RESUMO
Revealing unknown cues that regulate oligodendrocyte progenitor cell (OPC) function in remyelination is important to optimise the development of regenerative therapies for multiple sclerosis (MS). Platelets are present in chronic non-remyelinated lesions of MS and an increase in circulating platelets has been described in experimental autoimmune encephalomyelitis (EAE) mice, an animal model for MS. However, the contribution of platelets to remyelination remains unexplored. Here we show platelet aggregation in proximity to OPCs in areas of experimental demyelination. Partial depletion of circulating platelets impaired OPC differentiation and remyelination, without altering blood-brain barrier stability and neuroinflammation. Transient exposure to platelets enhanced OPC differentiation in vitro, whereas sustained exposure suppressed this effect. In a mouse model of thrombocytosis (Calr+/-), there was a sustained increase in platelet aggregation together with a reduction of newly-generated oligodendrocytes following toxin-induced demyelination. These findings reveal a complex bimodal contribution of platelet to remyelination and provide insights into remyelination failure in MS.
Assuntos
Plaquetas , Diferenciação Celular , Células Precursoras de Oligodendrócitos , Remielinização , Animais , Células Precursoras de Oligodendrócitos/fisiologia , Remielinização/fisiologia , Camundongos , Plaquetas/fisiologia , Encefalomielite Autoimune Experimental/patologia , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Modelos Animais de Doenças , Oligodendroglia/fisiologia , FemininoRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease affecting the central nervous system (CNS) in animals that parallels several clinical and molecular traits of multiple sclerosis in humans. Herpes simplex virus type 1 (HSV-1) infection mainly causes cold sores and eye diseases, yet eventually, it can also reach the CNS, leading to acute encephalitis. Notably, a significant proportion of healthy individuals are likely to have asymptomatic HSV-1 brain infection with chronic brain inflammation due to persistent latent infection in neurons. Because cellular senescence is suggested as a potential factor contributing to the development of various neurodegenerative disorders, including multiple sclerosis, and viral infections may induce a premature senescence state in the CNS, potentially increasing susceptibility to such disorders, here we examine the presence of senescence-related markers in the brains and spinal cords of mice with asymptomatic HSV-1 brain infection, EAE, and both conditions. Across all scenarios, we find a significant increases of senescence biomarkers in the CNS with some differences depending on the analyzed group. Notably, some senescence biomarkers are exclusively observed in mice with the combined conditions. These results indicate that asymptomatic HSV-1 brain infection and EAE associate with a significant expression of senescence biomarkers in the CNS.
Assuntos
Encéfalo , Senescência Celular , Herpes Simples , Herpesvirus Humano 1 , Esclerose Múltipla , Animais , Camundongos , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/metabolismo , Esclerose Múltipla/virologia , Esclerose Múltipla/patologia , Esclerose Múltipla/metabolismo , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/patogenicidade , Herpes Simples/virologia , Herpes Simples/patologia , Feminino , Camundongos Endogâmicos C57BL , Encefalomielite Autoimune Experimental/virologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Fenótipo , Sistema Nervoso Central/virologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Medula Espinal/virologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Biomarcadores/metabolismo , Encefalite por Herpes Simples/virologia , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/metabolismoRESUMO
This study aims to evaluate and compare cellular therapy with human Wharton's jelly (WJ) mesenchymal stem cells (MSCs) and neural precursors (NPs) in experimental autoimmune encephalomyelitis (EAE), a preclinical model of Multiple Sclerosis. MSCs were isolated from WJ by an explant technique, differentiated to NPs, and characterized by cytometry and immunocytochemistry analysis after ethical approval. Forty-eight rats were EAE-induced by myelin basic protein and Freund's complete adjuvant. Forty-eight hours later, the animals received intraperitoneal injections of 250 ng/dose of Bordetella pertussis toxin. Fourteen days later, the animals were divided into the following groups: a. non-induced, induced: b. Sham, c. WJ-MSCs, d. NPs, and e. WJ-MSCs plus NPs. 1 × 105. Moreover, the cells were placed in a 10 µL solution and injected via a stereotaxic intracerebral ventricular injection. After ten days, the histopathological analysis for H&E, Luxol, interleukins, and CD4/CD8 was carried out. Statistical analyses demonstrated a higher frequency of clinical manifestation in the Sham group (15.66%) than in the other groups; less demyelination was seen in the treated groups than the Sham group (WJ-MSCs, p = 0.016; NPs, p = 0.010; WJ-MSCs + NPs, p = 0.000), and a lower cellular death rate was seen in the treated groups compared with the Sham group. A CD4/CD8 ratio of <1 showed no association with microglial activation (p = 0.366), astrocytes (p = 0.247), and cell death (p = 0.577) in WJ-MSCs. WJ-MSCs and NPs were immunomodulatory and neuroprotective in cellular therapy, which would be translated as an adjunct in demyelinating diseases.
Assuntos
Encefalomielite Autoimune Experimental , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Esclerose Múltipla , Animais , Encefalomielite Autoimune Experimental/terapia , Encefalomielite Autoimune Experimental/patologia , Ratos , Esclerose Múltipla/terapia , Esclerose Múltipla/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Feminino , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Neurais , Modelos Animais de Doenças , Geleia de Wharton/citologiaRESUMO
Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-ß or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-ß and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-ß-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-ß. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.
Assuntos
Antígeno CD11b , Encefalomielite Autoimune Experimental , Interferon gama , Camundongos Endogâmicos C57BL , Células Mieloides , Baço , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Camundongos , Interferon gama/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Baço/imunologia , Antígeno CD11b/metabolismo , Feminino , Glicoproteína Mielina-Oligodendrócito/toxicidade , Glicoproteína Mielina-Oligodendrócito/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Fatores de Transcrição Forkhead/metabolismo , Modelos Animais de DoençasRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model of multiple sclerosis (MS). Oxidative stress and chronic inflammation play a major role in the pathogenesis of MS and EAE. Melatonin, a neurohormone, has potent anti-inflammatory properties. The aim of our study was to assess the therapeutic properties of melatonin alone or in combination with interferon ß-1b (IFNß-1b) or glatiramer acetate (GA) on EAE. EAE was induced in male Sprague-Dawley rats with an intraperitoneal injection of a homogenate of spinal cord and pig brain. At day 10 post immunization, rats were euthanized, and their brains were immediately excised and processed to measure oxidative stress markers and membrane fluidity. In addition, proinflammatory cytokines were quantified in plasma. Melatonin alone or in combination with GA and IFNß-1b inhibited the disease process of EAE and the synthesis of proinflammatory cytokines, caused a significant decrement in oxidative stress markers, and preserved the membrane fluidity in the motor cortex, midbrain, and spinal cord. The cumulative index score was significantly reduced in EAE rats treated with melatonin alone or in combination with GA and IFNß-1b. In conclusion, our findings provide preclinical evidence for the use of melatonin as an adjuvant therapeutic treatment for MS.
Assuntos
Encefalomielite Autoimune Experimental , Melatonina , Esclerose Múltipla , Animais , Biomarcadores , Citocinas , Encefalomielite Autoimune Experimental/patologia , Acetato de Glatiramer/farmacologia , Acetato de Glatiramer/uso terapêutico , Interferon beta-1b/uso terapêutico , Interferon beta , Masculino , Melatonina/farmacologia , Melatonina/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Ratos , Ratos Sprague-Dawley , SuínosRESUMO
BACKGROUND: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. PURPOSE: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. RESULTS: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. CONCLUSIONS: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.
Assuntos
Anticorpos Monoclonais/imunologia , Ácido Glutâmico/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Axônios/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Ácido Glutâmico/administração & dosagem , Ácido Glutâmico/farmacologia , Glutationa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Associada a Mielina/imunologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Receptores de Glutamato/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Multiple sclerosis (MS) is an immune-mediated and degenerating disease in which myelin sheaths are damaged as a result of chronic progressive inflammation of the central nervous system. Tibolone [(7α,17α)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-in-3-one], a synthetic estrogenic compound with tissue-specific actions and used for menopausal hormone therapy, shows neuroprotective and antioxidant properties both in vivo and in vitro. In the present study, we analyzed whether tibolone plays a therapeutic role in experimental autoimmune encephalomyelitis (EAE) mice, a commonly used model of MS. Female C57BL/6 mice were induced with the myelin oligodendrocyte glycoprotein MOG35-55 and received s.c. tibolone (0.08 mg kg-1 ) injection every other day from the day of induction until death on the acute phase of the disease. Reactive gliosis, Toll like receptor 4 (TLR4), high mobility group box protein 1 (HMGB1), inflammasome parameters, activated Akt levels and myelin were assessed by a real-time polymerase chain reaction, immunohistochemistry, and western blot analysis. Our findings indicated that, in the EAE spinal cord, tibolone reversed the astrocytic and microglial reaction, and reduced the hyperexpression of TLR4 and HMGB1, as well as NLR family pyrin domain containing 3-caspase 1-interleukin-1ß inflammasome activation. At the same time, tibolone attenuated the Akt/nuclear factor kappa B pathway and limited the white matter demyelination area. Estrogen receptor expression was unaltered with tibolone treatment. Clinically, tibolone improved neurological symptoms without uterine compromise. Overall, our data suggest that tibolone may serve as a promising agent for the attenuation of MS-related inflammation.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Neurite (Inflamação)/prevenção & controle , Norpregnenos/uso terapêutico , Animais , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Feminino , Inflamação/patologia , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Neurite (Inflamação)/patologia , Fármacos Neuroprotetores/uso terapêutico , Indução de RemissãoRESUMO
Multiple Sclerosis (MS) is a neuroinflammatory and chronic Central Nervous System (CNS) disease that affects millions of people worldwide. The search for more promising drugs for the treatment of MS has led to studies on Sildenafil, a phosphodiesterase type 5 Inhibitor (PDE5I) that has been shown to possess neuroprotective effects in the Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. We have previously shown that Sildenafil improves the clinical score of EAE mice via modulation of apoptotic pathways, but other signaling pathways were not previously covered. Therefore, the aim of the present study was to further investigate the effects of Sildenafil treatment on autophagy and nitrosative stress signaling pathways in EAE. 24 female C57BL/6 mice were divided into the following groups: (A) Control - received only water; (B) EAE - EAE untreated mice; (C) SILD - EAE mice treated with 25mg/kg of Sildenafil s.c. The results showed that EAE mice presented a pro-nitrosative profile characterized by high tissue nitrite levels, lowered levels of p-eNOS and high levels of iNOS. Furthermore, decreased levels of LC3, beclin-1 and ATG5, suggests impaired autophagy, and decreased levels of AMPK in the spinal cord were also detected in EAE mice. Surprisingly, treatment with Sildenafil inhibited nitrosative stress and augmented the levels of LC3, beclin-1, ATG5, p-CREB and BDNF and decreased mTOR levels, as well as augmented p-AMPK. In conclusion, we propose that Sildenafil alleviates EAE by activating autophagy via the eNOS-NO-AMPK-mTOR-LC3-beclin1-ATG5 and eNOS-NO-AMPK-mTOR-CREB-BDNF pathways in the spinal cord.
Assuntos
Autofagia/efeitos dos fármacos , Encefalomielite Autoimune Experimental/patologia , Inibidores da Fosfodiesterase 5/farmacologia , Citrato de Sildenafila/farmacologia , Medula Espinal/efeitos dos fármacos , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Estresse Nitrosativo/efeitos dos fármacosRESUMO
Multiple sclerosis is an autoimmune disease that affects the myelinated central nervous system (CNS) neurons and triggers physical and cognitive disabilities. Conventional therapy is based on disease-modifying drugs that control disease severity but can also be deleterious. Complementary medicines have been adopted and evidence indicates that yeast supplements can improve symptoms mainly by modulating the immune response. In this investigation, we evaluated the therapeutic potential of Saccharomyces cerevisiae and its selenized derivative (Selemax) in experimental autoimmune encephalomyelitis (EAE). Female C57BL/6 mice submitted to EAE induction were orally supplemented with these yeasts by gavage from day 0 to day 14 after EAE induction. Both supplements determined significant reduction in clinical signs concomitantly with diminished Th1 immune response in CNS, increased proportion of Foxp3+ lymphocytes in inguinal and mesenteric lymph nodes and increased microbiota diversity. However, Selemax was more effective clinically and immunologically; it reduced disease prevalence more sharply, increased the proportion of CD103+ dendritic cells expressing high levels of PD-L1 in mesenteric lymph nodes and reduced the intestinal inflammatory process more strongly than S. cerevisiae. These results suggest a clear gut-brain axis modulation by selenized S. cerevisiae and suggest their inclusion in clinical trials.
Assuntos
Suplementos Nutricionais , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Saccharomyces cerevisiae/imunologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/patologia , Tolerância Imunológica , Contagem de Linfócitos , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Multiple sclerosis is a chronic and demyelinating disease of the central nervous system (CNS), most prevalent in women, and with an important social and economic cost worldwide. It is triggered by self-reacting lymphocytes that infiltrate the CNS and initiate neuroinflammation. Further, axonal loss and neuronal death takes place, leading to neurodegeneration and brain atrophy. The murine model for studying MS, experimental autoimmune encephalomyelitis (EAE), consists in immunizing mice with myelin-derived epitopes. APCs activate encephalitogenic T CD4 and CD8 lymphocytes that migrate mainly to the spinal cord resulting in neuroinflammation. Most of the knowledge on the pathophysiology and treatment of MS was obtained from EAE experiments, as Th17 cells, anti-alpha4 blocking Abs and the role of microbiota. Conversely, recent technology breakthroughs, such as CyTOF and single-cell RNA-seq, promise to revolutionize our understanding on the mechanisms involved both in MS and EAE. In fact, the importance of specific cellular populations and key molecules in MS/EAE is a constant matter of debate. It is well accepted that both Th1 and Th17 T CD4 lymphocytes play a relevant role in disease initiation after re-activation in situ. What is still under constant investigation, however, is the plasticity of the lymphocyte population, and the individual contribution of both resident and inflammatory cells for the progression or recovery of the disease. Thus, in this review, new findings obtained after single-cell analysis of blood and central nervous system infiltrating cells from MS/EAE and how they have contributed to a better knowledge on the cellular and molecular mechanisms of neuroinflammation are discussed.