A Neuroimmune Signaling Pathway Drives Chronic Headache

A new study in mice finds that CCL2-CCR2 signaling in macrophages and T cells sensitizes trigeminal ganglion neurons, driving chronic headache in migraine models. With synergistic effects on CGRP, the pathway could be a new therapeutic target for migraine.

The meninges, as the only pain-sensitive structures in the skull, are a key location for the generation of headache pain. But exactly how neurons in the meninges become sensitized to drive the pain is less well known.

One of the challenges to a better understanding is that the meninges contain multiple cell types, including neurons, immune cells, and blood vessel cells. How these cell types interact with each other to cause headache pain, and via which molecules and signaling pathways, remains uncertain.

But now, a new study has uncovered a key signaling pathway linking neurons and immune cells in the meninges that drives the development of headache pain.

Yu-Qing Cao and colleagues at Washington University School of Medicine in St Louis, US, found that, in mouse models of migraine, trigeminal neurons release C-C motif ligand 2 (CCL2), an immune system protein known as a chemokine. CCL2 then activates its receptor, CCR2, on macrophages and T cells. That results in enhanced calcitonin gene-related peptide (CGRP) signaling in the neurons.

Blocking this neuroimmune pathway reversed headache pain in the animals. And, co-administration of antibodies against CCR2 and CGRP was more effective in reversing the pain than the individual antibodies were.

“This paper is important because it introduces a new therapeutic target for migraine, but also one that is consistent with the framework used to develop new drugs in the migraine field. This paper also underscores the importance of inflammation in migraine because of the identification of the macrophages as a target,” said Michael Moskowitz, Massachusetts General Hospital and Harvard Medical School, Boston, US, who was not part of the research.

The study appeared in Brain on June 7, 2023.

Increased CCL2 and CCR2 during headache
Cao said she and her co-authors had learned a lot about the role of CCL2-CCR2 signaling from the pain field, where researchers have shown that it contributes to chronic pain after tissue or nerve injury. There was reason to believe this type of pathway could be involved in migraine, too.

“There is already a lot of work on neuroimmune interactions in migraine, particularly around chemokine release and immune cell activation. That’s why we became interested in CCL2 as a driver of sensitization in migraine,” said Cao.

While the team also suspected CCL2-CCR2 signaling in particular might play a role in migraine, there was little direct evidence to show this, apart from a snippet of data from human tissue.

“Interestingly, sustained elevation of CCL2 mRNA [messenger RNA] is found in the cranial periosteum of chronic migraine patients, suggesting that the CCL2-CCR2 pathway might be involved in the development of chronic migraine,” first author Sun Ryu told Migraine Science Collaborative via email.

The group’s first experiments tested whether chronic headache is accompanied by changes in levels of CCL2 and CCR2 mRNA. Here, the investigators injected mice with nitroglycerin (NTG), a known migraine trigger in people, once every two days for a total of 10 days. They found increased levels of CCL2 and CCR2 mRNA in both dura and trigeminal ganglion tissues after NTG administration.

A knockout blow
The team then investigated how CCL2-CCR2 signaling might contribute to chronic headache.

To do so, they used genetically modified mice (“knockout” mice) missing the gene that makes CCL2 or missing the gene for CCR2. They measured the knockout animals’ behavioral responses to painful mechanical stimuli applied to facial skin before and after NTG administration. The idea was to see how the absence of CCL2 or CCR2 would affect headache pain.

Normally, wild-type mice (animals with the usual presence of the CCL2 and CCR2 genes) show mechanical pain hypersensitivity after receiving NTG. However, this was not the case in the knockout mice.

“If you look at the knockout data, you see the mice don’t respond to NTG at all. This is really functionally important as it shows a clear role of CCL2-CCR2 signaling in chronic migraine. However, this is global knockout data, so the mechanism is not very specific,” said Cao. She was referring to how the genetic manipulation eliminated the genes throughout the body, rather than only in a particular cell type of interest. That makes it difficult to know exactly how CCL2-CCR2 signaling contributed to headache.

Cao was keen to verify the knockout data because deleting a gene can cause compensatory changes that make it difficult to know exactly what the specific contribution of the gene is. So the researchers used pharmacological tools to block CCL2-CCR2 signaling in mice after they received NTG.

They found that pain hypersensitivity in response to NTG went down after the animals received either a CCR2 antagonist or an antibody that blocks CCL2 signaling.

“The antibody results are important because they verified what we saw in the knockout mice. The antibodies don’t cross the blood-brain barrier, so it’s most likely peripheral CCL2-CCR2 signaling that’s being affected,” said Cao.

The researchers would find a similar role for peripheral CCL2-CCR2 signaling in a different migraine model in which mice are subjected to restraint stress, followed by administration of sodium nitroprusside. This is a substance that releases nitric oxide, a known contributor to migraine.

Linking neurons and immune cells
Next, Cao’s team wanted to learn more about the cellular mechanisms underlying the effects of CCL2-CCR2 signaling on headache pain.

“Our first hypothesis was that CCL2 would be released from neurons and then act on CCR2 expressed on neurons – we thought everything would take place in primary sensory neurons,” said Cao.

However, the investigators were surprised to find that CCL2-CCR2 signaling involved crosstalk between neurons and immune cells.

To make that discovery, the group used a method called RNAscope, which allows researchers to visualize single RNA molecules in individual cells. In this case, they used the method to visualize CCL2 and CCR2 mRNA in different cell types in the trigeminal ganglion.

They found CCL2 mRNA in trigeminal neurons, as well as in cells associated with dural blood vessels (mural cells). And, CCL2 mRNA levels were higher in individual neurons after NTG.

However, CCR2 mRNA was present in immune cells, including subsets of macrophages and T cells, in the trigeminal ganglion and dura, but not in trigeminal neurons themselves.

“It was this RNAscope data that really showed us that we were looking at neuroimmune interactions here and not just neuronal sensitization in the migraine models,” said Cao.

The researchers went on to selectively eliminate CCR2 in T cells or macrophages to investigate how that affected headache pain. They found that this prevented pain hypersensitivity in mice after NTG administration. Control experiments showed that deleting CCR2 in neurons had no effect on pain, solidifying a role for the immune cells.

Summary of the contributions of peripheral CCL2-CCR2 and CGRP signalling pathways to NTG-induced sensitization. Image and caption from Ryu et al.

Links to CGRP
The neuropeptide CGRP has a well-known role in driving migraine pain by sensitizing trigeminal neurons. Cao and her team suspected that CCL2-CCR2 signaling may interact with CGRP signaling to contribute to neuronal sensitization.

So they went back to the CCR2 global knockout mice and tested whether the shutdown of CCR2 signaling in these animals affected CGRP in the NTG model. While NTG increased both CGRP and CGRP receptor levels in wild-type mice, it did not do so in CCR2 knockout mice. This suggested that CCR2 signaling is required for CGRP levels to increase during migraine attacks.

To test the idea further, the researchers gave anti-CGRP and/or CCL2-blocking antibodies to mice after NTG. While low doses of either antibody alone did not reduce headache pain, the combination of the two robustly did so.

“This shows that inhibition of peripheral CGRP signaling and CCL2-CCR2 signaling is a more effective therapy than targeting either pathway alone,” said Ryu.

The researchers would also find links between CCL2-CCR2 signaling and pituitary adenylate cyclase-activating polypeptide (PACAP), another intriguing target for migraine treatment. Here, too, unlike control animals, CCR2 knockout mice did not show increased trigeminal neuron responsiveness to PACAP in response to NTG.

Clinical applications: Exciting but far off
Moskowitz emphasized how important the new findings could be for people with migraine who don’t benefit from CGRP antibodies.

“We’ve seen major successes in using CGRP antibodies to treat migraine. However, there’s a significant population of people with migraine who don’t respond to them. The synergistic effects of CGRP and CCR2 signaling have potentially very important clinical implications for these patients in particular,” he said.

According to Moskowitz, CCR2 antibodies have strong clinical potential since they are already in Phase 2 clinical trials for cancer. So, much of the drug development has already been done, potentially making the antibodies easier to implement in future clinical trials for migraine.

But many questions remain before the potential from the new study can be fulfilled. One limitation Moskowitz pointed to is the absence of electrophysiology experiments – testing the electrical excitability of neurons – which makes it difficult to know if the changes in CCL2-CCR2 signaling are actually driving the headache pain in the NTG model.

“This paper is a tour de force,” he said. “But what’s lacking is the electrophysiology, meaning there are black boxes between the molecular and behavioral findings.”

For her part, Cao pointed to the need for a deeper understanding of how CCL2-CCR2 signaling interacts with CGRP signaling, among other gaps in knowledge.

“There are also big open questions about cellular interactions. What happens to infiltrating macrophages in the trigeminal ganglia and the dura?” she said, referring to the entry of macrophages into tissues after injury. “How are T cells affected, and what do they release? And then how does this change the sensory neurons? The involvement of both macrophages and T cells in migraine pathophysiology is certainly something we’re excited to pursue,” said Cao.

Fred Schwaller, PhD, is a freelance science writer based in Germany. Follow him on Twitter @SchwallerFred

Reference
Peripheral CCL2-CCR2 signalling contributes to chronic headache-related sensitization.
Ryu et al.
Brain. 2023 Jun 7;awad191. Online ahead of print.

Featured image: Colorized scanning electron micrograph of a T lymphocyte. Credit: NIAID.