Rosella Mechelli

CD161highCD8+T cells bear pathogenetic potential in multiple sclerosis

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines – an event that may favour autoimmunity – while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. Objectives: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. Methods: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. Results: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. Conclusion: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood–brain barrier, may contribute to the development of the autoimmune responses in MS.

TET2 gene expression and 5-hydroxymethylcytosine level in multiple sclerosis peripheral blood cells

Aberrant DNA methylation can lead to genome destabilization and to deregulated gene expression. Recently, 5-hydroxymethylcytosine (5hmC), derived from oxidation of 5-methylcytosine (5mC) by the Ten-Eleven Translocation (TET) enzymes, has been detected. 5hmC is now considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the DNA methylation/demethylation machinery in MS. We assessed the expression of enzymes involved in DNA methylation/demethylation in peripheral blood mononuclear cells (PBMCs) from 40 subjects with MS and 40 matched healthy controls. We performed also, DNA methylation analysis of specific promoters and analysis of global levels of 5mC and 5hmC. We show that TET2 and DNMT1 expression is significantly down-regulated in MS PBMCs and it is associated with aberrant methylation of their promoters. Furthermore, 5hmC is decreased in MS PBMCs, probably as a result of the diminished TET2 level.

A "candidate-interactome" aggregate analysis of genome-wide association data in multiple sclerosis

Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a “candidate interactome” (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms.

Altered intestinal permeability in patients with relapsing–remitting multiple sclerosis: A pilot study:

Background: Alterations of intestinal permeability (IP) may contribute to the pathophysiology of immune-mediated diseases. Objective: We investigated the possible association between IP changes and multiple sclerosis (MS). Methods: We studied 22 patients with relapsing–remitting multiple sclerosis (RRMS) and 18 age- and sex-matched healthy donors (HDs), including five twin pairs (one concordant, and four discordant for disease). Measurement of lactulose (L) and mannitol (M; two non-metabolized sugars) levels in urine samples, after an oral load, allowed to quantify gut dysfunction. Results: The proportion of participants with increased IP was significantly higher in patients than in HDs (16/22 (73%) versus 5/18 (28%); p = 0.001). Accordingly, the L/M urinary ratio showed significantly higher values in patients than in controls (p = 0.0284). Urinary mannitol concentration was significantly lower in patients than in controls (p = 0.022), suggesting a deficit of absorption from intestinal lumen. Such changes did not appear related to patients’ clinical–radiological features. Conclusion: The relatively high proportion of IP changes in RR-MS patients seems to confirm our work hypothesis and warrants more work to confirm the result on a larger sample, and to understand the implications for related immunological disturbances and intestinal microbiota alterations. Our finding may also have relevance for oral treatments, recently introduced in clinical practice.

Multiple sclerosis etiology: beyond genes and environment

Multiple sclerosis (MS) is a disorder of the CNS with inflammatory and neurodegenerative components. The etiology is unknown, but there is evidence for a role of both genetic and environmental factors. Among the heritable factors, MHC class II genes are strongly involved, as well as genes coding for others molecules of immunological relevance, genes controlling neurobiological pathways and genes of unknown function. Among nonheritable factors, many infectious agents (mainly viruses) and environmental factors (e.g., smoke, sun exposition and diet) seem to be of etiologic importance. Here, we report and discuss recent findings in MS on largely unexplored fields: the alternative splicing of mRNAs and regulatory noncoding RNAs, the major sources of transcriptome diversity; and epigenetic changes with special attention paid to DNA methylation and histone acetylation, the main regulators of gene expression.

CD28 ligation in the absence of TCR stimulation up-regulates IL-17A and pro-inflammatory cytokines in relapsing-remitting multiple sclerosis T lymphocytes.

CD28 is a crucial costimulatory receptor necessary full T cell activation. The role of CD28 in multiple sclerosis (MS) has been evaluated as the source of costimulatory signals integrating those delivered by TCR. However, CD28 is also able to act as a unique signaling receptor and to deliver TCR-independent autonomous signals, which regulate the expression and production of pro-inflammatory cytokines and chemokines. By comparing the cytokine/chemokine profiles of CD4(+) T cells from relapsing-remitting multiple sclerosis (RRMS) patients and healthy donors (HD), we found that CD28 engagement without TCR strongly up-regulates IL-8 and IL-6 expression in RRMS compared to HD. More interestingly, in RRMS but not in HD, CD28 stimulation selectively induces the expression of IL-17A by cooperating with IL-6-mediated signals. By using specific inhibitory drugs, we also identify the phosphatidylinositol 3 kinase (PI3K) as the critical regulator of CD28 proinflammatory functions in MS.

Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis

BackgroundHere, we evaluated the hypothesis that CD8+ T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology.MethodsThe percentage of autoreactive CD8+ T effector cells specific for various apoptotic T cell-associated self-epitopes (apoptotic epitopes) were detected in the peripheral blood and cerebrospinal fluid (CSF) by both enzyme-linked immunospot and dextramers of class I molecules complexed with relevant apoptotic epitopes. Moreover, the capacity of dextramer+ CD8+ T cells to produce interferon (IFN)-γ and/or interleukin (IL)-17 in response to the relevant apoptotic epitopes was evaluated by the intracellular cytokine staining. Cross-presentation assay of apoptotic T cells by dendritic cells was also evaluated ex vivo.ResultsWe found that polyfunctional (IFN-γ and/or IL-17 producing) autoreactive CD8+ T cells specific for apoptotic epitopes were represented in MS patients with frequencies significantly higher than in healthy donors. These autoreactive CD8+ T cells with a strong potential to produce IFN-γ or IL-17 in response to the relevant apoptotic epitopes were significantly accumulated in the CSF from the same patients. In addition, the frequencies of these autoreactive CD8+ T cells correlated with the disease disability. Cross-presentation assay revealed that caspase-cleaved cellular proteins are required to activate apoptotic epitope-specific CD8+ T cells ex vivo.ConclusionTaken together, these data indicate that apoptotic epitope-specific CD8+ T cells with strong inflammatory potential are recruited at the level of the inflammatory site, where they may be involved in MS immunopathology through the production of high levels of inflammatory cytokines.

Epstein-Barr virus nuclear antigen-1 B-cell epitopes in multiple sclerosis twins.

Background: Compared with quantitative observations, the search for qualitative changes that may characterize the immune response to Epstein–Barr virus (EBV) in multiple sclerosis (MS) has been less intense. Objective: To examine the B-cell epitopes of antibodies against the Epstein–Barr nuclear antigen-1 (EBNA-1) and their relevance for MS, through a study in disease-discordant identical twins. Methods: We evaluated the antibodies to all unique, maximally overlapping octapeptides of EBNA-1 in 12 pairs of monozygotic (MZ) twins (9 MS-discordant, 3 healthy), 3 non-twin patients and 2 healthy subjects. All except one of the patients were untreated. The EBV serology of these individuals had been assessed in advance using commercially available and in-house enzyme-linked immunosorbent assay (ELISA) kits, including assays for antibodies against select peptides of EBNA-1: EBNA-72 (GAGGGAGAGG) and EBNA-206 (EADYFEYHQEGGPDGE). Results: The glycine–alanine rich domain of EBNA-1 was immunodominant in all subjects. Compared with healthy individuals, and similarly to what has been described in infectious mononucleosis (IM) patients, affected co-twins and non-twin patients had a significantly increased response to another EBNA-1 epitope (aa. 401–411). Conclusion: In a study that controls for confounders, our data focus an EBNA-1 specificity that may be associated with MS pathogenesis.