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Impact of depression and infection diseases on the behavior of T cells from multiple sclerosis (MS)
Federal University of state of Rio de Janeiro (UNIRIO)
Department of Microbiology and Parasitology
Cleonice Alves de Melo Bento
Cleonice Alves de Melo Bento
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Both psychological stress and infection diseases have been associated with severity of Multiple sclerosis (MS), a demyelinating autoimmune disease mediated by T cells directed against proteins from myelin sheath of central nervous system leading to neurological deficits in young adults. Like to other autoimmune diseases, the development and severity of MS depends on the influence of environmental factors that modulate the behavior of T cells, such as infections and depression. Recent studies published by our research team demonstrated the ability of different pathogen-associated molecular patterns (PAMPs) in favoring directly the expansion of T cell subsets implicated in the brain lesions in MS patients. On the other hand, the addition serotonin, a neurotransmitter whose production is reduced during depression, attenuated the production of Th17-related cytokines but enhanced the function of regulatory T cells in MS patients. These results suggest that both pathogens and depression should negatively impact MS progression.
What is the aim of the project?
The present study aim to investigate if depression is associated with imbalance of T cell subsets in MS patients. Moreover, we will analyze if depression treatment with serotonin reuptake inhibitors can modify the clinical activity of MS.
What techniques and methods are used?
Clinical follow up: For our study, peripheral blood samples (20 mL) will be collected from patients with MS with or without depression during the outpatient follow-up. At the time of blood collection, clinical information related to disease activity will be collected, such as the degree of neurological disability, determined by the EDSS scale, and number of active brain lesions identified by magnetic resonance imaging. These steps of the study will be always performed with along research team neurologist. Cell cultures: For this study, peripheral blood mononuclear cells (PBMC) from depressed and non-depressed MS patients will be obtained through ficoll-hypque gradient. Then, the PBMC will be stimulated in the presence of polyclonal activator of T cells, PAMPs or myelin basic protein. After 3 days, different T cell phenotypes will be analyzed by Cytometry. Immunofluorescence labeling and flow cytometry: At the end of cell culturing with different stimuli, the PBMC will be additionally activated with phorbol-12-myristate-13-acetate (PMA, 20 ng/mL) plus ionomycin (600 ng/mL) in the presence of brefeldin A (10 μg/mL) for 4 h. For the fluorescence labeling, mouse anti-human fluorescent monoclonal antibodies (mAbs) directed against CD4-FITC/PE-Cy5.5/APC, FoxP3-PE, CD25-APC, CD39-APC, CD152-APC, IL-17-PECy7, IL-10-AlexaFluor 488, IFN--APC, and all isotype-control antibodies will be used to quantify the percentage of different T cell subsets. Briefly, various mAbs combinations directed toward surface markers will be added to the PBMC (2 X 10 5 /tube) and incubated for 30 min at room temperature in the dark. The cells will be washed with PBS solution and then permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA) at 4 °C for 20 min. After washing, the antibodies for intracellular staining (anti- IL-17, anti-IFN-γ, anti-IL-10, anti-FoxP3), or the corresponding anti-IgG1 isotype control, will be added in various combinations and incubated for 30 min at 4 °C. The cells will be analyzed in the Attune (Thermo Fischer™, San Diego, USA) and Flowjo software (Accuri™, Ann Arbor, MI, USA). Isotype control antibodies and single-stained samples will be periodically used to check the settings and gates on the flow cytometer. After acquisition of 100,000 to 200,000 events, lymphocytes will be gated based on forward and side scatter properties after the exclusion of dead cells, by using propidium iodide, and doublets. In some experiments, the analysis of T cell viability will be determined using 7-aminoactinomycin D (7-AAD). Additionally, the gated cells might be negative for CD14 marker. Enzyme-Linked Immunosorbent Assay (ELISA) technique Quantification of cytokines in the plasma and supernatants collected from PBMC activated from depressed and non-depressed MS patients will be performed by ELISA technique using OptEIA ELISA kits (BD, Pharmigen, San Diego, CA), according to manufacturer’s instructions. Each ELISA will be conducted by using pairs of antibodies against IFN-, IL-6, GM-CSF, IL-1β, TNF-α, IL-17, IL-21, IL-22 and IL-10. The reaction will be revealed with streptavidin-horseradish peroxidase, using 3,3’,5,5’-tetramethylbenzidine (TMB) as a substrate. Recombinant human cytokines, at concentrations ranging from 3.5–500 pg/mL, will be used to construct standard curves.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
The student should participate in the scientific meetings of the laboratory whose objective is to discuss scientific papers published in important journals in the area of applied immunology. Moreover, the student will not only follow the experiments but also he/she will help in the analysis of the data obtained by the techniques proposed for the study such as the cell cultures, immunofluorescence labeling and flow citometry and ELISA techniques. Finally, the student will accompany the neurologist doctor responsible for outpatient follow-up of patients with multiple sclerosis enrolled in the study.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
No, The student needs to know how the immune system works to protect the host against pathogens as well the involvement of the immune cells in the autoimmune diseases.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report - The student’s name will be mentioned in a future publication
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
We expect a knowledgeable about immune response and basic knowledge about use of a research laboratory, such as handling pipettes and working aseptically. In addition, we expected that the student is able to read scientific articles.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Students in biomedical fields
- Serotonin decreases the production of Th1/Th17 cytokines and elevates the frequency of regulatory CD4 + T-cell subsets in multiple sclerosis patients. Sacramento PM; Monteiro C; Dias ASO; Kasahara TM; Ferreira TB; Hygino J; Wing AC; Andrade RM; Rueda F; Sales MC; Vasconcelos CC; Bento CAM. Eur J Immunol. 2018 Aug;48(8):1376-1388. doi: 10.1002/eji.201847525. Epub 2018 Jun 6.
- Different interleukin-17-secreting Toll-like receptor + T-cell subsets are associated with disease activity in multiple sclerosis. Ferreira TB; Hygino J; Wing AC; Kasahara TM; Sacramento PM; Camargo S; Rueda F; Alves-Leon SV; Alvarenga R; Vasconcelos CC; Agrawal A; Gupta S; Bento CAM. Immunology. 2018 Jun;154(2):239-252. doi: 10.1111/imm.12872. Epub 2017 Dec 26.
- Vitamin D modulates different IL-17-secreting T cell subsets in multiple sclerosis patients. da Costa DS; Hygino J; Ferreira TB; Kasahara TM; Barros PO; Monteiro C; Oliveira A; Tavares F; Vasconcelos CC; Alvarenga R; Bento CA. J Neuroimmunol. 2016 Oct 15;299:8-18. doi: 10.1016/j.jneuroim.2016.08.005. Epub 2016 Aug 6.
- Combined exercise training reduces fatigue and modulates the cytokine profile of T-cells from multiple sclerosis patients in response to neuromediators. Alvarenga-Filho H; Sacramento PM; Ferreira TB; Hygino J; Abreu JE; Carvalho SR; Wing AC; Alvarenga RM; Bento CA. J Neuroimmunol. 2016 Apr 15;293:91-9. doi: 10.1016/j.jneuroim.2016.02.014. Epub 2016 Feb 26.
- Interleukin-17- and interleukin-22-secreting myelin-specific CD4(+) T cells resistant to corticoids are related with active brain lesions in multiple sclerosis patients. Wing AC; Hygino J; Ferreira TB; Kasahara TM; Barros PO; Sacramento PM; Andrade RM; Camargo S; Rueda F; Alves-Leon SV; Vasconcelos CC; Alvarenga R; Bento CA. Immunology. 2016 Feb;147(2):212-20. doi: 10.1111/imm.12552. Epub 2015 Dec 2.
- Endogenous interleukin-6 amplifies interleukin-17 production and corticoid-resistance in peripheral T cells from patients with multiple sclerosis. Ferreira TB; Hygino J; Barros PO; Teixeira B; Kasahara TM; Linhares UC; Lopes LM; Vasconcelos CC; Alvarenga R; Wing AC; Andrade RM; Andrade AF; Bento CA. Immunology. 2014 Dec;143(4):560-8. doi: 10.1111/imm.12334.
- Low sensitivity to glucocorticoid inhibition of in vitro Th17- related cytokine production in multiple sclerosis patients is related to elevated plasma lipopolysaccharide levels. Teixeira B; Bittencourt VC; Ferreira TB; Kasahara TM; Barros PO; Alvarenga R; Hygino J; Andrade RM; Andrade AF; Bento CA. Clin Immunol. 2013 Aug;148(2):209-18. doi: 10.1016/j.clim.2013.05.012. Epub 2013 May 28.
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