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) and ablated (TRPV1cre::DTAfl/wt) mice by crossing lox-stop-lox-diphtheria toxin mice (DTAfl/fl) with TRPV1cre/wt mice.</li>
	<li>Using CO2 inhalation, euthanize a na&#239;ve littermate control (TRPV1wt::DTAfl/wt) mouse.</li>
	<li>Collect the spleen in a 1.5 mL microcentrifuge tube containing 500 &#181;L of sterile phosphate-buffered saline (PBS).</li>
	<li>Homogenize the spleen using a pestle.</li>
	<li>Dilute the cells with 1 mL of sterile PBS.</li>
	<li>Filter the mixture through a 50 &#181;m cell strainer into a 15 mL conical tube.</li>
	<li>Top up the volume (10 mL) with sterile PBS.</li>
	<li>Collect 10 &#181;L and count the cells using a hemocytometer.</li>
	<li>Centrifuge the cells (500 &#215; g, 5 min) and remove the supernatant.</li>
	<li>Resuspend the cells at a concentration of 108 cells/mL in supplemented RPMI 1640 medium.</li>
	<li>Follow the manufacturer&#39;s instructions to magnetically purify spleen NK cells using a mouse NK cell isolation kit. Confirm NK cell purity using flow cytometry11.</li>
	<li>Collect 10 &#181;L and count the cells using a hemocytometer.</li>
	<li>Centrifuge the cells (500 &#215; g, 5 min) and remove the supernatant.</li>
	<li>Resuspend the NK cells in supplemented RPMI 1640 culture medium (containing IL-2 and IL-15).</li>
	<li>Culture 2 &#215; 106 NK cells/mL in a 96-well U-bottom plate for 48 h.</li>
</ol>
2. DRG neuron isolation and culture
<ol>
	<li>At 24 h prior to the end of the NK cell stimulation (step 1.15), coat a 96-well plate with 100 &#181;L/well of laminin (1 ng/mL) and incubate for 45 min (37 &#176;C).</li>
	<li>After incubation, remove the solution and allow the wells to air-dry in a&#160;biosafety cabinet.</li>
	<li>Using CO2 inhalation, euthanize nociceptor neuron intact (littermate control; TRPV1wt::DTAfl/wt) or ablated (TRPV1cre::DTAfl/wt) mice. 
	NOTE: CO2 inhalation is preferred to cervical dislocation as it avoids disrupting the spinal nerves connected to the DRG.</li>
	<li>Secure the mouse on the board in a prone position, lift the skin, and incise the skin along the dorsal column. Refer to Perner et al. for a detailed video12.</li>
	<li>Cut off the lumbosacral joint and separate the sacrum from the lumbar spine with scissors.</li>
	<li>Separate the spine by cutting the muscles and ribs on both sides of the spine in the cranial direction until the base of the skull is reached.</li>
	<li>Using scissors, cut off the spine at the atlanto-occipital joint.</li>
	<li>Remove muscle and fatty tissue from the spine.</li>
	<li>Pin and open the vertebral column to remove the spinal cord and gain access to the DRG. Look for the DRG in the intervertebral foramina connected to the spinal cord through the dorsal root but separated from the meninges.</li>
	<li>Harvest the DRGs into a 15 mL conical tube filled with 10 mL of ice-cold supplemented DMEM.</li>
	<li>Centrifuge the preparation (200 &#215; g, 5 min, room temperature) and remove the supernatant.</li>
	<li>Add 250 &#181;L of PBS containing Collagenase IV (1 mg/mL) and Dispase II (2.4 U/mL).</li>
	<li>Incubate the DRG with Collagenase IV/Dispase II (C/D) solution (80 min, 37 &#176;C, mild agitation). When pooling ganglia from several mice, maintain a ratio of 125 &#181;L of C/D solution per ganglion.</li>
	<li>To inactivate the enzymes, add 5 mL of supplemented DMEM medium.</li>
	<li>Centrifuge the solution (200 &#215; g, 5 min) and gently remove the supernatant using a pipette.</li>
	<li>To avoid unwanted cell loss, leave ~100 &#181;L of the supernatant.</li>
	<li>Add 1 mL of supplemented DMEM medium.</li>
	<li>Using a pipette gun and three glass Pasteur pipettes of decreasing sizes, gently triturate (~10 up/down per Pasteur pipette size) the DRG into a single-cell preparation.</li>
	<li>In a biosafety cabinet, dilute bovine serum albumin (BSA) in sterile PBS to a final concentration of 15%. Store 1 mL aliquots at &#8722;20 &#176;C.</li>
	<li>Create a BSA gradient in a 15 mL conical tube by adding 2 mL of sterile PBS and slowly dispensing 1 mL of 15% BSA solution at the bottom of the tube. Avoid disrupting the gradient by gently removing the pipette.</li>
	<li>Using a P200 pipette, slowly pipette the triturated ganglia suspension (which contains the neurons) onto the side of the tube into the gradient.</li>
	<li>Centrifuge the neuron-containing BSA gradient (200 &#215; g, 12 min, room temperature). Set the acceleration and deceleration of the centrifuge to the minimum speed. 
	NOTE: After centrifugation, the neurons will be at the bottom of the tube while the cell debris will be trapped in the BSA gradient.</li>
	<li>Gently remove all of the supernatant using a pipette.</li>
	<li>Resuspend the cells in 500 &#181;L of Neurobasal.</li>
	<li>Plate 104 neurons per well (200 &#181;L of neurobasal/well) in a laminin-coated, flat-bottom 96-well plate. 
	NOTE: On average, an investigator will be able to fill ~8 wells per mouse.</li>
	<li>Culture the neurons (37 &#176;C, overnight) to allow attachment.</li>
</ol>
3. Co-culture and flow cytometry
<ol>
	<li>Slowly remove the neurobasal from the neuron culture.</li>
	<li>After 48 h stimulation with IL-2 and IL-15 (see steps 1.14-1.15), resuspend the NK cells and add 105 NK cells/well to the neuron culture (96-well flat bottom plate). 
	NOTE: On average, the investigator will be able to fill ~6 wells per mouse.</li>
	<li>Co-culture the cells in supplemented neurobasal MX (37 &#176;C, 48 h).</li>
	<li>Collect the cells, wash with PBS (3x), and centrifuge (500 &#215; g, 5 min, 4 &#176;C).</li>
	<li>Discard the supernatant and stain the cells with Viability Dye eFlour-780 (1:1,000, 15 min, 4 &#176;C).</li>
	<li>Collect the cells, wash with PBS (3x), and centrifuge (500 &#215; g, 5 min, 4 &#176;C).</li>
	<li>Block nonspecific sites on the cells with CD16/32 (1:100, 15 min, 4 &#176;C).</li>
	<li>Collect the cells, wash with PBS (3x), and centrifuge (500 &#215; g, 5 min, 4 &#176;C).</li>
	<li>Stain (15 min, 4 &#176;C) the cells with BV421 anti-NK-1.1 (1:100), fluorescein isothiocyanate (FITC) anti-NKp46 (1:100), and phycoerythrin (PE) anti-GM-CSF (1:100).</li>
	<li>Collect the cells, wash with PBS (3x), and centrifuge (500 &#215; g, 5 min, 4 &#176;C).</li>
	<li>Discard the supernatant, resuspend the cells in FACS buffer (500 &#181;L) and immunophenotype the NK cells by flow cytometry11. See Figure 1A for the gating strategy.</li>
</ol>

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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. A., 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=Interleukin-1beta-mediated+induction+of+Cox-2+in+the+CNS+contributes+to+inflammatory+pain+hypersensitivity.">Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity.</a> Nature. 410 (6827), 471-475 (2001).</li><li>Godinho-Silva, 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=Light-entrained+and+brain-tuned+circadian+circuits+regulate+ILC3s+and+gut+homeostasis.">Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasis.</a> Nature. 574, 254-258 (2019).</li><li>Talbot, J., 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=Feeding-dependent+VIP+neuron-ILC3+circuit+regulates+the+intestinal+barrier.">Feeding-dependent VIP neuron-ILC3 circuit regulates the intestinal barrier.</a> Nature. 579, 575-580 (2020).</li><li>Raulet, D. 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. J., 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=Natural+Killer+Cells+Degenerate+Intact+Sensory+Afferents+following+Nerve+Injury.">Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.</a> Cell. 176, 716-728 (2019).</li><li>Perner, C., Sokol, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+and+culture+of+murine+dorsal+root+ganglia+neurons+to+study+neuropeptide+release.">Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.</a> STAR Protocols. 2, 100333 (2021).</li><li>Goswami, S. 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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The authors have no conflicts of interest to disclose.

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. Here we devised a simple co-culture protocol that allows investigators to study how neurons control NK cell function, and reciprocally, how NK cells could modulate the function of DRG neurons. Flow cytometry was used to immunophenotype how neurons modulate NK cell activation. Alternatively, investigators could use qPCR or RNA-sequencing to measure transcript changes in FACS-purified cells or analyze the secretion of secreted factors, such as cytokines or neuropeptides, using ELISAs. Raw data are deposited and freely available on www.talbotlab.com.
Here we found that TRPV1+ nociceptor neurons, secondary to the action of capsaicin, block NK cell activation (GM-CSF expression). While capsaicin used at a high concentration may impair NK cell functions14, we ruled out this possibility experimentally as NK cell activity was not impacted by capsaicin (1 &#181;M) exposure in the absence of peptidergic neurons (TRPV1cre::DTAfl/wt). As capsaicin leads to calcium influx and their subsequent release in neuropeptides, these data suggest that NK cell activation is therefore likely secondary to the action of soluble factors being released by neurons. Therefore, such a co-culture approach was useful to study how the release of neuropeptides may modulate NK cell function. In addition to soluble factors, the local cell-cell interactions between the two cell types are also likely to modulate their functions, something that should be tested experimentally (e.g., comparing the impact of conditioned medium to the one of co-culture).
Overall, such an interplay suggests that within the tissue microenvironment, NK cell function is likely tuned by nociceptor neuron terminals. These data stress the importance of assessing inflammatory responses holistically by simultaneously studying the role of neurons and innate immune cells (i.e., NK cells). For instance, NK cell-neuron crosstalk likely plays an important biological role in contexts spanning from nerve injury, tissue injury, bacterial or viral infection to malignancies. Future work, including the use of this co-culture method, is needed to assess the impact of neuron-NK cell interplay in health and disease.

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.

<table><tbody><tr><td>Anti-mouse CD16/32</td><td>Jackson Laboratory</td><td>Cat no: 017769</td><td></td></tr><tr><td>B-27</td><td>Jackson Laboratory</td><td>Cat no: 009669</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) culture grade</td><td>World Precision Instruments</td><td>Cat no: 504167</td><td></td></tr><tr><td>BV421 anti-mouse NK-1.1</td><td>Fisher Scientific</td><td>Cat no: 12430112</td><td></td></tr><tr><td>Cell strainer (50 &amp;mu;m)</td><td>Fisher Scientific</td><td>Cat no: A3160702</td><td></td></tr><tr><td>Collagenase IV</td><td>Fisher Scientific</td><td>Cat no: 15140148</td><td></td></tr><tr><td>Diphteria toxin&lt;sup&gt;fl/fl&lt;/sup&gt;</td><td>Fisher Scientific</td><td>Cat no: SH3057402</td><td></td></tr><tr><td>Dispase II</td><td>Fisher Scientific</td><td>Cat no: 13-678-20B</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Fisher Scientific</td><td>Cat no: 07-200-95</td><td></td></tr><tr><td>EasySep Mouse NK Cell Isolation Kit</td><td>Sigma</td><td>Cat no: CLS2595</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>Cat no: C0130</td><td></td></tr><tr><td>FACSAria III</td><td>Sigma</td><td>Cat no: 04942078001</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma</td><td>Cat no: 806552</td><td></td></tr><tr><td>FITC anti-mouse NKp46</td><td>Sigma</td><td>Cat no: L2020</td><td></td></tr><tr><td>Flat bottom 96-well plate</td><td>Sigma</td><td>Cat no: 03690</td><td></td></tr><tr><td>Glass Pasteur pipette</td><td>Sigma</td><td>Cat no: 470236-274</td><td></td></tr><tr><td>Glial cell line-derived neurotrophic factor (GDNF)</td><td>VWR</td><td>Cat no: 02-0131</td><td></td></tr><tr><td>Laminin</td><td>Cedarlane</td><td>Cat no: 03-50/31</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>Cat no: A14867-01</td><td></td></tr><tr><td>Mouse recombinant IL-15</td><td>Gibco</td><td>Cat no: 22400-089</td><td></td></tr><tr><td>Mouse recombinant IL-2</td><td>Gibco</td><td>Cat no: 21103-049</td><td></td></tr><tr><td>Nerve Growth Factor (NGF)</td><td>Life Technologies</td><td>Cat no: 13257-019</td><td></td></tr><tr><td>Neurobasal media</td><td>PeproTech</td><td>Cat no: 450-51-10</td><td></td></tr><tr><td>PE anti-mouse GM-CSF</td><td>PeproTech</td><td>Cat no: 212-12</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>PeproTech</td><td>Cat no: 210-15</td><td></td></tr><tr><td>Pestles</td><td>Stem Cell Technology</td><td>Cat no: 19855</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Biolegend</td><td>Cat no: 108732</td><td>Clone PK136</td></tr><tr><td>RPMI 1640 media</td><td>Biolegend</td><td>Cat no: 137606</td><td>Clone 29A1.4</td></tr><tr><td>TRPV1&lt;sup&gt;Cre&lt;/sup&gt;</td><td>Biolegend</td><td>Cat no: 505406</td><td>Clone MP1-22E9</td></tr><tr><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></tr><tr><td>Viability Dye eFlour-780</td><td>Becton Dickinson</td><td></td><td></td></tr></tbody></table>

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 FACS-purified and their activation levels were immunophenotyped using flow cytometry (Figure 1A).
When cultured alone, NK cells expressed basal levels of GM-CSF and NKp46. When co-cultured with DRG neurons harvested from nociceptor intact (TRPV1wt::DTAfl/wt) mice, capsaicin stimulation decreased NK cells expression of GM-CSF. Capsaicin had no impact on NK cell activation when co-cultured with DRG neurons harvested from nociceptor ablated (TRPV1cre::DTAfl/wt) mice (Figure 1A,B). It is worth noting that TRPV1cre mediated cell ablation using diphtheria toxin A (DTA) will eliminate all TRPV1+ neurons, peptidergic and non-peptidergic alike (MrgD+ cells)13,14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63800/63800fig01.jpg" /> 
Figure 1: TRPV1+ neurons control NK cell activation. 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 FACS-purified and their activation levels were immunophenotyped using flow cytometry (A). When cultured alone, NK cells expressed basal levels of GM-CSF and NKp46. GM-CSF expression decreased when NK cells were co-cultured with DRG neurons harvested from nociceptor intact (TRPV1wt::DTAfl/wt) mice and stimulated with capsaicin (A,B). The level of NKp46 were unaffected (A,B). Capsaicin had no impact on NK cell activation when co-cultured with DRG neurons harvested from nociceptor ablated (TRPV1cre::DTAfl/wt) mice (A,B). Representative FACS plot are shown (A). Data are presented as mean &#177; S.E.M (B). The experiment was repeated three times and similar conclusions were obtained. Number of animals tested is shown; n = 5 biological replicates/group (B). P values are shown in the figure and determined by one-way ANOVA post-hoc Bonferroni (B). Abbreviations: TRPV1 = transient receptor potential cation channel subfamily V member 1; NK = natural killer; DTA = diphtheria toxin A; DRG = dorsal root ganglion; FACS = fluorescence-activated cell sorting; GM-CSF = granulocyte-macrophage colony-stimulating factor; Veh = vehicle; Cap = capsaicin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63800/63800fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td colspan="3">Composition</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase (C/D)</td>
			<td colspan="3">Sterile PBS supplemented with collagenase (1 mg/mL) and Dispase II (2.4 U/mL)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td colspan="3">Sterile DMEM supplemented with FBS (10%), penicillin (100 I.U.) and streptomycin (100 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Supplemented RPMI 1640</td>
			<td colspan="3">Sterile RMPI 1640 supplemented with penicillin (100 I.U.), streptomycin (100 &#181;g/mL), fetal bovine serum (10%), L-glutamine (300 ng/mL), mouse recombinant IL-2 (200 U/mL), and mouse recombinant IL-15 (10 ng/mL)</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td colspan="3">Sterile Neurobasal supplemented with penicillin (100 I.U.), streptomycin (100 &#181;g/mL), L-glutamine (200 &#956;M), B-27 (2%), NGF (50 ng/mL), and GDNF (2 ng/mL)</td>
		</tr>
		<tr>
			<td>Neurobasal MX</td>
			<td colspan="3">Sterile Neurobasal supplemented with penicillin (100 I.U.), streptomycin (100 &#181;g/mL), L-glutamine (200 &#956;M), B-27 (2%), NGF (50 ng/mL), GDNF (2 ng/mL), mouse recombinant IL-2 (200 U/mL), and mouse recombinant IL-15 (10 ng/mL)</td>
		</tr>
		<tr>
			<td>FACS sorting buffer</td>
			<td colspan="3">Sterile PBS supplemented with FBS (2%) and EDTA (1 mM)</td>
		</tr>
	</tbody>
</table>
Table 1: List of solutions and media used in this protocol. Abbreviations: NGF = nerve growth factor; GDNF = glial cell-derived neurotrophic factor; FBS = fetal bovine serum; PBS = phosphate-buffered saline.

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 ...

teasing-out-interplay-between-natural-killer-cells-nociceptor

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.1K Views.  Université de Montréal. Gezien het feit dat nociceptorneuronen verschillende neuropeptiden afscheiden en dat NK-cel de receptor voor deze neuropeptiden tot expressie bracht, besloten we een co-kweekmethode te ontwikkelen om het samenspel tussen de NK-cel en de nociceptorneuronen te bestuderen. Een dergelijke reductionistische methode zou nuttig kunnen zijn om te bestuderen hoe nociceptorneuronen de antitumorfunctie van NK-cellen beheersen, en het zou ook interessant kunnen zijn om te bestuderen hoe NK-cellen de elim...

Video: Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Biology

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Medicine

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Immunology and Infection

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Isolating Sensory Neurons and Co-Culturing with Natural Killer Cells

Source:Ahmadi, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63800">Teasing out the interplay between natural killer cells and nociceptor neurons.</a> J. Vis. Exp. (2022).
This video describes the isolation of DRG neurons through enzymatic digestion, mechanical dissociation, and gradient centrifugation. The DRG cells are co-cultured with natural killer cells on an extracellular matrix to study the interaction between immune cells and sensory neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. NK ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Cells of the Innate Immune Response

The innate immune response is an immediate and non-specific response against pathogens, acting swiftly to prevent the spread of infections. The primary cells involved in this response are phagocytes and natural killer (NK) cells.
Phagocytes
Phagocytes police the peripheral tissues by removing cellular debris and responding to the invasion of foreign substances or pathogens. Many phagocytes attack and remove microorganisms even before lymphocytes detect them. The human body has two general classes of phagocytes &#8211; microphages and macrophages.
Microphages include the neutrophils and eosinophils that generally circulate in the blood. These phagocytes leave the bloodstream and enter peripheral tissues subjected to injury or infection. Neutrophils are abundant, mobile, and quick to phagocytize cellular debris or invading bacteria. The less abundant eosinophils target and destroy foreign substances or pathogens coated with antibodies or complement proteins.
Macrophages are large, active phagocytes primarily derived from circulating blood monocytes. They can be classified as wandering or fixed macrophages. The wandering macrophages move throughout the body, whereas the fixed macrophages reside permanently within specific tissues, such as the Kupffer cells in the liver, where they monitor and capture pathogens that bypass the initial defense lines. However, this distinction is not absolute. During certain infections, fixed macrophages may lose their attachments and start moving around the damaged tissue.
This extensive network of phagocytes is known as the monocyte-macrophage system, also referred to as the reticuloendothelial system.
Phagocytosis is a complex multi-step process that is initiated when phagocytes recognize and bind to foreign particles through surface receptors. This binding triggers the phagocyte to extend its membrane around the particle, encapsulating it within a compartment known as a phagosome. Subsequently, the phagosome merges with lysosomes to form a phagolysosome. Here, the foreign particle is broken down and destroyed by enzymes. The remnants are then expelled from the phagocyte, concluding the process of phagocytosis.
Natural Killer Cells
Natural killer (NK) cells are large granular lymphocytes comprising about 5&#8211;10% of lymphocytes in the blood and lymph. NK cells differ from phagocytes in their mode of action. Instead of engulfing pathogens, they directly recognize and eliminate the infected or cancerous cells. They do so by detecting changes in the surface proteins of these cells. Infected cells often display reduced levels of major histocompatibility complex (MHC) class I molecules,&#160; which are responsible for presenting antigens to T cells. NK cells can also recognize stress-induced molecules on infected cells through activating receptors. Upon recognition, NK cells release cytotoxic granules containing perforin and granzymes, which induce apoptosis in the target cells. This mechanism allows NK cells to rapidly respond to a variety of infections and prevent the spread of disease.

The two essential cells involved in the innate immune responses are phagocytes and natural killer, or NK cells.
Phagocytes &#8212; microphages and ...

Education

JoVE Core

Anatomy and Physiology

Unit 1

The Lymphatic and Immune System

KEY TERMS AND CONCEPTS

Nociception

Nociception&mdash;the ability to feel pain&mdash;is essential for an organism&rsquo;s survival and overall well-being. Noxious stimuli such as piercing pain from a sharp object, heat from an open flame, or contact with corrosive chemicals are first detected by sensory receptors, called nociceptors, located on nerve endings. Nociceptors express ion channels that convert noxious stimuli into electrical signals. When these signals reach the brain via sensory neurons, they are perceived as pain. Thus, pain helps the organism avoid noxious stimuli.

The immune system plays an essential role in pain pathology. Upon encountering noxious stimuli, immune cells such as mast cells and macrophages present at the site of injury release inflammatory chemicals such as cytokines, chemokines, histamines, and prostaglandins. These chemicals attract other immune cells such as monocytes and T cells to the injury site. They also stimulate nociceptors, resulting in hyperalgesia&mdash;a more intense response to a previously painful stimulus, or allodynia&mdash;a painful response to a normally innocuous stimulus such as light touch. Such pain sensitization helps protect the injured site during healing.

In some cases, pain outlives its role as an acute warning system if sensitization fails to resolve over time. Chronic pain&mdash;persistent or recurrent pain lasting longer than three months&mdash;often accompanies inflammatory conditions such as rheumatoid arthritis. Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen reduce pain by inhibiting the synthesis of the inflammatory molecules prostaglandins. However, NSAIDs and opioids currently used to combat pain suffer from severe side effects and the potential for addiction. Therefore, understanding the mechanisms underlying pain pathology can help develop more effective drugs to suppress pain perception with less severe negative side effects.

Pain and Nociception in Animals

Nociception&mdash;the ability to feel pain&mdash;is essential for an organism&rsquo;s survival and overall well-being. Noxious stimuli such as piercing ...

Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons

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