T Lymphocyte Serotonin 5-HT7 Receptor Is Dysregulated in Natalizumab-Treated Multiple Sclerosis Patients

Abstract

Serotonin (5-HT) is known as a potent immune cell modulator in autoimmune diseases and should be protective in the pathogenesis of multiple sclerosis (MS). Nevertheless, there is limited knowledge about receptors involved in 5-HT effects as well as induced mechanisms. Among 5-HT receptors, the 5-HT₇ receptor is able to activate naïve T cells and influence the inflammatory response; however, its involvement in the disease has never been studied so far. In this study, we collected blood sample from three groups: acute relapsing MS patients (ARMS), natalizumab-treated MS patients (NTZ), and control subjects. We investigated the 5-HT₇ expression on circulating lymphocytes and evaluated the effects of its activation on cytokine production with peripheral blood mononuclear cell (PBMC) cultures. We found a significant increase in the 5-HT₇ surface expression on T lymphocytes and on the different CD4⁺ T cell subsets exclusively in NTZ-treated patients. We also showed that the selective agonist 5-carboxamidotryptamine (5-CT)-induced 5-HT₇R activation significantly promotes the production of IL-10, a potent immunosuppressive cytokine in PBMCs. This study provides for the first time a dysregulation of 5-HT₇ expression in NTZ-MS patients and its ability to promote IL-10 release, suggesting its protective role. These findings strengthen the evidence that 5-HT₇ may play a role in the immuno-protective mechanisms of NTZ in MS disease and could be considered as an interesting therapeutic target in MS.

Keywords: 5-HT7 receptor; PBMCs (peripheral blood mononuclear cells); T lymphocyte (T-cell); interleukin-10 (IL-10); multiple sclerosis; natalizumab treatment; serotonin.

Figure 1

Experimental design and analyses performed. Abbreviations: PBMCs: peripheral blood mononuclear cells; RT: reverse transcription; qPCR: quantitative polymerase chain reaction; mRNA: messenger ribonucleic acid; Htr7: serotonin 5-HT₇ receptor gene.

Figure 2

5-HT₇ expression level on the surface of circulating lymphocytes from control, ARMS, and NTZ patients. Geometric mean fluorescence intensity (Geo MFI) of 5-HT₇ surface expression and histogram representation of the cytometry analysis on CD45⁺ CD19⁺ B and CD45⁺ CD3⁺ T lymphocytes (A,B), on CD3⁺ CD8⁺ and CD3⁺ CD4⁺ T cells (C,D), and CD4⁺ T cell subsets (E,F). Proportions of circulating cells analyzed by flow cytometry are shown for B cells (G), CD8⁺ (H), and CD4⁺ T cells (I) from control, ARMS, and NTZ patients. Results are given as mean ± SEM. B cells compared with T cells for each group. *: compare with control. δδδ p < 0.001; * p < 0.05; ** p < 0.01.

Figure 3

Plasma immunological context and Htr7 gene expression in PBMCs from control, ARMS, and NTZ patients. Cytokines assays: IL-1β (A), IFN-γ (B), IL-17 (C), IL-4 (D), and IL-10 (E) from plasma samples of control, ARMS, and NTZ patients. Htr7 was analyzed by RT-qPCR. The relative expression of Htr7 mRNA levels, normalized to Gapdh and Hprt1 of control subjects, is shown (F). Results are given as mean ± SEM. ** p < 0.01 compared with control.

Figure 4

Effects of 5-HT₇ receptor agonist 5-CT on PBMC cytokine releases compared to LPS stimulation. Cytokines assays: IL-1β (A), IFN-γ (B), IL-17 (C), IL-4 (D), and IL-10 (E) were performed from PBMC supernatants of control, ARMS, and NTZ patients. Htr7 was analyzed by RT-qPCR. The relative expression of Htr7 mRNA levels, normalized to Gapdh and Hprt1 of non-treated PBMCs from the control, is shown (F). Results are given as mean ± SEM. *: compare the values under the bars. $: compare with the respective non-treated (NT) condition. Compare with both NT and 5-CT conditions. * p < 0.05; ^$ p < 0.05; ^$$ p < 0.01; ^$$$ p < 0.001; ^δδ p < 0.01; ^δδδ p < 0.001.