Cellular mechanisms of neuroimmune interaction in multiple sclerosis: ionotropic glutamate receptors of T-lymphocytes

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Abstract


Using flow cytometry and real-time RT-PCR, we studied the effect of the blockade of glutamate NMDA receptors on the quantitative distribution of IFNγ- and IL-4-producing CD4+ and CD8+ T-lymphocytes and the expression of transcription factor of genes T-bet and GATA-3 in immune cells derived from healthy individuals and multiple sclerosis patients (MS). From the results of the study, it follows that blockade of NMDA receptors leads to a decrease in the proportion of studied effector subpopulations of T-lymphocytes, as well as differential modulation of gene expression of transcription factors (TBX21, GATA3). The severity of the negative effect of receptor blockade differs in different subpopulations of CD4+ and CD8+ T cells, which leads to a shift in the cytokine balance towards “proinflammatory” type 1 T-cells, to a greater extent in patients with MS. Thus, glutamate can regulate the Th1/Th2 and Tc1/Tc2 сytokine balance by modulating the activity of NMDA receptors of T lymphocytes in MS.

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Introduction. The heterogeneity of pathogenic processes in MS is due, among others, to a breakdown in the connections between the nervous and immune systems, in which neurotransmitters, such glutamate (Glu), play a significant role. Functionally active Glu receptors are found on human T-lymphocytes and their participation in the regulation of numerous processes in immune cells has been proven. However, the question of the role of glutamate and its receptors in the mechanisms, important for MS, are not understood. It is known that the terminal differentiation of CD4+ and CD8+ T-lymphocytes into effector cells is largely determined by the cytokine microenvironment, as well as the expression of transcription factors specific for a particular subpopulation. Based on this, the purpose of the study was to investigate the functional significance of the ionotropic NMDA receptors in the maintaining Th1/Th2 and Tc1/Tc2 cytokine balance in multiple sclerosis. Material and methods. The study involved 12 healthy individuals and 15 patients with relapsing-remitting MS (remission stage). An intracellular staining of marker cytokines was applied for evaluation of quantitative distribution the subsets of T-lymphocyte in cultures of peripheral blood mononuclear cells activated by phorbol-myristate-acetate (PMA, 25 ng/ml) and ionomycin (ion, 1 μg/ml) and treated with antagonist of NMDA receptor (+)-MK801 (10-4 M) with subsequent analysis by flow cytometry. CD4+ T cells (Th) producing IFNγ were defined as Th1, and IL-4 as Th2. IFN-γ-producing CD8+ T cells were identified as Tc1, and IL-4 - producing as Tc2. The mRNA level of genes coding the transcription factors (TBX21 and GATA3) was evaluated using real-time RT-PCR. Results and discussion. The blockade of NMDA receptors on PMA/ion-activated T-cells is accompanied by a decrease in the proportion of IFNγ- and IL-4-producing cells in both groups of donors (table). The intracellular level of IL-4 in CD4+ T-lymphocytes of patients with MS to compared with healthy donors decreases by 2.7 times stronger. (+)-MK801 causes the inhibition of the production of IFNγ in CD4+ cells to a lesser extent in patients with MS. Similar trends were observed in the population of CD8+ T-lymphocytes, while the sensitivity of IFNγ production to the blockade of NMDA receptors in MS was 3.2 times lower than in healthy donors. Analysis of the ratios of Th1/Th2 and Tc1/Tc2 showed that blockade of NMDA receptors leads to a shift of the cytokine balance towards the cellular level of immunity (Th1 and Tc1 cells), more significantly in MS. According to obtained data the blockade of NMDA receptors in the conditions of pharmacological activation of T-cells induces an increase in the relative mRNA level of the TBX21 while simultaneously suppressing the expression of the GATA3 (table). The consequence of this change in gene expression is an increase in the ratio of TBX21/GATA3 and shift in the balance towards the pattern of proinflammatory cytokines. Thus, the results of the study indicate the involvement of NMDA glutamate receptors in the mechanisms of maintaining a balance between the effector subpopulations of T lymphocytes. The obtained data on the effects of blockade on T-lymphocytes of patients with MS emphasize the importance of Glu and its receptors in the pathogenesis of the disease and the relevance of their further research.

About the authors

U Sh Kuzmina

IBG UFRC RAS

K Z Bakhtiyarova

Bashkir State Medical University, Ufa

Yu V Vakhitova

IBG UFRC RAS

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Copyright (c) 2019 Kuzmina U.S., Bakhtiyarova K.Z., Vakhitova Y.V.

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