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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Nociceptor neurons and NK cells actively interact in an inflammatory context. A co-culture approach enables studying this interplay.

Abstract

Somatosensory neurons have evolved to detect noxious stimuli and activate defensive reflexes. By sharing means of communication, nociceptor neurons also tune host defenses by controlling the activity of the immune system. The communication between these systems is mostly adaptive, helping to protect homeostasis, it can also lead to, or promote, the onset of chronic diseases. Both systems co-evolved to allow for such local interaction, as found in primary and secondary lymphoid tissues and mucosa. Recent studies have demonstrated that nociceptors directly detect and respond to foreign antigens, immune cell-derived cytokines, and microbes.

Nociceptor activation not only results in pain hypersensitivity and itching, but lowers the nociceptor firing threshold, leading to the local release of neuropeptides. The peptides that are produced by, and released from, the peripheral terminals of nociceptors can block the chemotaxis and polarization of lymphocytes, controlling the localization, duration, and type of inflammation. Recent evidence shows that sensory neurons interact with innate immune cells via cell-cell contact, for example, engaging group 2D (NKG2D) receptors on natural killer (NK) cells.

Given that NK cells express the cognate receptors for various nociceptor-produced mediators, it is conceivable that nociceptors use neuropeptides to control the activity of NK cells. Here, we devise a co-culture method to study nociceptor neuron-NK cell interactions in a dish. Using this approach, we found that lumbar nociceptor neurons decrease NK cell cytokine expression. Overall, such a reductionist method could be useful to study how tumor-innervating neurons control the anticancer function of NK cells and how NK cells control the elimination of injured neurons.

Introduction

The cell bodies of sensory neurons originate in the dorsal root ganglia (DRG). The DRG are located in the peripheral nervous system (PNS), between the dorsal horn of the spinal cord and the peripheral nerve terminals. The pseudo-unipolar nature of DRG neurons allows the transfer of information from the peripheral branch, which innervates the target tissue, to the central branch, which carries the somatosensory information to the spinal cord1. Using specialized ion channel receptors, first-order neurons sense threats posed by pathogens, allergens, and pollutants2, leading to the influx of cations (Na+, Ca2....

Protocol

The Institutional Animal Care and Use Committees of Université de Montréal (#22053, #22054) approved all animal procedures. See Table 1 for a list of solutions and their composition and the Table of Materials for a list of materials, equipment, and reagents used in this protocol.

1. NK cell isolation, culture, and stimulation

  1. Generate nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) a.......

Representative Results

NK cells were magnetically purified from littermate control (TRPV1wt::DTAfl/wt) mice splenocytes and stimulated (48 h) with IL-2 and IL-15. The NK cells were then cultured alone or co-cultured with DRG neurons harvested from nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) or ablated (TRPV1cre::DTAfl/wt) mice. The cells were then exposed to the TRPV1 agonist capsaicin (1 µM) or its vehicle. After 24 h of co-culture, the NK cells were F.......

Discussion

Davies et al.11 found that injured neurons upregulate RAE1. Via cell-cell contact, NKG2D-expressing NK cells were then able to identify and eliminate RAE1+ neurons, which in turn limit chronic pain11. Given that NK cells also express various neuropeptide receptors, and that those neuropeptides are known for their immunomodulatory capabilities, it appears increasingly important to study the interaction between NK cells and nociceptor neurons in vitro<.......

Acknowledgements

This work was supported by The New Frontiers in Research Fund (NFRFE201901326), the Canadian Institutes of Health Research (162211, 461274, 461275), the Canadian Foundation for Innovation (37439), Canada Research Chair program (950-231859), Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-06824), and the Fonds de Recherche du Québec Nature et technologies (253380).

....

Materials

NameCompanyCatalog NumberComments
Anti-mouse CD16/32Jackson LaboratoryCat no: 017769
B-27Jackson LaboratoryCat no: 009669
Bovine Serum Albumin (BSA) culture gradeWorld Precision InstrumentsCat no: 504167
BV421 anti-mouse NK-1.1Fisher ScientificCat no: 12430112
Cell strainer (50 μm)Fisher ScientificCat no: A3160702
Collagenase IVFisher ScientificCat no: 15140148
Diphteria toxinfl/flFisher ScientificCat no: SH3057402
Dispase IIFisher ScientificCat no: 13-678-20B
Dulbecco's Modified Eagle Medium (DMEM)Fisher ScientificCat no: 07-200-95
EasySep Mouse NK Cell Isolation KitSigmaCat no: CLS2595
Ethylenediaminetetraacetic acid (EDTA)SigmaCat no: C0130
FACSAria IIISigmaCat no: 04942078001
Fetal bovine serum (FBS)SigmaCat no: 806552
FITC anti-mouse NKp46SigmaCat no: L2020
Flat bottom 96-well plateSigmaCat no: 03690
Glass Pasteur pipetteSigmaCat no: 470236-274
Glial cell line-derived neurotrophic factor (GDNF)VWRCat no: 02-0131
LamininCedarlaneCat no: 03-50/31
L-GlutamineGibcoCat no: A14867-01
Mouse recombinant IL-15GibcoCat no: 22400-089
Mouse recombinant IL-2GibcoCat no: 21103-049
Nerve Growth Factor (NGF)Life TechnologiesCat no: 13257-019
Neurobasal mediaPeproTechCat no: 450-51-10
PE anti-mouse GM-CSFPeproTechCat no: 212-12
Penicillin and StreptomycinPeproTechCat no: 210-15
PestlesStem Cell TechnologyCat no: 19855
Phosphate Buffered Saline (PBS)BiolegendCat no: 108732Clone PK136
RPMI 1640 mediaBiolegendCat no: 137606Clone 29A1.4
TRPV1CreBiolegendCat no: 505406Clone MP1-22E9
Tweezers and dissection tools.BiolegendCat no: 65-0865-14
U-Shaped-bottom 96-well plateBiolegendCat no: 101319
Viability Dye eFlour-780Becton Dickinson

References

  1. Berta, T., Qadri, Y., Tan, P. H., Ji, R. R. Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain. Expert Opinion on Therapeutic Targets. 21 (7), 695-703 (2017).
  2. Baral, P., et al.

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