Name: teasing out the interplay between natural killer cells and nociceptor neurons
Uploaded: 2022-06-30T13:00:00
Description: Watch this Scientific Journal Video about Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons at JoVE.com

This work was supported by&#160;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 Qu&#233;bec Nature et technologies (253380).

The Institutional Animal Care and Use Committees of Universit&#233; de Montr&#233;al (#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
<ol>
	<li>Generate nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) a...

<ol>
	<li>Berta, T., Qadri, Y., Tan, P. H., Ji, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+dorsal+root+ganglia+and+primary+sensory+neurons+for+the+treatment+of+chronic+pain.">Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain.</a> Expert Opinion on Therapeutic Targets. 21 (7), 695-703 (2017).</li><li>Baral, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptor+sensory+neurons+suppress+neutrophil+and+gammadelta+T+cell+responses+in+bacterial+lung+infections+and+lethal+pneumonia.">Nociceptor sensory neurons suppress neutrophil and gammadelta T cell responses in bacterial lung infections and lethal pneumonia.</a> Nature Medicine. 24, 417-426 (2018).</li><li>Binshtok, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors+are+interleukin-1beta+sensors.">Nociceptors are interleukin-1beta sensors.</a> Journal of Neuroscience. 28 (52), 14062-14073 (2008).</li><li>Chesne, J., Cardoso, V., Veiga-Fernandes, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-immune+regulation+of+mucosal+physiology.">Neuro-immune regulation of mucosal physiology.</a> Mucosal Immunology. 12 (1), 10-20 (2019).</li><li>Samad, T. 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H., Gasser, S., Gowen, B. G., Deng, W., Jung, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ligands+for+the+NKG2D+activating+receptor.">Regulation of ligands for the NKG2D activating receptor.</a> Annual Review of Immunology. 31, 413-441 (2013).</li><li>Vivier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+or+adaptive+immunity?+The+example+of+natural+killer+cells.">Innate or adaptive immunity? The example of natural killer cells.</a> Science. 331, 44-49 (2011).</li><li>Davies, A. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+mouse+TRPV1-lineage+neurons+revealed+by+RNA-Seq+transcriptome+analysis.">Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis.</a> Journal of Pain. 15, 1338-1359 (2014).</li><li>Mishra, S. K., Tisel, S. M., Orestes, P., Bhangoo, S. K., Hoon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPV1-lineage+neurons+are+required+for+thermal+sensation.">TRPV1-lineage neurons are required for thermal sensation.</a> EMBO J. 30, 582-593 (2011).</li><li>Kim, H. 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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<...

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+) and the generation of an action potential3,4,5.
These neurons also send antidromic action potential toward the periphery, where the initial danger sensing had occurred, which leads to the local release of neuropeptides1,4. Therefore, the nociceptor neurons serve as a protective mechanism, alerting the host to environmental danger4,5,6,7.&#160;
To communicate with second-order neurons, the nociceptors release various neurotransmitters (e.g., glutamate) and neuropeptides (e.g., calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP))6,7.These peptides act on capillaries and promote plasma extravasation, edema, and the local influx and modulation of immune cells2,4,7.
The somatosensory and immune systems utilize a shared communication system composed of cytokines and neuropeptides, and their respective cognate receptors4. While this bidirectional communication helps protect from danger and preserve homeostasis, it can also contribute to disease pathophysiology4.&#160;
NK cells are classified as innate lymphoid cells and are specialized to eliminate virally infected cells. NK cell function is governed by a balance of stimulatory and inhibitory receptors, including the activating receptor NKG2D8. The endogenous ligand of NKG2D, retinoic acid early inducible1 (RAE1), is expressed by cells undergoing stress such as tumorigenesis and infection8,9.
Recent investigations have shown that peripheral nerve injury drives sensory neurons to express maladaptive molecules such as stathmin 2 (STMN2) and RAE1. Thus, via cell-cell contact, NKG2D-expressing NK cells were activated by interaction with RAE1-expressing neurons. In turn, NK cells were able to eliminate injured nociceptor neurons and blunt pain hypersensitivity normally associated with nerve injury10. In addition to the NKG2D-RAE1 axis, NK cells express the cognate receptors for various nociceptor-produced mediators. It is therefore possible that these mediators modulate NK cell activity. This paper presents a co-culture method to investigate the biology of the nociceptor neuron-NK cell interaction. This approach will help advance the understanding of how nociceptor neurons modulate innate immune cell responses to injury, infection, or malignancy.

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			<td>Anti-mouse CD16/32</td>
			<td>Jackson Laboratory</td>
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			<td>BV421 anti-mouse NK-1.1</td>
			<td>Fisher Scientific</td>
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			<td>Cell strainer (50 &#956;m)</td>
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			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Fisher Scientific</td>
			<td>Cat no: 07-200-95</td>
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			<td>EasySep Mouse NK Cell Isolation Kit</td>
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			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
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			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
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			<td>Flat bottom 96-well plate</td>
			<td>Sigma</td>
			<td>Cat no: 03690</td>
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			<td>Sigma</td>
			<td>Cat no: 470236-274</td>
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			<td>Glial cell line-derived neurotrophic factor (GDNF)</td>
			<td>VWR</td>
			<td>Cat no: 02-0131</td>
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			<td>Laminin</td>
			<td>Cedarlane</td>
			<td>Cat no: 03-50/31</td>
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			<td>L-Glutamine</td>
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			<td>Cat no: A14867-01</td>
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			<td>Mouse recombinant IL-15</td>
			<td>Gibco</td>
			<td>Cat no: 22400-089</td>
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			<td>Mouse recombinant IL-2</td>
			<td>Gibco</td>
			<td>Cat no: 21103-049</td>
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			<td>Nerve Growth Factor (NGF)</td>
			<td>Life Technologies</td>
			<td>Cat no: 13257-019</td>
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			<td>Neurobasal media</td>
			<td>PeproTech</td>
			<td>Cat no: 450-51-10</td>
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			<td>PE anti-mouse GM-CSF</td>
			<td>PeproTech</td>
			<td>Cat no: 212-12</td>
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			<td>Penicillin and Streptomycin</td>
			<td>PeproTech</td>
			<td>Cat no: 210-15</td>
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			<td>Pestles</td>
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			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Biolegend</td>
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			<td>RPMI 1640 media</td>
			<td>Biolegend</td>
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			<td>TRPV1Cre</td>
			<td>Biolegend</td>
			<td>Cat no: 505406</td>
			<td>Clone MP1-22E9</td>
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			<td>Tweezers and dissection tools.</td>
			<td>Biolegend</td>
			<td>Cat no: 65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>U-Shaped-bottom 96-well plate</td>
			<td>Biolegend</td>
			<td>Cat no: 101319</td>
			<td></td>
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			<td>Viability Dye eFlour-780</td>
			<td>Becton Dickinson</td>
			<td></td>
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teasing out the interplay between natural killer cells and nociceptor neurons

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.

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 &#181;M) or its vehicle. After 24 h of co-culture, the NK cells were F...

Watch this Scientific Journal Video about Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons at JoVE.com

Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons

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 ...

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