IL-22, GM-CSF and IL-17 in peripheral CD4⁺ T cell subpopulations during multiple sclerosis relapses and remission. Impact of corticosteroid therapy

<div><p>Multiple sclerosis (MS) is thought to be a Th17-mediated dysimmune disease of the central nervous system. However, recent publications have questioned the pathogenicity of IL-17 per se and rather suggest the implication of other Th17-related inflammatory mediators. Therefore, we studied the expression of GM-CSF, IL-22, IL-24, IL-26 and CD39 in peripheral blood mononuclear cells (PBMCs) from MS patients during relapses, remission and following corticosteroid treatment. We performed qPCR to measure mRNA levels from <i>ex vivo</i> or <i>in vitro</i>-stimulated PBMCs. Cytokine levels were determined by ELISA. We used flow cytometry to assess GM-CSF⁺, IL-22⁺ and CD39⁺ cells in relationship to IL-17⁺ CD4⁺ T cells. Our results showed that IL-22 mRNA and IL-22⁺CD4⁺ lymphocytes are increased in circulating cells of relapsing MS patients compared to remitting patients while GM-CSF was unchanged. We have further shown that 12.9, 39 and 12.4% of Th17 cells from MS patients during relapses expressed IL-22, GM-CSF and CD39 respectively. No changes in these proportions were found in stable MS patients. However, the majority of GM-CSF⁺ or IL-22⁺ T cells did not co-express IL-17. GM-CSF mRNA, but not IL-22 mRNA, was dramatically decreased <i>ex vivo</i> by ivMP. Our results contribute to a better characterisation of Th17, Th22 and ThGM-CSF cells in the setting of MS and according to disease activity.</p></div

FACS analysis of GM-CSF, IL-22 and CD39 coexpression with IL-17 in CD4⁺ T cells.

<p>PBMCs were fixed and stained after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor. CD4⁺ cells were gated among the CD3⁺ population. Representative dot plots of (A) GM-CSF, (B) IL-22 and (C) CD39 costaining with IL-17. Numbers in each quadrant represent the average percentage of positive CD4⁺ T cells in relapsing MS patients.</p

IL-22 mRNA and IL-22-producing cells in PBMCs from relapsing and stable MS patients and from healthy controls (HC).

<p>Quantitative-PCR to measure IL-22 mRNA expression (A) <i>ex vivo</i> (Relapsing MS: n = 56, Stable MS: n = 16, HC: n = 37) and (B) after 4h of stimulation by PMA/ionomycin (Relapsing MS: n = 14, Stable MS: n = 10, HC: n = 12). (C) PBMCs were stained for IL-22, CD3 and CD4 after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor and analysed by flow cytometry (Relapsing MS: n = 15, Stable MS: n = 12, HC: n = 14). Scatter dot plot shows the percentage of IL-22⁺CD4⁺ T cells. The horizontal lines of scatter plots represent the median value in all subgroups. * and *** indicate respectively p-values ≤ 0.05 and ≤ 0.001.</p

GM-CSF and IL-22 before and after intravenous methylprednisolone (ivMP) treatment in paired samples of relapsing MS patients.

<p>Quantitative-PCR to measure GM-CSF and IL-22 mRNA expression <i>ex vivo</i> (A, n = 13 and D, n = 17) and (B, n = 8 and E, n = 8) after 4h stimulation by PMA/ionomycin. Results are expressed relative to the mean of the relapsing patients set at 1. PBMCs were stained for GM-CSF (C, n = 8) or IL-22 (F, n = 15), CD3 and CD4 after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor and analysed by flow cytometry. The horizontal lines of scatter plots represent the median value in all subgroups. (G) PBMCs of HC (n = 5) were treated with methylprednisolone (MP) for 24h and stimulated 2h with PHA. Error bars represent the standard error of the mean. * and *** indicate respectively p-values ≤ 0.05 and ≤ 0.001.</p

Main demographic features of MS patients and healthy control groups.

<p>Main demographic features of MS patients and healthy control groups.</p

GM-CSF expression analysis in PBMCs of MS patients and HC.

<p>GM-CSF expression analysis in PBMCs of MS patients and HC.</p

IL-22 expression analysis in PBMCs of MS patients and HC.

<p>IL-22 expression analysis in PBMCs of MS patients and HC.</p

Method of ROI delineation and pixel-wise texture analysis from the gray level co-occurrence matrix (GLCM).

<p>a) Axial-transverse post-contrast T1-W image showing multiple enhanced lesions. b) T2-W image in similar slice location revealing additional hyper-intense unenhanced lesions. c) Segmentation on the same image as in b) of the largest active lesion as well as the contralateral mirrored area within NAWM. d) Corresponding DWI with gradient factor b_o = 0 s.mm^-2. e) Corresponding DWI with gradient factor b = 1000 s.mm^-2. f) ADC parametric map registered on anatomical T2-W image with superimposition of the ROIs drawn on c. g) Zoom of ADC mapped image on largest enhanced lesion (after erasing ROIs’ contours). h-m) Parametrical maps of the following texture parameter: h) contrast, i) correlation, j) homogeneity, k) sum average, l) sum variance and m) difference variance with mean value estimated on a 3x3 sliding window and normalized on the 0–255 range. Individual texture parameters revealed different local and regional statistical properties of the gray levels between MS lesions and NAWM and between enhanced and unenhanced MS lesions.</p

Overview of adverse events in 275 patients from the safety analysis set.

<p>N, number of patients; n, number of AEs; AE, adverse event;</p><p>*, patients could appear in more than one category;</p>†<p>, not related, unlikely related or assessment missing;</p>‡<p>, likely related or definitely related;</p>#<p>, two AEs in one patient were reported as leading to death.</p

Serious adverse events occurring in more than one patient by body system in 275 patients.

<p>N, number of patients; n, number of events; %, percentage based on N = 275.</p