Upregulation of IL-17, but not of IL-9, in circulating cells of CIS and relapsing MS patients. Impact of corticosteroid therapy on the cytokine network
Introduction
Multiple Sclerosis (MS) is considered to be a chronic autoimmune disease resulting in inflammation and demyelination in the central nervous system (CNS). A key event in disease pathogenesis involves CNS invasion by activated circulating myelin-reactive T-cells (Sospedra and Martin, 2005). In recent years, considerable attention has been focused on the T helper cells that produce interleukin (IL)-17 (Th17) as these cells were shown to be involved in autoimmune CNS diseases, such as experimental autoimmune encephalomyelitis (EAE) (Langrish et al., 2005). IL-17A and IL-23 are expressed in active and chronic-active MS lesions (Lock et al., 2002, Li et al., 2007, Tzartos et al., 2008, Montes et al., 2009). Recently, several authors have correlated the proportion of CD4+CD45RO+ IL-17A+ cells to clinical and radiological disease activities (Durelli et al., 2009, Kebir et al., 2009). Human Th17 cells obtained from relapsing MS patients express activation markers as well as endothelial adhesion molecules and are able to cross an in vitro blood–brain-barrier model (Brucklacher-Waldert et al., 2009, Kebir et al., 2009). Finally, as reciprocal developmental pathways exist between Th17 and CD4+CD25+ Foxp3+ T regulatory cells (Tregs), an impaired balance between these cell types is thought to determine the development of autoimmune disease (Korn et al., 2007, Eisenstein and Williams, 2009). IL-9 has emerged as one of the cytokines involved in the regulation of the Th17/Treg balance (Elyaman et al., 2009). It is involved in Th17 cell differentiation and can also be produced by Th17 cells in certain conditions. Upon IL-1β and TGF-β stimulation, IL-17A+IL-9+-dual producers were indeed identified in patients suffering from type 1 diabetes, but not in healthy controls (Beriou et al., 2010).
Because peripheral blood is thought to be only a trafficking compartment for regulatory and effector cells, the search for peripheral biomarkers of disease activity in MS could be hampered by a lack of sensitivity in detecting a disease process that mainly involves the CNS. However, a systemic increase in IL-1β, TGF-β and CCL20, all Th17-related molecules, in combination with other cytokine measurements, distinguished MS patients from controls (Furlan et al., 2005). As other autoimmune processes could theoretically induce the same peripheral immune signature, peripheral blood markers may also lack specificity for MS (Kunz and Ibrahim, 2009). Nevertheless, blood remains the only readily available biological sample in patients and could reveal pertinent information regarding triggers of autoimmune disease activity and drug effects on the immune system (Harris and Sadiq, 2009).
Corticosteroids are used in the therapy of numerous inflammatory, autoimmune and allergic diseases. High dose intravenous methylprednisolone (ivMP) is the treatment of choice for acute demyelinating events. Corticosteroids are known to reduce the number of circulating monocytes and lymphocytes, by modulating cell apoptosis. They also reduce vessel permeability, by down-regulating the expression of adhesion molecules on endothelial cells. Finally, they regulate the transcription of numerous genes, either positively or negatively, thereby affecting the balance between pro- and anti-inflammatory cytokines (Sloka and Stefanelli, 2005). Only a few studies have compared the effects of ivMP on the global cytokine expression of peripheral blood mononuclear cells (PBMCs), notably on IL-17A and IL-9 expression, in patients with relapsing clinically definite MS (CDMS) or in patients presenting with a clinically isolated syndrome (CIS) (Crockard et al., 1995, Gayo et al., 1998, Wandinger et al., 1998, Kahl et al., 2002, Liu et al., 2009).
The aim of our study was to analyze the expression levels of IL-17A and IL-9 in PBMCs of patients with CIS or relapsing CDMS during active clinical disease. The effects of ivMP treatment on the expression of these markers were analyzed by serial blood sampling, either ex vivo at the mRNA level or by Flow Cytometry, and following in vitro stimulation with myelin antigens of PBMCs. In addition the levels of other Th17-, Th1- or Th2-related cytokines were also assessed, as well as that of Foxp3 and IL-10.
Section snippets
Patients and controls
For real-time PCR experiments, blood samples were collected from 22 patients, who presented with a mono- or multifocal neurological deficit that had lasted for more than 24 h, was not associated with fever or infection, and was compatible with MS. Twenty-three patients met the criteria for a CIS. Ten patients had already been diagnosed with CDMS, according to the McDonald criteria (McDonald et al., 2001). Informed consent was obtained from all study subjects. The study was approved by the local
Upregulation of IL-17A, but not of IL-9, in CD3+ T cells from patients in relapse
The frequency range of CD3+-IL-17A-producing cells was larger in relapsing patients [0.053–0.736%] in comparison with healthy controls [0.08–0.27%]. There was no significant difference in the mean frequency of IL-9-producing cells between patients and controls (Fig. 1B). Compared to healthy controls (0/9), a higher proportion of CIS/CDMS patients (6/14) had more than 0.3% of CD3+ cells expressing IL-17A during a relapse (p = 0.048) (Fig. 1B). This is reflected in the lower IL-9+CD3+/IL-17A+CD3+
Discussion
There is accumulating experimental evidence showing an increase in the proportion of IL-17A-expressing cells in the periphery during active demyelinating disease in patients with CIS and relapsing MS (Brucklacher-Waldert et al., 2009, Durelli et al., 2009, Kebir et al., 2009, Edwards et al., 2011). Long considered a Th2 cytokine, recent experimental data suggests that IL-9 is involved in Th17 cell differentiation (Elyaman et al., 2009, Nowak et al., 2009). Moreover, Th9 cells can themselves
Acknowledgments
We thank Professors J.-C. Renauld, P. Coulie and P. Vanderbruggen as well as L. Dumoutier for invaluable discussions (Ludwig Institute for Cancer Research and de Duve Institute). We thank V. Stroobant (Ludwig Institute) for producing the PLP and MOG peptides. We thank A. Guillet, J.M. Freyermuth and Réjane Rousseau (Université Catholique de Louvain) for help with the statistical analysis. This work was financially supported by grants from the “Fonds de la Recherche Scientifique Médicale” (no.
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