Expression of miR-155 in monocytes of people with migraine: association with phenotype, disease severity and inflammatory profile

Abstract

Background: miR-155 is involved in the generation and maintenance of inflammation and pain, endothelial function and immune system homeostasis, all functions that are relevant for migraine. The present study aims to assess the levels of miR-155 in migraine subtypes (episodic and chronic) in comparison to age- and sex-matched healthy controls.

Methods: This is a cross-sectional, controlled, study involving three study groups: I) episodic migraine (n = 52, EM), II) chronic migraine with medication overuse (n = 44, CM-MO), and III) healthy controls (n = 32, HCs). We assessed the interictal gene expression levels of miR-155, IL-1β, TNF-α, and IL-10 in peripheral blood monocytes using rtPCR. The monocytic differentiation toward the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes was assessed in circulating monocytes with flow cytometry analysis and cell sorting.

Results: miR-155 gene expression was higher in CM-MO group (2.68 ± 2.47 Relative Quantification - RQ) when compared to EM group (1.46 ± 0.85 RQ, p = 0.006) and HCs (0.44 ± 0.18 RQ, p = 0.001). In addition, miR-155 gene expression was higher in EM group when compared to HCs (p = 0.001). A multivariate analysis confirmed the difference between EM and CM-MO groups after correction for age, sex, smoking habit, preventive treatment, aura, presence of psychiatric or other pain conditions. We found higher gene expression of IL-1β, TNF-α, and lower gene expression of IL-10 in migraine participants when compared to HCs (p = 0.001 for all comparisons). TNF-α and IL-10 genes alterations were more prominent in CM-MO when compared to EM participants (p = 0.001). miR-155 positively correlated with IL-1β (p = 0.001) and TNF-α (p = 0.001) expression levels. Finally, in people with CM-MO, we described an up-regulated percentage of events in both M1 and M2 monocytic profiles.

Conclusions: Our study shows for the first time a specific profile of activation of miR-155 gene expression levels in monocytes of selected migraine subpopulations, more pronounced in subjects with CM-MO. Interestingly, mir-155 expression correlated with markers of activation of the inflammatory and immune systems. The CM-MO subpopulation showed a peculiar increase of both pro-inflammatory and anti-inflammatory monocytes which worths further investigation.

Trial registration: www.

Clinicaltrials: gov . (NCT05891808).

Keywords: Epigenetics; MiR-155; Migraine pathophysiology; Migraine spectrum; Monocytes differentiation; Neuroinflammation; microRNAs.

Fig. 1

miR-155 expression levels in peripheral blood monocytes among the three study groups. Legend: CM-MO: chronic migraine with medication overuse, EM: episodic migraine, HCs: healthy controls. RQ Relative Quantification: 2 − ΔΔCt = 2 − (ΔCt gene − ΔCt housekeeping gene); Ct cycle threshold. Box-plot: the range between the upper and lower border of the box indicates the interquartile range (IQR), spanning from the 25^th to the 75^th percentile. Within the box, the line indicates the median, and the cross denotes the mean. The upper and lower whiskers extend to the maximum and minimum values, excluding outliers. Symbols positioned above the upper whisker represent outliers, defined statistically as values beyond the 75^th percentile plus 1.5 times the IQR. Kruskal–Wallis Test was used for intergroup comparisons

Fig. 2

Square matrix showing the correlation analysis among miR-155 and the other study variables. Legend: miR-155 is expressed as “Relative Quantification (RQ)”, Age and Disease duration are expressed in “Years”, MHDs: Monthly Headache Days, MMDs: Monthly Migraine Days, MDDs: Days of acute drugs intake per month, NRS: 0 to 10 numeric rating scale. Correlation matrix: Spearman’s rank correlation coefficients are plot in an heatmap where the direction of the relationship between two variables is displayed by color (red for positive correlations, and blue for negative correlations, with intensity of color representing the strength of correlation from 1 to -1). Blank cells represent correlations that did not reach statistical significance

Fig. 3

mRNA expression levels of pro-inflammatory (IL-1 β, TNF-α) and anti-inflammatory (IL-10) in peripheral blood monocytes among the three study groups. Legend: CM-MO: chronic migraine with medication overuse, EM: episodic migraine, HCs: healthy controls. RQ: Relative Quantification: 2 − ΔΔCt = 2 − (ΔCt gene − ΔCt housekeeping gene); Ct: cycle threshold. Box-plot: the range between the upper and lower border of the box indicates the interquartile range (IQR), spanning from the 25^th to the 75^th percentile. Within the box, the line indicates the median, and the cross denotes the mean. The upper and lower whiskers extend to the maximum and minimum values, excluding outliers. Symbols positioned above the upper whisker represent outliers, defined statistically as values beyond the 75^th percentile plus 1.5 times the IQR. Kruskal–Wallis Test was used for intergroup comparisons

Fig. 4

Percentage of events recorded for the different monocytic sub-populations among the three study groups. Legend: Panel A: percentage of M1 monocytes (CD80 +) sub-populations. Panel B: percentage of M2 monocytes (CD163 +) sub-populations. CM-MO: chronic migraine with medication overuse, EM: episodic migraine, HCs: healthy controls, classical: “classical” monocytes (CD14 + /CD16– expression), non-classical: “non classical-intermediate” monocytes (CD14 + /CD16 + expression), M1: pro-inflammatory “M1” monocytes (CD80 + expression), M2: anti-inflammatory “M2” monocytes (CD163 + expression). Box-plot: the range between the upper and lower border of the box indicates the interquartile range (IQR), spanning from the 25^th to the 75^th percentile. Within the box, the line indicates the median, and the cross denotes the mean. The upper and lower whiskers extend to the maximum and minimum values, excluding outliers. Symbols positioned above the upper whisker represent outliers, defined statistically as values beyond the 75^th percentile plus 1.5 times the IQR. Kruskal–Wallis Test was used for intergroup comparisons