RESUMO
Formyl peptide receptor 2 (FPR2) is a receptor for formylated peptides and specific pro-resolving mediators, and is involved in various inflammatory processes. Here, we aimed to elucidate the role of FPR2 in dendritic cell (DC) function and autoimmunity-related central nervous system (CNS) inflammation by using the experimental autoimmune encephalomyelitis (EAE) model. EAE induction was accompanied by increased Fpr2 mRNA expression in the spinal cord. FPR2-deficient (Fpr2 KO) mice displayed delayed onset of EAE compared to wild-type (WT) mice, associated with reduced frequencies of Th17 cells in the inflamed spinal cord at the early stage of the disease. However, FPR2 deficiency did not affect EAE severity after the disease reached its peak. FPR2 deficiency in mature DCs resulted in decreased expression of Th17 polarizing cytokines IL6, IL23p19, IL1ß, and thereby diminished the DC-mediated activation of Th17 cell differentiation. LPS-activated FPR2-deficient DCs showed upregulated Nos2 expression and nitric oxide (NO) production, as well as reduced oxygen consumption rate and impaired mitochondrial function, including decreased mitochondrial superoxide levels, lower mitochondrial membrane potential and diminished expression of genes related to the tricarboxylic acid cycle and genes related to the electron transport chain, as compared to WT DCs. Treatment with a NO inhibitor reversed the reduced Th17 cell differentiation in the presence of FPR2-deficient DCs. Together, by regulating DC metabolism, FPR2 enhances the production of DC-derived Th17-polarizing cytokines and hence Th17 cell differentiation in the context of neuroinflammation.
Assuntos
Diferenciação Celular , Células Dendríticas , Encefalomielite Autoimune Experimental , Camundongos Knockout , Receptores de Formil Peptídeo , Células Th17 , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Camundongos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/metabolismo , Feminino , Medula Espinal/imunologia , Medula Espinal/metabolismoRESUMO
Neuroinflammation and neurodegeneration are hallmarks of multiple sclerosis (MS). Bromodomain-containing protein 4 (BRD4), a bromodomain and extra-terminal domain (BET) protein family member, is indispensable for the transcription of pro-inflammatory genes. Therefore, inhibiting BRD4 may be a prospective therapeutic approach for modulating the inflammatory response and regulating the course of MS. dBET1, a newly synthesized proteolysis-targeting chimera (PROTAC), exhibits effectively degrades of BRD4. However, the precise effects of dBET1 on MS require further investigation. Therefore, we assessed the effect of dBET1 in experimental autoimmune encephalomyelitis (EAE), a typical MS experimental model. Our findings revealed that BRD4 is mainly expressed in astrocytes and neurons of the spinal cords, and is up-regulated in the spinal cords of EAE mice. The dBET1 attenuated lipopolysaccharide-induced expression of astrocytic pro-inflammatory mediators and inhibited deleterious molecular activity in astrocytes. Correspondingly, dBET1, used in preventive and therapeutic settings, alleviated the behavioral symptoms in EAE mice, as demonstrated by decreased demyelination, alleviated leukocyte infiltration, reduced microglial and astrocyte activation, and diminished inflammatory mediator levels. In addition, dBET1 corrected the imbalance in peripheral T cells and protected blood-brain barrier integrity in EAE mice. The underlying mechanism involved suppressing the phosphoinositide-3-kinase/protein kinase B, mitogen-activated protein kinase /extracellular signal-regulated kinase, and nuclear factor kappa B pathways. In summary, our data strongly suggests that dBET1 is a promising treatment option for MS.
Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Medula Espinal , Fatores de Transcrição , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/imunologia , Feminino , Esclerose Múltipla/tratamento farmacológico , Proteólise/efeitos dos fármacos , Humanos , Lipopolissacarídeos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Células Cultivadas , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Proteínas que Contêm BromodomínioRESUMO
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
Proinflammatory T-lymphocytes recruited into the brain and spinal cord mediate multiple sclerosis (MS) and currently there is no cure for MS. IFN-γ-producing Th1 cells induce ascending paralysis in the spinal cord while IL-17-producing Th17 cells mediate cerebellar ataxia. STAT1 and STAT3 are required for Th1 and Th17 development, respectively, and the simultaneous targeting of STAT1 and STAT3 pathways is therefore a potential therapeutic strategy for suppressing disease in the spinal cord and brain. However, the pharmacological targeting of STAT1 and STAT3 presents significant challenges because of their intracellular localization. We have developed a STAT-specific single-domain nanobody (SBT-100) derived from camelids that targets conserved residues in Src homolog 2 (SH2) domains of STAT1 and STAT3. This study investigated whether SBT-100 could suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show that SBT-100 ameliorates encephalomyelitis through suppressing the expansion of Th17 and Th1 cells in the brain and spinal cord. Adoptive transfer experiments revealed that lymphocytes from SBT-100-treated EAE mice have reduced capacity to induce EAE, indicating that the immunosuppressive effects derived from the direct suppression of encephalitogenic T-cells. The small size of SBT-100 makes this STAT-specific nanobody a promising immunotherapy for CNS autoimmune diseases, including multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental , Anticorpos de Domínio Único , Células Th17 , Animais , Feminino , Camundongos , Camelídeos Americanos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/uso terapêutico , Medula Espinal/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Th1/imunologia , Células Th1/efeitos dos fármacos , Células Th17/imunologia , Células Th17/efeitos dos fármacosRESUMO
Spinal cord injury triggers a strong innate inflammatory response in both non-regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet, their role in the repair process, particularly in a regenerative context, remains largely unknown. Here, we show that, following rapid recruitment to the injured spinal cord, neutrophils mostly reverse migrate throughout the zebrafish body. In addition, promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil-specific RNA-seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf-α expression in macrophage/microglia populations. Conversely, blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether, these findings provide new insights into the role of neutrophils in spinal cord regeneration, emphasizing the significant impact of their immune profile on the outcome of the repair process.
Assuntos
Neutrófilos , Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Medula Espinal , Peixe-Zebra , Animais , Neutrófilos/metabolismo , Neutrófilos/imunologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/metabolismo , Regeneração da Medula Espinal/fisiologia , Medula Espinal/imunologia , Medula Espinal/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Microglia/metabolismo , Microglia/imunologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Receptores CXCR4/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Infiltração de Neutrófilos/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
T cells play an important role in the acquired immune response, with regulatory T cells (Tregs) serving as key players in immune tolerance. Tregs are found in nonlymphoid and damaged tissues and are referred to as "tissue Tregs". They have tissue-specific characteristics and contribute to immunomodulation, homeostasis, and tissue repair through interactions with tissue cells. However, important determinants of Treg tissue specificity, such as antigen specificity, tissue environment, and pathology, remain unclear. In this study, we analyzed Tregs in the central nervous system of mice with ischemic stroke and experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. The gene expression pattern of brain Tregs in the EAE model was more similar to that of ischemic stroke Tregs in the brain than to that of spinal cord Tregs. In addition, most T-cell receptors (TCRs) with high clonality were present in both the brain and spinal cord. Furthermore, Gata3+ and Rorc+ Tregs expressed TCRs recognizing MOG in the spinal cord, suggesting a tissue environment conducive to Rorc expression. Tissue-specific chemokine/chemokine receptor interactions in the spinal cord and brain influenced Treg localization. Finally, spinal cord- or brain-derived Tregs had greater anti-inflammatory capacities in EAE mice, respectively. Taken together, these findings suggest that the tissue environment, rather than pathogenesis or antigen specificity, is the primary determinant of the tissue-specific properties of Tregs. These findings may contribute to the development of novel therapies to suppress inflammation through tissue-specific Treg regulation.
Assuntos
Encéfalo , Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Medula Espinal , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Camundongos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Medula Espinal/imunologia , Medula Espinal/patologia , Medula Espinal/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Modelos Animais de DoençasRESUMO
Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein-Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.
Assuntos
Encéfalo , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Cadeias HLA-DRB1 , Esclerose Múltipla , Medula Espinal , Animais , Humanos , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , Camundongos , Cadeias HLA-DRB1/genética , Linfócitos T CD8-Positivos/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Encéfalo/patologia , Encéfalo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Linfócitos T CD4-Positivos/imunologia , FemininoRESUMO
BACKGROUND: Pediatric acute transverse myelitis (ATM) accounts for 20-30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease. METHODS: We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response. RESULTS: We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis. CONCLUSIONS: Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.
Assuntos
Astrócitos , Mielite Transversa , Humanos , Mielite Transversa/imunologia , Animais , Feminino , Astrócitos/metabolismo , Astrócitos/imunologia , Criança , Camundongos , Masculino , Adolescente , Plasmócitos/imunologia , Plasmócitos/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/sangue , Camundongos Endogâmicos C57BL , Células Cultivadas , Pré-Escolar , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Medula Espinal/metabolismo , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20 ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration.
Assuntos
Animais Recém-Nascidos , Proliferação de Células , Modelos Animais de Doenças , Ratos Wistar , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Proliferação de Células/fisiologia , Ratos , Medula Espinal/patologia , Medula Espinal/imunologia , Astrócitos/patologia , FemininoRESUMO
BACKGROUND: A wide array of histone deacetylase (HDAC) inhibitors and aryl hydrocarbon receptor (AHR) agonists commonly arrest experimental autoimmune encephalomyelitis (EAE). However, it is not known whether HDAC inhibition is linked to the AHR signaling pathway in EAE. METHODS: We investigated how the pan-HDAC inhibitor SB939 (pracinostat) exerted immunoregulatory action in the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE mouse model by evaluating changes in of signal transducer and activator of transcription 3 (STAT3) acetylation and the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and AHR in inflamed spinal cords during EAE evolution. We proved the involvement of IDO1 and the AHR in SB939-mediated immunosuppression using Ido1-/- and Ahr-/- mice. RESULTS: Administration with SB939 halted EAE progression, which depended upon IDO1 expression in neurons of the central nervous system (CNS). Our in vitro and in vivo studies demonstrated that SB939 sustained the interleukin-6-induced acetylation of STAT3, resulting in the stable transcriptional activation of Ido1. The therapeutic effect of SB939 also required the AHR, which is expressed mainly in CD4+ T cells and macrophages in CNS disease lesions. Finally, SB939 was shown to markedly reduce the proliferation of CD4+ T cells in inflamed neuronal tissues but not in the spleen or draining lymph nodes. CONCLUSIONS: Overall, our results suggest that IDO1 tryptophan metabolites produced by neuronal cells may act on AHR in pathogenic CD4+ T cells in a paracrine fashion in the CNS and that the specific induction of IDO1 expression in neurons at disease-afflicted sites can be considered a therapeutic approach to block the progression of multiple sclerosis without affecting systemic immunity.
Assuntos
Encefalomielite Autoimune Experimental , Inibidores de Histona Desacetilases , Indolamina-Pirrol 2,3,-Dioxigenase , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios , Fator de Transcrição STAT3 , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Feminino , Medula Espinal/patologia , Medula Espinal/metabolismo , Medula Espinal/imunologia , Medula Espinal/efeitos dos fármacos , Glicoproteína Mielina-Oligodendrócito/imunologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Progressão da Doença , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Fragmentos de Peptídeos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Interleucina-6/metabolismo , Interleucina-6/genéticaRESUMO
Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1-G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age-matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.
Assuntos
Esclerose Lateral Amiotrófica , Camundongos Transgênicos , Microglia , Proteoma , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/genética , Microglia/metabolismo , Microglia/imunologia , Animais , Proteoma/metabolismo , Camundongos , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/imunologia , Modelos Animais de Doenças , Fagocitose/fisiologia , Humanos , Feminino , Camundongos Endogâmicos C57BL , MasculinoRESUMO
In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), early pathological features include immune cell infiltration into the central nervous system (CNS) and blood-brain barrier (BBB) disruption. We investigated the role of junctional adhesion molecule-A (JAM-A), a tight junction protein, in active EAE (aEAE) pathogenesis. Our study confirms JAM-A expression at the blood-brain barrier and its luminal redistribution during aEAE. JAM-A deficient (JAM-A-/-) C57BL/6J mice exhibited milder aEAE, unrelated to myelin oligodendrocyte glycoprotein-specific CD4+ T-cell priming. While JAM-A absence influenced macrophage behavior on primary mouse brain microvascular endothelial cells (pMBMECs) under flow in vitro, it did not impact T-cell extravasation across primary mouse brain microvascular endothelial cells. At aEAE onset, we observed reduced lymphocyte and CCR2+ macrophage infiltration into the spinal cord of JAM-A-/- mice compared to control littermates. This correlated with increased CD3+ T-cell accumulation in spinal cord perivascular spaces and brain leptomeninges, suggesting JAM-A absence leads to T-cell trapping in central nervous system border compartments. In summary, JAM-A plays a role in immune cell infiltration and clinical disease progression in aEAE.
Assuntos
Barreira Hematoencefálica , Encefalomielite Autoimune Experimental , Células Endoteliais , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Encefalomielite Autoimune Experimental/imunologia , Camundongos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , Medula Espinal/patologia , Medula Espinal/imunologia , Medula Espinal/metabolismo , Linfócitos T CD4-Positivos/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: Spinal cord injury (SCI) is a traumatic neurological disorder with limited therapeutic options. Tumor protein p53-inducible nuclear protein 2 (TP53INP2) is involved in the occurrence and development of various diseases, and it may play a role during SCI via affecting inflammation and neuronal apoptosis. This study investigated the associated roles and mechanisms of TP53INP2 in SCI. METHODS: Mouse and lipopolysaccharide (LPS)-induced SCI BV-2 cell models were constructed to explore the role of TP53INP2 in SCI and the associated mechanisms. Histopathological evaluation of spinal cord tissue was detected by hematoxylin and eosin staining. The Basso, Beattie, and Bresnahan score was used to measure the motor function of the mice, while the spinal cord water content was used to assess spinal cord edema. The expression of TP53INP2 was measured using RT-qPCR. In addition, inflammatory factors in the spinal cord tissue of SCI mice and LPS-treated BV-2 cells were measured using enzyme-linked immunosorbent assay. Apoptosis and related protein expression levels were detected by flow cytometry and western blot analysis, respectively. RESULTS: TP53INP2 levels increased in SCI mice and LPS-treated BV-2 cells. The results of in vivo and in vitro experiments showed that TP53INP2 knockdown inhibited the inflammatory response and neuronal apoptosis in mouse spinal cord tissue or LPS-induced BV-2 cells. CONCLUSIONS: After spinal cord injury, TP53INP2 was upregulated, and TP53INP2 knockdown inhibited the inflammatory response and apoptosis.
Assuntos
Apoptose , Inflamação , Traumatismos da Medula Espinal , Animais , Masculino , Camundongos , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Inflamação/patologia , Inflamação/metabolismo , Inflamação/genética , Inflamação/imunologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Medula Espinal/patologia , Medula Espinal/metabolismo , Medula Espinal/imunologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/genéticaRESUMO
We have recently characterized experimental autoimmune encephalomyelitis (EAE) induced in DA rats with spinal cord homogenate without complete Freund's adjuvant (CFA). The main advantage of this multiple sclerosis model is the lack of CFA-related confounding effects which represent the major obstacles in translating findings from EAE to multiple sclerosis. Here, antigen specificity of the cellular and humoral immune response directed against the central nervous system was explored. The reactivity of T and B cells to myelin basic protein, myelin oligodendrocyte glycoprotein, and ß-synuclein was detected. Having in mind that reactivity against ß-synuclein was previously associated with autoimmunity against the brain, the infiltration of immune cells into different brain compartments, i.e. pons, cerebellum, hippocampus, and cortex was determined. T cell infiltration was observed in all structures examined. This finding stimulated investigation of the effects of immunization on DA rat behavior using the elevated plus maze and the open field test. Rats recovered from EAE displayed increased anxiety-like behavior. These data support CFA-free EAE in DA rats as a useful model for multiple sclerosis research.
Assuntos
Encefalomielite Autoimune Experimental , Medula Espinal , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Ratos , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Modelos Animais de Doenças , Glicoproteína Mielina-Oligodendrócito/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteína Básica da Mielina/imunologia , Proteína Básica da Mielina/metabolismo , Encéfalo/patologia , Encéfalo/imunologia , Encéfalo/metabolismo , Feminino , Encefalite/imunologia , Encefalite/etiologia , Encefalite/patologia , Encefalite/metabolismo , Adjuvante de Freund/imunologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/patologiaRESUMO
We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3- (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1ß, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.
Assuntos
Encefalomielite Autoimune Experimental , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Ratos , Resposta a Proteínas não Dobradas/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Modelos Animais de Doenças , Feminino , Citocinas/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígeno Sialil Lewis X , Antígeno Sialil Lewis X/análogos & derivados , Células Th17 , Animais , Encefalomielite Autoimune Experimental/imunologia , Camundongos , Células Th17/imunologia , Antígeno Sialil Lewis X/metabolismo , Polissacarídeos/metabolismo , Interleucina-17/metabolismo , Interleucina-17/imunologia , Oligossacarídeos , Carboidrato Sulfotransferases , Células Th1/imunologia , Sulfotransferases/metabolismo , Sulfotransferases/genética , Sulfotransferases/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Feminino , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Movimento Celular/imunologiaRESUMO
The concept of immune privilege suggests that the central nervous system is isolated from the immune system. However, recent studies have highlighted the borders of the central nervous system as central sites of neuro-immune interactions. Although the nervous and immune systems both function to maintain homeostasis, under rare circumstances, they can develop pathological interactions that lead to neurological or psychiatric diseases. Here we discuss recent findings that dissect the key anatomical, cellular and molecular mechanisms that enable neuro-immune responses at the borders of the brain and spinal cord and the implications of these interactions for diseases of the central nervous system.
Assuntos
Encéfalo , Sistema Imunitário , Neuroimunomodulação , Encéfalo/imunologia , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Sistema Imunitário/imunologia , Sistema Imunitário/fisiologia , Sistema Imunitário/fisiopatologia , Neuroimunomodulação/imunologia , Neuroimunomodulação/fisiologia , Medula Espinal/imunologia , Medula Espinal/fisiologia , Medula Espinal/fisiopatologia , Humanos , Doenças do Sistema Nervoso/imunologia , Doenças do Sistema Nervoso/fisiopatologia , Doenças do Sistema Nervoso/psicologiaRESUMO
Development of a novel, animal model for multiple sclerosis (MS) with reproducible and predictable lesion placement would enhance the discovery of effective treatments. Therefore, we would like to combine the advantages of the demyelination model with experimental autoimmune encephalomyelitis (EAE) to provide a local autoimmune encephalomyelitis (LAE) inside rat brain. We induced a demyelinating lesion by immunizing male Wistar rats, followed by blood-brain barrier opening protein (vascular endothelial growth factor) by stereotactic injection. We confirmed the immunization against myelin epitopes and minor neurological impairment. Histological assessment confirmed the lesion development after both 3- and 7 days post-injection. Our approach was sufficient to develop a demyelinating lesion with high reproducibility and low morbidity.
Assuntos
Encéfalo/patologia , Encefalomielite Autoimune Experimental/etiologia , Animais , Anticorpos/imunologia , Bovinos , Encefalomielite Autoimune Experimental/patologia , Injeções Intraventriculares , Masculino , Ratos , Ratos Wistar , Medula Espinal/imunologiaRESUMO
The aim of this study is to investigate the potential preventive and therapeutic effects of nobiletin by evaluating the expression of cytokines associated with inflammatory reactions in an autoimmune encephalomyelitis mouse model. A total of 60 male C57BL/6 mice aged between 8 and 10 weeks were used. Mice were divided into six groups (n = 10 mice per group): control, EAE, low-prophylaxis, high-prophylaxis, low-treatment and high-treatment. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG) and pertussis toxin. Nobiletin was administered in low (25 mg/kg) and high (50 mg/kg) doses, intraperitoneally. The prophylactic and therapeutic effects of nobiletin on brain tissue and spinal cord were evaluated by expression of interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), IL-6, IL-10 and transforming growth factor-beta (TGF-ß) using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). Prophylactic and therapeutic use of nobiletin inhibited EAE-induced increase of TNF-α, IL-1ß and IL-6 activities to alleviate inflammatory response in brain and spinal cord. Moreover, nobiletin supplement dramatically increased the IL-10, TGF-ß and IFNγ expressions in prophylaxis and treatment groups compared with the EAE group in the brain and spinal cord. The results obtained from this study show that prophylactic and therapeutic nobiletin modulates expressions of proinflammatory and antiinflammatory cytokines in brain and spinal cord dose-dependent manner in EAE model. These data demonstrates that nobiletin has a potential to attenuate inflammation in EAE mouse model. These experimental findings need to be supported by clinical studies.