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+) 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.
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.
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.
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
2. DRG neuron isolation and culture
3. Co-culture and flow cytometry
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 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.
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 µM) 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 ± 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. Please click here to view a larger version of this figure.
Solution | Composition | ||
Collagenase/Dispase (C/D) | Sterile PBS supplemented with collagenase (1 mg/mL) and Dispase II (2.4 U/mL) | ||
DMEM | Sterile DMEM supplemented with FBS (10%), penicillin (100 I.U.) and streptomycin (100 µg/mL) | ||
Supplemented RPMI 1640 | Sterile RMPI 1640 supplemented with penicillin (100 I.U.), streptomycin (100 µg/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) | ||
Neurobasal | Sterile Neurobasal supplemented with penicillin (100 I.U.), streptomycin (100 µg/mL), L-glutamine (200 μM), B-27 (2%), NGF (50 ng/mL), and GDNF (2 ng/mL) | ||
Neurobasal MX | Sterile Neurobasal supplemented with penicillin (100 I.U.), streptomycin (100 µg/mL), L-glutamine (200 μM), 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) | ||
FACS sorting buffer | Sterile PBS supplemented with FBS (2%) and EDTA (1 mM) |
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.
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 µM) 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 authors have nothing to disclose.
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).
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 |