María José Mansilla

Heat Shock Protein 70: Roles in Multiple Sclerosis

Heat shock proteins (HSP) have long been considered intracellular chaperones that possess housekeeping and cytoprotective functions. Consequently, HSP overexpression was proposed as a potential therapy for neurodegenerative diseases characterized by the accumulation or aggregation of abnormal proteins. Recently, the discovery that cells release HSP with the capacity to trigger proinflammatory as well as immunoregulatory responses has focused attention on investigating the role of HSP in chronic inflammatory autoimmune diseases such as multiple sclerosis (MS). To date, the most relevant HSP is the inducible Hsp70, which exhibits both cytoprotectant and immunoregulatory functions. Several studies have presented contradictory evidence concerning the involvement of Hsp70 in MS or experimental autoimmune encephalomyelitis (EAE), the MS animal model. In this review, we dissect the functions of Hsp70 and discuss the controversial data concerning the role of Hsp70 in MS and EAE.

Beneficial effect of tolerogenic dendritic cells pulsed with MOG autoantigen in experimental autoimmune encephalomyelitis.

Treatment with tolerogenic dendritic cells (TolDC) is a promising, cell‐based strategy to regulate autoimmune diseases such as multiple sclerosis (MS) in an antigen‐specific way. This technique involves the use of TolDC from MS patients cultured in the presence of vitamin D3 (VitD3) and pulsed with myelin peptides to induce a stable hyporesponsiveness in myelin‐specific autologous T cells.

Implication of the toll-like receptor 4 pathway in the response to interferon-β in multiple sclerosis

Interferon‐beta (IFNβ) has demonstrated beneficial effects reducing disease activity in multiple sclerosis (MS) patients, but a relatively large proportion of patients do not respond to treatment. Here we aimed to investigate the roles of the Toll‐like receptor 4 (TLR4) and the type I IFN pathways in the response to IFNβ in MS patients.

Treatment with MOG-DNA vaccines induces CD4+CD25+FoxP3+ regulatory T cells and up-regulates genes with neuroprotective functions in experimental autoimmune encephalomyelitis

BackgroundDNA vaccines represent promising therapeutic strategies in autoimmune disorders such as multiple sclerosis (MS). However, the precise mechanisms by which DNA vaccines induce immune regulation remain largely unknown. Here, we aimed to expand previous knowledge existing on the mechanisms of action of DNA vaccines in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), by treating EAE mice with a DNA vaccine encoding the myelin oligodendrocyte glycoprotein (MOG), and exploring the therapeutic effects on the disease-induced inflammatory and neurodegenerative changes.MethodsEAE was induced in C57BL6/J mice by immunization with MOG35-55 peptide. Mice were intramuscularly treated with a MOG-DNA vaccine or vehicle in prophylactic and therapeutic approaches. Histological studies were performed in central nervous system (CNS) tissue. Cytokine production and regulatory T cell (Treg) quantification were achieved by flow cytometry. Gene expression patterns were determined using microarrays, and the main findings were validated by real-time PCR.ResultsMOG-DNA treatment reduced the clinical and histopathological signs of EAE when administered in both prophylactic and therapeutic settings. Suppression of clinical EAE was associated with dampening of antigen (Ag)-specific proinflammatory Th1 and Th17 immune responses and, interestingly, expansion of Treg in the periphery and upregulation in the CNS of genes encoding neurotrophic factors and proteins involved in remyelination.ConclusionsThese results suggest for the first time that the beneficial effects of DNA vaccines in EAE are not limited to anti-inflammatory mechanisms, and DNA vaccines may also exert positive effects through hitherto unknown neuroprotective mechanisms.

Tolerance Induction in Experimental Autoimmune Encephalomyelitis Using Non-myeloablative Hematopoietic Gene Therapy With Autoantigen

Experimental autoimmune encephalomyelitis (EAE) constitutes a paradigm of antigen (Ag)-specific T cell driven autoimmune diseases. In this study, we transferred bone marrow cells (BMCs) expressing an autoantigen (autoAg), the peptide 40-55 of the myelin oligodendrocytic glycoprotein (MOG(40-55)), to induce preventive and therapeutic immune tolerance in a murine EAE model. Transfer of BMC expressing MOG(40-55) (IiMOG-BMC) into partially myeloablated mice resulted in molecular chimerism and in robust protection from the experimental disease. In addition, in mice with established EAE, transfer of transduced BMC with or without partial myeloablation reduced the clinical and histopathological severity of the disease. In these experiments, improvement was observed even in the absence of engraftment of the transduced hematopoietic cells, probably rejected due to the previous immunization with the autoAg. Splenocytes from mice transplanted with IiMOG-BMC produced significantly higher amounts of interleukin (IL)-5 and IL-10 upon autoAg challenge than those of control animals, suggesting the participation of regulatory cells. Altogether, these results suggest that different tolerogenic mechanisms may be mediating the preventive and the therapeutic effects. In conclusion, this study demonstrates that a cell therapy using BMC expressing an autoAg can induce Ag-specific tolerance and ameliorate established EAE even in a nonmyeloablative setting.

Cryopreserved vitamin D-3-tolerogenic dendritic cells pulsed with autoantigens as a potential therapy for multiple sclerosis patients

BackgroundTolerogenic dendritic cells (tolDC) have been postulated as a potent immunoregulatory therapy for autoimmune diseases such as multiple sclerosis (MS). In a previous study, we demonstrated that the administration of antigen-specific vitamin D3 (vitD3) tolDC in mice showing clinical signs of experimental autoimmune encephalomyelitis (EAE; the animal model of MS) resulted in abrogation of disease progression. With the purpose to translate this beneficial therapy to the clinics, we have investigated the effectivity of vitD3-frozen antigen-specific tolDC pulsed with myelin oligodendrocyte glycoprotein 40-55 peptide (f-tolDC-MOG) since it would reduce the cost, functional variability and number of leukapheresis to perform to the patients.MethodsMice showing EAE clinical signs were treated with repetitive doses of f-tolDC-MOG. Tolerogenic mechanisms induced by the therapy were analysed by flow cytometry and T cell proliferation assays.ResultsTreatment with f-tolDC-MOG was effective in ameliorating clinical signs of mice with EAE, inhibiting antigen-specific reactivity and inducing Treg. In addition, the long-term treatment was well tolerated and leading to a prolonged maintenance of tolerogenicity mediated by induction of Breg, reduction of NK cells and activation of immunoregulatory NKT cells.ConclusionsThe outcomes of this study show that the use of antigen-specific f-tolDC promotes multiple and potent tolerogenic mechanisms. Moreover, these cells can be kept frozen maintaining their tolerogenic properties, which is a relevant step for their translation to the clinic. Altogether, vitD3 f-tolDC-MOG is a potential strategy to arrest the autoimmune destruction in MS patients.

Baseline Differences in Minor Lymphocyte Subpopulations may Predict Response to Fingolimod in Relapsing-Remitting Multiple Sclerosis Patients.

Fingolimod, oral treatment for relapsing–remitting multiple sclerosis (RRMS), is an agonist of sphingosine and its metabolite S1P that binds their receptors, blocking the egress of lymphocytes from lymph nodes. The aim of this study was immunomonitoring of minor peripheral lymphocyte subpopulations in RRMS patients under treatment with fingolimod and correlation with treatment response.

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients

Abstract Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.

Multiparametric flow cytometric analysis of whole blood reveals changes in minor lymphocyte subpopulations of multiple sclerosis patients

Abstract Objective: The objective of this study is to characterise the functionally relevant minor lymphocyte subpopulations in whole blood of multiple sclerosis (MS) patients and their potential utility as biomarkers for treatment follow up. Material and methods: Peripheral blood from 40 healthy donors (HD) and 66 MS patients [23 relapsing–remitting (RRMS) without treatment, 27 RRMS undergoing treatment (16 IFN-β, 11 natalizumab), and 16 progressive forms (eight secondary progressive and eight primary progressive)] was analysed by multiparametric flow cytometry. Results: Untreated MS patients showed a decrease in early effector memory (CD45RA−CCR7−CD27+) CD4+ and CD8+ T cells and an increase in Th17 lymphocytes in peripheral blood compared with HD. Regarding the effect of treatment, whereas no differences in relative percentages of cellular subpopulations were observed in patients under IFN-β treatment, those under treatment with natalizumab had an increased percentage of early effector memory CD4+ (CD45RA−CCR7−CD27+), central memory CD8+ (CD45RA−CCR7+CD27+) T cells, recent thymic emigrants (CD4+ CD45RA+CCR7+CD27+CD31+PTK7+) and transitional B cells (CD19+CD27−CD24hiCD38hi). Conclusions: Multiparametric flow cytometry analysis of whole blood is a robust, reproducible, and sensitive technology to monitor the effect of MS treatments even in minor lymphocyte subpopulations that might represent useful biomarkers of treatment response.

Myeloid-Derived Suppressor Cells are Generated during Retroviral Transduction of Murine Bone Marrow:

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b+ Gr-1hi) and monocytic (CD11b+Gr-1lo) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.