5-HT7 Receptor Implication in Inflammatory Mechanisms in Multiple Sclerosis
Condition: Multiple Sclerosis, Acute Relapsing; Multiple Sclerosis
MeSH terms: Multiple Sclerosis; Multiple Sclerosis, Relapsing-Remitting; Sclerosis
Study type: Observational

Summary: 
      Multiple Sclerosis is a chronic autoimmune disease associated with inflammatory response
      harmful for the Central Nervous System. Immunological imbalance is involved with Th1 and Th17
      cells in correlation with a disturbance of regulators mechanisms as Treg cells. Despite years
      of research, the mechanisms involved remain unclear.

      Serotonin (5-HT) seems to be a therapeutic target to treat multiple sclerosis. Indeed,
      several studies have shown the anti-inflammatory potential of this neurotransmitter and also
      its vulnerability in inflammatory context. Moreover, a recent study has shown that 5-HT can
      reduced CD4 T cells proliferation and pro-inflammatory cytokines released in vitro.

      5-HT protector effects have also demonstrated in Experimental Autoimmune Encephalomyelitis
      mouse model (EAE) with an inflammatory response reduction and also a decreased of spinal cord
      lesions.

      The latest receptor discovered, the 5-HT7 receptor, has been identify as a promise target to
      treat neurological disorders associated with inflammatory context. Present in humans and
      mice, this receptor spreads on the surface of a large number of cells, such as T-lymphocytes,
      macrophages, dendritic cells and also neurons, astrocytes and microglia.

      Given the importance of the positive cells for 5-HT7 receptor, in the inflammatory context
      observed in multiple sclerosis, The investigator propose to study the receptor expression in
      blood samples from multiple sclerosis patient.
    

Detailed description: 
      The expression modulation of 5-HT7 receptor will be investigate in correlation with the
      immunological context, in different group of MS patients:

        -  Group 1: MS patients with an acute relapse

        -  Group 2: MS stable patients treated with Natalizumab

        -  Group 3: Healthy people (from Etablissement Français du Sang)

             1. 5-HT7 receptor expression evaluation on circulating leukocyte cells Each group
                presents a different inflammatory context. The investigator wish to determine the
                immunological context influence on the 5-HT7 receptor expression and which cells
                express it. To analyze the 5-HT7 expression on circulating leukocyte cells, The
                investigator will perform flow cytometry studies from the whole blood of the three
                groups mentioned above. In a first step, the investigator will analyze the overall
                rate of 5-HT7 circulating positive cells by the use of an antibody against 5-HT7
                receptor whose specificity was validated in our research team. In a second step,
                the investigator will precise which cell type, among the lymphocytes implicated in
                the MS pathology, express the 5-HT7 receptor. The investigator will use antibodies
                against each population t helper cell: Th1 , Th2, Th17 and Treg. The investigator
                will also perform immunostaining on Cytospin (thin layer of blood cells on
                coverslips) to visualize co-labeling and confirm the results of flow cytometry.

             2. Correlation between 5-HT7 receptor expression and mRNA quantity In addition, a
                correlation has been shown between an increase of 5-HT7 positive cells and an mRNA
                quantity up regulated. For the three groups, the investigator will determine the
                5-HT7 mRNA quantity on circulating leukocyte cells. In a first step, the
                investigator will check correlation between the 5-HT7 receptor expression rate and
                the mRNA quantity on circulating leukocyte cells, using quantitative Polymerase
                Chain Reaction (qPCR). In a second step, the investigator will perform cell sorting
                to isolate Th1, Th2, Th17 and Treg cells and specifically extract 5-HT7 mRNA. In
                this way, the investigator can address the 5-HT7 receptor role on one or more
                specific cell types on the MS.

             3. 5-HT7 receptor modulation according to the immunological context In order to
                understand the 5-HT7 receptor potential role on immune response moderation, the
                investigator will identify the immune context by ELISA dosage from serum samples.
                The investigator will investigate on pro-inflammatory markers like IL1b, IFNg, IL17
                and anti-inflammatory markers like IL10 and IL4. These results will be correlated
                with the previous results concerning the 5-HT7 receptor expression/quantity for
                each patient groups.

             4. 5-HT7 receptor role on lymphocyte functions in vitro From blood samples of the
                three groups mentioned above, the investigator will isolate peripheral blood
                mononuclear cells (PBMC) in vitro, to evaluate the 5-HT7 receptor role on their
                function to produce and release cytokines. The investigator will stimulate PBMC
                with one or more 5-HT7 ligands, and will perform ELISA dosage from supernatant with
                pro-inflammatory markers like IL1b, IFNg, IL17 and anti-inflammatory markers like
                IL10 and IL4. The cell immunolabelling will be perform to visualize 5-HT7 receptor
                on the cell surface.

             5. Identification of 5-HT7 receptor isoforms There are three forms of 5-HT7 receptor
                (5-HT7a, 5-HT7b, 5-HT7d). However, no difference has yet been described for their
                pharmacological function. The investigator propose to identify these different
                isoforms by qPCR. As previously described, in a first step, on circulating
                leukocyte cells and in a second step, after cell sorting to isolate Th1, Th2, Th17
                and Treg cells.
    

Intervention name(s): blood sampling
Intervention type(s): Other
Intervention description(s): Analyse of expression modulation of 5-HT7 receptor in correlation with the immunological context, in 3 differents groups : 2 with multiple sclerosis patients, one healthy volunteers group
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Man and Woman 18 to 50 years old
      
Biospecimen description: 
      one sampling to assess nvestigate the expression modulation of 5-HT7 receptor
    
Keywords: Multiple sclerosis; serotonin; 5-HT7 receptor; inflammation; lymphocytes; cytokines