RESUMO
Seasonal changes in disease activity have been observed in multiple sclerosis, an autoimmune disorder that affects the CNS. These epidemiological observations suggest that environmental factors influence the disease course. Here, we report that melatonin levels, whose production is modulated by seasonal variations in night length, negatively correlate with multiple sclerosis activity in humans. Treatment with melatonin ameliorates disease in an experimental model of multiple sclerosis and directly interferes with the differentiation of human and mouse T cells. Melatonin induces the expression of the repressor transcription factor Nfil3, blocking the differentiation of pathogenic Th17 cells and boosts the generation of protective Tr1 cells via Erk1/2 and the transactivation of the IL-10 promoter by ROR-α. These results suggest that melatonin is another example of how environmental-driven cues can impact T cell differentiation and have implications for autoimmune disorders such as multiple sclerosis.
Assuntos
Melatonina/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Luz , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Recidiva , Estações do Ano , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Inflammatory bowel disease (IBD) spans a range of chronic conditions affecting the gastrointestinal (GI) tract, which are marked by intermittent flare-ups and remissions. IBD results from microbial dysbiosis or a defective mucosal barrier in the gut that triggers an inappropriate immune response in a genetically susceptible person, altering the immune-microbiome axis. In this review, we discuss the regulatory roles of miRNAs, small noncoding RNAs with gene regulatory functions, in the stability and maintenance of the gut immune-microbiome axis, and detail the challenges and recent advances in the use of miRNAs as putative therapeutic agents for treating IBD.
Assuntos
Doenças Inflamatórias Intestinais , MicroRNAs , Humanos , MicroRNAs/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/terapia , Disbiose , Homeostase , Doença CrônicaRESUMO
Mouse Ly49+CD8+ regulatory T cells (Tregs) can subdue autoreactive CD4+ T cells to suppress autoimmunity. Recently, Li et al. demonstrated that killer-cell immunoglobulin-like receptor (KIR)+CD8+ T cells are the human equivalent of Ly49+CD8+ regulatory T cells and kill pathogenic CD4+ T cells, which can be increased in certain human autoimmune diseases and viral infections.
Assuntos
Doenças Autoimunes , Autoimunidade , Animais , Linfócitos T CD8-Positivos , Humanos , Camundongos , Receptores KIR , Linfócitos T ReguladoresRESUMO
IL-17-producing Th17 cells are implicated in the pathogenesis of rheumatoid arthritis (RA) and TNF-α, a proinflammatory cytokine in the rheumatoid joint, facilitates Th17 differentiation. Anti-TNF therapy ameliorates disease in many patients with rheumatoid arthritis (RA). However, a significant proportion of patients do not respond to this therapy. The impact of anti-TNF therapy on Th17 responses in RA is not well understood. We conducted high-throughput gene expression analysis of Th17-enriched CCR6+CXCR3-CD45RA- CD4+ T (CCR6+ T) cells isolated from anti-TNF-treated RA patients classified as responders or nonresponders to therapy. CCR6+ T cells from responders and nonresponders had distinct gene expression profiles. Proinflammatory signaling was elevated in the CCR6+ T cells of nonresponders, and pathogenic Th17 signature genes were up-regulated in these cells. Gene set enrichment analysis on these signature genes identified transcription factor USF2 as their upstream regulator, which was also increased in nonresponders. Importantly, short hairpin RNA targeting USF2 in pathogenic Th17 cells led to reduced expression of proinflammatory cytokines IL-17A, IFN-γ, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as transcription factor T-bet. Together, our results revealed inadequate suppression of Th17 responses by anti-TNF in nonresponders, and direct targeting of the USF2-signaling pathway may be a potential therapeutic approach in the anti-TNF refractory RA.
Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores Estimuladores Upstream/genética , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Biomarcadores , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , RNA Interferente Pequeno/genética , Receptores CCR6/metabolismo , Receptores CXCR3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The purpose of this study was to examine whether myeloid dendritic cells (mDCs) from patients with multiple sclerosis (MS) and healthy controls (HCs) become similarly tolerogenic when exposed to IL-27 as this may represent a potential mechanism of autoimmune dysregulation. Our study focused on natural mDCs that were isolated from HCs and MS patient peripheral blood mononuclear cells (PBMCs). After a 24-h treatment with IL-27 ± lipopolysaccharide (LPS), the mDCs were either harvested to identify IL-27-regulated gene expression or co-cultured with naive T-cells to measure how the treated DC affected T-cell proliferation and cytokine secretion. mDCs isolated from HCs but not untreated MS patients became functionally tolerogenic after IL-27 treatment. Although IL-27 induced both HC and untreated MS mDCs to produce similar amounts of IL-10, the tolerogenic HC mDCs expressed PD-L2, IDO1, and SOCS1, while the non-tolerogenic untreated MS mDCs expressed IDO1 and IL-6R. Cytokine and RNA analyses identified two signature blocks: the first identified genes associated with mDC tolerizing responses to IL-27, while the second was associated with the presence of MS. In contrast to mDCs from untreated MS patients, mDCs from HCs and IFNb-treated MS patients became tolerogenic in response to IL-27. The genes differentially expressed in the different donor IL-27-treated mDCs may contain targets that regulate mDC tolerogenic responses.
Assuntos
Interleucina-27 , Esclerose Múltipla , Humanos , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas , Interleucina-27/metabolismo , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Linfócitos T/metabolismoRESUMO
MicroRNAs are a class of evolutionarily conserved, short non-coding RNAs that post-transcriptionally modulate the expression of multiple target genes. They are implicated in almost every biological process, including pathways involved in immune homeostasis, such as immune cell development, central and peripheral tolerance, and T helper cell differentiation. Alterations in miRNA expression and function can lead to major dysfunction of the immune system and mediate susceptibility to autoimmune disease. Here, we discuss the role of miRNAs in the maintenance of immune tolerance to self-antigens and the gain or loss of miRNA functions on tissue inflammation and autoimmunity.
Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Animais , Autoantígenos/metabolismo , Autoimunidade/imunologia , Diferenciação Celular/genética , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental , Homeostase , Humanos , Sistema Imunitário , Tolerância Imunológica , Inflamação , Camundongos , MicroRNAs/genética , Esclerose Múltipla/imunologia , Células Th17/imunologiaRESUMO
OBJECTIVE: To investigate miR-155 in the SOD1 mouse model and human sporadic and familial amyotrophic lateral sclerosis (ALS). METHODS: NanoString microRNA, microglia and immune gene profiles, protein mass spectrometry, and RNA-seq analyses were measured in spinal cord microglia, splenic monocytes, and spinal cord tissue from SOD1 mice and in spinal cord tissue of familial and sporadic ALS. miR-155 was targeted by genetic ablation or by peripheral or centrally administered anti-miR-155 inhibitor in SOD1 mice. RESULTS: In SOD1 mice, we found loss of the molecular signature that characterizes homeostatic microglia and increased expression of miR-155. There was loss of the microglial molecules P2ry12, Tmem119, Olfml3, transcription factors Egr1, Atf3, Jun, Fos, and Mafb, and the upstream regulators Csf1r, Tgfb1, and Tgfbr1, which are essential for microglial survival. Microglia biological functions were suppressed including phagocytosis. Genetic ablation of miR-155 increased survival in SOD1 mice by 51 days in females and 27 days in males and restored the abnormal microglia and monocyte molecular signatures. Disease severity in SOD1 males was associated with early upregulation of inflammatory genes, including Apoe in microglia. Treatment of adult microglia with apolipoprotein E suppressed the M0-homeostatic unique microglia signature and induced an M1-like phenotype. miR-155 expression was increased in the spinal cord of both familial and sporadic ALS. Dysregulated proteins that we identified in human ALS spinal cord were restored in SOD1(G93A) /miR-155(-/-) mice. Intraventricular anti-miR-155 treatment derepressed microglial miR-155 targeted genes, and peripheral anti-miR-155 treatment prolonged survival. INTERPRETATION: We found overexpression of miR-155 in the SOD1 mouse and in both sporadic and familial human ALS. Targeting miR-155 in SOD1 mice restores dysfunctional microglia and ameliorates disease. These findings identify miR-155 as a therapeutic target for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genética , Idoso , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Animais , Apolipoproteínas E/farmacologia , Apolipoproteínas E/uso terapêutico , Células Cultivadas , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/química , MicroRNAs/genética , Microglia/efeitos dos fármacos , Microglia/metabolismo , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligorribonucleotídeos Antissenso/uso terapêutico , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
IL-27-induced type 1 regulatory T (Tr1) cells suppress autoimmunity by producing IL-10. Signal transducer and activator of transcription (STAT) 1 and STAT3 have been described as key transcription factors that promote IL-10 secretion from Tr1 cells induced by IL-27. However, the molecular pathways for negatively regulating Tr1 cell differentiation remain elusive. Here, we show that IL-27 induces metallothioneins (MTs) that in turn prevent Tr1 cell development. MT expression leads to the reduction of STAT1 and STAT3 phosphorylation under Tr1 differentiation condition, resulting in impaired IL-10 production. Accordingly, Tr1 cells derived from MT-deficient mice showed an increased ability to produce IL-10 and potently suppress experimental autoimmune encephalomyelitis upon adoptive transfer. Moreover, activation of STAT1 and/or STAT3 can overcome the suppression of IL-10 by MTs, indicating a dynamic balance between STATs and MTs in regulating IL-10 during Tr1 cell differentiation.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Interleucina-17/metabolismo , Metalotioneína/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Separação Celular , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Humanos , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/imunologiaRESUMO
BACKGROUND: Fingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function. METHODS: T cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3ß. RESULTS: We showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients. CONCLUSIONS: These results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cloridrato de Fingolimode/farmacologia , Imunossupressores/farmacologia , Esclerose Múltipla/metabolismo , Fator 1 de Transcrição de Linfócitos T/biossíntese , Regulação para Cima/fisiologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Feminino , Cloridrato de Fingolimode/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Masculino , Esclerose Múltipla/tratamento farmacológico , Regulação para Cima/efeitos dos fármacosRESUMO
IL-9-producing Th9 cells have been associated with autoimmune diseases, such as experimental autoimmune encephalitis. However, the factors that negatively regulate Th9 cells during autoimmune inflammation are unclear. In this article, we show that IFN-γ inhibits Th9 differentiation both in vitro and in vivo. This suppressive activity was dependent on the transcription factor STAT-1. In addition to its direct inhibitory effect on Th9 differentiation, IFN-γ suppressed Th9 cells through the induction of IL-27 from dendritic cells. In vitro, treatment of naive CD4(+) T cells with IL-27 suppressed the development of Th9 cells, which was partially dependent on the transcription factors STAT-1 and T-bet. Moreover, IL-27 treatment completely abrogated the encephalitogenicity of Th9 cells in the experimental autoimmune encephalomyelitis model. Thus, our results identify a previously unknown mechanism by which IFN-γ limits Th9-mediated autoimmune inflammation through dendritic cell modulation of IL-27.
Assuntos
Células Dendríticas/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inflamação/tratamento farmacológico , Interferon gama/farmacologia , Interleucina-9/imunologia , Interleucinas/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/imunologia , Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/complicações , Inflamação/imunologia , Interferon gama/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-9/biossíntese , Interleucinas/biossíntese , Interleucinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/farmacologia , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
IFN-γ-producing Th1 and IL-17-producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that microRNA-155 (Mir-155) expression is elevated in CD4(+) T cells during EAE, and Mir-155(-/-) mice had a delayed course and reduced severity of disease and less inflammation in the CNS. The attenuation of EAE in Mir-155(-/-) mice was associated with a decrease in Th1 and Th17 responses in the CNS and peripheral lymphoid organs. The T cell-intrinsic function of Mir-155(-/-) was demonstrated by the resistance of Mir-155(-/-) CD4(+) T cell-repleted Rag-1(-/-) mice to EAE. Finally, we found that anti-Mir-155 treatment reduced clinical severity of EAE when given before and after the appearance of clinical symptoms. These findings demonstrate that Mir-155 confers susceptibility to EAE by affecting inflammatory T cell responses and identify Mir-155 as a new target for therapeutic intervention in multiple sclerosis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/genética , MicroRNAs/genética , Subpopulações de Linfócitos T/imunologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Inativação Gênica , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dendritic cells (DCs) play a central role in determining the induction of T cell responses. IL-27 production by DCs favors induction of IL-10-producing regulatory T cells, whereas osteopontin (OPN) promotes pathogenic IL-17 T cell responses. The regulatory mechanisms in DCs that control these two cells types are not understood well. Here, we show that IFN-gamma induces IL-27 while inhibiting OPN expression in DCs both in vitro and in vivo and that engagement of IFN-gammaR expressed by DCs leads to suppression of IL-17 production while inducing IL-10 from T cells. DCs modified by IFN-gamma acquire IL-27-dependent regulatory function, promote IL-10-mediated T cell tolerance, and suppress autoimmune inflammation. Thus, our results identify a previously unknown pathway by which IFN-gamma limits IL-17-mediated autoimmune inflammation through differential regulation of OPN and IL-27 expression in DCs.
Assuntos
Autoimunidade , Encefalomielite Autoimune Experimental/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Osteopontina/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Técnicas de Cocultura , Camundongos , Modelos Biológicos , Peptídeos/química , Fenótipo , Baço/citologia , Fatores de TempoRESUMO
The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3(+) T(reg), Tr1 cells, and IL-17-producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3(+) T(reg) compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3(+) T(reg) in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3(+) T(reg) in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3(+) T(reg) that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3(+) T(reg) differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders.
Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Compartimento Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Indóis/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Tiazóis/metabolismo , Tretinoína/farmacologiaRESUMO
Leptin-deficient ob/ob mice are overweight, develop insulin resistance, and serve as a model for type 2 diabetes (T2D). Studies suggest that inflammatory pathways are linked to the development of insulin resistance and T2D both in animals and humans. We asked whether the induction of regulatory T cells (Tregs) could alleviate the pathological and metabolic abnormalities in ob/ob mice. We induced TGF-beta-dependent CD4(+) latency-associated peptide (LAP)-positive Tregs by oral administration of anti-CD3 antibody plus beta-glucosylceramide. We found a decrease in pancreatic islet cell hyperplasia, fat accumulation in the liver, and inflammation in adipose tissue, accompanied by lower blood glucose and liver enzymes. In addition, treated animals had decreased CD11b(+)F4/80(+) macrophages and TNF-alpha in adipose tissue. Adoptive transfer of orally induced CD4(+)LAP(+) Tregs ameliorated metabolic and cytokine abnormalities. Our results demonstrate the importance of inflammation in T2D and identify a unique immunological approach for treatment of T2D by the induction of Tregs.
Assuntos
Tecido Adiposo/imunologia , Resistência à Insulina , Linfócitos T Reguladores/imunologia , Animais , Glicemia/metabolismo , Proliferação de Células , Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/imunologia , Hiperplasia/imunologia , Inflamação/imunologia , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Células T Matadoras Naturais/imunologia , Pâncreas/imunologia , Linfócitos T Reguladores/citologiaRESUMO
Transforming growth factor ß (TGF-ß) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-ß receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources. For complete details on the use and execution of this protocol, please refer to Garo et al.1.
RESUMO
Microglia play a critical role in brain homeostasis and disease progression. In neurodegenerative conditions, microglia acquire the neurodegenerative phenotype (MGnD), whose function is poorly understood. MicroRNA-155 (miR-155), enriched in immune cells, critically regulates MGnD. However, its role in Alzheimer's disease (AD) pathogenesis remains unclear. Here, we report that microglial deletion of miR-155 induces a pre-MGnD activation state via interferon-γ (IFN-γ) signaling, and blocking IFN-γ signaling attenuates MGnD induction and microglial phagocytosis. Single-cell RNA-sequencing analysis of microglia from an AD mouse model identifies Stat1 and Clec2d as pre-MGnD markers. This phenotypic transition enhances amyloid plaque compaction, reduces dystrophic neurites, attenuates plaque-associated synaptic degradation and improves cognition. Our study demonstrates a miR-155-mediated regulatory mechanism of MGnD and the beneficial role of IFN-γ-responsive pre-MGnD in restricting neurodegenerative pathology and preserving cognitive function in an AD mouse model, highlighting miR-155 and IFN-γ as potential therapeutic targets for AD.
Assuntos
Doença de Alzheimer , MicroRNAs , Camundongos , Animais , Doença de Alzheimer/metabolismo , Interferon gama/metabolismo , Microglia/metabolismo , Transdução de Sinais/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Placa Amiloide/metabolismoRESUMO
Interleukin (IL)-27 exerts an anti-inflammatory effect on human and mice CD4(+) T cells by inducing IL-10-producing T regulatory 1 cells through induction of IL-21. However, the role of IL-27 and how it regulates IL-21 from human CD8(+) T cells is unclear. Here, we show that the IL-27 receptor is expressed on human CD8(+) T cells and stimulation of human naïve CD8(+) T cells in the presence of IL-27 leads to an increase in IL-21 and interferon (IFN)-γ production. IL-21 induction in IL-27-stimulated human CD8(+) T cells correlates specifically with expression of the transcription factor T-bet. IL-27 stimulation of naïve CD8(+) T cells induces a double-positive T-bet(+) IL-21(+) expressing CD8(+) T-cell population. Furthermore, IL-27 stimulation of human naïve CD8(+) T cells greatly increases expression of granzyme B. Antibody-mediated neutralization of IL-21 abrogates IL-27-induced granzyme B expression. Moreover, direct addition of IL-21 greatly amplifies granzyme B expression in human naïve CD8(+) T cells. Our findings identify IL-27-induced IL-21 as a key autocrine regulator of granzyme B expression in human CD8(+) T cells.
Assuntos
Comunicação Autócrina , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Granzimas/biossíntese , Interleucinas/metabolismo , Interleucinas/farmacologia , Animais , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Indução Enzimática/imunologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , CamundongosRESUMO
Dendritic cell (DC)-expressed CD40 is shown to play crucial roles in eliciting effector T cell responses, primarily the proinflammatory CD4(+) Th subsets and cytotoxic CD8(+) T cells that eliminate various infections and tumors, respectively. In contrast, DCs are also implied in the generation of regulatory T cells (Tregs) that counteract the functions of the proinflammatory Th subsets and exacerbate infections. However, the role of DC-expressed CD40 in the generation of Tregs is unknown. In this study, we generated bone marrow-derived DCs from mice (on a BALB/c background) expressing different levels of CD40 and tested their relative efficiency in generating Tregs. We observed that low levels of CD40 expression were required for efficient Treg generation. DCs expressing low levels of CD40 induced Tregs, whereas DCs expressing high levels of CD40 induced effector T cells, possibly CD8(+)CD40(+) T cells with a contraregulatory activity; the adoptive transfer of the former DC exacerbated whereas the latter significantly reduced Leishmania donovani infection in BALB/c mice. Similarly, priming of mice with leishmanial Ag-pulsed DCs expressing high levels of CD40 induced host protection against L. donovani challenge infection. In contrast, priming with the low CD40-expressing DC resulted in aggravated infection as compared with the control mice. The results establish that CD40 can play differential roles in Treg differentiation and determine the course of infection. We demonstrate that the knowledge can be efficiently used in adoptive cell transfer therapy against an infectious disease.
Assuntos
Antígenos de Protozoários/fisiologia , Antígenos CD40/fisiologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/parasitologia , Transferência Adotiva , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Antígenos CD40/biossíntese , Antígenos CD40/deficiência , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Células Dendríticas/transplante , Predisposição Genética para Doença , Imunofenotipagem , Leishmaniose Visceral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Linfócitos T Reguladores/metabolismoRESUMO
A disequilibrium between immunosuppressive Tregs and inflammatory IL-17-producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying the Treg and Th17 imbalance in CNS autoimmunity remain largely unclear. Identifying the factors that drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. miR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell-intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg responses and dampening inflammatory T cell responses. Analogous to our findings in mice, miR-92a was elevated in CD4+ T cells from patients with MS, and miR-92a silencing in patients' T cells promoted Treg development but limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.