Background & Aims

The importance of bidirectional neuroimmune interactions is increasingly recognised in migraine pathogenesis. Release of calcitonin gene-related peptide (CGRP) from sensory terminals on nociceptors plays a critical role in altering vascular permeability and recruiting peripheral immune cells to the meninges, contributing to pain (Burstein et al., 2015), and anti-CGRP immunotherapy has recently been approved to treat migraine (de Vries Lentsch et al., 2022). Downstream immune mediators, such as cytokines, matrix metalloproteinases, and kynurenine pathway (KP) metabolites, have been implicated in initiating migraine attacks (Geng et al., 2022; Imamura et al., 2008; Leira et al., 2007; Tuka et al., 2021), as have changes in circulating immune cells and platelets (Lee et al., 2022; Zeller et al., 2004). Despite this, immune cell subsets and their functional states have been poorly characterised to date. Therefore, this study aimed to perform a comprehensive analysis across migraine phases.

Methods

A quantitative proteomic analysis was performed on 10 migraineurs (n=5 chronic/n=5 episodic) and 10 age-matched healthy controls by SWATH-mass spectrometry (Collins et al., 2017). Next, mass cytometry, ELISA and KP analysis were performed on blood collected from 38 migraineurs (n=24 chronic/n=14 episodic) and 22 healthy controls. Migraineurs were classified into migraine phases (interictal: n=8; preictal: n=11; ictal: n=11; postictal: n=8) based on migraine diary data and interviews at time of blood collection. Using a panel of 39-heavy metal conjugated antibodies for all major leukocyte populations, cells were stained and acquired on a Helios mass cytometer, with analysis performed by manual gating. Plasma levels of CGRP and matrix metalloproteinase-9 (MMP-9) were quantified using ELISA (ABclonal). Plasma KP metabolite levels were quantified using gas/high-performance liquid chromatography (Staats-Pires et al, 2020).

Results

Proteomic analysis uncovered the differential expression of multiple soluble proteins in plasma related to vascular dysfunction and cell-cell adhesion in migraineurs. Migraineurs had an increase in neutrophils (P<0.05), and a higher proportion of neutrophil-platelet aggregates compared to controls (P<0.01). Neutrophils expressed higher levels of matrix metalloproteinase-9 (MMP-9) in ictal and interictal periods that were significantly decreased during preictal and postictal periods (P<0.05). Moreover, combined preictal and ictal plasma MMP-9 levels were significantly increased relative to the interictal period (P<0.05). Surprisingly, plasma CGRP levels were lower in migraineurs relative to controls (P<0.05), but across migraine phases CGRP showed a similar trend to MMP-9, increasing during preictal and ictal periods. The immune-mediated KP showed a shift towards neurotoxic quinolinic acid (QUIN) production and away from the protective kynurenic acid during the preictal phase (P<0.05).

Conclusions

These results strongly support a role for interactions between immune, vascular, and metabolic pathways in migraine pathogenesis, with dysregulation of vascular homeostasis and endothelial cell function likely mediated by circulating immune cells and their mediators. In particular, neutrophil-platelet aggregates and their release of MMP-9 into the plasma during the interictal period may be a crucial step in driving vascular dysregulation initiating a migraine attack. Similarly, temporally coincident immune-mediated KP metabolism towards the neurotoxic QUIN branch may directly activate trigeminal nociceptors, as has been suggested in other chronic pain conditions (Gunn et al., 2020). Further investigation is required to link these observational changes to an underlying mechanism contributing to migraine pathogenesis, and thus underpin the therapeutic potential of targeting MMP-9 and QUIN, or indeed neutrophil-platelet aggregates, to prevent migraine attack onset.

References

Burstein, R., Noseda, R., Borsook, D., 2015. Migraine: multiple processes, complex pathophysiology. J. Neurosci. Off. J. Soc. Neurosci. 35, 6619–6629. https://doi.org/10.1523/JNEUROSCI.0373- 15.2015

Collins BC, Hunter CL, Liu Y, et al., (2017) Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry. Nat Commun. 8(1):291. doi: 10.1038/s41467-017-00249-5.

de Vries Lentsch, S., Garrelds, I.M., Danser, A.H.J., Terwindt, G.M., MaassenVanDenBrink, A., 2022.
Serum CGRP in migraine patients using erenumab as preventive treatment. J. Headache Pain 23, 1–7. https://doi.org/10.1186/s10194-022-01483-z

Geng, C., Yang, Z., Xu, P., Zhang, H., 2022. Aberrations in peripheral inflammatory cytokine levels in migraine: A systematic review and meta-analysis. J. Clin. Neurosci. 98, 213–218. https://doi.org/10.1016/j.jocn.2022.02.026

Gunn J, Hill MM, Cotten BM, Deer TR. 2020 An Analysis of Biomarkers in Patients with Chronic Pain. Pain Physician. 23(1):E41-E49.

Imamura, K., Takeshima, T., Fusayasu, E., Nakashima, K., 2008. Increased Plasma Matrix Metalloproteinase-9 Levels in Migraineurs. Headache J. Head Face Pain 48, 135–139. https://doi.org/10.1111/j.1526-4610.2007.00958.x

Lee, S.-H., Kim, J.-H., Kwon, Y.-S., Sohn, J.-H., 2022. Role of Peripheral Inflammatory Markers in Patients with Acute Headache Attack to Differentiate between Migraine and Non-Migraine Headache. J. Clin. Med. 11, 6538. https://doi.org/10.3390/jcm11216538

Leira, R., Sobrino, T., Rodríguez-Yáñez, M., Blanco, M., Arias, S., Castillo, J., 2007. MMP-9 Immunoreactivity in Acute Migraine. Headache J. Head Face Pain 47, 698–702. https://doi.org/10.1111/j.1526-4610.2006.00641.x

Staats Pires A, Heng B, Tan VX, Latini A, Russo MA, Santarelli DM, Bailey D, Wynne K, O’Brien JA, Guillemin GJ, Austin PJ. (2020) Kynurenine, Tetrahydrobiopterin, and Cytokine Inflammatory Biomarkers in Individuals Affected by Diabetic Neuropathic Pain. Front Neurosci. 14:890. doi: 10.3389/fnins.2020.00890.

Tuka, B., Nyari, A., Cseh, E.K., Kortesi, T., Vereb, D., Tomosi, F., Kecskemeti, G., Janaky, T., Tajti, J., Vecsei, L., 2021. Clinical relevance of depressed kynurenine pathway in episodic migraine patients: potential prognostic markers in the peripheral plasma during the interictal period. J Headache Pain 22, 60. https://doi.org/10.1186/s10194-021-01239-1

Zeller, J.A., Frahm, K., Baron, R., Stingele, R., Deuschl, G., 2004. Platelet-leukocyte interaction and platelet activation in migraine: a link to ischemic stroke? J. Neurol. Neurosurg. Psychiatry 75, 984–987. https://doi.org/10.1136/jnnp.2003.019638

Presenting Author

Paul Austin

Poster Authors

Paul Austin

PhD

University of Sydney

Lead Author

E-mail

Jayden O'Brien

University of Sydney

Lead Author

E-mail

Benjamin Heng

PhD

Macquarie University

Lead Author

E-mail

Charlie Anh

PhD

Macquarie University

Lead Author

E-mail

Ananda Staats-Pires

PhD

Macquarie University

Lead Author

E-mail

Ashleigh Wake

The University of Sydney

Lead Author

E-mail

Rebecca Wong

BSc

Baker Institute

Lead Author

E-mail

Noemi Meylakh BA

BSc

The University of Sydney

Lead Author

E-mail

Vaughan G Macefield BSc

PhD

Monash University

Lead Author

E-mail

Luke Henderson

PhD

The University of Sydney

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Migraine