RESUMO
BACKGROUND: Alpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), "augmentation therapy", provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT. METHODS: In an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo. RESULTS: Both elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4+ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17. CONCLUSIONS: Compared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.
Assuntos
Fragmentos Fc das Imunoglobulinas/administração & dosagem , Exposição por Inalação/efeitos adversos , Elastase Pancreática/toxicidade , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Fumaça/efeitos adversos , alfa 1-Antitripsina/administração & dosagem , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/administração & dosagem , Enfisema Pulmonar/imunologia , Suínos , NicotianaRESUMO
BACKGROUND: Inducible CD4+CD25+ regulatory T (iTreg) cells can become pathogenic effector cells, enhancing lung allergic responses. OBJECTIVE: We aimed to define the underlying cellular and molecular pathways activated by TGF-ß, which determine the suppressor or enhancing activities of iTreg cells. METHODS: Sensitized wild-type and CD8-deficient (CD8-/-) mice were challenged with allergen. Isolated CD4+CD25- T cells were activated by using anti-CD3/anti-CD28. To generate suppressor iTreg cells, cells were then differentiated in the presence of TGF-ß, whereas IL-17-producing effector T cells were additionally exposed to IL-6. After TGF-ß, Smad3 and TGF-ß-activated kinase 1 (TAK1) kinase levels were monitored. The consequences of inhibiting either kinase were determined in vitro and after transfer into CD8-/- recipients. Quantitative PCR and chromatin immunoprecipitation were used to monitor gene expression and histone modifications at the retinoic acid-related orphan receptor γt (Rorγt) locus. RESULTS: In wild-type mice, iTreg cells suppressed lung allergic responses linked to Smad3-dependent forkhead box P3 (Foxp3) expression and IL-10 production. In the presence of IL-6, iTreg cells converted to TH17 cells, mediating a neutrophil-dependent enhancement of lung allergic responses in CD8-/- mice. Conversion was regulated by TAK1. Inhibition or silencing of TAK1 prevented expression of Rorγt and TH17 differentiation through histone modifications of Rorγt; Foxp3 expression and iTreg cell-mediated suppression remained intact. In the same cell, TGF-ß induced coexpression of Smad3 and TAK1 proteins; in the presence of IL-6, expression of Smad3 and Foxp3 but not TAK1 decreased. CONCLUSION: TGF-ß regulates iTreg cell outcomes through 2 distinct signal transduction pathways: one Smad3 dependent and the other TAK1 dependent. The balance of these pathways has important implications in TH17-mediated autoimmune diseases and neutrophil-dependent asthma.
Assuntos
MAP Quinase Quinase Quinases/imunologia , Hipersensibilidade Respiratória/imunologia , Proteína Smad3/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Diferenciação Celular/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
The impact of naturally occurring regulatory T cells (nTregs) on the suppression or induction of lung allergic responses in mice depends on the nuclear environment and the production of the pro-inflammatory cytokine interleukin 6 (IL-6). These activities were shown to be different in nTregs derived from wild-type (WT) and CD8-deficient mice (CD8-/-), with increased IL-6 levels in nTregs from CD8-/- mice in comparison to WT nTregs. Thus, identification of the molecular mechanisms regulating IL-6 production is critical to understanding the phenotypic plasticity of nTregs. Electrophoretic mobility shift assays (EMSA) were performed to determine transcription factor binding to four Il-6 promoter loci using nuclear extracts from nTregs of WT and CD8-/- mice. Increased transcription factor binding for each of the Il-6 loci was identified in CD8-/- compared to WT nTregs. The impact of transcription factor binding and a novel short tandem repeat (STR) on Il-6 promoter activity was analyzed by luciferase reporter assays. The Il-6 promoter regions closer to the transcription start site (TSS) were more relevant to the regulation of Il-6 depending on NF-κB, c-Fos, and SP and USF family members. Two Il-6 promoter loci were most critical for the inducibility by lipopolysaccharide (LPS) and tumor necrosis factor α (TNFα). A novel STR of variable length in the Il-6 promoter was identified with diverging prevalence in nTregs from WT or CD8-/- mice. The predominant GT repeat in CD8-/- nTregs revealed the highest luciferase activity. These novel regulatory mechanisms controlling the transcriptional regulation of the Il-6 promoter are proposed to contribute to nTregs plasticity and may be central to disease pathogenesis.
Assuntos
Hipersensibilidade/imunologia , Interleucina-6/imunologia , Pneumopatias/imunologia , Linfócitos T Reguladores/metabolismo , Adaptação Fisiológica/imunologia , Animais , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Citocinas/imunologia , Interleucina-10/imunologia , Interleucina-6/genética , Pneumopatias/genética , Pneumopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Regiões Promotoras Genéticas , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Despite advances in the treatment of asthma, optimization of symptom control remains an unmet need in many patients. These patients, labeled severe asthma, are responsible for a substantial fraction of the disease burden. In these patients, research is needed to define the cellular and molecular pathways contributing to disease which in large part are refractory to corticosteroid treatment. The causes of steroid-resistant asthma are multifactorial and result from complex interactions of genetics, environmental factors, and innate and adaptive immunity. Adaptive immunity, addressed here, integrates the activities of distinct T-cell subsets and by definition is dynamic and responsive to an ever-changing environment and the influences of epigenetic modifications. These T-cell subsets exhibit different susceptibilities to the actions of corticosteroids and, in some, corticosteroids enhance their functional activation. Moreover, these subsets are not fixed in lineage differentiation but can undergo transcriptional reprogramming in a bidirectional manner between protective and pathogenic effector states. Together, these factors contribute to asthma heterogeneity between patients but also in the same patient at different stages of their disease. Only by carefully defining mechanistic pathways, delineating their sensitivity to corticosteroids, and determining the balance between regulatory and effector pathways will precision medicine become a reality with selective and effective application of targeted therapies.
Assuntos
Hipersensibilidade/imunologia , Imunomodulação , Ativação Linfocitária/imunologia , Pneumonia/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Pneumonia/genética , Pneumonia/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
BACKGROUND: CD8+ type 2 cytotoxic T (TC2) cells undergo transcriptional reprogramming to IL-13 production in the presence of IL-4 to become potent, steroid-insensitive, pathogenic effector cells in asthmatic patients and in mice in a model of experimental asthma. However, no studies have described the effects of hypoxia exposure on TC2 cell differentiation. OBJECTIVE: We determined the effects of hypoxia exposure on IL-13-producing CD8+ TC2 cells. METHODS: CD8+ transgenic OT-1 cells differentiated with IL-2 and IL-4 (TC2 cells) were exposed to normoxia (21% oxygen) or hypoxia (3% oxygen), and IL-13 production in vitro was monitored. After differentiation under these conditions, cells were adoptively transferred into CD8-deficient mice, and lung allergic responses, including airway hyperresponsiveness to inhaled methacholine, were assessed. The effects of pharmacologic inhibitors of hypoxia-inducible factor (HIF) 1α and HIF-2α were determined, as were responses in HIF-1α-deficient OT-1 cells. RESULTS: Under hypoxic conditioning, CD8+ TC2 cell differentiation was significantly enhanced, with increased numbers of IL-13+ T cells and increased production of IL-13 in vitro. Adoptive transfer of TC2 cells differentiated under hypoxic conditioning restored lung allergic responses in sensitized and challenged CD8-deficient recipients to a greater degree than seen in recipients of TC2 cells differentiated under normoxic conditioning. Pharmacologic inhibition of HIF-1α or genetic manipulation to reduce HIF-1α expression reduced the hypoxia-enhanced differentiation of TC2 cells, IL-13 production, and the capacity of transferred cells to restore lung allergic responses in vivo. IL-4-dependent, hypoxia-mediated increases in HIF-1α and TC2 cell differentiation were shown to be mediated through activation of Janus kinase 1/3 and GATA-3. CONCLUSIONS: Hypoxia enhances CD8+ TC2 cell-dependent airway hyperresponsiveness and inflammation through HIF-1α activation. These findings coupled with the known insensitivity of CD8+ T cells to corticosteroids suggests that activation of the IL-4-HIF-1α-IL-13 axis might play a role in the development of steroid-refractory asthma.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Hipóxia/imunologia , Inflamação/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Broncoconstritores , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Pulmão/imunologia , Pulmão/fisiopatologia , Cloreto de Metacolina , Camundongos Transgênicos , Hipersensibilidade Respiratória/fisiopatologiaRESUMO
BACKGROUND: Regulatory T (Treg) cells play an essential role in the maintenance of immune homeostasis in allergic diseases. OBJECTIVES: We sought to define the mechanisms underlying induction of tolerance to peanut protein and prevention of the development of peanut allergy. METHODS: High or low doses of peanut extract were administered to pups every day for 2 weeks before peanut sensitization and challenge. After challenge, symptoms, Treg cell numbers, and forkhead box protein 3 (Foxp3), TH2 and TH17 cytokine, and Tgfß expression in mesenteric lymph node (MLN) CD4+ T cells and jejunum were monitored. Treg cell suppressive activity and Foxp3 methylation in MLN CD4+ T cells were assayed. RESULTS: Feeding high but not low doses of peanut before sensitization induced tolerance, as demonstrated by prevention of diarrhea and peanut-specific IgE responses, increases in the percentage of CD4+CD25+FoxP3+ cells in MLNs, and Foxp3 mRNA and protein expression in CD4+ cells from MLNs or jejunum. Feeding high doses of peanut before sensitization decreased percentages of CD3+CD4+IL-13+ and CD3+CD4+IL-17+ cells in MLNs and decreased Il13 and Il17a and increased Tgfß mRNA expression in the jejunum; numbers of CD103+ dendritic cells in MLNs were significantly increased. Treg cell suppression was shown to be antigen specific. Foxp3 methylation was increased in peanut extract-sensitized and challenged mice, whereas in tolerized mice levels were significantly reduced. CONCLUSIONS: Feeding high doses of peanut to pups induced tolerance to peanut protein. Foxp3 demethylation was associated with tolerance induction, indicating that Treg cells play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis.
Assuntos
Arachis/química , Fatores de Transcrição Forkhead/imunologia , Tolerância Imunológica/efeitos dos fármacos , Jejuno/imunologia , Hipersensibilidade a Amendoim , Extratos Vegetais/farmacologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Citocinas/imunologia , Desmetilação/efeitos dos fármacos , Modelos Animais de Doenças , Jejuno/patologia , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade a Amendoim/imunologia , Hipersensibilidade a Amendoim/patologia , Hipersensibilidade a Amendoim/prevenção & controle , Extratos Vegetais/química , Extratos Vegetais/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
BACKGROUND: Regulatory T cells attenuate development of asthma in wild-type (WT) mice, with both naturally occurring regulatory T (nTreg) cells and inducible regulatory T (iTreg) cells exhibiting suppressive activity. When transferred into CD8-deficient (CD8-/-) recipients, both cell types enhanced development of allergen-induced airway hyperresponsiveness. OBJECTIVE: We sought to determine whether the pathways leading to enhancement of lung allergic responses by transferred nTreg and iTreg cells differed. METHODS: nTreg cells (CD4+CD25+) were isolated from WT mice and iTreg cells were generated from WT CD4+CD25- T cells after activation in the presence of TGF-ß and transferred into sensitized CD8-/- recipients before challenge. Development of airway hyperresponsiveness, cytokine levels, and airway inflammation were monitored. RESULTS: Transfer of nTreg cells enhanced lung allergic responses, as did transfer of iTreg cells. Although anti-IL-13 reduced nTreg cell-mediated enhancement, it was ineffective in iTreg cell-mediated enhancement; conversely, anti-IL-17, but not anti-IL-13, attenuated the enhancement by iTreg cells. Recovered iTreg cells from the lungs of CD8-/- recipients were capable of IL-17 production and expressed high levels of signature genes of the TH17 pathway, RORγt and Il17, whereas reduced expression of the Treg cell key transcription factor forkhead box p3 (Foxp3) was observed. In vitro exogenous IL-6-induced IL-17 production in iTreg cells, and in vivo conversion of transferred iTreg cells was dependent on recipient IL-6. CONCLUSIONS: iTreg cells, similar to nTreg cells, exhibit functional plasticity and can be converted from suppressor cells to pathogenic effector cells, enhancing lung allergic responses, but these effects were mediated through different pathways.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Citocinas/biossíntese , Citocinas/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Hipersensibilidade/imunologia , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Glucocorticoid-induced TNFR family-related protein (GITR)-mediated activation of JNK was shown to regulate the suppressive activity of CD4(+)CD25(+) naturally occurring T regulatory cells (nTregs) in wild-type (WT) hosts. In this study, CD4(+)CD25(+) T cells were shown to be capable of becoming pathogenic effector cells in sensitized and challenged CD8(-/-) recipient mice. Only GITR-expressing CD4(+)CD25(+) T cells, but neither GITR knocked-in CD4(+)CD25(-) T cells nor GITR-silenced CD4(+)CD25(+) T cells, enhanced development of lung allergic responses. Inhibition of JNK in WT nTregs or nTregs from GITR(-/-)and JNK2(-/-) mice failed to enhance lung allergic responses in sensitized and challenged CD8(-/-) recipient mice. The failure to enhance responses was associated with increased bronchoalveolar lavage fluid levels of IL-10 and TGF-ß and decreased levels of IL-5, IL-6, and IL-13. In contrast, nTregs from JNK1(-/-) mice, similar to WT nTregs, were fully effective in enhancing responses. Thus, GITR stimulation of nTregs and signaling through JNK2, but not JNK1, triggered the loss of regulatory function while concomitantly gaining pathogenic CD4(+) T effector cell function responsible for exacerbating asthma-like immunopathology.
Assuntos
Asma/imunologia , Pulmão/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 9 Ativada por Mitógeno/imunologia , Linfócitos T Reguladores/imunologia , Animais , Asma/genética , Asma/patologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/imunologia , Proteína Quinase 9 Ativada por Mitógeno/genética , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
Histamine H(4) receptor (H(4)R)-deficient mice (H(4)R(-/-)), H(4)R antagonist-treated wild-type (WT) mice, and WT mice depleted of basophils failed to develop early (EPR) or late phase (LPR) nasal responses following allergen sensitization and challenge. Basophil transfer from WT but not H(4)R(-/-) mice restored the EPR and LPR in H(4)R(-/-) mice. Following passive sensitization with OVA-specific IgE, FcεRI(-/-) recipients of WT basophils plus OVA and histamine developed an EPR and LPR. OVA-IgE passively sensitized FcεRI(-/-) recipients of H(4)R(-/-) basophils and OVA and histamine challenge failed to develop an EPR or LPR, and basophils were not detected in nasal tissue. In contrast, recipients of basophils from IL-13(-/-) and IL-4(-/-)/IL-13(-/-) mice developed an EPR but not an LPR. These results demonstrate the development of allergic rhinitis proceeded in two distinct stages: histamine release from FcεRI-activated mast cells, followed by histamine-mediated recruitment of H(4)R-expressing basophils to the nasal cavity and activation through FcεRI.
Assuntos
Basófilos/imunologia , Mastócitos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Histamínicos/imunologia , Receptores de IgE/imunologia , Rinite Alérgica Perene/imunologia , Transferência Adotiva , Animais , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Feminino , Antagonistas dos Receptores Histamínicos/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Modelos Imunológicos , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/genética , Receptores Histamínicos H4 , Receptores de IgE/deficiência , Receptores de IgE/genética , Rinite Alérgica , Rinite Alérgica Perene/genética , Rinite Alérgica Perene/terapiaRESUMO
The functional plasticity of CD8(+) T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness and inflammation, and yet remains mechanistically undefined. We demonstrate that CD8(+) T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8(+) T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the stepwise molecular conversion of CD8(+) T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA polymerase II (Pol II) recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage after TCR stimulation, indicating that IL-4-induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro, an atopic (IL-4) environment poises CD8(+) T cells via stepwise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Epigênese Genética/imunologia , Imunofenotipagem , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Imunofenotipagem/métodos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Hipersensibilidade Respiratória/metabolismoRESUMO
BACKGROUND: Janus kinases (JAKs) are regulators of signaling through cytokine receptors. The importance of JAK1/3 signaling on TH2 differentiation and development of lung allergic responses has not been investigated. OBJECTIVE: We sought to examine a selective JAK1/3 inhibitor (R256) on differentiation of TH subsets in vitro and on development of ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in an experimental model of asthma. METHODS: A selective JAK1/3 inhibitor was used to assay the importance of this pathway on induction of TH1, TH2, and TH17 differentiation in vitro. In vivo, the effects of inhibiting JAK1/3 signaling were examined by administering the inhibitor during the sensitization or allergen challenge phases in the primary challenge model or just before provocative challenge in the secondary challenge model. Airway inflammation and AHR were examined after the last airway challenge. RESULTS: In vitro, R256 inhibited differentiation of TH2 but not TH1 or TH17 cells, which was associated with downregulation of signal transducer and activator of transcription (STAT) 6 and STAT5 phosphorylation. However, once polarized, TH2 cells were unaffected by the inhibitor. In vivo, R256 administered during the OVA sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and TH2 cytokine production without changes in TH1 and TH17 cytokine levels, indicating that selective blockade of TH2 differentiation was critical. Inhibitor administration after OVA sensitization but during the challenge phases in the primary or secondary challenge models similarly suppressed AHR, airway eosinophilia, and mucus hypersecretion without any reduction in TH2 cytokine production, suggesting the inhibitory effects were downstream of TH2 cytokine receptor signaling pathways. CONCLUSIONS: Targeting the TH2-dependent JAK/STAT activation pathway represents a novel therapeutic approach for the treatment of asthma.
Assuntos
Diferenciação Celular , Janus Quinase 1/metabolismo , Janus Quinase 3/metabolismo , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Transdução de Sinais , Células Th2/citologia , Células Th2/metabolismo , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacologia , Hipersensibilidade Respiratória/patologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Naturally occurring Foxp3(+)CD4(+)CD25(+) T regulatory cell (nTreg)-mediated suppression of lung allergic responses is abrogated following ligation of glucocorticoid-induced tumor necrosis receptor (GITR) family-related protein. In vitro stimulation of nTregs with GITR ligand increased phosphorylation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated protein kinase (ERK) or p38 MAPK. SP600125, a known JNK inhibitor, prevented GITR-mediated phosphorylation of JNK. Activation of JNK was associated with increases in the upstream mitogen-activated protein kinase kinase 7 (MKK7) and the downstream transcription factor NF-κß. Phosphorylated c-Jun (p-c-Jun), indicative of the activation of JNK, was detected in the immunoprecipitates of nTregs from wild-type but not JNK- or GITR-deficient mice. Treatment with an inhibitor of JNK phosphorylation resulted in complete reversal of all GITR-induced changes in nTreg phenotype and function, with full restoration of suppression of in vivo lung allergic responses and in vitro proliferation of activated CD4(+)CD25(-) T cells. Thus, regulation of JNK phosphorylation plays a central role in T regulatory cell function with therapeutic implications for the treatment of asthma and autoimmune diseases.
Assuntos
Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Tolerância Imunológica/fisiologia , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Linfócitos T Reguladores/metabolismo , Animais , Antracenos/farmacologia , Asma/genética , Asma/imunologia , Asma/metabolismo , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Tolerância Imunológica/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , MAP Quinase Quinase 7/genética , MAP Quinase Quinase 7/imunologia , MAP Quinase Quinase 7/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Reguladores/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.
Assuntos
Artrite Experimental/prevenção & controle , Doenças Autoimunes/prevenção & controle , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doença Crônica , Modelos Animais de Doenças , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
Naturally occurring CD4(+)CD25(+)Foxp3(+) T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-ß. nTregs from CD8(-/-) mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-ß, and high levels of IL-6. Administration of anti-IL-6 or anti-IL-6R to wild-type recipients prior to transfer of CD8(-/-) nTregs restored suppression. nTregs from IL-6(-/-) mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8(+) cells. Transfer of nTregs from CD8(-/-) donors reconstituted with CD8(+) T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8-T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.
Assuntos
Antígenos CD8/fisiologia , Imunofenotipagem , Terapia de Imunossupressão , Interleucina-6/biossíntese , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD8/genética , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Interleucina-10/antagonistas & inibidores , Interleucina-6/deficiência , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/fisiologia , Hipersensibilidade Respiratória/genética , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
The NLRP3 inflammasome is an intracellular, multiprotein complex that promotes the auto-catalytic activation of caspase-1 and the subsequent maturation and secretion of the pro-inflammatory cytokines, IL-1ß and IL-18. Persistent activation of the NLRP3 inflammasome has been implicated in the pathophysiology of a number of inflammatory and autoimmune diseases, including neuroinflammation, cardiovascular disease, non-alcoholic steatohepatitis, lupus nephritis and severe asthma. Here we describe the preclinical profile of JT002, a novel small molecule inhibitor of the NLRP3 inflammasome. JT002 potently reduced NLRP3-dependent proinflammatory cytokine production across a number of cellular assays and prevented pyroptosis, an inflammatory form of cell death triggered by active caspase-1. JT002 demonstrated in vivo target engagement at therapeutically relevant concentrations when orally dosed in mice and prevented body weight loss and improved inflammatory and fibrotic endpoints in a model of Muckle-Wells syndrome (MWS). In two distinct models of neutrophilic airway inflammation, JT002 treatment significantly reduced airway hyperresponsiveness and airway neutrophilia. These results provide a rationale for the therapeutic targeting of the NLRP3 inflammasome in severe asthma and point to the use of JT002 in a variety of inflammatory disorders.
Assuntos
Doenças Cardiovasculares , Nefrite Lúpica , Animais , Camundongos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Caspase 1RESUMO
BACKGROUND: Notch signaling pathways govern immune function and the regulation of Th1 and Th2 differentiation. We previously demonstrated essential interactions between Notch on CD4+ T cells and Jagged1 on antigen-presenting cells in Th2 differentiation for the full development of allergen-induced airway hyperresponsiveness (AHR) and allergic airway inflammation. METHODS: Bone marrow-derived dendritic cells (BMDCs) were differentiated and incubated with different preparations of ovalbumin (OVA), including lipopolysaccharide (LPS)-depleted and LPS-spiked preparations. In some experiments recipient mice also received soluble Jagged1-Fc in addition to allergen-pulsed BMDCs. Ten days following transfer of BMDCs, mice were exposed to three airway challenges with OVA, and airway responsiveness to inhaled methacholine, airway inflammation and cytokine production were monitored 48 h later. Notch ligand expression was assessed by real-time PCR. RESULTS: Induction of Jagged1 expression on antigen-pulsed BMDCs was dependent on low-dose endotoxin. In vivo, transfer of endotoxin-free, antigen-pulsed BMDCs failed to induce AHR or airway eosinophilia on allergen challenge. However, administration of exogenous Jagged1-Fc together with endotoxin-free, allergen-pulsed BMDCs fully restored the responses to allergen challenge. CONCLUSIONS: These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.
Assuntos
Antígenos/imunologia , Asma/imunologia , Asma/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Dendríticas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipopolissacarídeos/imunologia , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Transferência Adotiva , Animais , Modelos Animais de Doenças , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1 , Ligantes , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Receptores Notch/metabolismo , Proteínas Serrate-JaggedRESUMO
Naturally occurring Foxp3(+)CD4(+)CD25(+) T cells isolated from lungs of naive mice regulate lung allergic airway hyperresponsiveness, inflammation, levels of Th2 cytokines, and mucus production. OVA-specific (alphabetaTCR(+)) CD4(+)CD25(+) T cells suppressed ragweed-induced airway hyperresponsiveness and inflammation as did anti-TCR-treated OVA-specific CD4(+)CD25(+) T cells, suggesting that Ag-specificity was not required for expression of regulatory activities. Suppression was associated with increased levels of IL-10 and TGF-beta; decreased levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid; and reduced recruitment and activation of CD8(+) T cells in the airways. Following intratracheal administration, OVA-specific CD4(+)CD25(+) T cells were identified in both the airway lumens and lung parenchyma, and in some instances in close proximity to host CD8(+) T cells. These results demonstrate that the regulatory activities of naturally occurring Foxp3(+)CD4(+)CD25(+) T cells on lung allergic responses are Ag-nonspecific and thus, independent of Ag-specific recognition.
Assuntos
Epitopos/imunologia , Hipersensibilidade/imunologia , Pneumopatias/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Ambrosia/imunologia , Animais , Linfócitos T CD8-Positivos , Citocinas/análise , Inflamação , Camundongos , Camundongos Endogâmicos , Ovalbumina/imunologiaRESUMO
Jagged1, a Notch ligand, and Notch have been implicated in Th2 differentiation, but their role in initiating IL-4 production and Th2 differentiation in vivo and the development of allergic airway responses has not been defined. In this study, we show that Jagged1 is up-regulated on bone marrow-derived dendritic cells (BMDCs) pulsed with allergen and that the transfer of these BMDCs before allergen challenge induces airway hyperresponsiveness (AHR) and eosinophilic airway inflammation. Treatment of CD4(+) T cells with a gamma-secretase inhibitor (GSI), which inhibits Notch signaling, resulted in decreased cytokine production when the cells were cocultured with allergen-pulsed, Jagged1-expressing BMDCs and, after the transfer of allergen-pulsed BMDCs, IL-4-deficient (IL-4(-/-)) recipients of GSI-treated naive CD4(+) T cells developed lower levels of AHR, reduced numbers of eosinophils, and lower Th2 cytokine levels when challenged with allergen. In vivo treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation, whereas the transfer of BMDC transfected with Jagged1 small interfering RNA (siRNA) cells into WT or IL-4(-/-) mice before transfer of CD4(+) T cells resulted in decreased AHR, inflammation, and Th2 cytokines, indicating the critical role for Jagged1 expression on APCs. These data identify the essential role of the interactions between Notch on CD4(+) T cells and Jagged1 on APCs in the initiation of IL-4 production and Th2 differentiation for the development of AHR and allergic airway inflammation.
Assuntos
Hiper-Reatividade Brônquica/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Interleucina-4/biossíntese , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Animais , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Linfócitos T CD4-Positivos/transplante , Proteínas de Ligação ao Cálcio/administração & dosagem , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/transplante , Eosinofilia/imunologia , Eosinofilia/metabolismo , Eosinofilia/patologia , Feminino , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Interleucina-4/deficiência , Interleucina-4/fisiologia , Proteína Jagged-1 , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/toxicidade , Receptores Notch/administração & dosagem , Proteínas Serrate-Jagged , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologiaRESUMO
Suppressing the abnormalities associated with asthma has been difficult to accomplish using immunotherapy or vaccination once the disease is established. The effector cells necessary for effective immunization/vaccination and immunotherapy of asthma are also not well understood. Therefore, we vaccinated allergen (OVA)-sensitized mice to determine whether therapeutic immunization could suppress airway hyperresponsiveness (AHR) and inflammation and to identify key immune effector cells and cytokines. Mice were immunized with a vaccine comprised of Ag and cationic liposome-DNA complexes (CLDC), a vaccine which has previously been shown to elicit strong CD4(+) and CD8(+) T cell responses and activation of Th1 immunity. We showed that immunization with the OVA-CLDC vaccine significantly suppressed AHR, eosinophilia, goblet cell metaplasia, and Th2 cytokine production. In contrast, immunization with CLDC alone suppressed eosinophilia and Th2 cytokine production, but failed to suppress AHR and goblet cell changes. Using adoptive transfer experiments, we found that suppression of AHR was mediated by Ag-specific CD8(+) T cells and was dependent on IFN-gamma production by the transferred T cells. Thus, we conclude that generation of strong, allergen-specific CD8(+) T cell responses by immunization may be capable of suppressing AHR and allergic airway inflammation, even in previously sensitized and challenged mice.
Assuntos
Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/prevenção & controle , Linfócitos T CD8-Positivos/imunologia , Imunossupressores/administração & dosagem , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/imunologia , Vacinas de DNA/imunologia , Transferência Adotiva , Animais , Asma/imunologia , Asma/patologia , Asma/prevenção & controle , Hiper-Reatividade Brônquica/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Cátions/administração & dosagem , Cátions/metabolismo , Citocinas/fisiologia , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Feminino , Antígenos H-2/administração & dosagem , Antígenos H-2/genética , Antígenos H-2/imunologia , Humanos , Imunossupressores/imunologia , Lipossomos , Cloreto de Metacolina/administração & dosagem , Cloreto de Metacolina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
BACKGROUND: Although implicated in the disease, the specific contributions of FcepsilonRI and IL-13 to the pathogenesis of peanut-induced intestinal allergy are not well defined. OBJECTIVES: We sought to determine the contributions of FcepsilonRI, IL-13, and mast cells to the development of intestinal mucosal responses in a murine model of peanut-induced intestinal allergy. METHODS: Sensitized wild-type (WT), FcepsilonRI-deficient (FcepsilonRI(-/-)), and mast cell-deficient (Kit(W-sh/W-sh)) mice received peanut orally every day for 1 week. Bone marrow-derived mast cells (BMMCs) from WT, FcepsilonRI(-/-), IL-4(-/-), IL-13(-/-), and IL-4/IL-13(-/-) mice were differentiated and transferred into WT, FcepsilonRI(-/-), and Kit(W-sh/W-sh) recipients. BMMCs from WT and UBI-GFP/BL6 mice were differentiated and transferred into WT and Kit(W-sh/W-sh) mice. Blockade of IL-13 was achieved by using IL-13 receptor alpha2 (IL-13Ralpha2)-IgG fusion protein. RESULTS: FcepsilonRI(-/-) mice showed decreased intestinal inflammation (mast cell and eosinophil numbers) and goblet cell metaplasia and reduced levels of IL4, IL6, IL13, and IL17A mRNA expression in the jejunum. Transfer of WT BMMCs to FcepsilonRI(-/-) recipients restored their ability to develop intestinal allergic responses unlike transfer of FcepsilonRI(-/-), IL-13(-/-), or IL-4/IL-13(-/-) BMMCs. FcepsilonRI(-/-) mice exhibited lower IL-13 levels and treatment of WT mice with IL-13 receptor alpha2 prevented peanut-induced intestinal allergy and inflammation. CONCLUSIONS: These data indicate that the development of peanut-induced intestinal allergy is mediated through a mast cell-dependent IgE-FcepsilonRI-IL-13 pathway. Targeting IL-13 might be a potential treatment for IgE-mediated peanut-induced allergic responses in the intestine.