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Nociceptor neurons and NK cells actively interact in an inflammatory context. A co-culture approach enables studying this interplay.
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.
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....
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
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.......
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<.......
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).
....Name | Company | Catalog Number | Comments |
Anti-mouse CD16/32 | Jackson Laboratory | Cat no: 017769 | |
B-27 | Jackson Laboratory | Cat no: 009669 | |
Bovine Serum Albumin (BSA) culture grade | World Precision Instruments | Cat no: 504167 | |
BV421 anti-mouse NK-1.1 | Fisher Scientific | Cat no: 12430112 | |
Cell strainer (50 μm) | Fisher Scientific | Cat no: A3160702 | |
Collagenase IV | Fisher Scientific | Cat no: 15140148 | |
Diphteria toxinfl/fl | Fisher Scientific | Cat no: SH3057402 | |
Dispase II | Fisher Scientific | Cat no: 13-678-20B | |
Dulbecco's Modified Eagle Medium (DMEM) | Fisher Scientific | Cat no: 07-200-95 | |
EasySep Mouse NK Cell Isolation Kit | Sigma | Cat no: CLS2595 | |
Ethylenediaminetetraacetic acid (EDTA) | Sigma | Cat no: C0130 | |
FACSAria III | Sigma | Cat no: 04942078001 | |
Fetal bovine serum (FBS) | Sigma | Cat no: 806552 | |
FITC anti-mouse NKp46 | Sigma | Cat no: L2020 | |
Flat bottom 96-well plate | Sigma | Cat no: 03690 | |
Glass Pasteur pipette | Sigma | Cat no: 470236-274 | |
Glial cell line-derived neurotrophic factor (GDNF) | VWR | Cat no: 02-0131 | |
Laminin | Cedarlane | Cat no: 03-50/31 | |
L-Glutamine | Gibco | Cat no: A14867-01 | |
Mouse recombinant IL-15 | Gibco | Cat no: 22400-089 | |
Mouse recombinant IL-2 | Gibco | Cat no: 21103-049 | |
Nerve Growth Factor (NGF) | Life Technologies | Cat no: 13257-019 | |
Neurobasal media | PeproTech | Cat no: 450-51-10 | |
PE anti-mouse GM-CSF | PeproTech | Cat no: 212-12 | |
Penicillin and Streptomycin | PeproTech | Cat no: 210-15 | |
Pestles | Stem Cell Technology | Cat no: 19855 | |
Phosphate Buffered Saline (PBS) | Biolegend | Cat no: 108732 | Clone PK136 |
RPMI 1640 media | Biolegend | Cat no: 137606 | Clone 29A1.4 |
TRPV1Cre | Biolegend | Cat no: 505406 | Clone MP1-22E9 |
Tweezers and dissection tools. | Biolegend | Cat no: 65-0865-14 | |
U-Shaped-bottom 96-well plate | Biolegend | Cat no: 101319 | |
Viability Dye eFlour-780 | Becton Dickinson |
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