RESUMO
CD8 T cells play a crucial role in immune responses to virus infections and tumors. Naïve CD8 T lymphocytes after TCR stimulation undergo differentiation into CTLs and memory cells, which are essential sources of IFN-γ. We investigated IFN-γ production by CD8 T cell subsets found in nonimmune mice. A minor fraction of in vitro TCR-stimulated CD8 T cells produce IFN-γ, and it is regulated at the transcriptional level. Antigen inexperienced C57BL/6 mice present the coexistence of 2 populations. The main population exhibits a CD44low CD122low profile, which is compatible with naïve lymphocytes. The minor expresses a phenotype of immunologic memory, CD44hi CD122hi . Both subsets are able to produce IL-2 in response to TCR activation, but only the memory-like population is responsible for IFN-γ production. Similar to memory CD8 T cells, CD44hi CD8+ T cells also present a higher level of the transcriptional factor Eomes and a lower level of T-bet (Tbx21) mRNA than CD44low CD8+ T cells. The presence of the CD44hi CD8+ T cell population in nonimmune OT-I transgenic mice reveals that the population is generated independently of antigenic stimulation. CpG methylation is an efficient epigenetic mechanism for gene silencing. DNA methylation at posttranscriptional CpG sites in the Ifng promoter is higher in CD44low CD8+ T cells than in CD44hi CD8+ T cells. Thus, memory-like CD8 T cells have a distinct epigenetic pattern in the Ifng promoter and can rapidly produce IFN-γ in response to TCR stimulation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Interferon gama/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Ilhas de CpG/imunologia , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Interferon gama/genética , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologiaRESUMO
UNLABELLED: DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands. Unmethylated CpGs are related to transcriptionally active structure, whereas methylated CpG recruits methyl-binding proteins that promote chromatin compaction. DNA methylation can influence the expression of cytokines and affect the development of periodontal disease. OBJECTIVES: The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-γ) and interleukin-10 (IL-10) genes in periodontal tissues. DESIGN: Methylation-specific polymerase chain reaction (MSP) and DNA sequencing analysis were used to verify the DNA methylation status of the IFN-γ and IL-10 genes, respectively, in samples from subjects without periodontitis and individuals with chronic periodontitis. Histological sections stained by hematoxylin-eosin were used for histopathological evaluation of samples. RESULTS: The methylation status of the IFN-γ and IL-10 genes was similar among the groups. Most of the samples were positive for IFN-γ methylation. Only 11% of the periodontitis group showed unmethylated DNA. Considering the IL-10 gene, no unmethylated sample was observed. The profile of total or partial methylation was detected in CpGs evaluated. CONCLUSIONS: The results showed evidence that methylation of IFN-γ and IL-10 genes is a usual feature on periodontal tissues. Further studies are needed to determine the functional relevance of these alterations.
Assuntos
Periodontite Crônica , Metilação de DNA/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Transcrição Gênica/genética , Sequência de Bases , Estudos de Casos e Controles , Cromatina/genética , Cromatina/imunologia , Cromatina/metabolismo , Periodontite Crônica/genética , Periodontite Crônica/imunologia , Periodontite Crônica/metabolismo , Ilhas de CpG/imunologia , Citosina/metabolismo , Humanos , Interferon gama/genética , Interleucina-10/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/imunologia , Análise de Sequência de DNA , Transcrição Gênica/imunologiaRESUMO
TCR signaling leads to the activation of kinases such as inducible tyrosine kinase (Itk), a key regulatory protein in T-lymphocyte activation and function. The homolog of Itk in B cells is Bruton's tyrosine kinase, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was twofold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of WT, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itk's kinase, pleckstrin-homology, and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T-cell function.
Assuntos
Linfócitos B/imunologia , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição TFII/metabolismo , Animais , Linfócitos B/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Genes fos/genética , Genes fos/imunologia , Humanos , Células Jurkat , Leucossialina/imunologia , Leucossialina/metabolismo , Camundongos , Fosforilação/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Linfócitos T/metabolismoRESUMO
Leprosy is a complex infectious disease influenced by genetic and environmental factors. The genetic contributing factors are considered heterogeneous and several genes have been consistently associated with susceptibility like PARK2, tumor necrosis factor (TNF), lymphotoxin-alpha (LTA) and vitamin-D receptor (VDR). Here, we combined a case-control study (374 patients and 380 controls), with meta-analysis (5 studies; 2702 individuals) and biological study to test the epidemiological and physiological relevance of the interleukin-10 (IL-10) genetic markers in leprosy. We observed that the -819T allele is associated with leprosy susceptibility either in the case-control or in the meta-analysis studies. Haplotypes combining promoter single-nucleotide polymorphisms also implicated a haplotype carrying the -819T allele in leprosy susceptibility (odds ratio (OR)=1.40; P=0.01). Finally, we tested IL-10 production in peripheral blood mononuclear cells stimulated with Mycobacterium leprae antigens and found that -819T carriers produced lower levels of IL-10 when compared with non-carriers. Taken together, these data suggest that low levels of IL-10 during the disease outcome can drive patients to a chronic and unprotective response that culminates with leprosy.
Assuntos
Predisposição Genética para Doença/genética , Interleucina-10/genética , Hanseníase/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Antígenos de Bactérias/imunologia , Estudos de Casos e Controles , Doença Crônica , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Marcadores Genéticos/genética , Marcadores Genéticos/imunologia , Predisposição Genética para Doença/epidemiologia , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Hanseníase/epidemiologia , Hanseníase/imunologia , Hanseníase/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Epidemiologia Molecular/métodos , Mycobacterium leprae/imunologia , Regiões Promotoras Genéticas/imunologiaRESUMO
In populations exposed to Leishmania braziliensis, certain subjects develop skin ulcers, whereas others are naturally protected against cutaneous leishmaniasis. We have evaluated which cytokines are most crucial in the development of skin lesions. We found that active lesions occur in subjects with polarized Th2 or mixed Th1/Th2 responses, both associated with elevated IL-10 production. IL-10 was strongly associated (p = 0.004, odd ratio (OR) = 6.8, confidence interval = 1.9-25) with lesions, excluding IFN-gamma, IL-12, TNF, IL-13, and IL-4 from the regression model. IL-10 was produced by blood monocytes and CD4(+)CD25(+) T lymphocytes (mostly Foxp3(+)). However, we did not observe any difference between the number of these cells present in the blood of subjects with active lesions and those present in resistant subjects. Genetic analysis of the IL10-819C/T polymorphism, located in the IL10 promoter, showed that the C allele increased the risk of lesions (OR = 2.5 (1.12-5.7), p = 0.003). Functional analysis of these variants showed allele-specific binding of nuclear factors. The IL10-819C/C genotype was associated with higher levels of IL-10 than C/T and T/T genotypes. These observations demonstrate an important role for IL-10 in skin lesions in humans infected with L. braziliensis, and identify circulating monocytes and Tregs as principal sources of IL-10 in these patients.
Assuntos
Interleucina-10/genética , Interleucina-10/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Monócitos/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Animais , Citocinas/genética , Citocinas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Fatores de Risco , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Mannan-binding lectin (MBL) is an innate pattern recognition molecule known to play a key role in pathogen clearance. As MBL2 gene polymorphism is associated to an increased susceptibility to infection, we aimed to determine genetic variations in the MBL2 gene in rheumatic heart disease (RHD). Genetic variations in the promoter and exon 1 region of the MBL2 gene were analyzed in 107 patients with RHD and 105 controls by real-time polymerase chain reaction. The frequency of MBL2* A/A genotype was significantly higher in RHD patients (71/107, 66.36% vs 52/105, 49.52%, pAssuntos
Frequência do Gene
, Predisposição Genética para Doença
, Lectina de Ligação a Manose/genética
, Polimorfismo Genético
, Cardiopatia Reumática/genética
, Adulto
, Idoso
, Estudos de Casos e Controles
, Éxons/imunologia
, Feminino
, Haplótipos
, Humanos
, Masculino
, Lectina de Ligação a Manose/imunologia
, Pessoa de Meia-Idade
, Regiões Promotoras Genéticas/imunologia
, Cardiopatia Reumática/imunologia
RESUMO
HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carbidopa/imunologia , Epitopos de Linfócito T/imunologia , Epitopos/imunologia , Vírus da Varíola das Aves Domésticas/imunologia , Levodopa/imunologia , Animais , Linhagem Celular , Galinhas , Combinação de Medicamentos , Epitopos/genética , Epitopos de Linfócito T/genética , Feminino , Vírus da Varíola das Aves Domésticas/genética , Regulação Viral da Expressão Gênica , Antígenos HIV/genética , Antígenos HIV/imunologia , Imunidade Celular/imunologia , Levodopa/genética , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Regiões Promotoras Genéticas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Recombinação Genética , Linfócitos T Citotóxicos/imunologia , Vaccinia virus/imunologiaRESUMO
A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.