RESUMO
Regulatory T cells (T(reg) cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in T(reg) cell development. Mice that lacked all Nr4a factors could not produce T(reg) cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the T(reg) cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4(+) T cell fates in the thymus and thus contribute to immune homeostasis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Esteroides/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Autoimunidade/genética , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genes Precoces , Homeostase , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Timócitos/metabolismoRESUMO
The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.
Assuntos
Capsídeo , Quirópteros , Suscetibilidade a Doenças , Retroviridae , Animais , Humanos , Camundongos , Austrália , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Quirópteros/virologia , Retroviridae/classificação , Retroviridae/metabolismo , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/virologia , Linhagem Celular , Especificidade da Espécie , Fatores de Restrição Antivirais/metabolismoRESUMO
Colonization with a mixture of Clostridium species has been shown to induce accumulation of induced regulatory T (iTreg) cells in the colon. Transforming growth factor-ß (TGF-ß) is an essential factor for iTreg cell induction; however, the relationship between Clostridium species and TGF-ß remains to be clarified. Here we demonstrated that a gram-positive probiotic bacterial strain, Clostridium butyricum (C. butyricum), promoted iTreg cell generation in the intestine through induction of TGF-ß1 from lamina propria dendritic cells (LPDCs). C. butyricum-mediated TGF-ß1 induction was mainly Toll-like receptor 2 (TLR2) dependent, and the ERK-AP-1 kinase pathway played an important role. In addition, the autocrine TGF-ß-Smad3 transcription factor signal was necessary for robust TGF-ß expression in DCs, whereas Smad2 negatively regulated TGF-ß expression. Smad2-deficient DCs expressed higher concentrations of TGF-ß and were tolerogenic for colitis models. This study reveals a novel mechanism of TGF-ß induction by Clostridia through a cooperation between TLR2-AP-1 and TGF-ß-Smad signaling pathways.
Assuntos
Clostridium butyricum/imunologia , Células Dendríticas/imunologia , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/biossíntese , Animais , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Colite/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/citologia , Mucosa/imunologia , Regiões Promotoras Genéticas/genética , Linfócitos T Reguladores/imunologia , Receptor 2 Toll-Like/imunologia , Fator de Transcrição AP-1/imunologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Peripheral nerve injury to dorsal root ganglion (DRG) neurons develops intractable neuropathic pain via induction of neuroinflammation. However, neuropathic pain is rare in the early life of rodents. Here, we aimed to identify a novel therapeutic target for neuropathic pain in adults by comprehensively analyzing the difference of gene expression changes between infant and adult rats after nerve injury. METHODS: A neuropathic pain model was produced in neonatal and young adult rats by spared nerve injury. Nerve injury-induced gene expression changes in the dorsal root ganglion (DRG) were examined using RNA sequencing. Thymic stromal lymphopoietin (TSLP) and its siRNA were intrathecally injected. T cells were examined using immunofluorescence and were reduced by systemic administration of FTY720. RESULTS: Differences in changes in the transcriptome in injured DRG between infant and adult rats were most associated with immunological functions. Notably, TSLP was markedly upregulated in DRG neurons in adult rats, but not in infant rats. TSLP caused mechanical allodynia in adult rats, whereas TSLP knockdown suppressed the development of neuropathic pain. TSLP promoted the infiltration of T cells into the injured DRG and organized the expressions of multiple factors that regulate T cells. Accordingly, TSLP caused mechanical allodynia through T cells in the DRG. CONCLUSION: This study demonstrated that TSLP is causally involved in the development of neuropathic pain through T cell recruitment.
Assuntos
Neuralgia , Linfopoietina do Estroma do Timo , Ratos , Animais , Gânglios Espinais , Hiperalgesia/etiologia , Linfócitos T , Citocinas , NeurôniosRESUMO
A few long noncoding RNAs (long ncRNAs, lncRNAs) exhibit trophoblast cell type-specific expression patterns and functional roles in mouse placenta. However, the cell- and stage-specific expression patterns and functions of most placenta-derived lncRNAs remain unclear. In this study, we explored mouse placenta-associated lncRNAs using a combined bioinformatic and experimental approach. We used the FANTOM5 database to survey lncRNA expression in mouse placenta and found that 1600012P17Rik (MGI: 1919275, designated P17Rik), a long intergenic ncRNA, was the most highly expressed lncRNA at gestational day 17. Polymerase chain reaction analysis confirmed that P17Rik was exclusively expressed in placenta and not in any of the adult organs examined in this study. In situ hybridization analysis revealed that it was highly expressed in spongiotrophoblast cells and to a lesser extent in glycogen trophoblast cells, including migratory glycogen trophoblast cells invading the decidua. Moreover, we found that it is a polyadenylated lncRNA localized mainly to the cytoplasm of these trophoblast cells. As these trophoblast cells also expressed the neighboring protein-coding gene, pappalysin 2 (Pappa2), we investigated the effects of P17Rik on Pappa2 expression using Pappa2-expressing MC3T3-E1 cells and found that P17Rik transfection increased the messenger RNA (mRNA) and protein levels of Pappa2. These results indicate that mouse placenta-specific lncRNA P17Rik modulates the expression of the neighboring protein-coding gene Pappa2 in spongiotrophoblast and glycogen trophoblast cells of mouse placenta during late gestation.
Assuntos
RNA Longo não Codificante , Trofoblastos , Animais , Feminino , Glicogênio/metabolismo , Hibridização In Situ , Camundongos , Gravidez , Proteína Plasmática A Associada à Gravidez/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Trofoblastos/metabolismoRESUMO
OBJECTIVES: We sought to clarify the presence of radiographic thymus variants using a scoring system, and their association with clinical and immunological features in RA patients. METHODS: A total of 387 RA patients were randomly selected from all patients visiting our department who underwent chest CT scanning, with exclusion of patients with thymoma or thymic cyst, or age < 30 years. Thymus size and attenuation score in axial CT images were quantitatively interpreted and assessed. Associations between immunophenotype data and clinical and serological features were analysed in a subset of patients. RESULTS: Thymic enlargement was found in 76 (19.6%) patients, and a thymus attenuation score ≥ 2 was found in 50 (12.9%) patients. The score was significantly associated with antibodies to ACPA positivity. Thymic enlargement was significantly associated with the proportions of CD4+ effector memory T cells. CONCLUSION: Radiographic thymus variants were frequently observed in RA patients and may reflect an abnormal immune response involved in the pathogenesis of RA.
Assuntos
Artrite Reumatoide/diagnóstico , Autoanticorpos/sangue , Células T de Memória/imunologia , Timoma/diagnóstico , Timo/diagnóstico por imagem , Neoplasias do Timo/diagnóstico , Tomografia Computadorizada por Raios X/métodos , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/complicações , Autoanticorpos/imunologia , Estudos Transversais , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Imunidade Celular , Imunofenotipagem , Masculino , Células T de Memória/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Timoma/complicações , Timoma/imunologia , Neoplasias do Timo/complicações , Neoplasias do Timo/imunologiaRESUMO
Transforming growth factor-ß (TGF-ß) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-ß through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-ß via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-ß in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-ß via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.
Assuntos
Proteína Smad2/imunologia , Proteína Smad3/imunologia , Proteínas com Domínio T/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Sítios de Ligação , Diferenciação Celular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad3/deficiência , Células Th17/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.
Assuntos
Linfócitos B/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , Células Th2/metabolismo , Adolescente , Adulto , Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos B/patologia , Antígenos CD4/biossíntese , Criança , Pré-Escolar , Dermatomiosite/imunologia , Progressão da Doença , Feminino , Humanos , Memória Imunológica , Interleucinas/metabolismo , Masculino , Comunicação Parácrina , Receptores CXCR5/biossíntese , Células Th1/imunologia , Células Th1/patologia , Equilíbrio Th1-Th2 , Células Th17/imunologia , Células Th17/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease accompanied by lymphocyte infiltration into joint synovium. While T cells are considered to be important for its pathogenesis, the features that are the most relevant to disease and how they change after treatment remain unclear. The aim of this study was to clarify the characteristics of T cells in RA, comprehensively. METHODS: We enrolled a total of 311 patients with RA and 73 healthy participants, and carefully classified them by disease state, constructed multiple cohorts and analysed clinical samples from them in a stepwise manner. We performed immunophenotyping with multiple evaluation axes, and two independent transcriptome analyses complementary to each other. RESULTS: We identified that 'effector memory-Tfh' subset was specifically expanded in the peripheral blood (PB) of patients with RA in correlation with disease activity, and reverted after treatment. Besides, we revealed distinct features of T cells in synovial fluid (SF) that the expression of Tfh/Tph-related genes and pro-inflammatory cytokines and chemokines, including CXCL13, were significantly enriched, whereas these phenotype were Th1-like. Finally, we identified specific pathways, such as mTORC1, IL-2-stat5, E2F, cell cycle and interferon-related genes, that were significantly enriched in SF, in particular, as well as PB of untreated patients with RA, and notably, these features reverted after treatment. CONCLUSION: Our multi-dimensional investigation identified disease relevant T-cell subsets and gene signatures deeply involved in pathogenesis of RA. These findings could aid in our understanding of essential roles of T cells in RA and will facilitate to development better diagnostic and therapeutic interventions.
Assuntos
Artrite Reumatoide/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Artrite Reumatoide/genética , Quimiocina CXCL13/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/imunologiaRESUMO
Adoptive T-cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (TSCM ) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM -like cells (iTSCM ) by coculturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9-hDLL1 cells, interleukin (IL)-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iTSCM cells. Epstein-Barr virus-specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein-Barr virus transformed-tumor model mice. Thus, adoptive T-cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.
Assuntos
Imunoterapia Adotiva/métodos , Neoplasias , Células-Tronco/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Humanos , Memória Imunológica , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologiaRESUMO
T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Interleucina-12/imunologia , Interleucinas/imunologia , Fator de Transcrição STAT4/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Interleucina-12/metabolismo , Interleucinas/metabolismo , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT4/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Mucosal immunoglobulin A (IgA) secreted by local plasma cells (PCs) is a critical component of mucosal immunity. Although IgA class switching can occur at mucosal sites, high-affinity PCs are optimally generated in germinal centers (GCs) in a T cell-dependent fashion. However, how CD4(+) helper T cells induce mucosal-homing IgA-PCs remains unclear. Here, we show that transforming growth factor beta1 (TGFbeta1) and interleukin 21 (IL-21), produced by follicular helper T cells (Tfh), synergized to generate abundant IgA-plasmablasts (PBs). In the presence of IL-21, TGFbeta1 promoted naive B cell proliferation and differentiation and overrode IL-21-induced IgG class switching in favor of IgA. Furthermore, TGFbeta1 and IL-21 downregulated CXCR5 while upregulating CCR10 on plasmablasts, enabling their exit from GCs and migration toward local mucosa. This was supported by the presence of CCR10(+)IgA(+)PBs in tonsil GCs. These findings show that Tfh contribute to mucosal IgA. Thus, mucosal vaccines should aim to induce robust Tfh responses.
Assuntos
Quimiotaxia de Leucócito , Imunoglobulina A/metabolismo , Mucosa/imunologia , Plasmócitos/imunologia , Linfócitos T/imunologia , Linfócitos B/imunologia , Diferenciação Celular , Proliferação de Células , Criança , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/classificação , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucinas/fisiologia , Reação em Cadeia da Polimerase , Receptores CCR10/metabolismo , Receptores CXCR5/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Padrões de Referência , Fator de Crescimento Transformador beta1/fisiologia , Regulação para CimaRESUMO
Chitinases are enzymes that cleave chitin, a component of the exoskeleton of many organisms including the house dust mite (HDM). Here we show that knockin mice expressing an enzymatically inactive acidic mammalian chitinase (AMCase), the dominant true chitinase in mouse lung, showed enhanced type 2 immune responses to inhaled HDM. We found that uncleaved chitin promoted the release of IL-33, whereas cleaved chitin could be phagocytosed and could induce the activation of caspase-1 and subsequent activation of caspase-7; this results in the resolution of type 2 immune responses, probably by promoting the inactivation of IL-33. These data suggest that AMCase is a crucial regulator of type 2 immune responses to inhaled chitin-containing aeroallergens.
Assuntos
Asma/imunologia , Asma/prevenção & controle , Asma/parasitologia , Quitinases/imunologia , Modelos Animais de Doenças , Pyroglyphidae/imunologia , Animais , Western Blotting , Quitinases/genética , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Introdução de Genes , Interleucina-33 , Interleucinas/imunologia , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Animais , Vasos Sanguíneos/metabolismo , Proliferação de Células , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , PerfusãoRESUMO
Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.
Assuntos
Manchas Café com Leite/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Neurofibromatose 1/genética , Neurofibromina 1/metabolismo , Mutação Puntual , Proteínas Adaptadoras de Transdução de Sinal , Sistema A de Transporte de Aminoácidos , Manchas Café com Leite/metabolismo , Manchas Café com Leite/fisiopatologia , Fator de Crescimento Epidérmico/metabolismo , Feminino , Genes Reporter , Estudos de Associação Genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neurofibromatose 1/metabolismo , Neurofibromatose 1/fisiopatologia , Neurofibromina 1/química , Neurofibromina 1/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/agonistas , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
OBJECTIVES: Multiomics study was conducted to elucidate the crucial molecular mechanisms of primary Sjögren's syndrome (SS) pathology. METHODS: We generated multiple data set from well-defined patients with SS, which includes whole-blood transcriptomes, serum proteomes and peripheral immunophenotyping. Based on our newly generated data, we performed an extensive bioinformatic investigation. RESULTS: Our integrative analysis identified SS gene signatures (SGS) dysregulated in widespread omics layers, including epigenomes, mRNAs and proteins. SGS predominantly involved the interferon signature and ADAMs substrates. Besides, SGS was significantly overlapped with SS-causing genes indicated by a genome-wide association study and expression trait loci analyses. Combining the molecular signatures with immunophenotypic profiles revealed that cytotoxic CD8 -T cells- were associated with SGS. Further, we observed the activation of SGS in cytotoxic CD8 T cells isolated from patients with SS. CONCLUSIONS: Our multiomics investigation identified gene signatures deeply associated with SS pathology and showed the involvement of cytotoxic CD8 T cells. These integrative relations across multiple layers will facilitate our understanding of SS at the system level.
Assuntos
Epigenômica , Perfilação da Expressão Gênica , Imunofenotipagem , Proteômica , RNA Mensageiro/metabolismo , Síndrome de Sjogren/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas ADAM/genética , Proteínas ADAM/imunologia , Proteínas ADAM/metabolismo , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Biologia Computacional , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Linfócitos T Citotóxicos/metabolismo , TranscriptomaRESUMO
Lipopolysaccharide (LPS)-induced acute lung injury (ALI) is known as a mouse model of acute respiratory distress syndrome; however, the function of T-cell-derived cytokines in ALI has not yet been established. We found that LPS challenge in one lung resulted in a rapid induction of innate-type pro-inflammatory cytokines such as IL-6 and TNF-α, followed by the expression of T-cell-type cytokines, including IL-17, IL-22 and IFN-γ. We discovered that IL-23 is important for ALI, since blockage of IL-23 by gene disruption or anti-IL-12/23p40 antibody treatment reduced neutrophil infiltration and inflammatory cytokine secretion into the bronchoalveolar lavage fluid (BALF). IL-23 was mostly produced from F4/80(+)CD11c(+) alveolar macrophages, and IL-23 expression was markedly reduced by the pre-treatment of mice with antibiotics, suggesting that the development of IL-23-producing macrophages required commensal bacteria. Unexpectedly, among T-cell-derived cytokines, IL-22 rather than IL-17 or IFN-γ played a major role in LPS-induced ALI. IL-22 protein levels were higher than IL-17 in the BALF after LPS instillation, and the major source of IL-22 was memory Th17 cells. Lung memory CD4(+) T cells had a potential to produce IL-22 at higher levels than IL-17 in response to IL-1ß plus IL-23 without TCR stimulation. Our study revealed an innate-like function of the lung memory Th17 cells that produce IL-22 in response to IL-23 and are involved in exaggeration of ALI.
Assuntos
Lesão Pulmonar Aguda/imunologia , Citocinas/imunologia , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Pulmão/imunologia , Células Th17/imunologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Lavagem Broncoalveolar , Citocinas/genética , Imunidade Inata/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Células Th17/patologiaRESUMO
Little is known about the functional differences between the human skin myeloid dendritic cell (DC) subsets, epidermal CD207(+) Langerhans cells (LCs) and dermal CD14(+) DCs. We showed that CD14(+) DCs primed CD4(+) T cells into cells that induce naive B cells to switch isotype and become plasma cells. In contrast, LCs preferentially induced the differentiation of CD4(+) T cells secreting T helper 2 (Th2) cell cytokines and were efficient at priming and crosspriming naive CD8(+) T cells. A third DC population, CD14(-)CD207(-)CD1a(+) DC, which resides in the dermis, could activate CD8(+) T cells better than CD14(+) DCs but less efficiently than LCs. Thus, the human skin displays three DC subsets, two of which, i.e., CD14(+) DCs and LCs, display functional specializations, the preferential activation of humoral and cellular immunity, respectively.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Células de Langerhans/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/imunologia , Epiderme/imunologia , Granzimas/metabolismo , Humanos , Memória Imunológica , Células de Langerhans/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Ativação Linfocitária , Pele/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1(-/-) ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.
Assuntos
Asma/imunologia , Pulmão/imunologia , Linfócitos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Papaína/efeitos adversos , Proteínas Repressoras/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/genética , Asma/genética , Linhagem Celular , Proliferação de Células/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Transcrição GATA3/metabolismo , Células HEK293 , Humanos , Interleucina-13/biossíntese , Interleucina-33 , Interleucina-5/biossíntese , Interleucinas , Pulmão/citologia , Pulmão/patologia , Linfócitos/citologia , MAP Quinase Quinase 1/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Oncogênica p21(ras)/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/imunologiaRESUMO
OBJECTIVES: To conduct a comprehensive quantitative proteomics analysis of novel serum protein biomarkers based on synovitis status associated with matrix metalloproteinase-3 (MMP-3) and to determine the clinical significance of these biomarkers in rheumatoid arthritis (RA). METHODS: Patients with untreated RA (n=28), primary Sjogren's syndrome (pSS) (n=30), and healthy controls (HCs) (n=30) were enrolled for the screening assay. A total of 1128 serum proteins were analyzed using the SOMAscan™ assay. Serum levels of MMP-3 and interleukin (IL)-16 were measured using a latex turbidimetric immunoassay and ELISA at baseline and 12weeks after treatment with methotrexate (MTX) for MTX-naïve RA patients (n=28) or with the biologics tocilizumab (TCZ) (n=7), abatacept (ABT) (n=11) or infliximab (n=22) for MTX-inadequate response (IR) RA patients. Correlation analysis was conducted using Spearman's rank correlation method. RESULTS: Proteomics showed that serum IL-16 levels were most positively correlated with those of MMP-3 (ρ=0.51, p<0.01) and were significantly increased in patients with untreated active RA compared to HCs (p<0.01) or those with pSS (p<0.01). IL-16 levels decreased following treatment in both the MTX-naïve and MTX-IR groups. Regarding clinical response, fluctuations in IL-16 levels were positively associated with changes in clinical indicators, particularly the Clinical Disease Activity Index (ρ=0.89, p<0.01) in the TCZ and ABT-treated group. However, no similar correlation was noted in MMP-3 and acute phase reactants in any groups. CONCLUSIONS: IL-16 was a more effective clinical parameter than MMP-3, C-reactive protein, or erythrocyte sedimentation rate in both MTX-naive and MTX-IR RA patients. IL-16 might be a useful biomarker for evaluating clinical response in RA patients.