RESUMEN
Experimental autoimmune encephalomyelitis (EAE) serves as a model for studying multiple sclerosis, with immunization strategies utilizing MOG35-55 peptide, emulsified in adjuvant enriched with mycobacterium tuberculosis (Mtb). This study examined the effects of Bacillus Calmette-Guérin (BCG) as an adjuvant, alongside the impact of MOG35-55 peptide doses and their residual counter ions on EAE development. We found that BCG can be effectively used to induce EAE with similar incidence and severity as heat-killed H37Ra, contingent upon the appropriate MOG35-55 peptide dose. Different immunization doses of MOG35-55 peptide significantly affect EAE development, with higher doses leading to a paradoxical reduction in disease activity, probably due to peripheral tolerance mechanisms. Furthermore, doses of MOG35-55 peptides with acetate showed a more pronounced effect on disease development compared to those containing trifluoroacetic acid (TFA), suggesting the potential influence of residual counter ions on EAE activity. We highlighted the feasibility of applying BCG to the establishment of EAE for the first time. Our findings emphasized the importance of MOG peptide dosage and composition in modulating EAE development, offering insights into the mechanisms of autoimmunity and tolerance. This could have implications for autoimmune disease research and the design of therapeutic strategies.
RESUMEN
Emerging evidence underscores the importance of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), but the precise mechanisms remain ambiguous. This study intends to elucidate the involvement of a novel subset of follicular CD8+ T cells (CD8+CXCR5+ T) in MS and an experimental autoimmune encephalomyelitis (EAE) murine model. The expansion of CD8+CXCR5+ T cells was observed in both MS patients and EAE mice during the acute phase. In relapsing MS patients, higher frequencies of circulating CD8+CXCR5+ T cells were positively correlated with new gadolinium-enhancement lesions in the central nervous system (CNS). In EAE mice, frequencies of CD8+CXCR5+ T cells were also positively correlated with clinical scores. These cells were found to infiltrate into ectopic lymphoid-like structures in the spinal cords during the peak of the disease. Furthermore, CD8+CXCR5+ T cells, exhibiting high expression levels of ICOS, CD40L, IL-21, and IL-6, were shown to facilitate B cell activation and differentiation through a synergistic interaction between CD40L and IL-21. Transferring CD8+CXCR5+ T cells into naïve mice confirmed their ability to enhance the production of anti-MOG35-55 antibodies and contribute to the disease progression. Consequently, CD8+CXCR5+ T cells may play a role in CNS demyelination through heightening humoral immune responses.
Asunto(s)
Linfocitos T CD8-positivos , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ratones , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Humanos , Femenino , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Receptores CXCR5/metabolismo , Masculino , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Adulto , Persona de Mediana Edad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Inmunoglobulinas/metabolismo , Inmunoglobulinas/inmunología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patologíaRESUMEN
We have previously performed preclinical studies with the oxidized mannan-conjugated peptide MOG35-55 (OM-MOG35-55) in vivo (EAE mouse model) and in vitro (human peripheral blood) and demonstrated that OM-MOG35-55 suppresses antigen-specific T cell responses associated with autoimmune demyelination. Based on these results, we developed different types of dendritic cells (DCs) from the peripheral blood monocytes of patients with multiple sclerosis (MS) or healthy controls presenting OM-MOG35-55 or MOG-35-55 to autologous T cells to investigate the tolerogenic potential of OM-MOG35-55 for its possible use in MS therapy. To this end, monocytes were differentiated into different DC types in the presence of IL-4+GM-CSF ± dexamethasone (DEXA) ± vitamin D3 (VITD3). At the end of their differentiation, the DCs were loaded with peptides and co-cultured with T cells +IL-2 for 4 antigen presentation cycles. The phenotypes of the DC and T cell populations were analyzed using flow cytometry and the secreted cytokines using flow cytometry or ELISA. On day 8, the monocytes had converted into DCs expressing the typical markers of mature or immature phenotypes. Co-culture of T cells with all DC types for 4 antigen presentation cycles resulted in an increase in memory CD4+ T cells compared to memory CD8+ T cells and a suppressive shift in secreted cytokines, mainly due to increased TGF-ß1 levels. The best tolerogenic effect was obtained when patient CD4+ T cells were co-cultured with VITD3-DCs presenting OM-MOG35-55, resulting in the highest levels of CD4+PD-1+ T cells and CD4+CD25+Foxp3+ Τ cells. In conclusion, the tolerance induction protocols presented in this work demonstrate that OM-MOG35-55 could form the basis for the development of personalized therapeutic vaccines or immunomodulatory treatments for MS.
Asunto(s)
Células Dendríticas , Tolerancia Inmunológica , Esclerosis Múltiple , Glicoproteína Mielina-Oligodendrócito , Humanos , Glicoproteína Mielina-Oligodendrócito/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Esclerosis Múltiple/tratamiento farmacológico , Tolerancia Inmunológica/efectos de los fármacos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Adulto , Femenino , Mananos/farmacología , Masculino , Diferenciación Celular/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Cultivadas , Persona de Mediana Edad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismoRESUMEN
Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a "near cure") with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Hormona Inhibidora de la Liberación de MSH , Factores Inhibidores de la Migración de Macrófagos , Esclerosis Múltiple , Animales , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Hormona Inhibidora de la Liberación de MSH/metabolismo , Hormona Inhibidora de la Liberación de MSH/uso terapéutico , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Médula EspinalRESUMEN
Multiple Sclerosis (MS) is a serious autoimmune disease. The patient in an advanced state of the disease has restrained mobility and remains handicapped. It is therefore understandable that there is a great need for novel drugs and vaccines for the treatment of MS. Herein we summarise two major approaches applied for the treatment of the disease using peptide molecules alone or conjugated with mannan. The first approach focuses on selective myelin epitope peptide or peptide mimetic therapy alone or conjugated with mannan, and the second on immune-therapy by preventing or controlling disease through the release of appropriate cytokines. In both approaches the use of cyclic peptides offers the advantage of increased stability from proteolytic enzymes. In these approaches, the synthesis of myelin epitope peptides conjugated to mannan is of particular interest as this was found to protect mice against experimental autoimmune encephalomyelitis, an animal model of MS, in prophylactic and therapeutic protocols. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation. The aim of the studies of these peptide epitope analogs is to understand their molecular basis of interactions with human autoimmune T-cell receptor and a MS-associated human leucocyte antigen (HLA)-DR2b. This knowledge will lead the rational design to new beneficial non-peptide mimetic analogs for the treatment of MS. Some issues of the use of nanotechnology will also be addressed as a future trend to tackle the disease. We highlight novel immunomodulation and vaccine-based research against MS based on myelin epitope peptides and strategies developed in our laboratories.
RESUMEN
Myelin peptide-mannan conjugates have been shown to be potential vaccines in the immunotherapy of multiple sclerosis. The conjugates are comprised from the epitope peptide and the polysaccharide mannan which transfers as a carrier the antigenic peptide to dendritic cells that process and present antigenic peptides at their surface in complex with MHC class I or class II resulting in T-cell stimulation. The conjugation of antigenic peptide with mannan occurs through the linker (Lys-Gly)5, which connects the peptide with the oxidized mannose units of mannan. This study describes novel methods for the quantification of the vaccine ingredient peptide within the conjugate, a prerequisite for approval of clinical trials in the pursuit of multiple sclerosis therapeutics. Myelin peptides, such as MOG35-55, MBP83-99, and PLP131-145 in linear or cyclic form, as altered peptide ligands or conjugated to appropriate carriers, possess immunomodulatory properties in experimental models and are potential candidates for clinical trials.
RESUMEN
Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood-brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfbret/ret ), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfbret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti-ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfbret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfbret/ret ;2D2tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder.
Asunto(s)
Barrera Hematoencefálica/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Homeostasis/inmunología , Leucocitos/inmunología , Pericitos/inmunología , Animales , Barrera Hematoencefálica/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Homeostasis/genética , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Leucocitos/patología , Ratones , Ratones Transgénicos , Pericitos/patología , Proteínas Proto-Oncogénicas c-sis/deficiencia , Proteínas Proto-Oncogénicas c-sis/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Multiple sclerosis (MS) is considered a chronic disease of the central nervous system, with a strong neurodegenerative component. The exact mechanism of MS is not clear. However, the therapeutic strategies for controlling MS are based on immune modulation and inflammation control. Regarding this, the present study was conducted to investigate the influence of snake venom on the suppression of the immune system after the induction of experimental autoimmune encephalomyelitis (EAE) in mice. For this purpose, C57BL/6 female mice, divided into three groups, were selected to be induced by EAE. Groups 2 and 3 received flank injection with the emulsion of myelin oligodendrocyte glycoprotein (MOG 35-55), as well as complete Freund adjuvant, followed by the administration of pertussis toxin. Furthermore, the treatment group, as an immune-modulator, received cobra venom (CV) after EAE induction. The mice were then evaluated daily based on clinical symptoms, weight changes (within 26 days), histopathological analysis, and serum levels of interleukin 27 (IL-27) for neurological motor deficits. The clinical signs of MOG-EAE in C57BL/6 mice began 9-14 days post-immunization. Histopathological results also revealed that CV-treated EAE mice, compared to the untreated EAE group, witnessed a significant reduction in the intensity of inflammatory cells in parenchymal sections. Furthermore, the increase of IL-27 levels was significant in the CV-treated group (P=0.001), compared with those in the EAE and control groups. Based on results obtained in the present study, it may be concluded that Naja naja oxiana snake venom is a potential immunomodulatory agent that can be effective in the treatment of MS.
Asunto(s)
Antiinflamatorios/farmacología , Venenos Elapídicos/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Interleucinas/sangre , Esclerosis Múltiple/tratamiento farmacológico , Naja naja , Animales , Antiinflamatorios/administración & dosificación , Venenos Elapídicos/administración & dosificación , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito/farmacologíaRESUMEN
The impact of immunization with Anisakis simplex larval antigen on the occurrence and progression of experimental autoimmune encephalomyelitis (EAE) induced in mice was studied. C57BL/6J mice were immunized with the MOG35-55 peptide and one batch was treated with A. simplex total larval antigen on days 1, 8, 10 and 12 after EAE induction. Significantly higher values were obtained in the EAE clinical parameters of the antigen-treated group. Likewise, there was a significant decrease in the weights of the animals. Anisakis-treatment produced a significant decrease in anti-MOG35-55 specific IgG1 on day 21. On day 14 there was an increase in serum IL-2, IL-6, IL-10, IL-17A, and TGF-ß in the treated group. On day 21, a decrease in IL-4, IL-6, TNF-α, TGF-ß was observed. All brain determinations were made on day 21. The treatment decreased values of IL-6, IL-10, IL-17A and TNF-α. A. simplex antigen caused a significantly higher incidence of EAE and an advance in the appearance of the disease manifestations. However, treatment with the antigen was able to cause a decrease in proinflammatory cytokines (IL-6, IL-17A, and TNF-α) in nervous tissue that could establish a future preventive scenario for myelin damage.
Asunto(s)
Anisakis/inmunología , Antiinflamatorios no Esteroideos/uso terapéutico , Antígenos/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Animales , Peso Corporal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Citocinas/sangre , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Inmunoglobulina G/inmunología , Larva/inmunología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Recent studies have shown the ability of electrochemical methods to sense and determine, even at very low concentrations, the presence and quantity of molecules or analytes including pharmaceutical samples. Furthermore, analytical methods, such as high-pressure liquid chromatography (HPLC), can also detect the presence and quantity of peptides at very low concentrations, in a simple, fast, and efficient way, which allows the monitoring of conjugation reactions and its completion. Graphite/SiO2 film electrodes and HPLC methods were previously shown by our group to be efficient to detect drug molecules, such as losartan. We now use these methods to detect the conjugation efficiency of a peptide from the immunogenic region of myelin oligodendrocyte to a carrier, mannan. The HPLC method furthermore confirms the stability of the peptide with time in a simple one pot procedure. Our study provides a general method to monitor, sense and detect the presence of peptides by effectively confirming the conjugation efficiency. Such methods can be used when designing conjugates as potential immunotherapeutics in the treatment of diseases, including multiple sclerosis.
RESUMEN
The aim of this study was to investigate potential therapeutic effects of IFN-γ primed human umbilical cord mesenchymal stem cell (IFN-γ-hUCMSCs) transplantation on experimental autoimmune encephalomyelitis (EAE) in mice. In this study, EAE mouse model was established by MOG35-55 immunization method. Outcomes of the EAE mice in terms of body weight and clinical symptoms were analyzed. Electromyography (EMG) was performed to evaluate nerve conduction. ELISA was applied to quantify inflammatory cytokine levels in serum. Our results showed that IFN-γ could up-regulate protein expression of indoleamine 2, 3-dioxygenease 1 (IDO1), an important molecule released by MSCs to exert their immune suppressive activity (p < 0.01). In this study treatment efficacy for EAE was compared between transplantation of hUCMSCs alone and the IFN-γ-hUCMSCs which were cultured in the presence of IFN-γ for 48 h prior to be harvested for transplantation. Compared with hUCMSCs alone and control (PBS transfusion) group, transplantation of the IFN-γ-hUCMSCs could significantly alleviate the body weight loss and clinical symptoms of EAE mice (p < 0.05). Consistently EMG latency was significantly improved in treatment groups (p < 0.001), and the IFN-γ-hUCMSCs group was even better than the hUCMSCs group (p < 0.05). Moreover, the concentrations of IL-17A and TNF-α in serum of the mice treated by IFN-γ-hUCMSCs were significantly lower than hUCMSCs alone and controls, respectively (p < 0.05). In few of the roles of IL-17A and TNF-α in the pathogenesis of EAE, IFN-γ-hUCMSCs treatment associated-suppression of IL-17A and TNF-α expression may contribute in part to their therapeutic effects on EAE. In sum, our study highlights a great clinical potential of IFN-γ-hUCMSCs for multiple sclerosis (MS) treatment.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Encefalomielitis Autoinmune Experimental/terapia , Interferón gamma/administración & dosificación , Trasplante de Células Madre Mesenquimatosas/métodos , Animales , Células Cultivadas , Trasplante de Células Madre de Sangre del Cordón Umbilical/tendencias , Encefalomielitis Autoinmune Experimental/fisiopatología , Potenciales Evocados Motores/efectos de los fármacos , Potenciales Evocados Motores/fisiología , Femenino , Humanos , Trasplante de Células Madre Mesenquimatosas/tendencias , Ratones , Ratones Endogámicos C57BL , Resultado del Tratamiento , Cordón Umbilical/citología , Cordón Umbilical/fisiología , Cordón Umbilical/trasplanteRESUMEN
BACKGROUND: Interleukin-6 (IL-6) is a pleiotropic and multifunctional cytokine that plays a critical role in induction of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Although EAE has always been considered a peripherally elicited disease, Il6 expression exclusively within central nervous system is sufficient to induce EAE development. Neurons, astrocytes, and microglia can secrete and respond to IL-6. METHODS: To dissect the relevance of each cell source for establishing EAE, we generated and immunized conditional Il6 knockout mice for each of these cell types with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide dissolved in complete Freund's adjuvant (CFA) and supplemented with Mycobacterium tuberculosis. RESULTS AND CONCLUSIONS: The combined results reveal a minor role for Il6 expression in both astrocytes and microglia for symptomatology and neuropathology of EAE, whereas neuronal Il6 expression was not relevant for the variables analyzed.
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Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Interleucina-6/metabolismo , Animales , Receptor 1 de Quimiocinas CX3C/metabolismo , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/genética , Femenino , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Inflamación/genética , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/metabolismo , Médula Espinal/patologíaRESUMEN
Central neuropathic pain is the main symptom caused by spinal cord lesion in relapsing-remitting multiple sclerosis (RRMS), but its management is still not effective. The transient receptor potential ankyrin 1 (TRPA1) is a pain detecting ion channel involved in neuropathic pain development. Thus, the aim of our study was to evaluate the role of TRPA1 in central neuropathic nociception induced by relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) mouse model. In this model, we observed the development of similar clinical conditions of RRMS in C57BL/6 female mice through RR-EAE using MOG35-55 antigen and Quil A adjuvant. At the thirty-fifth day post-induction, C57BL/6 female mice demonstrated alteration in the RR-EAE score without motor impairment, mechanical and cold allodynia. Also, significative changes in demyelinating (Mog and olig-1) and neuroinflammatory (Iba1, Gfap and Tnfa) markers were observed, but this model did not alter Trpa1 RNA expression levels in the spinal cord. The hydrogen peroxide and 4-hydroxynonenal levels (TRPA1 agonists) were increased in RR-EAE induced mice, as well as the NADPH oxidase activity. The intragastric treatment of RR-EAE induced mice with TRPA1 antagonists (HC-030031 and A-967079) and antioxidant (α-lipoic acid and apocynin) caused an antiallodynic effect. Moreover, the intrathecal administration of TRPA1 antisense oligonucleotide, HC-030031, α-lipoic acid, and apocynin transiently attenuated mechanical and cold allodynia. Thus, TRPA1 plays a key role in the induction of neuropathic pain in this model of RR-EAE and can be a possible target for investigating the development of pain in RRMS patients.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Nocicepción/fisiología , Canal Catiónico TRPA1/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/complicaciones , Femenino , Hiperalgesia/etiología , Ratones , Ratones Endogámicos C57BL , Neuralgia/etiologíaRESUMEN
Multiple sclerosis (MS) is an autoimmune disease that involves demyelination of axons in the central nervous system (CNS) and affects patients worldwide. It has been demonstrated that ligand-activated aryl hydrocarbon receptor (Ahr) ameliorates experimental autoimmune encephalomyelitis (EAE), a murine model of MS, by increasing CD4+FoxP3+ T cells. Recent evidence indicates that AT-rich interactive domain-containing protein 5a (Arid5a) is required for EAE pathogenesis by stabilizing Il6 and OX40 mRNAs. However, the differential modulation of Ahr and Arid5a in autoimmunity as a therapeutic strategy is unexplored. Herein, an in silico, in vitro and in vivo approach identified Flavipin (3,4,5-trihydroxy-6-methylphthalaldehyde) as an Ahr agonist that induces the expression of Ahr downstream genes in mouse CD4+ T cells and CD11b+ macrophages. Interestingly, Flavipin inhibited the stabilizing function of Arid5a and its counteracting effects on Regnase-1 on the 3' untranslated region (3'UTR) of target mRNAs. Furthermore, it inhibited the stabilizing function of Arid5a on Il23a 3'UTR, a newly identified target mRNA. In EAE, Flavipin ameliorated disease severity, with reduced CD4+IL-17+ T cells, IL-6 and TNF-α and increased CD4+FoxP3+ T cells. Moreover, EAE amelioration was concomitant with reduced CD4+OX40+ and CD4+CD45+ T cells in the CNS. RNA interference showed that the modulatory effects of Flavipin on pro- and anti-inflammatory mediators in CD4+ T cells and macrophages were Ahr- and/or Arid5a-dependent. In conclusion, our findings reveal differential modulation of Ahr and Arid5a as a new therapeutic strategy for MS.
RESUMEN
Multiple sclerosis (MS) is the common demyelinating disease of human central nervous system. Among mouse models available to study MS, including the cuprizone application and lysolecithin-injection models, experimental autoimmune encephalomyelitis (EAE) model is widely used so that chronic EAE model of C57BL/6J can reflect the autoimmune pathogenesis of MS well. Here we introduce the EAE model based on C57BL/6J mice, which is generated by injection of myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) as an antigen. After immunization with complete Freund's adjuvant, clinical signs and changes in body weight are observed one or two weeks later. The EAE model will continue to be useful for development of therapeutics for MS.
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Multiple sclerosis (MS) is a debilitating autoimmune disease affecting over 2.3 million people worldwide, and it is characterized by inflammation and demyelination of nerve cells. The currently available biomarkers for the diagnosis and management of MS have inherent limitations, therefore, additional new biomarkers are needed. We studied the microRNA (miRNA) profile of splenocytes of mice having experimental autoimmune encephalomyelitis (EAE), a model of human MS. A miRNA-microarray analysis revealed increased expression of nine miRNAs (let-7e, miR-23b, miR-31, miR-99b, miR-125a, miR-146b, miR-155, miR-193b, and miR-221) following EAE development. Interestingly, serum levels of miR-99b, miR-125a, and miR-146b were significantly higher in EAE mice compared to normal mice. Bioinformatics analysis revealed the experimentally validated as well as predicted gene targets of specific miRNAs that are important for disease progression in MS. Specifically, we observed inverse correlation in the levels of miR-99b versus LIF, and between miR-125a versus BDNF and LIF. Our results suggest that above-mentioned miRNAs may play a crucial role in the pathogenesis of MS, and that miR-99b, miR-125a, and miR-146b in particular may serve as useful biomarkers for disease activity.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is one of the most popular animal models of multiple sclerosis (MS). There are a number of EAE models, being actively induced EAE in strains such as C57Bl/6 mice very robust and reproducible. We herewith present details of the materials and methods for active EAE. Mice are immunized with an emulsion of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) + complete Freund's adjuvant (supplemented with Mycobacterium tuberculosis), and treated with Bordetella pertussis toxin, to induce EAE. Sham-EAE mice are immunized with bovine serum albumin instead of MOG35-55.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/diagnóstico , Inmunización , Ratones , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/química , Fragmentos de Péptidos/química , Toxina del Pertussis/efectos adversos , Toxina del Pertussis/inmunología , Fenotipo , Médula Espinal/metabolismo , Evaluación de SíntomasRESUMEN
Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme that produces prostaglandin E2 (PGE2). In our previous study, we investigated the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, using mPGES-1-deficient (mPGES-1-/-) and wild-type (wt) mice. We found that mPGES-1 facilitated inflammation, demyelination, and paralysis and was induced in vascular endothelial cells and macrophages and microglia around inflammatory foci. Here, we investigated the role of interleukin-1ß (IL-1ß) in the intercellular mechanism stimulated by mPGES-1 in EAE spinal cords in the presence of inflammation. We found that the area invaded by CD4-positive (CD4âº) T cells was extensive, and that PGE2 receptors EP1-4 were more induced in activated CD4⺠T cells of wt mice than in those of mPGES-1-/- mice. Moreover, IL-1ß and IL-1 receptor 1 (IL-1r1) were produced by 65% and 48% of CD4⺠T cells in wt mice and by 44% and 27% of CD4⺠T cells in mPGES-1-/- mice. Furthermore, interleukin-17 (IL-17) was released from the activated CD4⺠T cells. Therefore, mPGES-1 stimulates an intercellular interaction between CD4⺠T cells by upregulating the autocrine function of IL-1ß in activated CD4⺠T cells, which release IL-17 to facilitate axonal and myelin damage in EAE mice.
Asunto(s)
Comunicación Autocrina , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Prostaglandina-E Sintasas/metabolismo , Animales , Femenino , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Prostaglandina-E Sintasas/genética , Receptores Tipo I de Interleucina-1/metabolismoRESUMEN
BACKGROUND: While many groups use experimental autoimmune encephalomyelitis (EAE) as a model to uncover therapeutic targets and understand the pathology underlying multiple sclerosis (MS), EAE protocol variability introduces discrepancies in central nervous system (CNS) pathogenesis and clinical disease, limiting the comparability between studies and slowing much-needed translational research. OPTIMIZED METHOD: Here we describe a detailed, reliable protocol for chronic EAE induction in C57BL/6 mice utilizing two injections of myelin oligodendrocyte glycoprotein (35-55) peptide mixed with complete Freund's adjuvant and paired with pertussis toxin. RESULTS: The active MOG35-55-EAE protocol presented here induces ascending paralysis in 80-100% of immunized mice. We observe: (1) consistent T cell immune activation, (2) robust CNS infiltration by peripheral immune cells, and (3) perivascular demyelinating lesions concurrent with axon damage in the spinal cord and various brain regions, including the optic nerve, cortex, hippocampus, internal capsule, and cerebellum. COMPARISON WITH EXISTING METHOD(S): Lack of detailed protocols, combined with variability between laboratories, make EAE results difficult to compare and hinder the use of this model for therapeutic development. We provide the most detailed active MOG35-55-EAE protocol to date. With this protocol, we observe high disease incidence and a consistent, reliable disease course. The resulting pathology is MS-like and includes optic neuritis, perivascular mononuclear infiltration, CNS axon demyelination, and axon damage in both infiltrating lesions and otherwise normal-appearing white matter. CONCLUSIONS: By providing a detailed active MOG35-55-EAE protocol that yields consistent and robust pathology, we aim to foster comparability between pre-clinical studies and facilitate the discovery of MS therapeutics.
Asunto(s)
Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Adyuvante de Freund , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito , Animales , Combinación de Medicamentos , Encefalomielitis Autoinmune Experimental/inducido químicamente , Femenino , Humanos , Estudios Longitudinales , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Fragmentos de Péptidos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Traumatic brain injury (TBI) results in severe neurological impairments without effective treatments. Inflammation appears to be an important contributor to key pathogenic events such as secondary brain injury following TBI and therefore serves as a promising target for novel therapies. We have recently demonstrated the ability of a molecular construct comprised of the human leukocyte antigen (HLA)-DRα1 domain linked covalently to mouse (m)MOG-35-55 peptide (DRα1-MOG-35-55 construct) to reduce CNS inflammation and tissue injury in animal models of multiple sclerosis and ischemic stroke. The aim of the current study was to determine if DRα1-MOG-35-55 treatment of a fluid percussion injury (FPI) mouse model of TBI could reduce the lesion size and improve disease outcome measures. Neurodeficits, lesion size, and immune responses were determined to evaluate the therapeutic potential and mechanisms of neuroprotection induced by DRα1-MOG-35-55 treatment. The results demonstrated that daily injections of DRα1-MOG-35-55 given after FPI significantly reduced numbers of infiltrating CD74+ and CD86+ macrophages and increased numbers of CD206+ microglia in the brain concomitant with smaller lesion sizes and improvement in neurodeficits. Conversely, DRα1-MOG-35-55 treatment of TBI increased numbers of circulating CD11b+ monocytes and their expression of CD74 but had no detectable effect on cell numbers or marker expression in the spleen. These results demonstrate that DRα1-MOG-35-55 therapy can reduce CNS inflammation and significantly improve histological and clinical outcomes after TBI. Future studies will further examine the potential of DRα1-MOG-35-55 for treatment of TBI.