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Removing Damaged Nerves: A Potential Role for Natural Killer Cells in Neuropathic Pain

NK cells degenerate injured axons and may help to reduce pain from nerve injury in mice

by Dara Bree


12 March 2019


PRF News

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NK cells degenerate injured axons and may help to reduce pain from nerve injury in mice

Damage to peripheral nerves sets in motion a series of events that facilitate the removal of neuronal debris following Wallerian degeneration and promote the regeneration of axons. But this can also contribute to neuropathic pain. New research in mice now reveals an immune mechanism that complements Wallerian degeneration and may ease neuropathic pain.

 

The study, led by Seog Bae Oh at Seoul National University, South Korea, and largely funded by the National Research Foundation of Korea, reveals that in the early stages of peripheral nerve cut or crush injury, natural killer (NK) cells infiltrate damaged nerves, where they produce a cytotoxic environment that degenerates damaged axons. At the same time, NK cell activity correlated with the loss of sensation that can occur following this axonal degeneration, but also eased subsequent neuropathic pain hypersensitivity. Consistent with these findings, mice lacking NK cells displayed persistent pain-like behaviors following nerve injury.

 

“The exciting implication of this paper is that natural killer cells can contribute to Wallerian degeneration. Previously, macrophages were really the only cells that were recognized to play a role in this process,” said Halina Machelska, Charité-Universitätsmedizin, Berlin, Germany, who was not involved in the study. “The therapeutic potential of targeting NK cells to remove axonal debris and reduce pain is also exciting, and this study is an important first step in realizing that potential,” according to Machelska.

 

One important caveat, though, which Machelska offered and which the researchers acknowledge, is that the effects on pain from partial crush injury were modest, so how the findings truly bear on pain remains uncertain.

 

The research appeared online January 31 in Cell.

 

A new role for NK cells

The role of the immune system in pain has long held the interest of Seog Bae Oh, and also first author Alexander Davies, now at the University of Oxford, UK, who wondered specifically about how NK cells might contribute, with much of the work performed in Oh's lab in Seoul.

 

“There was clearly evidence of microglia and T cell activity in pain, but natural killer cells piqued my interest, as there was some scant evidence at the time suggesting an association between natural killer cell overactivity and some sort of nerve damage,” Davies told PRF.

 

NK cells are on constant surveillance for tumor cells and for virus-infected cells, and are classically activated by the cytokine interleukin-2 (IL-2). So the researchers began by examining how IL-2-activated NK cells affected dorsal root ganglia (DRG) neurons in vitro. They found that these activated NK cells fragmented the neurites of adult DRG neurons, compared to control inactivated NK cells.

 

They also confirmed previous reports by identifying retinoic acid early inducible protein 1 (RAE1) as an endogenous ligand in embryonic DRGs responsible for NK activation. Specifically, blocking NKG2D, the receptor for RAE1, with a receptor-blocking antibody significantly decreased DRG neurite fragmentation. Consistent with this result, experiments using small interfering RNA (siRNA) to knock down RAE1 caused a 20 percent reduction in DRG cytotoxicity.

 

“RAE1 is specifically upregulated by cells that are in trouble or damaged, and its induction and subsequent expression on the cell membrane of those cells licenses natural killer cells to destroy the damaged cell,” said Michael Costigan, co-lead on the study at Children's Hospital Boston and Harvard Medical School, US.

 

To examine this phenomenon in vivo, the team cut the spinal nerve of the fifth lumbar DRG in adult mice. They saw a pronounced upregulation of RAE1 in these DRG neurons that persisted for seven days, compared to uninjured cells. Using genetic approaches to label NK cells in mice in vivo, the researchers also observed a marked recruitment of NK cells to the site of spinal nerve injury as well as an increase in granzyme B, a protease essential for the cytotoxic functions of NK cells.

 

“What is interesting from our data is that in sensory neurons, RAE1 protein is localized to the distal site of the injured nerve axon and not to the cell body, which enables degeneration of the injured axon but crucially spares the cell body,” said Costigan.

 

Cutting or crushing?

Unsurprisingly, cutting the spinal nerve of the fifth lumbar DRG caused hind paw mechanical hypersensitivity in mice. Mice that were genetically bred to systemically lack NK cells also displayed this hypersensitivity up to 21 days post-injury which, interestingly, was not exacerbated by the NK cell depletion. This suggested that the role of these cells may not be in the initial development of neuropathic pain behaviors but rather down the line.

 

But a transection of a nerve is a severe and irreversible injury that results in permanent nerve damage. Based on their in vitro data, the researchers became interested in how nerves recover from injury and so considered different ways to cause partial damage.

 

“We initially looked at a sciatic nerve crush to model recovery from this type of injury, and in particular the role the NK cells might play in this,” Davies said. “However, with typical crush injury models you are already causing significant physical damage. We then came up with the idea of a partial crush paradigm which enabled us to see a gain and loss of axonal function in the same model.”

 

Using a partial crush model of the sciatic nerve in mice, the researchers saw an increase in RAE1 protein with accompanying NK cell recruitment in the nerve but not in the DRG of mice. This again suggested a localization of RAE1 to the injured site and not to the cell body.

 

Boosting NK signaling, dampening the pain

To investigate whether NK cells could selectively degenerate partially injured sensory axons in the partial crush model, the investigators boosted NK cell activity in vivo in mice by systemically administering an IL-2 antibody complex. Non-NK cell-stimulated control mice had residual sensitivity to hind paw pin prick stimulation one day after the partial crush injury, and near full recovery of sensitivity six days later. This suggested that a portion of nerve fibers were not fully axotomized by the injury.

 

In contrast, mice with NK cells stimulated by the antibody complex showed a robust reduction in pin prick sensitivity six days following partial crush injury, which was paralleled by a 10-fold increase in NK cell infiltration at the crush site, compared to controls. The lack of pin prick sensitivity in NK cell-stimulated mice suggests that boosting NK cell signaling can specifically remove injured axons and consequently may also mitigate the tactile hypersensitivity that is present when damaged axons remain.

 

“Aside from removing axonal debris, it struck me that NK cells may also reduce pain by releasing analgesic mediators. There is a broad range of research showing that immune cells, including macrophages which contribute to Wallerian degeneration, secrete opioid peptides, with evidence to show that this may also be relevant to human pain. Perhaps NK cells are acting in a similar way,” said Machelska.

 

Compared to a full crush injury, partial nerve crush resulted in lower withdrawal thresholds (after recovery of pin prick sensation) that persisted for at least 30 days post-injury, again suggesting the presence of remaining damaged fibers in the partial crush injury model. Stimulating NK cells with IL-2 complex after partial crush injury attenuated this increased mechanical sensitivity.

 

However, Machelska cautioned, “while there is a difference in hind paw withdrawal thresholds between control and NK cell-stimulated mice following partial crush injury, the magnitude of difference is very little, and from the point of view of pain relief, you could argue how relevant it is.”

 

Finally, the team overexpressed RAE1 in TRPV1-lineage thermal nociceptive DRG neurons in uninjured mice. This led to a loss of sensitivity specific to noxious heat, with no effect on mechanical thresholds. This suggests that uninjured neurons, when under stress, express ligands that can license axonal damage by NK cells. Peripheral neuropathies in human patients are associated with a dying back of peripheral sensory nerves, and these results suggest that targeting NK cells may have therapeutic potential for these disorders.

 

Pare back to grow back

Costigan and colleagues recognize the need to interpret some of their new findings cautiously, considering the size of the effects they observed, but they plan to address this in future studies.

 

“The effects are relatively subtle over multiple experiments, which may be due to the fact that NK cells have an ability to turn themselves off and become very unsensitized to this process,” Costigan said. “Therefore, we want to work further on these mechanisms to better understand them, but to also remember that interfering with the immune system carries its own inherent risks,” explained Costigan.

 

As for Davies, he is keen to investigate if NK cells have similar actions in people with nerve damage. “There are clinical reports that suggest NK cells are found in the nerve on occasion, but are they? And if so, how do they respond, and how can that be controlled to try and maximize the kind of benefits we think we see in mice? The immune systems of mice and men have many differences so much work remains to see if we can translate these findings into relevant human cases of neuropathic pain,” according to Davies.

 

In the meantime, the researchers offer an analogy to put the new findings into context.

 

“Imagine a landowner who has trees with unhealthy or dying branches,” Davies said. “To generate fresh growth in these trees he or she cuts back the tree to a healthy stump, a process known as coppicing. These trees then have the ability to regrow their branches, similar to our peripheral nerves. You could imagine that NK cells have this partnership with nerves and that when a nerve is damaged it signals to NK cells to essentially come and chop these branches off so they can grow back healthily again.”

 

Dara Bree is a postdoctoral fellow at Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, US.

 

Editor's note: The text in the second and sixth paragraphs of this news story has been amended to better reflect Dr. Oh's contribution to the study and to include information about the funding for the research. 

 

Featured image: High-magnification image of NK cell in contact with embryonic DRG neurite. Credit: Davies et al. Cell. 2019 Feb 07; 176(4):716-728.e18.

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