Loading...
1 results
Search Results
Now showing 1 - 1 of 1
- Immune monitoring of relapsing-remitting multiple sclerosis patients submitted to interferon beta treatmentPublication . Monteiro, Andreia Sofia dos Reis; Paiva, Artur Augusto; Fonseca, Ana Mafalda LoureiroTraditionally, the central nervous system (CNS) is described as an immune-privileged site that receives limited immune surveillance by peripheral lymphocytes under physiological conditions. The discovery of the CNS lymphatic system suggests that the CNS is an immune competent organ, closely interacting with the systemic immune compartment under physiological conditions, in which almost all pathological changes in the CNS elicit a prominent inflammatory reaction. Multiple Sclerosis (MS) is a chronic inflammatory disease of the CNS which affects the white and gray matter. MS is believed to be an autoimmune disorder, but the antigen specificity of the immune response is unknown. The pathological hallmark of chronic MS is demyelinated plaque or lesions, which results from an attack on the CNS by immune cells, relative preservation of axons, and the formation of astrocytic scars. Complex multifactorial factors are implicated, in which the environmental are hypothesized to interact with genetically susceptible individuals. In Portugal it is estimated that around 5000 people are affected with MS. The notion that MS is primarily a CD4+ T cell-mediated disease arises from the similarities between the experimental autoimmune encephalomyelitis (EAE) and MS, including the fact that T lymphocytes greatly outnumber B lymphocytes within MS lesions. Diagnosis of MS depends on clinical and paraclinical exams; there is no single diagnostic test to recognize the disease. 85% of patients present a relapse-remitting (RR) MS form, characterized by discrete episodes of neurological dysfunction (relapses or exacerbations) separated by clinical stable periods with lack of disease progression (remissions). Interferon (IFN)-β is the most widely prescribed treatment for MS. IFN-β is a highly pleiotropic cytokine which antagonizes the proinflammatory milieu by increasing production of anti-inflammatory factors. It inhibits leukocyte trafficking and regulates expression of the adhesion molecule. The mechanism of action of IFN-β is complex and multifactorial but has been shown to reduce the biological activity of RRMS in several clinical class I trials. CNS tissue is difficult to access and immune responses within this tissue cannot be easily monitored. Peripheral blood seems to mirror the immunological disturbances that underlie MS, which could represent the migration patterns between the periphery and other tissues according to the clinical phase of the disease. Based on this assumption, the main aim of this thesis was to characterize the circulating immune cell populations of RRMS patients submitted to IFN-β treatment in remission and relapse phases of the disease and compared with healthy subjects. Several studies point to significant alterations in peripheral blood homeostasis of different subpopulations of T cells, like γδ T cells or T helper (Th) 1, Th2, Th17 and T cytotoxic (Tc) 1, Tc2, Tc17 functional subsets; of B cells subpopulations; and of innate cells like monocytes and dendritic cells (DCs). First, we started with the selection of the RRMS patients and collected blood from each one after an informed consent was signed. Through direct immunofluorescence membrane and intracytoplasmic staining protocols, by flow cytometry, were identified and characterized the circulating cell subsets. For the functional assessment of the cells intracellular cytokines at single cell level were measured after in vitro stimulation. To evaluate gene expression, RNA isolation and quantitative real-time reverse transcriptase-polymerase chain reaction was performed. The systemic circulation of IFN-β-treated RRMS patients in remission showed lower frequency of the (myeloid dendritic cells) mDCs subset and higher frequency in the relapse phase, while the frequency of the (plasmacytoid dendritic cells) pDCs subset remains unchanged. Consequently, the mDCs/pDCs ratio decreases in remission and increases in relapse episodes. In remission RRMS patients, the DCs subsets increased their capability to interact with T cells revealed through the increased expression of the HLA-DR and decreased in relapse episodes. In RRMS, the mDCs/pDCs ratio and the activation status of both DCs subsets constitutes a good peripheral biomarker between phases. In circulation of remission RRMS patients, the total monocyte cells and intermediate monocyte (iMo) subset increased and the non-classical monocyte (ncMo) subset decreased. In the relapse phase, the ncMo subset remains decreased. The monocyte subsets present the same pattern of the expression of HLA-DR as the DCs subsets, increasing in remission and decreasing in relapse. The frequency of immature/transitional B cells increases in circulation of remission IFN-β treated RRMS patients. Inside the memory B cell subsets, there was an increase in CD27− B cell subset, more precisely the CD27−IgG+ cells, and a decrease in CD27−IgA+ cells. Relapse RRMS patients showed lower total B cells when compared with remission phase patients, accompanied by an increase in the CD27- memory B cell phases were the increase in the plasmablast B cell subset. The ratio between immature/transitional B cells and plasmablasts decreased in relapse when compared with remission RRMS. The T cell subsets exhibit a shift toward Th2 and Tc2 polarization with a reduction of the Th1 and Tc1 functional subset, in remission episodes. This is accompanied by a reduction in the production of proinflammatory cytokines, mainly IFNγ. Conversely, the frequency of the Th17, Tc17 subsets and the serum level of IL17 increased. In the relapse phase, the Th17 subset decreases the cytokines produced, while the Tc17 subset maintains high levels of tumor necrosis factor (TNF)-α production. The signature of cytokines produced by Th(c)1 and Th(c)17 cells was different. The present study demonstrates that the action mode of IFN-β on Th(c)1 and Th(c)17 cells promotes different results in systemic circulation of RRMS patients. IFN-β therapy supports the decrease in pro-inflammatory cytokines produced by Th(c)1 cells and increase the production of pro-inflammatory cytokines by the Th(c)17. Th17 subset perpetuates and promote the chronic inflammation in periphery in remission RRMS patients, through the production of IL-17 and Th1 type cytokines. No differences were found between the frequency of regulatory T and T follicular helper -like subsets. However, CXCR5+CD4+T cells exhibit a more proinflammatory activity, presenting higher frequencies of TNF-α+ cells in both phases of RRMS. CXCR5+CD8+T cells exhibited an increased ability to produce IL-2 (assuming a Th1 profile) in the remission phase of the disease, thus decreasing in relapsing episodes. The frequency of γδ T cells was the same between healthy subjects and RRMS patients. In remission phase, the central memory γδ T (TCM) subset decreased, and the naive compartment increased. In relapse RRMS patients, the terminally differentiated effector memory γδ T (TEMRA) subset decreased when compared with remission episodes. CCR5+ γδ TEMRA cells were significantly depleted, as a consequence of the migratory pattern in order to play effector functions. This subset presents as a possible participator in the demyelination process and an attractive peripheral blood biomarker between RRMS phases. The identification and characterization of circulating cells can contribute to clarify the pathophysiology of MS, their progression, and the function of each subset in this process. Some of the more relevant results obtained in this study could have the potential to be considered as peripheral biomarkers between remission and relapse RRMS patients treated with IFN-β, namely: the mDCs/pDCs ratio, the activation profile of DCs and monocyte subsets, the commitment of the Th17 cells with a Th1 signature, the CCR5+γδTEMRA cell subset and the ratio between immature/transitional B cells and plasmablasts. In both phases of RRMS, the ncMo subset was decreased and the IgM+CD27+ memory B cells and the compartment CD27- memory B cells increased in RRMS patients. The identification of peripheral markers that could reflect the clinical course of MS and the efficacy of treatment is a stimulating field of research and debate. In the future, further studies including larger cohorts of patients and a larger follow-up are needed in order to establish whether this immune shift correlates with a favorable clinical response.