RESUMO
T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.
Assuntos
Diferenciação Celular , Interleucina-12 , Linfócitos T Reguladores , Humanos , Interleucina-12/imunologia , Diferenciação Celular/imunologia , Linfócitos T Reguladores/imunologia , Fator de Transcrição STAT4/imunologia , Fator de Transcrição STAT4/genética , Receptores de Interleucina-12/imunologia , Receptores de Interleucina-12/genética , Feminino , MasculinoRESUMO
In kidney transplantation, deteriorated progression of rejection is considered to be a leading course of postoperative mortality. However, the conventional histologic diagnosis is limited in reading the rejection status at the molecular level, thereby triggering mismatched pathogenesis with clinical phenotypes. Here, by applying uniform manifold approximation and projection and Leiden algorithms to 2,611 publicly available microarray datasets of renal transplantation, we uncovered six rejection states with corresponding signature genes and revealed a high-risk (HR) state that was essential in promoting allograft loss. By identifying cell populations from single-cell RNA sequencing data that were associated with the six rejection states, we identified a T-cell population to be the pathogenesis-triggering cells associated with the HR rejection state. Additionally, by constructing gene regulatory networks, we identified that activated STAT4, as a core transcription factor that was regulated by PTPN6 in T cells, was closely linked to poor allograft function and prognosis. Taken together, our study provides a novel strategy to help with the precise diagnosis of kidney allograft rejection progression, which is powerful in investigating the underlying molecular pathogenesis, and therefore, for further clinical intervention.
Assuntos
Transplante de Rim , Insuficiência Renal , Fator de Transcrição STAT4 , Linfócitos T , Aloenxertos , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
Innate lymphoid cells (ILC) play a significant role in the intestinal immune response and T-bet+ CD127+ group 1 cells (ILC1) have been linked to the pathogenesis of human inflammatory bowel disease (IBD). However, the functional importance of ILC1 in the context of an intact adaptive immune response has been controversial. In this report we demonstrate that induced depletion of T-bet using a Rosa26-Cre-ERT2 model resulted in the loss of intestinal ILC1, pointing to a post-developmental requirement of T-bet expression for these cells. In contrast, neither colonic lamina propria (cLP) ILC2 nor cLP ILC3 abundance were altered upon induced deletion of T-bet. Mechanistically, we report that STAT1 or STAT4 are not required for intestinal ILC1 development and maintenance. Mice with induced deletion of T-bet and subsequent loss of ILC1 were protected from the induction of severe colitis in vivo. Hence, this study provides support for the clinical development of an IBD treatment based on ILC1 depletion via targeting T-bet or its downstream transcriptional targets.
Assuntos
Mucosa Intestinal/imunologia , Linfócitos/imunologia , Proteínas com Domínio T/imunologia , Animais , Citrobacter rodentium , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana , Infecções por Enterobacteriaceae/imunologia , Feminino , Imunidade Inata , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Tamoxifeno/farmacologia , Trichinella spiralis , Triquinelose/imunologiaRESUMO
BACKGROUND: Dysregulated receptor tyrosine kinases, such as the mesenchymal-epidermal transition factor (MET), have pivotal role in gliomas. MET and its interaction with the tumor microenvironment have been previously implicated in secondary gliomas. However, the contribution of MET gene to tumor cells' ability to escape immunosurveillance checkpoints in primary gliomas, especially in glioblastoma (GBM), which is a WHO grade 4 glioma with the worst overall survival, is still poorly understood. METHODS: We investigated the relationship between MET expression and glioma microenvironment by using multiomics data and aimed to understand the potential implications of MET in clinical practice through survival analysis. RNA expression data from a total of 1243 primary glioma samples (WHO grades 2-4) were assembled, incorporating The Cancer Genome Atlas, Chinese Glioma Genome Atlas, and GSE16011 data sets. RESULTS: Pearson's correlation test from the three data sets indicated that MET showed a robust correlation with programmed death-ligand 1 (PD-L1) and STAT pathways. Western blot analysis revealed that in GBM cell lines (N33 and LN229), PD-L1 and phosphorylated STAT4 were upregulated by MET activation treatment with hepatocyte growth factor and were downregulated on MET suppression by PLB-1001. Tumor tissue microarray analysis indicated a positive correlation between MET and PD-L1 and macrophage-associated markers. Chromatin immunoprecipitation-PCR assay showed enrichment of STAT4 in the PD-L1 DNA. Transwell co-culture and chemotaxis assays revealed that knockdown of MET in GBM cells inhibited macrophage chemotaxis. Moreover, we performed CIBERSORTx and single-cell RNA sequencing data analysis which revealed an elevated number of macrophages in glioma samples with MET overexpression. Kaplan-Meier survival analysis indicated that activation of the MET/STAT4/PD-L1 pathway and upregulation of macrophages were associated with shorter survival time in patients with primary GBM. CONCLUSIONS: These data indicated that the MET-STAT4-PD-L1 axis and tumor-associated macrophages might enforce glioma immune evasion and were associated with poor prognosis in GBM samples, suggesting potential clinical strategies for targeted therapy combined with immunotherapy in patients with primary GBM.
Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Macrófagos/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT4/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/imunologia , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Transdução de Sinais/imunologia , Evasão TumoralRESUMO
OBJECTIVE: To assess the role of STAT4 activation in driving pathogenic follicular helper T (Tfh) cell secretion of the cytokines interleukin-21 (IL-21) and interferon-γ (IFNγ) in murine and human lupus. METHODS: The effect of STAT4-dependent Tfh cell signaling on cytokine production and autoreactive B cell maturation was assessed temporally during the course of lupus in a murine model, with further assessment of Tfh cell gene transcription performed using RNA-Seq technology. STAT4-dependent signaling and cytokine production were also determined in circulating Tfh-like cells in patients with systemic lupus erythematosus (SLE), as compared to cells from healthy control subjects, and correlations with disease activity were assessed in the Tfh-like cells from SLE patients. RESULTS: IL-21- and IFNγ-coproducing Tfh cells expanded prior to the detection of potentially pathogenic IgG2c autoantibodies in lupus-prone mice. Tfh cells transcriptionally evolved during the course of disease with acquisition of a STAT4-dependent gene signature. Maintenance of Tfh cell cytokine synthesis was dependent upon STAT4 signaling, driven by type I IFNs. Circulating Tfh-like cells from patients with SLE also secreted IL-21 and IFNγ, with STAT4 phosphorylation enhanced by IFNß, in association with the extent of clinical disease activity. CONCLUSION: We identified a role for type I IFN signaling in driving STAT4 activation and production of IL-21 and IFNγ by Tfh cells in murine and human lupus. Enhanced STAT4 activation in Tfh cells may underlie pathogenic B cell responses in both murine and human lupus. These data indicate that STAT4 guides pathogenic cytokine and immunoglobulin production in SLE, demonstrating a potential therapeutic target to modulate autoimmunity.
Assuntos
Autoanticorpos/imunologia , Citocinas/imunologia , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fator de Transcrição STAT4/imunologia , Células T Auxiliares Foliculares/imunologia , Adulto , Animais , Formação de Anticorpos/imunologia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulinas , Interferon gama/imunologia , Interleucinas/imunologia , Masculino , Camundongos Endogâmicos MRL lpr , Pessoa de Meia-Idade , RNA-SeqRESUMO
Natural killer (NK) cells are innate lymphocytes that exhibit adaptive features, such as clonal expansion and memory, during viral infection. Although activating receptor engagement and proinflammatory cytokines are required to drive NK cell clonal expansion, additional stimulatory signals controlling their proliferation remain to be discovered. Here, we describe one such signal that is provided by the adrenergic nervous system, and demonstrate that cell-intrinsic adrenergic signaling is required for optimal adaptive NK cell responses. Early during mouse cytomegalovirus (MCMV) infection, NK cells up-regulated Adrb2 (which encodes the ß2-adrenergic receptor), a process dependent on IL-12 and STAT4 signaling. NK cell-specific deletion of Adrb2 resulted in impaired NK cell expansion and memory during MCMV challenge, in part due to a diminished proliferative capacity. As a result, NK cell-intrinsic adrenergic signaling was required for protection against MCMV. Taken together, we propose a novel role for the adrenergic nervous system in regulating circulating lymphocyte responses to viral infection.
Assuntos
Neurônios Adrenérgicos/imunologia , Infecções por Citomegalovirus/imunologia , Células Matadoras Naturais/imunologia , Transdução de Sinais/imunologia , Animais , Proliferação de Células/fisiologia , Citocinas/imunologia , Memória Imunológica/imunologia , Interleucina-12/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Receptores Adrenérgicos beta 2/imunologia , Fator de Transcrição STAT4/imunologia , Regulação para Cima/imunologiaRESUMO
Innate lymphoid cells (ILC) play a significant immunological role at mucosal surfaces such as the intestine. T-bet-expressing group 1 innate lymphoid cells (ILC1) are believed to play a substantial role in inflammatory bowel disease (IBD). However, a role of T-bet-negative ILC3 in driving colitis has also been suggested in mouse models questioning T-bet as a critical factor for IBD. We report here that T-bet deficient mice had a greater cellularity of NKp46-negative ILC3 correlating with enhanced expression of RORγt and IL-7R, but independent of signaling through STAT1 or STAT4. We observed enhanced neutrophilia in the colonic lamina propria (cLP) of these animals, however, we did not detect a greater risk of T-bet-deficient mice to develop spontaneous colitis. Furthermore, by utilizing an in vivo fate-mapping approach, we identified a population of T-bet-positive precursors in NKp46-negative ILC3s. These data suggest that T-bet controls ILC3 cellularity, but does do not drive a pathogenic role of ILC3 in mice with a conventional specific pathogen-free microbiota.
Assuntos
Imunidade Inata , Mucosa Intestinal/imunologia , Linfócitos/imunologia , Proteínas com Domínio T/imunologia , Animais , Colite/genética , Colite/imunologia , Colite/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Linfócitos/patologia , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Proteínas com Domínio T/genéticaRESUMO
Generation of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). As demonstrated in a number of lupus mouse models, recent evidence suggests that both GC and extrafollicular pathways contribute to the generation of autoantibodies also in human SLE, and that CD11c+ IgD- CD27- (double negative:DN) B cells play a central role in the latter pathway. In this mini-review, the author will first briefly summarize the features of CD11c+ DN B cells in human SLE, and discuss how the IL-12-STAT4 axis might contribute to the generation of autoantibodies in SLE. In addition, various types of CD4+ helper T cell subsets promoting the generation of autoantibodies in SLE will be described, and finally it will be discussed how these recent discoveries contribute to understanding of SLE pathogenesis and treatment of SLE patients.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Interleucina-12/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fator de Transcrição STAT4/imunologia , Animais , Humanos , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Cytokines are critical for guiding the differentiation of T lymphocytes to perform specialized tasks in the immune response. Developing strategies to manipulate cytokine-signaling pathways holds promise to program T cell differentiation toward the most therapeutically useful direction. Suppressor of cytokine signaling (SOCS) proteins are attractive targets, as they effectively inhibit undesirable cytokine signaling. However, these proteins target multiple signaling pathways, some of which we may need to remain uninhibited. SOCS3 inhibits IL-12 signaling but also inhibits the IL-2-signaling pathway. In this study, we use computational protein design based on SOCS3 and JAK crystal structures to engineer a mutant SOCS3 with altered specificity. We generated a mutant SOCS3 designed to ablate interactions with JAK1 but maintain interactions with JAK2. We show that this mutant does indeed ablate JAK1 inhibition, although, unexpectedly, it still coimmunoprecipitates with JAK1 and does so to a greater extent than with JAK2. When expressed in CD8 T cells, mutant SOCS3 preserved inhibition of JAK2-dependent STAT4 phosphorylation following IL-12 treatment. However, inhibition of STAT phosphorylation was ablated following stimulation with JAK1-dependent cytokines IL-2, IFN-α, and IL-21. Wild-type SOCS3 inhibited CD8 T cell expansion in vivo and induced a memory precursor phenotype. In vivo T cell expansion was restored by expression of the mutant SOCS3, and this also reverted the phenotype toward effector T cell differentiation. These data show that SOCS proteins can be engineered to fine-tune their specificity, and this can exert important changes to T cell biology.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Fator de Transcrição STAT4/imunologia , Fator de Transcrição STAT5/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Silenciamento de Genes , Janus Quinase 1/imunologia , Janus Quinase 2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Engenharia de Proteínas , Transdução de SinaisRESUMO
The interleukin (IL)-12/interferon(IFN)γ axis plays an important role in the control of mycobacterial diseases as demonstrated by the increased susceptibility to mycobacterial species in patients with an inborn error of the IL-12-dependent IFNγ immunity. Here, we report a novel mutation in the IL-12Rß1 gene in a female Pakistani patient who was born in a consanguineous marriage and developed severe bacille Calmette-Guérin (BCG) infection and recurrent tuberculosis. After reviewing the patient's clinical records, she was investigated for IL-12/IFNγ defects using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and DNA genetic Sanger sequencing. Quantification of secretory cytokines from the patient's peripheral blood mononuclear cells (PBMCs) revealed significantly reduced IFNγ production. Flow cytometric analysis revealed no surface expression of IL-12Rß1 on PHA-activated T lymphocytes. In addition, IL-12-induced impaired STAT4 phosphorylation in the patient's lymphocytes when compared with those from five healthy controls. The genetic analysis of IL-12Rß1 gene identified a novel nonsense mutation c.199G>T/p.E67* within exon 3, which encodes part of the cytokine-binding region (CBR). In silico analysis indicates that this novel nonsense mutation generates a truncated protein with an apparent inactivating effect. Our data expand the genetic spectrum of IL-12Rß1 deficiency. Moreover, our findings highlight the need for developing newborn screening for patients with primary immunodeficiency associated with mycobacterial infections in areas where BCG vaccination is mandatory in order to improve the treatment of patients, and consequently their quality of life.
Assuntos
Códon sem Sentido , Receptores de Interleucina-12 , Tuberculose , Adulto , Criança , Éxons , Feminino , Humanos , Fosforilação , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/imunologia , Recidiva , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Tuberculose/genética , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
Type 2 reaction (T2R) or erythema nodosum leprosum (ENL), a sudden episode of acute inflammation predominantly affecting lepromatous leprosy patients (LL), characterized by a reduced cellular immune response. This possibly indicates a close relationship between the onset of T2R and the altered frequency, and functional activity of T lymphocytes, particularly of memory subsets. This study performed ex vivo and in vitro characterizations of T cell blood subpopulations from LL patients with or without T2R. In addition, the evaluation of activity of these subpopulations was performed by analyzing the frequency of these cells producing IFN-γ, TNF, and IL-10 by flow cytometry. Furthermore, the expression of transcription factors, for the differentiation of T cells, were analyzed by quantitative real-time polymerase chain reaction. Our results showed an increased frequency of CD8+/TNF+ effector memory T cells (TEM) among T2Rs. Moreover, there was evidence of a reduced frequency of CD4 and CD8+ IFN-γ-producing cells in T2R, and a reduced expression of STAT4 and TBX21. Finally, a significant and positive correlation between bacteriological index (BI) of T2R patients and CD4+/TNF+ and CD4+/IFN-γ+ T cells was observed. Thus, negative correlation between BI and the frequency of CD4+/IL-10+ T cells was noted. These results suggest that CD8+/TNF+ TEM are primarily responsible for the transient alteration in the immune response to Mycobacterium leprae in ENL patients. Thus, our study improves our understanding of pathogenic mechanisms and might suggest new therapeutic approaches for leprosy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Eritema Nodoso/imunologia , Hanseníase Virchowiana/imunologia , Mycobacterium leprae/patogenicidade , Fator de Necrose Tumoral alfa/imunologia , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD8-Positivos/microbiologia , Estudos de Casos e Controles , Eritema Nodoso/genética , Eritema Nodoso/patologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica , Imunofenotipagem , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Hanseníase Virchowiana/genética , Hanseníase Virchowiana/patologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/imunologia , Cultura Primária de Células , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy. METHODS: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry. RESULTS: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients. CONCLUSION: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Fator de Transcrição STAT4/metabolismo , Fator de Transcrição STAT6/metabolismo , Adulto , Antígenos de Bactérias/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Citocinas/metabolismo , Humanos , Hanseníase/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/microbiologia , Pessoa de Meia-Idade , Mycobacterium leprae/patogenicidade , Fator de Transcrição STAT4/imunologia , Fator de Transcrição STAT6/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a prevalent form of cancer with 5-years survival rates around 57%, and metastasis is a leading cause of mortality. Host-derived immunological factors that affect HNSCC tumor development and metastasis are not completely understood. We investigated the role of host-derived signal transducer and activator of transcription 4 (STAT4) during experimental HNSCC using an aggressive and metastatic HNSCC cell line, LY2, which was orthotopically injected into the buccal sulcus of wild type (WT) and STAT4 deficient (Stat4-/-) BALB/c mice. Necropsies performed at terminal sacrifice revealed that Stat4-/- mice displayed comparable primary tumor growth to the WT mice. However, the rate and extent of lymph node and lung metastasis among Stat4-/- mice was significantly higher. Downstream analyses performed on primary tumors, draining lymph nodes, spleens and bone marrow revealed significant upregulation of lymphocytic immunosuppressive biomarkers as well as an accumulation of granulocytic MDSC subpopulations in draining lymph nodes of metastatic Stat4-/- mice. Further, we observed a significant decrease in TH1, TH17, and cytotoxic activity in tumor bearing Stat4-/- compared to WT mice. Our results demonstrate that STAT4 mediates resistance to HNSCC metastasis, and activation of STAT4 could potentially mitigate lymphatic metastasis in HNSCC patients.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Imunidade Celular , Fator de Transcrição STAT4/deficiência , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator de Transcrição STAT4/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Th1/patologia , Células Th17/patologiaRESUMO
Wealth of structural data on theurapeutic targets in complex with monoclonal antibodies (mAbs) and advances in molecular modeling algorithms present exciting opportunities in the field of novel biologic design. Interleukin 23 (IL23), a well-known drug target for autoimmune diseases, in complex with mAb 7G10 offers prospect to design potent lead antibodies by traversing the complete epitope-paratope interface. Herein, key interactions aiding antibody-based neutralization in IL23-7G10 complex are resolute through PyMOL, LigPlot+, Antibody i-Patch, DiscoTope and FoldX. Six amino acids Ser31, Val33, Asn55, Lys59 in heavy chain and His34, Ser93 in light chain are subjected to in silico mutagenesis with residues Met, Trp, Ile, Leu and Arg. A set of 431 mutant macromolecules are outlined. Binding affinities of these molecules with IL23 are estimated through protein-protein docking by employing ZDOCK, ClusPro and RosettaDock. Subsequently, the macromolecules revealed comparable result with 7G10 are cross validated through binding free-energy calculations by applying Molecular Mechanics/Poisson Boltzman Surface Area method in CHARMM. Thirty nine designed theoretical antibodies showed improved outcome in all evaluations; from these, top 10 molecules showed at least nine unit better binding affinity compared to the known mAb. These molecules have the potential to act as lead antibodies. Subsequent molecular dynamics simulations too favored prospective of best ranked molecule to have therapeutic implications in autoimmune and inflammatory diseases. Abbreviations: IL23: interleukin 23; IL17: interleukin17; Ab: antibody; Ag: antigen; mAbs: monoclonal antibodies; STAT3: signal transducer and activator of transcription 3; STAT4: signal transducer and activator of transcription 4; PDB: protein databank; MM/PBSA: molecular mechanics Poisson-Boltzmann surface area; Ag-Ab: antigen- antibody complex; SPC/E: extended simple point charge; SD: steepest descents; PME: particle mesh ewald; dG: binding free energies; Fv: variable fragment.
Assuntos
Anticorpos Monoclonais/imunologia , Doenças Autoimunes/imunologia , Inflamação/imunologia , Interleucina-23/química , Anticorpos Monoclonais/química , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Sítios de Ligação de Anticorpos , Biologia Computacional , Epitopos/química , Epitopos/imunologia , Humanos , Ligação de Hidrogênio , Inflamação/tratamento farmacológico , Inflamação/genética , Interleucina-23/imunologia , Simulação de Dinâmica Molecular , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT4/química , Fator de Transcrição STAT4/imunologiaRESUMO
IL-12 is a pro-inflammatory cytokine that induces the production of interferon-γ (IFNγ) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjögren's syndrome (pSS)(autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (nâ¯=â¯17) and pSS monocytes (nâ¯=â¯6) resulted in significant induction of IL12A mRNA (pâ¯<â¯0.05, pâ¯<â¯0.01, respectively) compared to PSC monocytes (nâ¯=â¯13) and at similar levels to HC monocytes (nâ¯=â¯8). PSC monocytes expressed significantly less IL-12p70 (108â¯pg/ml, mean) and IL-23 (358â¯pg/ml) compared to HC (458â¯pg/ml and 951â¯pg/ml, respectively) (pâ¯<â¯0.01, pâ¯<â¯0.05). Treg cells from patients with PBC (nâ¯=â¯16) and pSS (nâ¯=â¯3) but not PSC (nâ¯=â¯10) and HC (nâ¯=â¯8) responded to low dose (10â¯ng/ml) IL-12 stimulation by significant upregulation of IFNγ (mean 277 and 254â¯pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77â¯pg/ml, respectively)(pâ¯<â¯0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (pâ¯<â¯0.05) but not PSC and HC. In the liver of patients with PBC (nâ¯=â¯7) a significantly higher proportion of IL-12Rß2+Tregs (16% on average) was detected (pâ¯<â¯0.05) compared to other liver disease controls (5%)(nâ¯=â¯18) which also showed ex vivo high IFNG and TBET expression. CONCLUSION: Our data show an increased sensitivity of PBC and pSS Treg cells to low dose IL-12 stimulation, providing ongoing support for the importance of the IL12-IL-12Rß2-STAT4 pathway on Treg cells in disease pathogenesis and potentially treatment.
Assuntos
Colangite Esclerosante/imunologia , Interferon gama/imunologia , Subunidade p35 da Interleucina-12/imunologia , Cirrose Hepática Biliar/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Autoimunidade , Estudos de Casos e Controles , Diferenciação Celular , Colangite Esclerosante/genética , Colangite Esclerosante/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Interferon gama/genética , Interleucina-12/genética , Interleucina-12/imunologia , Subunidade p35 da Interleucina-12/genética , Interleucina-23/genética , Interleucina-23/imunologia , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Fenótipo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/patologia , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Lupus erythematosus (LE) is an autoimmune disease with a broad clinical spectrum ranging from cutaneous lesions to severe systemic manifestations. The pathogenesis of the disease and the immunological mechanisms for the heterogeneities in lupus remain unclear. The LE-specific cutaneous manifestations are generally divided into three categories: acute cutaneous LE (ACLE), subacute cutaneous LE (SCLE) and chronic cutaneous lupus erythematosus (CCLE). Clinically, lupus patients with skin lesions can be divided into two subsets based on the organs involved: cutaneous LE, such as DLE and SCLE, which appears only as a skin manifestation, and systemic lupus erythematosus (SLE), e.g., ACLE, which involves other organs, such as kidneys, joints, and the hematopoietic system. However, lupus is an aggressive disease, and cutaneous lupus and systemic lupus partially overlap. Fewer than 5% of DLE patients and approximately 50% of SCLE patients might develop major organ damage and then develop SLE in subsequent years. Furthermore, there are no predictive biomarkers in clinical use. To the best of our knowledge, increasing evidence from clinical trials has revealed that early intervention can either reduce or delay the onset of severe manifestations. Therefore, identification of certain biomarkers in skin lesions or circulation from patients with skin lesions to predict future flares and advise treatment is an unmet need. In this review, we comprehensively describe the subtypes of LE with pathological criteria and clinical manifestations; summarize the up-to-date evidence on certain cell distributions, such as keratinocytes, neutrophils, dendritic cells, T cells and B cells, in skin and peripheral blood; and discuss their pathogenic roles and their potential roles in predictive diagnosis and as therapeutic targets.
Assuntos
Linfócitos B/patologia , Queratinócitos/patologia , Lúpus Eritematoso Discoide/patologia , Lúpus Eritematoso Sistêmico/patologia , Pele/patologia , Linfócitos T/patologia , Corticosteroides/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Ensaios Clínicos como Assunto , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Rim/efeitos dos fármacos , Rim/imunologia , Rim/patologia , Lúpus Eritematoso Discoide/genética , Lúpus Eritematoso Discoide/imunologia , Lúpus Eritematoso Discoide/terapia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/terapia , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Pele/efeitos dos fármacos , Pele/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
OBJECTIVES: T follicular helper (Tfh) cells are critical in the development and progression of systemic lupus erythematosus (SLE). To assess the characteristics and mechanisms of differentiation of Tfh cells, we investigated the phenotype of T helper cells in patients with SLE and underlying epigenetic modifications by cytokine-induced signal transducer and activators of transcription (STAT) family factors. METHODS: Peripheral blood mononuclear cells from patients and healthy donors were analysed by flow cytometry. CD4+ T cells were isolated and cultured under various stimulations. Expression of characteristic markers and phosphorylation of STATs were analysed by flow cytometry and quantitative PCR. Histone modifications were analysed by chromatin immunoprecipitation (ChIP)-PCR. RESULTS: Differentiation of CD4+CXCR5+CXCR3+Bcl-6+T-bet+IL-21+IFN-γ+Tfh-Th1-like cells was induced by interleukin (IL)-12-induced activation of STAT1 and STAT4 simultaneously. The loci of Bcl-6 and T-bet at STAT binding sites were marked by bivalent histone modifications. After IL-12 stimulation, both STAT1 and STAT4 directly bound on BCL6 and TBX21 gene loci accompanied by suppression of repressive histone mark trimethylated histone 3 lysine 27. Levels of serum IL-12 and interferon (IFN)-γ, expression of IL-12 receptors and proportion of CXCR5+CXCR3+ activated Tfh-Th1-like cells were increased in patients with SLE. Furthermore, the level of pSTAT1, pSTAT4 and T-bet were higher in activated Tfh-Th1-like cells than non-Tfh-Th1 cells. CONCLUSION: Our findings suggest that IL-12-mediated co-activation of STAT1 and STAT4 alters histone modification, resulting in differentiation of Tfh-Th1-like cells that are characteristically expanded in patients with SLE. This could be one of the underlying mechanisms responsible for expansion of Tfh-Th1-like cells and potentially helpful towards development of cell-specific treatment for SLE.
Assuntos
Epigênese Genética/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Transcrição STAT/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Humanos , Imunofenotipagem , Interleucina-12/imunologia , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Fosforilação/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT4/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
A series of natural compounds have been implicated to be useful in regulating the pathogenesis of various autoimmune diseases. The present study demonstrated that the Scutellariae radix compounds baicalein and baicalin may serve as drugs for the treatment of autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease. Following the administration of baicalein and baicalin in vivo, T cellmediated autoimmune diseases in the mouse model were profoundly ameliorated: In the collageninduced arthritis model (CIA), the severity of the disease was reduced by baicalein and, consistently, baicalein was demonstrated to suppress T cell proliferation in CIA mice. In the dextran sodium sulfate (DSS)induced colitis model, the disease was attenuated by baicalin, and baicalin promoted colon epithelial cell (CEC) proliferation in vitro. The present study further revealed that the mRNA expression of signal transducer and activator of transcription (STAT)3 and STAT4 in the tyrosineprotein kinase JAKSTAT signaling pathway in T cells was downregulated by baicalein, contributing to its regulation of T cell proliferation. However, in the DSS model, the STAT4 transcription in CECs, which are the target cells of activated T cells in the gut, was downregulated by baicalin, suggesting that baicalein and baicalin mediated similar STAT expression in different cell types in autoimmune diseases. In conclusion, the similarly structured compounds baicalein and baicalin selectively exhibited therapeutic effects on autoimmune diseases by regulating cell proliferation and STAT gene expression, albeit in different cell types.
Assuntos
Artrite Experimental/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Colite/tratamento farmacológico , Flavanonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Sulfato de Dextrana/toxicidade , Masculino , Camundongos , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT4/imunologia , Transdução de Sinais/imunologiaRESUMO
The T-box transcription factors T-bet and Eomesodermin (Eomes) instruct discrete stages in NK cell development. However, their role in the immune response of mature NK cells against pathogens remains unexplored. We used an inducible deletion system to elucidate the cell-intrinsic role of T-bet and Eomes in mature NK cells during the course of mouse CMV infection. We show both T-bet and Eomes to be necessary for the expansion of virus-specific NK cells, with T-bet upregulation induced by IL-12 signaling and STAT4 binding to a conserved enhancer region upstream of the Tbx21 loci. Interestingly, our data suggest maintenance of virus-specific memory NK cell numbers and phenotype was dependent on T-bet, but not Eomes. These findings uncover a nonredundant and stage-specific influence of T-box transcription factors in the antiviral NK cell response.
Assuntos
Memória Imunológica/imunologia , Células Matadoras Naturais/imunologia , Proteínas com Domínio T/imunologia , Animais , Infecções por Citomegalovirus/imunologia , Interleucina-12/imunologia , Camundongos , Fator de Transcrição STAT4/imunologia , Regulação para Cima/imunologiaRESUMO
Follicular helper T (Tfh) cells promote germinal center (GC) B cell survival and proliferation and guide their differentiation and immunoglobulin isotype switching by delivering contact-dependent and soluble factors, including IL-21, IL-4, IL-9, and IFN-γ. IL-21 and IFN-γ are coexpressed by Tfh cells during viral infections, but transcriptional regulation of these cytokines is not completely understood. In this study, we show that the T helper type 1 cell (Th1 cell) transcriptional regulators T-bet and STAT4 are coexpressed with Bcl6 in Tfh cells after acute viral infection, with a temporal decline in T-bet in the waning response. T-bet is important for Tfh cell production of IFN-γ, but not IL-21, and for a robust GC reaction. STAT4, phosphorylated in Tfh cells upon infection, is required for expression of T-bet and Bcl6 and for IFN-γ and IL-21. These data indicate that T-bet is expressed with Bcl6 in Tfh cells and is required alongside STAT4 to coordinate Tfh cell IL-21 and IFN-γ production and for promotion of the GC response after acute viral challenge.