The biology of neuropathic pain

Unfortunately, humans are fragile individuals. Our backs hurt when we sleep on a bad mattress. We cannot carry a heavy object without enduring some aches the next day. And one explanation comes from the fragility of our nerves. They are composed of multitudes of fibers called axons. They are often susceptible to stretching, tension or even breakage that partially destroys them.

The immune system participates in the degeneration of injured nerves. This process is called Wallerian degeneration, during which macrophages clear the injury site from debris. That supports the subsequent regeneration of axons. However, this mechanism is not complete, and recurring nerve injuries can result in neuropathic pain, especially near the spinal cord. Different type of neuropathic pain and level of pain impact the patients. There is not a specific treatment to improve healing.

A novel study in mice explored the possible use of specific immune cells to treat chronic pain: natural killer (NK) cells. NK cells are largely studied considering their possible role as therapies against cancer, HIV or obesity. They are the “007” of the immune response: licensed to kill foreign operatives without the need to request approval from a fellow cell. NK cells rapidly identify infected and cancerous cells in the body and eliminate them. So, could NK cells employ a similar mechanism to ease neuropathic pain?

NK cells “kill me” signal expressed by sensory neurons cultured in vitro

Davies et al. highlighted a specific role for NK cells in the regeneration of a nerve following an injury. Previous investigations showed that in vitro culture of embryonic neurons with activated NK cells destroyed the axons. Surprisingly, in the presence of adult neurons, activated NK cells had no effect. As shown in the videos below, NK cells are more dynamic in the presence of embryonic sensory neurons.

Confocal Imaging of Embryonic DRG Neuron Cytotoxicity by IL-2-Stimulated NK Cells (Davies et al.)

Confocal Imaging of Adult DRG Neuron Resistance to Toxicity by IL-2-Stimulated NK Cells (Davies et al.)

NK cells employ different inhibitory and activating receptors to monitor their environment. NKG2D, a well-studied receptor of NK cells, can trigger their activation. NKG2D binds several ligands in humans: ULBP or MIC-A/B proteins. In mice, NKG2D interacts with proteins from the RAE1 (Retinoic Acid Early inducible protein 1) family.

Interestingly, the authors noticed that embryonic dissociated neurons expressed high amounts of RAE1, compared to adult neurons. That is why NK cells are able to only kill embryonic receptors. They validated their observation by blocking the expression of RAE-1 with a siRNA. They also performed experiments in the presence of a blocking antibody against NKG2D. Both experiences provoked a reduction of the specific lysis of neurons by activated NK cells.

Furthermore, the authors co-cultured adult sensory neurons in a microfluidic chamber. That enables proliferation and physical expansion of neurites. Interestingly, they observed that adult neurons can also express a higher amount of Raet1 mRNA (the gene that encodes RAE1 protein) that will result in de novo production of RAE1 protein over time. As expected, these cultured neurons were sensitive to NK cells killing. This observation was made in vitro and required confirmation in vivo.

NK cells are recruited in the site of injury in mice in vivo

To strengthen their observation, researchers developed a specific mouse model. The mice had particular NK cells expressing two proteins. First the GFP, a green fluorescent protein that allows tracking the migration of NK cells in the mice. Second, the receptor of the diphtheria toxin or DTR had the advantage to eliminate the NK cells with a diphtheria toxin injection. Overall, this particular mouse model confirms the direct role of NK cells in nerve injury.

They induced in the mice two distinct types of sciatic nerve injury. They cut or crushed the nerve that is connected to the back of the mice leg. In both cases, they observed an astonishing phenomenon. GFP-NK cells were migrating to the site of injury. They observed that neurons, similar to the in vitro observation, started to express RAE1 that leave them susceptible to NK cell attack. It appears that NK cells were reacting, migrating into the nerve and killing the damaged axons.

Within days after the nerve injury, experiments indicated that mice treated with IL-2, an activating cytokine of NK cells, had significantly reduced sensation in the affected paw. But once the damaged axons were cleared—thanks to NK cells—healthy ones began to grow back. Fifteen days after damaging the nerve, the mice’s paws regained sensation.

Other mice, whose NK cell were not activated, had a similar recovery timeline. However, their partially damaged axons were not completely eliminated. 30 days after the nerve injury, neurologic tests still showed high levels of touch-induced pain. It seems the partially damaged nerves may continue to carry pain signals to the brain. This observation is comparable to the human neuropathic pain that leads to chronic pain and hypersensitivity.

Imagining a better future for neuropathic pain with NK cells

Modulating the immune response often displays risk due to our incomplete knowledge. Ultimately, there is a need to better understand the function of NK cells and how to harness their potential. Nonetheless, the research presented by Davies et al. suggests that the modulation of NK cell function could efficiently eliminate damaged axons. This could have a positive impact in treating axonal degeneration. Healthy axonal regrowth would happen and potentially decreased chronic neuropathic pain. This could improve the lives of more than 8% of the world population who suffer from this pathology. It would replace pain killer prescription that has a lot of secondary effects.

 

Reference:
Davies et al. (2019) Natural killer cells degenerate intact sensory afferents following nerve injury. Cell. 174, 4. https://doi.org/10.1016/j.cell.2018.12.022
Videos available under CC BY 4.0.

Featured Image:
Neuron in tissue culture by GerryShaw, Wikimedia Commons.