IL-10 suppressor activity andex vivo Tr1 cell function are impaired in multiple sclerosis

Department of Neurology, University of Navarra, Pamplona, Spain.
European Journal of Immunology (Impact Factor: 4.03). 02/2008; 38(2):576-86. DOI: 10.1002/eji.200737271
Source: PubMed


T regulatory cells type 1 (Tr1 cells) are excellent candidates for cell therapy in multiple sclerosis (MS). The aim of our study was to assess the functional state of Tr1 cells and IL-10R signaling in patients with MS. Tr1 cells were induced in vitro by activation with anti-CD46 antibodies in controls and patients with MS. Cells were phenotyped by cytometry and suppression assays, and the expression of cytokines and transcription factors was evaluated by real-time PCR, ELISA, cytometry and Western blotting. We found that the activity of Tr1 cells and IL-10R signaling is impaired in MS patients since Tr1 cells isolated from MS patients produced less IL-10 than those obtained from controls. Indeed, the supernatants from Tr1 cells from controls did not suppress the proliferation of stimulated CD4(+) cells from patients with MS. Furthermore, the IL-10R signaling pathway was not fully active in CD4(+) cells from MS patients and these cells had higher baseline levels of SOCS3 transcripts than controls. Indeed, after in vitro IL-10 stimulation, the expression levels of the STAT1, STAT3 and IL-10RA genes were higher in MS patients than in controls. Moreover, Stat-3 phosphorylation was lower in controls than in patients after IL-10 stimulation. These results indicate that IL-10 regulatory function is impaired in patients with MS.

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Available from: Beatriz Moreno
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    • "In autoimmune diseases, the balance between the pro-inflammatory response and regulatory factors seems to be disrupted. As such, antigen specific effector T cells (Teff) are over-activated [6], whereas the function of regulatory lymphocytes (Treg) is altered [7-9], indicating impairment of peripheral tolerance. It could be expected that this impaired immune response would produce a chronic inflammatory process leading a progressive clinical course. "
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    ABSTRACT: Background Multiple Sclerosis (MS) is considered a T-cell-mediated autoimmune disease with a prototypical oscillatory behavior, as evidenced by the presence of clinical relapses. Understanding the dynamics of immune cells governing the course of MS, therefore, has many implications for immunotherapy. Here, we used flow cytometry to analyze the time-dependent behavior of antigen-specific effector (Teff) and regulatory (Treg) T cells and microglia in mice model of MS, Experimental Autoimmune Encephalomyelitis (EAE), and compared the observations with a mathematical cross-regulation model of T-cell dynamics in autoimmune disease. Results We found that Teff and Treg cells specific to myelin olygodendrocyte glycoprotein (MOG) developed coupled oscillatory dynamics with a 4- to 5-day period and decreasing amplitude that was always higher for the Teff populations, in agreement with the mathematical model. Microglia activation followed the oscillations of MOG-specific Teff cells in the secondary lymphoid organs, but they were activated before MOG-specific T-cell peaks in the CNS. Finally, we assessed the role of B-cell depletion induced by anti-CD20 therapy in the dynamics of T cells in an EAE model with more severe disease after therapy. We observed that B-cell depletion decreases Teff expansion, although its oscillatory behavior persists. However, the effect of B cell depletion was more significant in the Treg population within the CNS, which matched with activation of microglia and worsening of the disease. Mathematical modeling of T-cell cross-regulation after anti-CD20 therapy suggests that B-cell depletion may influence the dynamics of T cells by fine-tuning their activation. Conclusions The oscillatory dynamics of T-cells have an intrinsic origin in the physiological regulation of the adaptive immune response, which influences both disease phenotype and response to immunotherapy.
    Full-text · Article · Apr 2013 · BMC Systems Biology
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    • "Three cerebellar slices per group were used for analysis. Western-blots were performed as previously described [12]. Briefly, total protein (10 μg) from cerebellar slices was separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane and hybridized for 2 h or overnight with primary antibodies diluted in the same blocking buffer: mouse anti-CNPase (2′,3′-cyclic-nucleotide 3′-phosphodiesterase) 1∶500 (Abcam), rabbit anti-iNOS (inducible Nitric Oxide Synthase) 1∶200 (BD Bioscience). "
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    ABSTRACT: Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "Importantly, CD46 costimulation promotes Tr1-like Treg differentiation, characterized by secretion of large amounts of IL-10 and low levels of IFNγ [10], [11]. Defects in IL-10 production upon CD46 activation have been demonstrated in patients with MS [12], [13], [14], asthma [15] and rheumatoid arthritis [11]. "
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    ABSTRACT: The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)(2)D(3) or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS.
    Full-text · Article · Oct 2012 · PLoS ONE
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