RESUMO
Multiple sclerosis (MS) onset at an advanced age is associated with a higher risk of developing progressive forms and a greater accumulation of disability for which there are currently no effective disease-modifying treatments. Immunosenescence is associated with the production of the senescence-associated secretory phenotype (SASP), with IL-6 being one of the most prominent cytokines. IL-6 is a determinant for the development of autoimmunity and neuroinflammation and is involved in the pathogenesis of MS. Herein, we aimed to preclinically test the therapeutic inhibition of IL-6 signaling in experimental autoimmune encephalomyelitis (EAE) as a potential age-specific treatment for elderly MS patients. Young and aged mice were immunized with myelin oligodendrocyte protein (MOG)35-55 and examined daily for neurological signs. Mice were randomized and treated with anti-IL-6 antibody. Inflammatory infiltration was evaluated in the spinal cord and the peripheral immune response was studied. The blockade of IL-6 signaling did not improve the clinical course of EAE in an aging context. However, IL-6 inhibition was associated with an increase in the peripheral immunosuppressive response as follows: a higher frequency of CD4 T cells producing IL-10, and increased frequency of inhibitory immune check points PD-1 and Tim-3 on CD4+ T cells and Lag-3 and Tim-3 on CD8+ T cells. Our results open the window to further studies aimed to adjust the anti-IL-6 treatment conditions to tailor an effective age-specific therapy for elderly MS patients.
Assuntos
Encefalomielite Autoimune Experimental , Interleucina-6 , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Animais , Camundongos , Interleucina-6/metabolismo , Interleucina-6/antagonistas & inibidores , Feminino , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Envelhecimento/imunologia , Interleucina-10/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.
Assuntos
Inflamassomos/imunologia , Interleucina-1beta/imunologia , Esclerose Múltipla Crônica Progressiva/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Adulto , Animais , Biomarcadores/análise , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PrognósticoRESUMO
Although genome-wide association studies have identified a number of common variants associated with multiple sclerosis (MS) susceptibility, little is known about the relevance of rare variants. Here, we aimed to explore the role of rare variants in 14 MS risk genes (FCRL1, RGS1, TIMMDC1, HHEX, CXCR5, LTBR, TSFM, GALC, TRAF3, STAT3, TNFSF14, IFI30, CD40, and CYP24A1) by targeted resequencing in an Iberian population of 524 MS cases and 546 healthy controls. Four rare variants-enriched regions within CYP24A1, FCRL1, RGS1, and TRAF3 were identified as significantly associated with MS. Functional studies revealed significantly decreased regulator of G protein signaling 1 (RGS1) gene expression levels in peripheral blood mononuclear cells from MS patients with RGS1 rare variants compared to noncarriers, whereas no significant differences in gene expression were observed for CYP24A1, FCRL1, and TRAF3 between rare variants carriers and noncarriers. Immunophenotyping showed significant decrease in RGS1 expression in peripheral blood B lymphocytes from MS patients with RGS1 rare variants relative to noncarriers. Lastly, peripheral blood mononuclear cell from MS patients carrying RGS1 rare variants showed significantly lower induction of RGS1 gene expression by interferon-ß compared to MS patients lacking RGS1 variants. The presence of rare variants in RGS1 reinforce the ideas of high genetic heterogeneity and a role of rare variants in MS pathogenesis.
Assuntos
Predisposição Genética para Doença , Esclerose Múltipla/genética , Linfócitos B , Estudos de Casos e Controles , Análise Mutacional de DNA , Humanos , Leucócitos Mononucleares , Proteínas de Membrana/genética , Proteínas RGS/genética , Espanha , Fator 3 Associado a Receptor de TNF/genética , Vitamina D3 24-Hidroxilase/genéticaRESUMO
Bone morphogenetic proteins (BMPs) are secreted proteins that belong to the transforming growth factor-ß superfamily. In the adult brain, they modulate neurogenesis, favor astrogliogenesis, and inhibit oligodendrogenesis. Because BMPs may be involved in the failure of remyelination in multiple sclerosis (MS), we characterized the expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMP type II receptor (BMPRII); and phosphorylated SMAD (pSMAD) 1/5/8 in lesions of MS and other demyelinating diseases. A total of 42 MS lesions, 12 acute ischemic lesions, 8 progressive multifocal leukoencephalopathy lesions, and 10 central nervous system areas from four nonneuropathological patients were included. Lesions were histologically classified according to the inflammatory activity. The expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified by immunostaining, and colocalization studies were performed. In MS lesions, astrocytes, microglia/macrophages, and neurons expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8. Oligodendrocytes expressed BMP-2 and BMP-7 and pSMAD1/5/8. The percentage of cells that expressed BMPs, BMPRII, and pSMAD1/5/8 correlated with the inflammatory activity of MS lesions, and changes in the percentage of positive cells were more relevant in MS than in other white matter-damaging diseases. These data indicate that BMPs are increased in active MS lesions, suggesting a possible role in MS pathogenesis.
Assuntos
Astrócitos/metabolismo , Proteínas Morfogenéticas Ósseas/biossíntese , Regulação da Expressão Gênica , Esclerose Múltipla/metabolismo , Oligodendroglia/metabolismo , Substância Branca/metabolismo , Astrócitos/patologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Feminino , Humanos , Leucoencefalopatia Multifocal Progressiva/metabolismo , Leucoencefalopatia Multifocal Progressiva/patologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Oligodendroglia/patologia , Proteínas Smad/metabolismo , Substância Branca/fisiologiaRESUMO
Bone morphogenetic proteins (BMPs) are growth factors that represent the largest subgroup of signalling ligands of the transforming growth factor beta (TGF-ß) superfamily. Their participation in the proliferation, survival and cell fate of several cell types and their involvement in many pathological conditions are now well known. BMP expression is altered in multiple sclerosis (MS) patients, suggesting that BMPs have a role in the pathogenesis of this disease. MS is a demyelinating and neurodegenerative autoimmune disorder of the central nervous system (CNS). MS is a complex pathological condition in which genetic, epigenetic and environmental factors converge, although its aetiology remains elusive. Multifunctional molecules, such as BMPs, are extremely interesting in the field of MS because they are involved in the regulation of several adult tissues, including the CNS and the immune system. In this review, we discuss the extensive data available regarding the role of BMP signalling in neuronal progenitor/stem cell fate and focus on the participation and expression of BMPs in CNS demyelination. Additionally, we provide an overview of the involvement of BMPs as modulators of the immune system, as this subject has not been thoroughly explored even though it is of great interest in autoimmune disorders. Moreover, we describe the data on BMP signalling in autoimmunity and inflammatory diseases, including MS and its experimental models. Thus, we aim to provide an integrated view of the putative role of BMPs in MS pathogenesis and to open the field for the further development of alternative therapeutic strategies for MS patients.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Esclerose Múltipla/patologia , Autoimunidade/fisiologia , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Linhagem da Célula , Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Neuroglia , Neuroimunomodulação/fisiologia , Neurônios , Transdução de SinaisRESUMO
We characterised the expression of semaphorin (sema)3A, sema7A and their receptors in the immune and the central nervous system (CNS) at different stages of experimental autoimmune encephalomyelitis (EAE). We also studied their expression in neonatal and adult oligodendrocyte progenitor cell (OPC) and in mature oligodendrocyte cultures. Our results show that sema3A is increased in the CNS and decreased in the immune system upon EAE induction. However, sema7A expression is increased in both the CNS and the immune system during EAE. We also detected sema3A, sema7A and their receptors in neonatal and adult OPCs and in mature oligodendrocytes. These data suggest that sema3A and sema7A are involved in the pathogenesis of EAE, in the modulation of the immune response and in the neurodegeneration that take place in the CNS. Sema7A may represent an intriguing potential therapeutic target for the treatment of both the neurodegenerative and immune-mediated disease processes in MS.
Assuntos
Antígenos CD/imunologia , Encefalomielite Autoimune Experimental/imunologia , Semaforina-3A/imunologia , Semaforinas/imunologia , Animais , Antígenos CD/genética , Encéfalo/imunologia , Encéfalo/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Immunoblotting , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Oligodendroglia/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Semaforina-3A/genética , Semaforinas/genética , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Introduction: Little is known about the molecular profiling associated with the effect of cladribine in patients with multiple sclerosis (MS). Here, we aimed first to characterize the transcriptomic and proteomic profiles induced by cladribine in blood cells, and second to identify potential treatment response biomarkers to cladribine in patients with MS. Methods: Gene, protein and microRNA (miRNA) expression profiles were determined by microarrays (genes, miRNAs) and mass spectrometry (proteins) in peripheral blood mononuclear cells (PBMCs) from MS patients after in vitro treatment with cladribine in its active and inactive forms. Two bioinformatics approaches to integrate the three obtained datasets were applied: (i) a multiomics discriminant analysis (DIABLO - Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies); and (ii) a multi-stage integration of features selected in differential expression analysis on each dataset and then merged. Selected molecules from the in vitro study were quantified by qPCR ex vivo in PBMCs from MS patients receiving cladribine. Results: PBMCs treated in vitro with cladribine were characterized by a major downregulation of gene, protein, and miRNA expression compared with the untreated cells. An intermediate pattern between the cladribine-treated and untreated conditions was observed in PBMCs treated with cladribine in its inactive form. The differential expression analysis of each dataset led to the identification of four genes and their encoded proteins, and twenty-two miRNAs regulating their expression, that were associated with cladribine treatment. Two of these genes (PPIF and NHLRC2), and three miRNAs (miR-21-5p, miR-30b-5p, and miR-30e-5p) were validated ex vivo in MS patients treated with cladribine. Discussion: By using a combination of omics data and bioinformatics approaches we were able to identify a multiomics molecular profile induced by cladribine in vitro in PBMCs. We also identified a number of biomarkers that were validated ex vivo in PBMCs from patients with MS treated with cladribine that have the potential to become treatment response biomarkers to this drug.
Assuntos
MicroRNAs , Esclerose Múltipla , Humanos , Cladribina/farmacologia , Cladribina/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Leucócitos Mononucleares/metabolismo , Proteômica , MicroRNAs/metabolismo , BiomarcadoresRESUMO
The pathogenic role of the interleukin 21 (IL-21) in different autoimmune diseases, such as multiple sclerosis (MS), has been extensively studied. However, its pleiotropic nature makes it a cytokine that may exhibit different activity depending on the immunological stage of the disease. In this study, we developed a gene therapy strategy to block the interaction between IL-21 and its receptor (IL-21R) by using adeno-associated vectors (AAV) encoding a new soluble cytokine receptor (sIL21R) protein. We tested this strategy in a murine model of experimental autoimmune encephalomyelitis (EAE), obtaining different clinical effects depending on the time at which the treatment was applied. Although the administration of the treatment during the development of the immune response was counterproductive, the preventive administration of the therapeutic vectors showed a protective effect by reducing the number of animals that developed the disease, as well as an improvement at the histopathological level and a modification of the immunological profile of the animals treated with the AAV8.sIL21R. The beneficial effect of the treatment was also observed when inducing the expression of the therapeutic molecule once the first neurological signs were established in a therapeutic approach with a doxycyline (Dox)-inducible expression system. All these clinical results highlight the pleiotropicity of this cytokine in the different clinical stages and its key role in the EAE immunopathogenesis.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Camundongos , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Camundongos Endogâmicos C57BL , Esclerose Múltipla/genética , Esclerose Múltipla/terapia , Terapia Genética/métodos , Citocinas/genética , Receptores de Citocinas/genética , Receptores de Citocinas/uso terapêuticoRESUMO
The number of elderly multiple sclerosis (MS) patients is growing, mainly due to the increase in the life expectancy of the general population and the availability of effective disease-modifying treatments. However, current treatments reduce the frequency of relapses and slow the progression of the disease, but they cannot stop the disability accumulation associated with disease progression. One possible explanation is the impact of immunosenescence, which is associated with the accumulation of unusual immune cell subsets that are thought to have a role in the development of an early ageing process in autoimmunity. Here, we provide a recent overview of how senescence affects immune cell function and how it is involved in the pathogenesis of autoimmune diseases, particularly MS. Numerous studies have demonstrated age-related immune changes in experimental autoimmune encephalomyelitis models, and the premature onset of immunosenescence has been demonstrated in MS patients. Therefore, potential therapeutic strategies based on rejuvenating the immune system have been proposed. Senolytics and regenerative strategies using haematopoietic stem cells, therapies based on rejuvenating oligodendrocyte precursor cells, microglia and monocytes, thymus cells and senescent B and T cells are capable of reversing the process of immunosenescence and could have a beneficial impact on the progression of MS.
Assuntos
Encefalomielite Autoimune Experimental , Imunossenescência , Esclerose Múltipla , Idoso , Animais , Autoimunidade , Encefalomielite Autoimune Experimental/terapia , Humanos , Esclerose Múltipla/terapia , Linfócitos TRESUMO
Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.
Assuntos
Cladribina/farmacologia , Imunomodulação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina Quinase/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Pró-Fármacos/farmacologiaRESUMO
Gut microbiome studies in multiple sclerosis (MS) patients are unravelling some consistent but modest patterns of gut dysbiosis. Among these, a significant decrease of Clostridia cluster IV and XIVa has been reported. In the present study, we investigated the therapeutic effect of a previously selected mixture of human gut-derived 17 Clostridia strains, which belong to Clostridia clusters IV, XIVa, and XVIII, on the clinical outcome of experimental autoimmune encephalomyelitis (EAE). The observed clinical improvement was related to lower demyelination and astrocyte reactivity as well as a tendency to lower microglia reactivity/infiltrating macrophages and axonal damage in the central nervous system (CNS), and to an enhanced immunoregulatory response of regulatory T cells in the periphery. Transcriptome studies also highlighted increased antiinflammatory responses related to interferon beta in the periphery and lower immune responses in the CNS. Since Clostridia-treated mice were found to present higher levels of the immunomodulatory short-chain fatty acid (SCFA) butyrate in the serum, we studied if this clinical effect could be reproduced by butyrate administration alone. Further EAE experiments proved its preventive but slight therapeutic impact on CNS autoimmunity. Thus, this smaller therapeutic effect highlighted that the Clostridia-induced clinical effect was not exclusively related to the SCFA and could not be reproduced by butyrate administration alone. Although it is still unknown if these Clostridia strains will have the same effect on MS patients, gut dysbiosis in MS patients could be partially rebalanced by these commensal bacteria and their immunoregulatory properties could have a beneficial effect on MS clinical course.
Assuntos
Butiratos/administração & dosagem , Clostridiaceae/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Microbioma Gastrointestinal/fisiologia , Animais , Disbiose/imunologia , Disbiose/patologia , Disbiose/terapia , Encefalomielite Autoimune Experimental/patologia , Ácidos Graxos Voláteis/administração & dosagem , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP(+) cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-gamma (IFN-gamma), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols.
Assuntos
Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Celular/imunologia , Animais , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Bussulfano/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Vetores Genéticos , Imunidade Celular/efeitos dos fármacos , Imunossupressores/farmacologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Retroviridae/genética , Transdução Genética , Condicionamento Pré-Transplante , Irradiação Corporal TotalRESUMO
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.
Assuntos
Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Terapia Genética/métodos , Tolerância Imunológica/imunologia , Animais , Células da Medula Óssea , Transplante de Medula Óssea , Feminino , Citometria de Fluxo , Vetores Genéticos , Interleucina-10/metabolismo , Interleucina-5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Associada a Mielina/imunologia , RetroviridaeRESUMO
Previous studies in experimental autoimmune encephalomyelitis (EAE) models have shown that some probiotic bacteria beneficially impact the development of this experimental disease. Here, we tested the therapeutic effect of two commercial multispecies probiotics-Lactibiane iki and Vivomixx-on the clinical outcome of established EAE. Lactibiane iki improves EAE clinical outcome in a dose-dependent manner and decreases central nervous system (CNS) demyelination and inflammation. This clinical improvement is related to the inhibition of pro-inflammatory and the stimulation of immunoregulatory mechanisms in the periphery. Moreover, both probiotics modulate the number and phenotype of dendritic cells (DCs). Specifically, Lactibiane iki promotes an immature, tolerogenic phenotype of DCs that can directly induce immune tolerance in the periphery, while Vivomixx decreases the percentage of DCs expressing co-stimulatory molecules. Finally, gut microbiome analysis reveals an altered microbiome composition related to clinical condition and disease progression. This is the first preclinical assay that demonstrates that a commercial probiotic performs a beneficial and dose-dependent effect in EAE mice and one of the few that demonstrates a therapeutic effect once the experimental disease is established. Because this probiotic is already available for clinical trials, further studies are being planned to explore its therapeutic potential in multiple sclerosis patients.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Microbioma Gastrointestinal/imunologia , Tolerância Imunológica/imunologia , Esclerose Múltipla/tratamento farmacológico , Probióticos/uso terapêutico , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Camundongos , Esclerose Múltipla/imunologia , Probióticos/farmacologiaRESUMO
Bone morphogenetic proteins (BMPs) are secreted growth factors that belong to the transforming growth factor beta superfamily. BMPs have been implicated in physiological processes, but they are also involved in many pathological conditions. Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS); however, its etiology remains elusive. Some evidence points to BMPs as important players in the pathogenesis of inflammatory and autoimmune disorders. In the present work, we studied the expression of BMP2, BMP4, BMP5, BMP6, BMP7, BMP type II receptor, and noggin in the immune system during different phases of experimental autoimmune encephalomyelitis (EAE). Major changes in the expression of BMPs took place in the initial phases of EAE. Indeed, those changes mainly affected BMP6 (whose expression was abrogated), BMP2, and BMP7 (whose expression was increased). In addition, we showed that in vivo inhibition of the BMP signaling pathway with small molecules ameliorated the already established clinical symptoms of EAE, as well as the CNS histopathological features. At the immune level, we observed an expansion of plasmacytoid dendritic cells (pDCs) in mice treated with small molecules that inhibit the BMP signaling pathway. pDCs could play an important role in promoting the expansion of antigen-specific regulatory T cells. Altogether, our data suggest a role for BMPs in early immune events that take place in myelin oligodendrocyte glycoprotein (MOG)-induced EAE. In addition, the clinical outcome of the disease was improved when the BMP signaling pathway was inhibited in mice that presented established EAE symptoms.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Pirazóis/administração & dosagem , Quinolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/administração & dosagem , Feminino , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Genome-wide association studies and meta-analysis have contributed to the identification of more than 200 loci associated with multiple sclerosis (MS). However, a proportion of MS heritability remains unknown. We aimed to uncover new genetic variants associated with MS and determine their functional effects. For this, we resequenced the exons and regulatory sequences of 14 MS risk genes in a cohort of MS patients and healthy individuals (n = 1,070) and attempted to validate a selection of signals through genotyping in an independent cohort (n = 5,138). We identified three new MS-associated variants at C-X-C motif chemokine receptor 5 (CXCR5), Ts translation elongation factor, mitochondrial (TSFM) and cytochrome P450 family 24 subfamily A member 1 (CYP24A1). Rs10892307 resulted in a new signal at the CXCR5 region that explains one of the associations with MS within the locus. This polymorphism and three others in high linkage disequilibrium mapped within regulatory regions. Of them, rs11602393 showed allele-dependent enhancer activity in the forward orientation as determined by luciferase reporter assays. Immunophenotyping using peripheral blood mononuclear cells from MS patients associated the minor allele of rs10892307 with increased percentage of regulatory T cells expressing CXCR5. This work reports a new signal for the CXCR5 MS risk locus and points to rs11602393 as the causal variant. The expansion of CXCR5+ circulating regulatory T cells induced by this variant could cause its MS association.
RESUMO
BACKGROUND AND OBJECTIVES: Percentages of blood CD19+CD5+ B cells and CD8+perforin+ T lymphocytes can predict response to Interferon (IFN)-beta treatment in relapsing-remitting multiple sclerosis (RRMS) patients. We aimed to standardize their detection in a multicenter study, prior to their implementation in clinical practice. METHODS: Fourteen hospitals participated in the study. A reference centre was established for comparison studies. Peripheral blood cells of 105 untreated RRMS patients were studied. Every sample was analyzed in duplicate in the participating centre and in the reference one by flow cytometry. When needed, participating centres corrected fluorescence compensations and negative cut-off position following reference centre suggestions. Concordance between results obtained by participating centres and by reference one was evaluated by intraclass correlation coefficients (ICC) and Spearman correlation test. Centre performance was measured by using z-scores values. RESULTS: After results review and corrective actions implementation, overall ICC was 0.86 (CI: 0.81-0.91) for CD19+CD5+ B cell and 0.89 (CI: 0.85-0.93) for CD8+â¯perforin+ T cell quantification; Spearman r was 0.92 (0.89-0.95; pâ¯<0.0001) and 0.92 (0.88-0.95; pâ¯<0.0001) respectively. All centres obtained z-scores≤0.5 for both biomarkers. CONCLUSION: Homogenous percentages of CD19+CD5+ B cells and CD8â¯perforin+ T lymphocytes can be obtained if suitable compensation values and negative cut-off are pre-established.
Assuntos
Citometria de Fluxo , Interferon beta/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Esclerose Múltipla/sangueRESUMO
The commensal microbiota has emerged as an environmental risk factor for multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE) models have shown that the commensal microbiota is an essential player in triggering autoimmune demyelination. Likewise, the commensal microbiota modulates the host immune system, alters the integrity and function of biological barriers and has a direct effect on several types of central nervous system (CNS)-resident cells. Moreover, a characteristic gut dysbiosis has been recognized as a consistent feature during the clinical course of MS, and the MS-related microbiota is gradually being elucidated. This review highlights animal studies in which commensal microbiota modulation was tested in EAE, as well as the mechanisms of action and influence of the commensal microbiota not only in the local milieu but also in the innate and adaptive immune system and the CNS. Regarding human research, this review focuses on studies that show how the commensal microbiota might act as a pathogenic environmental risk factor by directing immune responses towards characteristic pathogenic profiles of MS. We speculate how specific microbiome signatures could be obtained and used as potential pathogenic events and biomarkers for the clinical course of MS. Finally, we review recently published and ongoing clinical trials in MS patients regarding the immunomodulatory properties exerted by some microorganisms. Because MS is a complex disease with a large variety of associated environmental risk factors, we suggest that current treatments combined with strategies that modulate the commensal microbiota would constitute a broader immunotherapeutic approach and improve the clinical outcome for MS patients.
Assuntos
Disbiose/imunologia , Encefalomielite Autoimune Experimental/imunologia , Microbioma Gastrointestinal/imunologia , Microbiota/imunologia , Esclerose Múltipla/imunologia , Animais , Humanos , CamundongosRESUMO
Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system that develops in genetically susceptible individuals and likely requires environmental triggers. The autoantigens and molecular mimics triggering the autoimmune response in multiple sclerosis remain incompletely understood. By using a brain-infiltrating CD4+ T cell clone that is clonally expanded in multiple sclerosis brain lesions and a systematic approach for the identification of its target antigens, positional scanning peptide libraries in combination with biometrical analysis, we have identified guanosine diphosphate (GDP)-l-fucose synthase as an autoantigen that is recognized by cerebrospinal fluid-infiltrating CD4+ T cells from HLA-DRB3*-positive patients. Significant associations were found between reactivity to GDP-l-fucose synthase peptides and DRB3*02:02 expression, along with reactivity against an immunodominant myelin basic protein peptide. These results, coupled with the cross-recognition of homologous peptides from gut microbiota, suggest a possible role of this antigen as an inducer or driver of pathogenic autoimmune responses in multiple sclerosis.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Fucose/metabolismo , Glucosiltransferases/metabolismo , Cadeias HLA-DRB3/metabolismo , Esclerose Múltipla/imunologia , Alelos , Sequência de Aminoácidos , Encéfalo/metabolismo , Células Clonais , Humanos , Esclerose Múltipla/líquido cefalorraquidiano , Proteína Básica da Mielina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Semaphorin 7A (sema7A) is classified as an immune semaphorin with dual functions in the immune system and in the central nervous system (CNS). These molecules are of interest due to their potential role in multiple sclerosis (MS), which is a chronic demyelinating and neurodegenerative disease of autoimmune origin. In this study, we elucidated the role of sema7A in neuroinflammation using both in vitro and in vivo experimental models. In an in vitro model of neuroinflammation, using cerebellar organotypic slice cultures, we observed that challenge with lipopolysaccharide (LPS) endotoxin did not affect demyelination or cell death in sema7A-deficient cultures compared to wild-type cultures. Moreover, the in vivo outcome of experimental autoimmune encephalomyelitis (EAE) in sema7A-deficient mice was altered in an antigen- and adjuvant-dose-dependent manner, while no differences were observed in the wild-type counterparts. Altogether, these results indicate that sema7A is involved in peripheral immunity and CNS inflammation in MS pathogenesis. Indeed, these data suggest that sema7A might be a potential therapeutic target to treat MS and autoimmune conditions.