RESUMO
Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17â¼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.
Assuntos
Colite/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Repressoras/imunologia , Serina-Treonina Quinases TOR/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular , Colite/genética , Colite/patologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/imunologia , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Fosfatidilinositol 3-Quinases/genética , Cultura Primária de Células , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Serina-Treonina Quinases TOR/genética , Células Th17/imunologia , Células Th17/patologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.
Assuntos
Linfócitos T Reguladores , Células Th17 , Camundongos , Animais , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Diferenciação Celular , Inflamação/metabolismo , Redução de Peso , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus that causes encephalitis followed by chronic demyelination in SJL mice and spontaneous seizures in C57BL/6 mice. Since earlier studies indicated a critical role of type I interferon (IFN-I) signaling in the control of viral replication in the central nervous system (CNS), mouse strain-specific differences in pathways induced by the IFN-I receptor (IFNAR) might determine the outcome of TMEV infection. METHODS: Data of RNA-seq analysis and immunohistochemistry were used to compare the gene and protein expression of IFN-I signaling pathway members between mock- and TMEV-infected SJL and C57BL/6 mice at 4, 7 and 14 days post-infection (dpi). To address the impact of IFNAR signaling in selected brain-resident cell types, conditional knockout mice with an IFNAR deficiency in cells of the neuroectodermal lineage (NesCre±IFNARfl/fl), neurons (Syn1Cre±IFNARfl/fl), astrocytes (GFAPCre±IFNARfl/fl), and microglia (Sall1CreER±IFNARfl/fl) on a C57BL/6 background were tested. PCR and an immunoassay were used to quantify TMEV RNA and cytokine and chemokine expression in their brain at 4 dpi. RESULTS: RNA-seq analysis revealed upregulation of most ISGs in SJL and C57BL/6 mice, but Ifi202b mRNA transcripts were only increased in SJL and Trim12a only in C57BL/6 mice. Immunohistochemistry showed minor differences in ISG expression (ISG15, OAS, PKR) between both mouse strains. While all immunocompetent Cre-negative control mice and the majority of mice with IFNAR deficiency in neurons or microglia survived until 14 dpi, lack of IFNAR expression in all cells (IFNAR-/-), neuroectodermal cells, or astrocytes induced lethal disease in most of the analyzed mice, which was associated with unrestricted viral replication. NesCre±IFNARfl/fl mice showed more Ifnb1, Tnfa, Il6, Il10, Il12b and Ifng mRNA transcripts than Cre-/-IFNARfl/fl mice. IFNAR-/- mice also demonstrated increased IFN-α, IFN-ß, IL1-ß, IL-6, and CXCL-1 protein levels, which highly correlated with viral load. CONCLUSIONS: Ifi202b and Trim12a expression levels likely contribute to mouse strain-specific susceptibility to TMEV-induced CNS lesions. Restriction of viral replication is strongly dependent on IFNAR signaling of neuroectodermal cells, which also controls the expression of key pro- and anti-inflammatory cytokines during viral brain infection.
Assuntos
Theilovirus , Animais , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Encéfalo , Sistema Nervoso Central , Citocinas , AnticorposRESUMO
Foxp3+ Treg cells, which are crucial for maintenance of self-tolerance, mainly develop within the thymus, where they arise from CD25+ Foxp3- or CD25- Foxp3+ Treg cell precursors. Although it is known that infections can cause transient thymic involution, the impact of infection-induced thymus atrophy on thymic Treg (tTreg) cell development is unknown. Here, we infected mice with influenza A virus (IAV) and studied thymocyte population dynamics post infection. IAV infection caused a massive, but transient thymic involution, dominated by a loss of CD4+ CD8+ double-positive (DP) thymocytes, which was accompanied by a significant increase in the frequency of CD25+ Foxp3+ tTreg cells. Differential apoptosis susceptibility could be experimentally excluded as a reason for the relative tTreg cell increase, and mathematical modeling suggested that enhanced tTreg cell generation cannot explain the increased frequency of tTreg cells. Yet, an increased death of DP thymocytes and augmented exit of single-positive (SP) thymocytes was suggested to be causative. Interestingly, IAV-induced thymus atrophy resulted in a significantly reduced T-cell receptor (TCR) repertoire diversity of newly produced tTreg cells. Taken together, IAV-induced thymus atrophy is substantially altering the dynamics of major thymocyte populations, finally resulting in a relative increase of tTreg cells with an altered TCR repertoire.
Assuntos
Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Timo/patologia , Animais , Atrofia , Biomarcadores , Sobrevivência Celular/imunologia , Imunofenotipagem , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Camundongos , Infecções por Orthomyxoviridae/virologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Timócitos/imunologia , Timócitos/metabolismoRESUMO
The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD2/genética , Antígenos CD2/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Metilação de DNA , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Memória Imunológica , Técnicas In Vitro , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Linfopenia/imunologia , Linfopenia/metabolismo , Linfopenia/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologiaRESUMO
Forkhead box P3 positive (Foxp3(+)) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκB(NS) drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκB(NS) deficiency leads to a substantial reduction of Foxp3(+) Treg cells in vivo and impaired Foxp3 induction upon transforming growth factor-ß (TGF-ß) treatment in vitro. Moreover, fewer Foxp3(+) Treg cells developed from IκB(NS)-deficient CD25(-)CD4(+) T cells adoptively transferred into immunodeficient recipients. Importantly, IκB(NS) was required for the transition of immature GITR(+)CD25(+)Foxp3(-) thymic Treg cell precursors into Foxp3(+) cells. In contrast to mice lacking c-Rel or Carma1, IκB(NS)-deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκB(NS) critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκB(NS) could modulate the Treg cell compartment.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Apoptose , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Proteínas I-kappa B/deficiência , Proteínas I-kappa B/genética , Imunomodulação , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Subunidade p50 de NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and in vitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-ß-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Acetilação , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/imunologia , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/imunologia , Histona Desacetilases/metabolismo , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/metabolismo , Lisina Acetiltransferase 5 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Linfócitos T Reguladores/metabolismo , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
During influenza A virus (IAV) infections, CD4+ T cell responses within infected lungs mainly involve T helper 1 (Th1) and regulatory T cells (Tregs). Th1-mediated responses favor the co-expression of T-box transcription factor 21 (T-bet) in Foxp3+ Tregs, enabling the efficient Treg control of Th1 responses in infected tissues. So far, the exact accumulation kinetics of T cell subsets in the lungs and lung-draining lymph nodes (dLN) of IAV-infected mice is incompletely understood, and the epigenetic signature of Tregs accumulating in infected lungs has not been investigated. Here, we report that the total T cell and the two-step Treg accumulation in IAV-infected lungs is transient, whereas the change in the ratio of CD4+ to CD8+ T cells is more durable. Within lungs, the frequency of Tregs co-expressing T-bet is steadily, yet transiently, increasing with a peak at Day 7 post-infection. Interestingly, T-bet+ Tregs accumulating in IAV-infected lungs displayed a strongly demethylated Tbx21 locus, similarly as in T-bet+ conventional T cells, and a fully demethylated Treg-specific demethylated region (TSDR) within the Foxp3 locus. In summary, our data suggest that T-bet+ but not T-bet- Tregs are epigenetically stabilized during IAV-induced infection in the lung.
Assuntos
Linfócitos T CD8-Positivos/virologia , Epigênese Genética , Fatores de Transcrição Forkhead/metabolismo , Pulmão/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Vírus da Influenza A/fisiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas com Domínio T/genéticaRESUMO
BACKGROUND: Transmaternal exposure to tobacco, microbes, nutrients, and other environmental factors shapes the fetal immune system through epigenetic processes. The gastric microbe Helicobacter pylori represents an ancestral constituent of the human microbiota that causes gastric disorders on the one hand and is inversely associated with allergies and chronic inflammatory conditions on the other. OBJECTIVE: Here we investigate the consequences of transmaternal exposure to H pylori in utero and/or during lactation for susceptibility to viral and bacterial infection, predisposition to allergic airway inflammation, and development of immune cell populations in the lungs and lymphoid organs. METHODS: We use experimental models of house dust mite- or ovalbumin-induced airway inflammation and influenza A virus or Citrobacter rodentium infection along with metagenomics analyses, multicolor flow cytometry, and bisulfite pyrosequencing, to study the effects of H pylori on allergy severity and immunologic and microbiome correlates thereof. RESULTS: Perinatal exposure to H pylori extract or its immunomodulator vacuolating cytotoxin confers robust protective effects against allergic airway inflammation not only in first- but also second-generation offspring but does not increase susceptibility to viral or bacterial infection. Immune correlates of allergy protection include skewing of regulatory over effector T cells, expansion of regulatory T-cell subsets expressing CXCR3 or retinoic acid-related orphan receptor γt, and demethylation of the forkhead box P3 (FOXP3) locus. The composition and diversity of the gastrointestinal microbiota is measurably affected by perinatal H pylori exposure. CONCLUSION: We conclude that exposure to H pylori has consequences not only for the carrier but also for subsequent generations that can be exploited for interventional purposes.
Assuntos
Infecções por Helicobacter/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Efeitos Tardios da Exposição Pré-Natal/microbiologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/microbiologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Tolerância Imunológica/imunologia , Camundongos Endogâmicos C57BL , GravidezRESUMO
E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Metilação de DNA , Fucosiltransferases/metabolismo , Inflamação/metabolismo , Pele/metabolismo , Animais , Células Cultivadas , Metilação de DNA/genética , Fucosiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Autoreactive Th1 and Th17 cells are believed to mediate the pathology of multiple sclerosis in the central nervous system (CNS). Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of the neuroinflammation. Previously, we have shown that only Th1 but not Th17 effectors activate microglia. However, it is not clear which cells are targets of Th17 effectors in the CNS. METHODS: To understand the effects driven by Th17 cells in the CNS, we induced experimental autoimmune encephalomyelitis in wild-type mice and CD4+ T cell-specific integrin α4-deficient mice where trafficking of Th1 cells into the CNS was affected. We compared microglial and astrocyte response in the brain and spinal cord of these mice. We further treated astrocytes with supernatants from highly pure Th1 and Th17 cultures and assessed the messenger RNA expression of neurotrophic factors, cytokines and chemokines, using real-time PCR. Data obtained was analyzed using the Kruskal-Wallis test. RESULTS: We observed in α4-deficient mice weak microglial activation but comparable astrogliosis to that of wild-type mice in the regions of the brain populated with Th17 infiltrates, suggesting that Th17 cells target astrocytes and not microglia. In vitro, in response to supernatants from Th1 and Th17 cultures, astrocytes showed altered expression of neurotrophic factors, pro-inflammatory cytokines and chemokines. Furthermore, increased expression of chemokines in Th1- and Th17-treated astrocytes enhanced recruitment of microglia and transendothelial migration of Th17 cells in vitro. CONCLUSION: Our results demonstrate the delicate interaction between T cell subsets and glial cells and how they communicate to mediate their effects. Effectors of Th1 act on both microglia and astrocytes whereas Th17 effectors preferentially target astrocytes to promote neuroinflammation.
Assuntos
Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Gliose/metabolismo , Mediadores da Inflamação/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , Animais , Astrócitos/patologia , Movimento Celular/fisiologia , Células Cultivadas , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/patologia , Gliose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Th1/patologia , Células Th17/patologiaRESUMO
Activated naive CD4(+) T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4(+) T cells, Th1 and Th17 cells. We could demonstrate that naive CD4(+) T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.
Assuntos
Diferenciação Celular/genética , Epigênese Genética , Células Th17/citologia , Animais , Metilação de DNA , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Foxp3⺠regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymus-derived, tTreg cells) and in the periphery, where they are converted from conventional CD4⺠T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4⺠T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3⺠Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1⺠tTreg cells outcompeted Nrp1⻠iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Tolerância Imunológica/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.
Assuntos
Antígenos CD28/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subpopulações de Linfócitos T/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Autoimunidade/genética , Antígenos CD28/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Sobrevivência Celular/genética , Seleção Clonal Mediada por Antígeno/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Homeostase , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genéticaRESUMO
Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.
Assuntos
Metilação de DNA , Fatores de Transcrição Forkhead/genética , Linfócitos T Reguladores/metabolismo , Timo/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Ilhas de CpG , Citosina/química , Regulação da Expressão Gênica , Ordem dos Genes , Masculino , Camundongos , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Timo/imunologiaRESUMO
Microglia act as sensors of inflammation in the central nervous system (CNS) and respond to many stimuli. Other key players in neuroinflammatory diseases are CD4+ T helper cell (Th) subsets that characteristically secrete IFN-γ (Th1) or IL-17 (Th17). However, the potential of a distinct cytokine milieu generated by these effector T cell subsets to modulate microglial phenotype and function is poorly understood. We therefore investigated the ability of factors secreted by Th1 and Th17 cells to induce microglial activation. In vitro experiments wherein microglia were cultured in the presence of supernatants derived from polarized Th1 or Th17 cultures, revealed that Th1-associated factors could directly activate and trigger a proinflammatory M1-type gene expression profile in microglia that was cell-cell contact independent, whereas Th17 cells or its associated factors did not have any direct influence on microglia. To assess the effects of the key Th17 effector cytokine IL-17A in vivo we used transgenic mice in which IL-17A is specifically expressed in astrocytes. Flow cytometric and histological analysis revealed only subtle changes in the phenotype of microglia suggesting only minimal effects of constitutively produced IL-17A on microglia in vivo. Neither IL-23 signaling nor addition of GM-CSF, a recently described effector molecule of Th17 cells, changed the incapacity of Th17 cells to activate microglia. These findings demonstrate a potent effect of Th1 cells on microglia, however, the mechanism of how Th17 cells achieve their effect in CNS inflammation remains unclear.
Assuntos
Microglia/fisiologia , Células Th1/metabolismo , Células Th17/metabolismo , Animais , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th1/imunologia , Células Th17/imunologiaRESUMO
It is emerging that CD4+Foxp3+ regulatory T (Treg) cells can produce the proinflammatory cytokine IFN-γ when stimulated in a Th1 cytokine environment. In this study, we report that Foxp3+ Treg cells readily produced IFN-γ in vivo in a highly inflammatory model of graft-versus-host disease (GVHD) and during a Th1-dominated immune response to intracellular bacteria. Moreover, stimulation in vitro via TCR in the presence of IL-12 alone was sufficient to induce IFN-γ production by Treg cells in a dose-dependent manner. Transfer of donor Treg cells can prevent lethal GVHD; therefore, we used this model as a robust readout for in vivo Treg function. Interestingly, >50% of allogeneic donor, but not residual recipient Foxp3+ Treg cells produced IFN-γ after transplantation, suggesting that this cytokine production was alloantigen specific. These IFN-γ producers were stable Foxp3+ Treg cells because methylation analysis of the Foxp3 gene locus of transferred and reisolated Treg cells during GVHD showed a fully demethylated Treg-specific-demethylated region. Next, we addressed whether IFN-γ production was supporting or rather impairing the immunosuppressive function of Treg cells during GVHD. Blocking of IFN-γ with specific mAb completely abolished the beneficial effect of donor Treg cells. We could further show that only wild-type Treg cells, but not Treg cells from IFN-γ-deficient donor mice, prevented GVHD. This indicated that Treg cell-intrinsic IFN-γ production was required for their protective function. In conclusion, our data show that IFN-γ produced by Foxp3+ Treg cells has essential immune-regulatory functions that are required for prevention of experimental GVHD.
Assuntos
Fatores de Transcrição Forkhead/biossíntese , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Interferon gama/biossíntese , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Células Cultivadas , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Imunidade Celular/genética , Interferon gama/deficiência , Interferon gama/metabolismo , Isoantígenos/biossíntese , Isoantígenos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologiaRESUMO
Foxp3(+) T regulatory (Treg) cells can be induced to produce interleukin (IL)-17 by in vitro exposure to proinflammatory cytokines, drawing into question their functional stability at sites of inflammation. Unlike their splenic counterparts, Treg cells from the inflamed central nervous system (CNS-Treg cells) during EAE resisted conversion to IL-17 production when exposed to IL-6. We show that the highly activated phenotype of CNS-Treg cells includes elevated expression of the Th1-associated molecules CXCR3 and T-bet, but reduced expression of the IL-6 receptor α chain (CD126) and the signaling chain gp130. We found a lack of IL-6 receptor on all CNS CD4(+) T cells, which was reflected by an absence of both classical and trans-IL-6 signaling in CNS CD4(+) cells, compared with their splenic counterparts. We propose that extinguished responsiveness to IL-6 (via down-regulation of CD126 and gp130) stabilizes the regulatory phenotype of activated Treg cells at sites of autoimmune inflammation.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Fatores de Transcrição Forkhead/imunologia , Interleucina-17/biossíntese , Interleucina-6/imunologia , Receptores de Interleucina-6/imunologia , Linfócitos T Reguladores/imunologia , Animais , Receptor gp130 de Citocina/biossíntese , Receptor gp130 de Citocina/imunologia , Regulação para Baixo , Camundongos , Receptores de Interleucina-6/biossínteseRESUMO
CCR6 is a chemokine receptor expressed on Th17 cells and regulatory T cells that is induced by T-cell priming with certain cytokines, but how its expression and stability are regulated at the molecular level is largely unknown. Here, we identified and characterized a noncoding region of the human CCR6 locus that displayed unmethylated CpG motifs (differentially methylated region [DMR]) selectively in CCR6(+) lymphocytes. CCR6 expression on circulating CD4(+) T cells was stable on cytokine-induced proliferation but partially down-regulated on T-cell receptor stimulation. However, CCR6 down-regulation was mostly transient, and the DMR within the CCR6 locus remained demethylated. Notably, in vitro induction of CCR6 expression with cytokines in T-cell receptor-activated naive CD4(+) T cells was not associated with a demethylated DMR and resulted in unstable CCR6 expression. Conversely, treatment with the DNA methylation inhibitor 5'-azacytidine induced demethylation of the DMR and led to increased and stable CCR6 expression. Finally, when cloned into a reporter gene plasmid, the DMR displayed transcriptional activity in memory T cells that was suppressed by DNA methylation. In summary, we have identified a noncoding region of the human CCR6 gene with methylation-sensitive transcriptional activity in CCR6(+) T cells that controls stable CCR6 expression via epigenetic mechanisms.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Receptores CCR6/genética , Linfócitos T/metabolismo , Separação Celular , Citometria de Fluxo , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.