Peripheral cytokine and monocyte phenotype associations in drug-resistant epilepsy

Abstract

Novel therapeutic targets are required to develop new treatments to lower the rates of drug-resistant epilepsy (DRE). This study assessed differences in plasma inflammatory biomarker concentrations and monocyte phenotype and function in patients with DRE versus psychogenic non-epileptic seizures (PNES). Luminex was used to analyse plasma samples from 21 DRE cases and 19 PNES controls for concentrations of selected cytokines and chitinase 3-like 1 (CHI3L1). Flow cytometry was used to quantify the percentage of monocytes expressing HLADR, CD14, CD16, CD11b, P2X7R and their median fluorescence intensity (MFI) ratio in 22 DRE and 11 PNES patients. Flow cytometry was used to assess P2X7 receptor (P2X7R) pore function via YO-PRO-1 uptake. No difference in cytokine and CHI3L1 concentrations was seen. Compared to PNES, DRE had a higher percentage of 'classical' monocytes (CD14++CD16-) and less 'non-classical' monocytes (CD14-CD16+). The percentage of 'classical' monocytes expressing P2X7R was increased in DRE compared to PNES. CD11b MFI ratio was increased in 'classical' and 'intermediate' (CD14+CD16+) monocytes. P2X7R pore function was similar between groups. Overall, while cytokine levels were similar between groups, the differences in monocyte phenotype indicates a more 'proinflammatory' circulating innate immune state in DRE. Thus, monocytes may be a novel therapeutic target for future research.

Keywords: Chitinase 3-like 1; Cytokine; Drug resistant epilepsy; Monocyte; P2X7 receptor; Psychogenic non-epileptic seizures.

Fig. 1

Monocyte gating strategy. (a) selection of live monocytes (DAPI negative) expressing HLADR and separation of those into classical (CD14++CD16−), intermediate (CD14+CD16+) and non-classical (CD14−CD16+) monocytes. (b) gating strategy for the selection of CD11b or P2X7R expression for each monocyte subset.

Fig. 2

Concentrations of various analytes in picograms/litre. Results obtained using Luminex to test plasma from drug resistant epilepsy patients and psychogenic non-epileptic controls—there were no differences found between the two cohorts. (a) ‘proinflammatory’ cytokines; (b) ‘immunomodulatory’ cytokines; (c), chemokines; (d), Chitinase-3 Like 1 protein. Abbreviations: DRE, drug resistant epilepsy; PNES, psychogenic non-epileptic seizures.

Fig. 3

Percentage of live monocytes expressing various cell surface markers. Median and interquartile ranges depicted. *p > 0.01 to 0.05, **p 0.001 to ≤ 0.01. (a) percentage of live monocytes expressing single cell surface markers; (b) percentage of HLADR positive live monocytes with classical (CD14++CD16−), intermediate (CD14+CD16+) and non-classical (CD14− CD16+) phenotypes; (c) percentage of subsets in b that express CD11b or P2X7R. Abbreviations: DRE, drug resistant epilepsy; PNES, psychogenic non-epileptic seizures.

Fig. 4

Median fluorescence intensity (MFI) ratios for various cell surface markers. MFI ratio = (MFI_{all stained sample} − MFI_{unstained sample})/MFI_{unstained sample}. Results are displayed on a log10 scale. Median and interquartile range are shown. *p > 0.01 to 0.05. (a) MFI ratio of live monocytes expressing single cell surface markers; (b) MFI ratio of HLADR positive live monocytes with classical (CD14++CD16−), intermediate (CD14+CD16+) and non-classical (CD14− CD16+) phenotypes. Abbreviations: DRE, drug resistant epilepsy; PNES, psychogenic non-epileptic seizures.

Fig. 5

Monocyte P2X7R functional pore assay comparison between drug resistant epilepsy and psychogenic non-epileptic seizure cohorts. Median fluorescence intensity ratio of monocytes treated with YO-PRO-1 Iodide (5 µM) with or without concomitant exposure to Bz-ATP (P2X7R agonist; 200 µM) and/or Az (P2X7R antagonist, 4 µM). Abbreviations: AZ, AZ 10606120 dihydrochloride; BZ, 2’(3’)-O-(4-Benzoylbenzoyl)adenosine 5’-triphosphate triethylammonium salt; DRE, drug resistant epilepsy; PNES, psychogenic non-epileptic seizure; YO-PRO, YO-PRO-1 Iodide.