RESUMO
Helper T (Th) cell subsets direct immune responses by producing signature cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular analysis of Th2 cell differentiation and maintenance of function has led to recent discoveries that have refined our understanding of Th2 cell biology. Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell identity. In the context of allergic diseases, memory-type pathogenic Th2 cells have been identified in both mice and humans. To better understand these disease-driving cell populations, we have developed a model called the pathogenic Th population disease induction model. The concept of defined subsets of pathogenic Th cells may spur new, effective strategies for treating intractable chronic inflammatory disorders.
Assuntos
Helmintíase/imunologia , Hipersensibilidade/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Epigênese Genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imunidade Humoral , Memória Imunológica , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA/genética , Distonia/genética , Feminino , Fator C1 de Célula Hospedeira/metabolismo , Proteínas Mad2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismoRESUMO
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Elementos Facilitadores Genéticos/genética , Neurônios/metabolismo , 5-Metilcitosina/metabolismo , Linhagem Celular , DNA/biossíntese , Replicação do DNA , Humanos , Masculino , Metilação , Poli(ADP-Ribose) Polimerases/metabolismo , Análise de Sequência de DNARESUMO
Mounting evidence suggests that nematode infection can protect against disorders of immune dysregulation. Administration of live parasites or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for immune disorders, including asthma. Human clinical trials of live parasite ingestion for the treatment of immune disorders have produced promising results, yet concerns persist regarding the ingestion of pathogenic organisms and the immunogenicity of protein components. Despite extensive efforts to define the active components of ES products, no small molecules with immune regulatory activity have been identified from nematodes. Here we show that an evolutionarily conserved family of nematode pheromones called ascarosides strongly modulates the pulmonary immune response and reduces asthma severity in mice. Screening the inhibitory effects of ascarosides produced by animal-parasitic nematodes on the development of asthma in an ovalbumin (OVA) murine model, we found that administration of nanogram quantities of ascr#7 prevented the development of lung eosinophilia, goblet cell metaplasia, and airway hyperreactivity. Ascr#7 suppressed the production of IL-33 from lung epithelial cells and reduced the number of memory-type pathogenic Th2 cells and ILC2s in the lung, both key drivers of the pathology of asthma. Our findings suggest that the mammalian immune system recognizes ascarosides as an evolutionarily conserved molecular signature of parasitic nematodes. The identification of a nematode-produced small molecule underlying the well-documented immunomodulatory effects of ES products may enable the development of treatment strategies for allergic diseases.
Assuntos
Inflamação/prevenção & controle , Nematoides/química , Traqueia/efeitos dos fármacos , Animais , Asma/fisiopatologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Hipersensibilidade/fisiopatologia , Inflamação/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Nematoides/patogenicidade , Ovalbumina/efeitos adversos , Bibliotecas de Moléculas Pequenas/farmacologia , Traqueia/fisiopatologiaRESUMO
Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-ß and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T(H)2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T(H)2 cytokine, and prevented binding of GATA-3 to this promoter. T(H)2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-ß to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.
Assuntos
Fator de Transcrição GATA3/metabolismo , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Células Th2/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Fator de Transcrição GATA3/biossíntese , Interferon gama/biossíntese , Interleucina-5/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pneumonia/imunologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Células Th2/imunologia , Células Th2/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Memory CD4(+) T helper (Th) cells provide long-term protection against pathogens and are essential for the development of vaccines; however, some antigen-specific memory Th cells also drive immune-related pathology, including asthma. The mechanisms regulating the pathogenicity of memory Th cells remain poorly understood. We found that interleukin-33 (IL-33)-ST2 signals selectively licensed memory Th2 cells to induce allergic airway inflammation via production of IL-5 and that the p38 MAP kinase pathway was a central downstream target of IL-33-ST2 in memory Th2 cells. In addition, we found that IL-33 induced upregulation of IL-5 by memory CD4(+) T cells isolated from nasal polyps of patients with eosinophilic chronic rhinosinusitis. Thus, IL-33-ST2-p38 signaling appears to directly instruct pathogenic memory Th2 cells to produce IL-5 and induce eosinophilic inflammation.
Assuntos
Asma/imunologia , Interleucina-5/imunologia , Interleucinas/imunologia , Receptores de Interleucina/imunologia , Células Th2/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Asma/patologia , Células Cultivadas , Humanos , Memória Imunológica/imunologia , Inflamação/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucina-5/biossíntese , Interleucinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pólipos Nasais/imunologia , Eosinofilia Pulmonar/imunologia , Interferência de RNA , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina/genética , Sinusite/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.
Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Complexo Repressor Polycomb 2/fisiologia , Subpopulações de Linfócitos T/citologia , Células Th1/citologia , Células Th2/citologia , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Fator de Transcrição GATA3/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Memória Imunológica , Testes de Liberação de Interferon-gama , Linfocinas/biossíntese , Linfocinas/genética , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Distant metastasis is the most important prognostic factor for head and neck cancer. This report presents the case of a 50-year-old man with distant metastasis of tongue carcinoma to the vastus lateralis muscle which presented to Nihon University Itabashi Hospital, Tokyo, Japan. Tumourectomy was performed with a diagnosis of tongue carcinoma (cT2N0M0, Stage II). Seven months later, radical neck dissection was performed for lymph node metastasis to a left supraclavicular lymph node. In addition, metastasis was then detected outside the neck dissection region. Tumourectomy and radiotherapy (50 Gy) were, therefore, added to the treatment regimen. However, left-sided vastus lateralis muscle metastasis was then observed. To the best of our knowledge, this is the first report of distant metastasis of oral squamous cell carcinoma to the vastus lateralis muscle.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias da Língua , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Humanos , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Esvaziamento Cervical , Estadiamento de Neoplasias , Músculo Quadríceps , Língua , Neoplasias da Língua/patologia , Neoplasias da Língua/cirurgiaRESUMO
BACKGROUND: Natural killer T (NKT) cells express a T-cell receptor that recognizes endogenous and environmental glycolipid antigens. Several subsets of NKT cells have been identified, including IFN-γ-producing NKT1 cells, IL-4-producing NKT2 cells, and IL-17-producing NKT17 cells. However, little is known about the factors that regulate their differentiation and respective functions within the immune system. OBJECTIVE: We sought to determine whether the polycomb repressive complex 2 protein enhancer of zeste homolog 2 (Ezh2) restrains pathogenicity of NKT cells in the context of asthma-like lung disease. METHODS: Numbers of invariant natural killer T (iNKT) 1, iNKT2, and iNKT17 cells and tissue distribution, cytokine production, lymphoid tissue localization, and transcriptional profiles of iNKT cells from wild-type and Ezh2 knockout (KO) iNKT mice were determined. The contribution of NKT cells to development of spontaneous and house dust mite-induced airways pathology, including airways hyperreactivity (AHR) to methacholine, was also assessed in wild-type, Ezh2 KO, and Ezh2 KO mice lacking NKT cells. RESULTS: Ezh2 restrains development of pathogenic NKT cells, which induce spontaneous asthma-like disease in mice. Deletion of Ezh2 increased production of IL-4 and IL-13 and induced spontaneous AHR, lung inflammation, mucus production, and IgE. Increased IL-4 and IL-13 levels, AHR, lung inflammation, and IgE levels were all dependent on iNKT cells. In house dust mite-exposed animals Ezh2 KO resulted in enhanced AHR that was also dependent on iNKT cells. CONCLUSION: Ezh2 is a central regulator of iNKT pathogenicity and suppresses the ability of iNKT cells to induce asthma-like pathology.
Assuntos
Asma/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Pulmão/imunologia , Células T Matadoras Naturais/imunologia , Animais , Asma/genética , Asma/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/patologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/imunologiaRESUMO
The anaerobic ammonium oxidation (anammox) process holds great promise for treating nitrogen-contaminated water; stable nitrite-nitrogen (NO2 --N) production is significant to anammox performance. In this study, partial hydrogenotrophic denitrification (PHD) was used to stably and efficiently produce NO2 --N from nitrate-nitrogen (NO3 --N). An investigation of the effects of initial pH on the PHD process revealed that a high NO2 --N production efficiency (77.9%) could be ensured by setting an initial pH of 10.5. A combined PHD-anammox process was run for more than three months with maximal ammonium-nitrogen (NH4 +-N), NO3 --N, and total dissolved inorganic nitrogen removal efficiencies of 93.4, 98.0, and 86.9%, respectively. The NO2 --N to NH4 +-N and NO3 --N to NH4 +-N ratios indicated that various bioprocesses were involved in nitrogen removal during the anammox stage, and a 16S rRNA gene amplicon sequencing was performed to further clarify the composition of microbial communities and mechanisms involved in the nitrogen removal process.
Assuntos
Desnitrificação , Nitrogênio , Reatores Biológicos , Oxirredução , RNA Ribossômico 16S/genéticaRESUMO
Memory CD4(+) T helper (Th) cells are central to long-term protection against pathogens, but they can also be pathogenic and drive chronic inflammatory disorders. How these pathogenic memory Th cells are maintained, particularly at sites of local inflammation, remains unclear. We found that ectopic lymphoid-like structures called inducible bronchus-associated lymphoid tissue (iBALT) are formed during chronic allergic inflammation in the lung, and that memory-type pathogenic Th2 (Tpath2) cells capable of driving allergic inflammation are maintained within the iBALT structures. The maintenance of memory Th2 cells within iBALT is supported by Thy1(+)IL-7-producing lymphatic endothelial cells (LECs). The Thy1(+)IL-7-producing LECs express IL-33 and T-cell-attracting chemokines CCL21 and CCL19. Moreover, ectopic lymphoid structures consisting of memory CD4(+) T cells and IL-7(+)IL-33(+) LECs were found in nasal polyps of patients with eosinophilic chronic rhinosinusitis. Thus, Thy1(+)IL-7-producing LECs control chronic allergic airway inflammation by providing a survival niche for memory-type Tpath2 cells.
Assuntos
Células Endoteliais/fisiologia , Rinite Alérgica/imunologia , Sinusite/imunologia , Estruturas Linfoides Terciárias/imunologia , Animais , Sobrevivência Celular , Interleucina-7/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estruturas Linfoides Terciárias/patologia , Células Th2/imunologia , Antígenos Thy-1/metabolismoRESUMO
Nitrate removal during anaerobic ammonium oxidation (anammox) treatment is a concern for optimization of the anammox process. This study demonstrated the applicability and long-term stability of the coupled anammox and hydrogenotrophic denitrification (CAHD) process as an alternative method for nitrate removal. Laboratory-scale fixed bed anammox reactors (FBR) supplied with H2 to support denitrification were operated under two types of synthetic water. The FBRs showed simultaneous NH4-N and NO3-N removal, indicating that the CAHD process can support NO3-N removal during the anammox process. Intermittent H2 supply (e.g. 5 mL/min for a 1-L reactor, 14/6-min on/off cycle) helped maintain the CAHD process without deteriorating its performance under long-term operation and resulted in a nitrogen removal rate of 0.21 kg-N/m3/d and ammonium, nitrate, and dissolved inorganic nitrogen removal efficiencies of 73.4%, 80.4%, and 77%, respectively. The microbial community structure related to the CAHD process was not influenced by changes in influent water quality, and included the anammox bacteria 'Candidatus Jettenia' and a Sulfuritalea hydrogenivorans-like species as the dominant bacteria even after long-term reactor operation, suggesting that these bacteria are key to the CAHD process. These results indicate that the CAHD process is a promising method for enhancing the efficiency of anammox process.
Assuntos
Compostos de Amônio/metabolismo , Desnitrificação , Nitrogênio/metabolismo , Eliminação de Resíduos Líquidos/métodos , Reatores Biológicos , Oxirredução , Águas ResiduáriasRESUMO
Epigenetic modifications, such as posttranslational modifications of histones, play an important role in gene expression and regulation. These modifications are in part mediated by the Trithorax group (TrxG) complex and the Polycomb group (PcG) complex, which activate and repress transcription, respectively. We herein investigate the role of Menin, a component of the TrxG complex in T helper (Th) cell differentiation and show a critical role for Menin in differentiation and maintenance of Th17 cells. Menin(-/-) T cells do not efficiently differentiate into Th17 cells, leaving Th1 and Th2 cell differentiation intact in in vitro cultures. Menin deficiency resulted in the attenuation of Th17-induced airway inflammation. In differentiating Th17 cells, Menin directly bound to the Il17a gene locus and was required for the deposition of permissive histone modifications and recruitment of the RNA polymerase II transcriptional complex. Interestingly, although Menin bound to the Rorc locus, Menin was dispensable for the induction of Rorc expression and permissive histone modifications in differentiating Th17 cells. In contrast, Menin was required to maintain expression of Rorc in differentiated Th17 cells, indicating that Menin is essential to stabilize expression of the Rorc gene. Thus, Menin orchestrates Th17 cell differentiation and function by regulating both the induction and maintenance of target gene expression.
Assuntos
Asma/imunologia , Epigênese Genética/imunologia , Interleucina-17/imunologia , Proteínas Proto-Oncogênicas/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cromatina/imunologia , Cromatina/metabolismo , Epigênese Genética/genética , Regulação da Expressão Gênica/imunologia , Histona-Lisina N-Metiltransferase/imunologia , Histona-Lisina N-Metiltransferase/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/imunologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ovalbumina/imunologia , Ovalbumina/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/imunologia , RNA Polimerase II/metabolismo , Células Th17/metabolismoRESUMO
Immunological memory is an important protective mechanism that enables host organisms to respond rapidly and vigorously to pathogens that have been previously encountered. In addition to the protective function, memory CD4+ T helper (Th) cells play a central role in the pathogenesis of chronic inflammatory disorders, including asthma. Recently, several investigators have identified phenotypically and functionally distinct memory Th2 cell subsets that produce IL-5. These memory Th2 cell subsets play an important role in the pathology of allergic inflammation and function as memory-type "pathogenic Th2 (Tpath2) cells" both in mice and humans. We review the role of lung Tpath2 cells in the development of allergic inflammation and, in the context of recent findings, propose a mechanism by which Tpath2 cells not only survive but also continue to function at the sites where antigens were encountered. A greater understanding of the functional molecules or signaling pathways that regulate the inflammatory niche for Tpath2 cells may aid in the design of more effective treatments for chronic inflammatory disorders.
Assuntos
Alérgenos/imunologia , Hipersensibilidade/imunologia , Células Th2/imunologia , Animais , Comunicação Celular , Doença Crônica , Células Endoteliais/metabolismo , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/metabolismo , Memória Imunológica , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/metabolismoRESUMO
GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , DNA Helicases/imunologia , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/imunologia , Complexos Multiproteicos/imunologia , Células Th2/citologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Bromodesoxiuridina , Imunoprecipitação da Cromatina , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Immunoblotting , Imunoprecipitação , Luciferases , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25-hydroxyvitamin D3 [25(OH)D3 ] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson-type reagents, 4-(4-diethylaminophenyl)-1,2,4-triazoline-3,5-dione (DEAPTAD) and 4-(6-quinolyl)-1,2,4-triazoline-3,5-dione, were designed and synthesized, then compared with the previous reagents, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) and 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D3 in neonatal blood samples by liquid chromatography/electrospray ionization-tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD-derivatized 25(OH)D3 was 60 and 2 times lower than those of the intact 25(OH)D3 and the PTAD derivative, respectively. 25(OH)D3 was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D3 from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 . By using DEAPTAD, a trace amount of 25(OH)D3 in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D3 in neonatal blood samples. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Calcifediol/sangue , Cromatografia Líquida/métodos , Indicadores e Reagentes/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Recém-NascidoRESUMO
To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4(+) T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ-producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Memória Imunológica/imunologia , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Animais , Antígenos CD1d/genética , Glicolipídeos/farmacologia , Memória Imunológica/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Knockout , Sulfoglicoesfingolipídeos/farmacologia , Células Th2RESUMO
Memory T-helper (Th) lymphocytes are crucial for the maintenance of acquired immunity to eliminate infectious pathogens. We have previously demonstrated that most memory Th lymphocytes reside and rest on stromal niches of the bone marrow (BM). Little is known, however, regarding the molecular basis for the generation and maintenance of BM memory Th lymphocytes. Here we show that CD69-deficient effector CD4 T lymphocytes fail to relocate into and persist in the BM and therefore to differentiate into memory cells. Consequently, CD69-deficient CD4 T cells fail to facilitate the production of high-affinity antibodies and the generation of BM long-lived plasma cells in the late phase of immune responses. Thus, CD69 is critical for the generation and maintenance of professional memory Th lymphocytes, which can efficiently help humoral immunity in the late phase. The deficit of immunological memory in CD69-deficient mice also highlights the essential role of BM for the establishment of Th memory.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Memória Imunológica/imunologia , Lectinas Tipo C/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Microscopia Confocal , Células Estromais/imunologia , Células Estromais/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
The determination of the urinary vitamin D3 metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D3 metabolites, 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, (2)H4-DAPTAD (d-DAPTAD). The urine samples were treated with ß-glucuronidase, purified with an Oasis hydrophilic-lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n = 5) and inter-assay (n = 3) relative standard deviations did not exceed 9.5%]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D3 and 24,25(OH)2D3 levels due to vitamin D3 administration.
Assuntos
Calcifediol/urina , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Vitamina D/análogos & derivados , Humanos , Isótopos/análise , Limite de Detecção , Espectrometria de Massas em Tandem/métodos , Vitamina D/urinaRESUMO
CD4 T cells play a key role in immunological memory. We have demonstrated that professional memory CD4 T cells reside and rest in the bone marrow (BM). However, the molecular mechanisms of their establishment in the BM and their maintenance remain unclear. We here show that memory CD4 T cells express high levels of CD49b and that CD49b-deficient or -blocked memory CD4 T-cell precursors fail to migrate from blood into the marrow of the bone, and they especially fail to transmigrate through sinusoidal endothelial cells of the BM. In the marrow, memory CD4 T cells and the precursors contact stromal cells expressing collagen II that are specific ligands for CD49b. Interestingly, memory CD4 T cells on day 117 of an immune response also dock on IL-7(+)/collagen XI(+) stromal cells, whereas memory precursors on day 12 do not. These results indicate that the collagen receptor CD49b is required for the migration of memory CD4 T-cell precursors into their survival niches of the bone marrow.