of the United States of America 115, E8057-E8066 (2018). https://doi.org/10.1073/pnas.1721815115
3Dourson, A. J. et al. Macrophage epigenetic memories of early life injury drive neonatal nociceptive priming. bioRxiv, 2023.2002.2013.528015 (2023). https://doi.org/10.1101/2023.02.13.528015
4Fragiadakis, G. K. et al. Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery. Anesthesiology 123, 1241-1255 (2015). https://doi.org/10.1097/aln.0000000000000887
5Fanucchi, S., Domínguez-Andrés, J., Joosten, L. A. B., Netea, M. G. & Mhlanga, M. M. The Intersection of Epigenetics and Metabolism in Trained Immunity. Immunity 54, 32-43 (2021). https://doi.org/https://doi.org/10.1016/j.immuni.2020.10.011
6Mitroulis, I., Hajishengallis, G. & Chavakis, T. Bone marrow inflammatory memory in cardiometabolic disease and inflammatory comorbidities. Cardiovasc Res (2023). https://doi.org/10.1093/cvr/cvad003
7Leitão, L. et al. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models. The FASEB Journal 33, 857-872 (2019). https://doi.org/https://doi.org/10.1096/fj.201800610RR
8Subramanian Vignesh, K., Landero Figueroa, J. A., Porollo, A., Caruso, J. A. & Deepe, G. S., Jr. Granulocyte macrophage-colony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival. Immunity 39, 697-710 (2013). https://doi.org/10.1016/j.immuni.2013.09.006
9Valtcheva, M. V. et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc 11, 1877-1888 (2016). https://doi.org/10.1038/nprot.2016.111

Presenting Author

Adam Dourson

Poster Authors

Adam Dourson

BSc

Washington University

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Systems (Physiology/Anatomy)