Circulating EZH2-positive T cells are decreased in multiple sclerosis patients

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) is a histone methyltransferase that serves as the catalytic subunit of the polycomb repressive complex 2, a protein complex that regulates gene expression by methylating nucleosomal histone H3 at lysine 27 (H3K27) on the promoter of its target genes [1]. The identification of a cytosolic methyltransferase EZH2-containing complex suggested that, in addition to its role methylating histones, EZH2 could also be involved in the regulation of extra-nuclear signaling pathways, in particular actin polymerization-dependent processes [2]. Recent investigation on its cytoplasmic role has revealed that EZH2 interacts with cytosolic proteins such as talin and the guanine nucleotide–exchange factor vav1 that link integrin molecules to the actin cytoskeleton, suggesting the potential implication of EZH2 in cell adhesion and migration processes [3]. Interestingly, mice lacking the EZH2 gene exhibited attenuated experimental autoimmune encephalomyelitis (EAE) disease progression due to the inability of EZH2-deficient cells, particularly neutrophils and dendritic cells, to reach the site of inflammation [3]. Taking into consideration the findings of EZH2 in EAE mice and its implication in important processes for the pathogenesis of multiple sclerosis such as cell adhesion and migration, we believe that EZH2 may also be playing a role in multiple sclerosis and contribute to the inflammatory component observed in the central nervous system (CNS) of patients. Hence, the purpose of the present study was to explore the role of EZH2 in the disease by measuring the gene expression levels of EZH2 and associated molecules in peripheral blood cells from untreated and treated multiple sclerosis patients and by characterizing the immune cell populations responsible for EZH2 expression.

Extensive literature exists about the role of EZH2 as histone methyltransferase and, particularly, about EZH2 involvement in a wide range of malignant tumors [8, 12, 13] due to its function as epigenetic silencer [14]. The identification of a cytosolic methyltransferase complex containing EZH2 suggested that, in addition to its nuclear role, EZH2 could also be involved in other important cellular processes such as cell adhesion and migration [2]. In an attempt to characterize more in depth the cytosolic role of EZH2, Gunawan et al. [2] recently reported that EZH2 was critical for regulating leukocyte migration to sites of inflammation in EAE mice, findings that opened a potential and attractive link between EZH2 and autoimmune disorders such as multiple sclerosis in which cell adhesion and migration are critical pathogenic mechanisms [15]. Despite this initial publication in the animal model of multiple sclerosis [3], to date, there are no studies of EZH2 in patients with multiple sclerosis. Aiming to explore the role of EZH2 in multiple sclerosis, we first determined mRNA expression levels in PBMC from untreated patients and healthy individuals and observed that EZH2, together with molecules reported to be associated with cytosolic EZH2 such as TLN1 and VAV1 [3], were all downregulated in multiple sclerosis patients regardless of whether they were having relapse-onset or progressive clinical forms, or whether they were in clinical remission or in acute relapse. The decreased expression of EZH2 in PBMC from patients with multiple sclerosis was replicated in an independent validation cohort of patients and controls. These initial observations at the gene expression level suggested an involvement of EZH2 in the disease, which was explored in additional experiments.

We first explored microRNAs, which are known to exert regulatory functions at the posttranscriptional level via binding to the 3′ untranslated region of target mRNAs [16]. In the search for potential microRNAs that on the one hand regulated EZH2 expression and on the other hand were involved in multiple sclerosis, the microRNAs miR-124 and miR-155 emerged as attractive candidates [8, 9]. In this context, expression levels for miR-124 were found increased in demyelinated hippocampi from postmortem brains of multiple sclerosis patients [10]. miR-155 expression was upregulated in peripheral blood monocytes and active lesions from patients [11]. In our study, when expression levels for these two microRNAs were determined in PBMC from a subgroup of untreated multiple sclerosis patients and controls, miR-124 but not miR-155 was significantly upregulated in patients, suggesting a potential functional relationship between decreased EZH2 mRNA expression levels and miR-124 upregulation in patients with multiple sclerosis. These findings warrant future studies to investigate the cell types that contribute to miR-124 upregulation.

In order to explore whether the expression of EZH2 and associated molecules was modulated by commonly used disease-modifying therapies in patients with multiple sclerosis, a cohort of patients treated with interferon-beta, glatiramer acetate, fingolimod, and natalizumab was also included in the study. Treatment with interferon-beta, glatiramer acetate, and natalizumab was associated with increased expression levels of EZH2 and TLN1. By contrast, fingolimod treatment was only associated with upregulated expression of TLN1. Although based on a small number of samples and in spite of the descriptive nature of the experiments, it is tempting to speculate that this finding may be due to the different mechanisms by which these drugs regulate leukocyte migration to the CNS, being the mechanism of action of fingolimod not exerted in peripheral blood but in the lymph nodes where it retains naïve and central memory lymphocytes [17‐20]. In view of these data, we hypothesize that the reduction in EZH2 expression observed in untreated patients with subsequent upregulation after treatment may indicate a migration capacity of PBMC expressing EZH2 to the CNS that is inhibited by the effect of treatment. These findings also open a new research avenue to investigate whether the increase in EZH2 expression observed after treatment is associated with the response to therapies and hence may differ between responders and non-responders to each particular therapeutic strategy.

Immunophenotyping of the major PBMC populations revealed restricted EZH2 expression in CD4+ and CD8+ T cells as well as CD56dim NK cells. Similar to the gene expression findings, the percentage of EZH2-positive T cells was reduced in untreated multiple sclerosis patients compared to controls, pointing to a role of EZH2 in this particular cell subset rather than in CD56dim NK cells, which showed similar percentages of EZH2-positive cells in patients and controls. It is worth highlighting that except for one patient and one control, the immunophenotyping cohort included new individuals, and hence, the decrease of EZH2-positive T cells observed in patients can also be considered as a new validation of the EZH2 expression findings at the protein level. Natalizumab, which was selected as control therapy because of its known effects reducing T cell trafficking into the CNS, increased the percentage of EZH2-positive T cells, but, contrary to gene expression results, differences did not reach statistical significance. Interestingly, further T cell immunophenotyping showed heterogeneity in the T cell subsets positive for EZH2 expression. In this context, most CD4+ T cells expressing EZH2 were effector memory T cells, a population that per se has the capacity to migrate to non-lymphoid tissues including the CNS [21]. In contrast, CD8+ T cells positive for EZH2 had a predominant central memory phenotype, suggesting that this particular subset can migrate to the CNS in MS and may later differentiate into effector memory populations in the sites of inflammation. Of note, EZH2-positive cells both in CD4+ and CD8+ T cells may have pathogenic potential through the secretion of TNF-α, a pro-inflammatory cytokine involved in the pathogenesis of multiple sclerosis [22]. Although caution should be taken when considering these data owing to the small sample size and high variability, these findings altogether warrant additional studies to deepen into the pathogenic capacity of T cells expressing EZH2 and explore whether EZH2 may become a therapeutic target in MS patients to reduce disease activity.

The potential for EZH2-positive T cells to migrate to the CNS was first suggested in the EAE study, which showed EZH2 expression in spinal cord tissue from EAE mice following a similar pattern to Cd3 expression over time. Confirmation of the migratory capacity of EZH2-positive T cells was provided by their detection in the CSF and brain lesions from multiple sclerosis patients. Noteworthy, the enrichment for CD4+ and CD8+ T cells expressing EZH2 in the CSF and chronic active lesions compared to the blood compartment suggested that the decrease of EZH2-positive T cells observed in untreated multiple sclerosis patients compared to healthy individuals was secondary to their migration into the CNS. This notion was further supported by the observation that other EZH2-expressing blood cell populations such as CD56dim NK cells, which did not depict differences between patients and controls in blood, were not enriched in the CSF. Although EZH2 expression in brain lesions from multiple sclerosis patients seemed restricted to CD4+ and CD8+ T cells, additional sources of EZH2 expression within the CNS cannot be totally ruled out, as evidenced by EZH2 immunohistochemistry. In contrast, EZH2 expression was not observed in brain tissue from non-neurological controls, a finding that confers specificity for EZH2 expression in the CNS of multiple sclerosis patients.

Finally, the implication of EZH2 in T cell adhesion, an important step for cell migration, was supported by the microarray findings conducted after EZH2 blocking with a histone deacetylase inhibitor that regulates EZH2 expression [23], which showed striking downregulation of cell adhesion molecules expressed in T cells such as SELL [24], ITGA4 [25], ITGAL [26], and PECAM1 [27]. The aggregate results from the study suggest a role for EZH2 in the migration of T cells into the CNS in patients with multiple sclerosis and also suggest a potential pathogenic capacity of EZH2-positive T cells that will need to be explored more in depth in future studies.