RESUMO
Transmissible gastroenteritis virus (TGEV) replicates in the small intestine and induces enteritis and watery diarrhea. Establishment of local immunity in the intestine would thus prevent TGEV transmission. CpG DNA has been reported as a promising mucosal adjuvant in some animals. The effects of oral immunization of CpG DNA together with inactivated TGEV (ITGEV) were investigated in this study. Pigs (6 weeks old) were orally immunized with ITGEV plus CpG DNA. The TGEV-specific IgA level in the intestinal tract and the TGEV-specific IgG level in serum significantly increased following immunization with ITGEV plus CpG DNA (P ≤ 0.05). Moreover, populations of IgA-secreting cells, CD3+ T lymphocytes and intraepithelial lymphocytes (IELs), in the intestine increased significantly after immunization with ITGEV plus CpG DNA (P ≤ 0.05). Furthermore, the expression of IL-6, IL-12 and interferon-γ (IFN-γ) in ligated intestine segments increased significantly after injection with ITGEV plus CpG DNA (P ≤ 0.05). Taken together, these data suggest that oral immunization of ITGEV plus CpG DNA elicits a local immune response. Further studies are required to determine whether this immunity provides protection against TGEV in pigs.
Assuntos
Adjuvantes Imunológicos , Suínos , Vírus da Gastroenterite Transmissível/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oftálmica , Animais , Anticorpos Antivirais/imunologia , Nucleotídeos de Citosina/administração & dosagem , Nucleotídeos de Citosina/imunologia , Sequência Rica em GC/imunologia , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/prevenção & controle , Nucleotídeos de Guanina/administração & dosagem , Nucleotídeos de Guanina/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Intestinos/imunologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Linfócitos T/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagemRESUMO
Recognition of microbial products by macrophages (Mphi) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon-gamma (IFN-gamma), both arginase and inducible nitric oxide synthase (iNOS) in murine Mphi. Unexpectedly, IFN-gamma, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre-incubation (1 hr) with IFN-gamma and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG-mediated arginase activity is dependent on IFN-gamma priming. The increase in arginase activity as a result of stimulation with CpG plus IFN-gamma was correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinase (ERK), but independent of c-Jun N-terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN-gamma, one of the mayor cytokines produced in response to CpG administration in vivo.
Assuntos
Arginase/biossíntese , Sequência Rica em GC/imunologia , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Animais , Antracenos/farmacologia , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Flavonoides/farmacologia , Imidazóis/farmacologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Piridinas/farmacologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
SUMMARY: The innate immune system is a critical element of protection from invading pathogens. The specific receptors that recognize various components of the pathogens trigger signals that result in the production of proinflammatory cytokines as well as the activation of antigen-presenting cells, which activate the adaptive immune system. The discovery of the Toll-like receptors (TLRs) as important components of pathogen recognition has brought new understanding of the key signaling molecules involves in innate immune activation. Interestingly, it appears that most TLRs can recognize self-ligands as well and that mechanisms are required to discriminate between self and non-self ligands. One of these mechanisms is the expression of all the nucleic acid-specific TLR in endosomal compartments and not on the cell surface. Inappropriate activation of TLRs by self-components can result in sterile inflammation or autoimmunity. For example, TLR7 and TLR9 activation by endogenous RNA and DNA, transported to the endosomes in the form of immune complexes or non-covalently associated with cationic peptides, could be an important mechanism involved in promoting diseases such as systemic lupus erythematosus and psoriasis. In this review, we discuss the rationale for self-recognition by TLR7 and TLR9 as an important part of the development of lupus and other autoimmune diseases. We describe novel inhibitors of these receptors and provide evidence to support their use as novel therapeutic agents for autoimmunity.
Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Autoantígenos/imunologia , DNA/imunologia , DNA/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Sequência Rica em GC/imunologia , Humanos , Imunidade Inata , Imunoterapia Ativa , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Camundongos , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , RNA/imunologia , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/genéticaRESUMO
To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-kappaB, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-kappaB (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative IkappaB alpha plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-kappaB-independent pathways.
Assuntos
Sangue Fetal/imunologia , Sequência Rica em GC/imunologia , Células-Tronco Hematopoéticas/imunologia , Interleucina-8/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Antígenos CD34/metabolismo , Butadienos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Sangue Fetal/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Nitrilas/farmacologia , Oligodesoxirribonucleotídeos/imunologia , Piridinas/farmacologia , Receptor Toll-Like 9/imunologiaRESUMO
Telomeres and subtelomeres are important to the virulence of a number of pathogens, as they harbour large diverse gene families associated with the maintenance of infection. Evasion of immunity by African trypanosomes involves the differential expression of variant surface glycoproteins (VSGs), which are encoded by a family of >1500 genes and pseudogenes. This silent archive is located subtelomerically and is activated by gene conversion into specialized transcription units, which themselves are subject to silencing by allelic exclusion. Current research addresses the role of telomeres in the conversion and silencing mechanisms and in the diversification of the VSG archive.
Assuntos
Variação Antigênica/genética , Telômero/fisiologia , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Variação Antigênica/fisiologia , Sequência de Bases , Sequência Rica em GC/genética , Sequência Rica em GC/imunologia , Inativação Gênica/imunologia , Dados de Sequência Molecular , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/imunologia , Proteínas de Ligação a Telômeros/fisiologia , Trypanosoma brucei brucei/fisiologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologiaRESUMO
BACKGROUND AND METHODS: Cytosin-guanosin dinucleotide (CpG) motifs of bacterial DNA are known to be potent activators of innate immunity. We have shown previously that administration of CpG containing oligodeoxynucleotide (CpG-ODN) to mice before the onset of dextran sodium sulphate induced colitis ameliorated colitis and inhibited induction of proinflammatory cytokines. To investigate the possible involvement of CD4(+) T cells in the prophylactic CpG-ODN effects, we used the SCID transfer model of colitis. RESULTS: CD4(+)CD62L(+) T cells from CpG-ODN treated donors did not induce significant intestinal inflammation in SCID recipients, in contrast with control cells. Additionally, cotransfer of these cells with CD4(+)CD62L(+) cells from normal mice protected recipient animals from colitis, indicating regulatory activity. Also, CD4(+)CD62L(+) cells from toll-like receptor 9 deficient animals induced a significantly more severe colitis in SCID recipients than cells from wild-type littermate controls, suggesting a similar protective role of "endogenous" bacterial DNA leading to a less "aggressive" phenotype of these cells. There was no detectable difference in regulatory T cell surface markers between aggressive and attenuated cell pools but attenuated cell pools showed reduced proliferation in vitro and in vivo and produced less interferon gamma, interleukin (IL)-5, and IL-6 after anti-CD3 stimulation. CONCLUSIONS: Collectively, our data support the concept that both endogenous bacterial DNA and exogenously supplied CpG motifs of bacterial DNA induce regulatory properties in CD4(+) T cells. Therefore, bacterial DNA derived from the normal gut flora may contribute essentially to the homeostasis between effector and regulatory immune mechanisms in healthy individuals to protect them from chronic intestinal inflammation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Sequência Rica em GC/imunologia , Oligonucleotídeos/imunologia , Animais , Células Cultivadas , Colite/metabolismo , Modelos Animais de Doenças , Interferon gama/imunologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Selectina L/imunologia , Linfonodos/imunologia , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Reto/imunologia , Redução de Peso/fisiologiaRESUMO
The serum hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), found in 95% of patients. However, nearly every patient with PBC, including those who are AMA-negative, has an elevation in serum IgM. This hyper-IgM is neither representative of other Ig isoforms, nor is due to the levels of AMA. In fact, we have recently reported that the hyper-IgM is an innate immune response and can be induced with CpG-B with concurrent up-regulation of toll-like receptor 9 (TLR9). Based on these observations, we performed a two-tier study. First, we quantitated TLR9 genotypes in patients with PBC and controls and correlated these data with the B cell response to CpG-B. Second, based on these data, we performed an extensive TLR9 genotyping in a large cohort of patients and controls. We report herein that the 2848 AA TLR9 genotype is associated with enhanced gene expression and higher frequency of intracellular IgM(+) B cells following CpG stimulation. Interestingly, however, despite the functional association, there is no difference in the distribution of TLR9 genotypes between patients and controls. Our data emphasize the importance of dissecting the innate immune response in PBC.
Assuntos
Autoanticorpos/imunologia , Imunoglobulina M/imunologia , Cirrose Hepática Biliar/genética , Glicoproteínas de Membrana/genética , Oligodesoxirribonucleotídeos/farmacologia , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Adulto , Idoso , Células Cultivadas , Feminino , Sequência Rica em GC/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Cirrose Hepática Biliar/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Oligodesoxirribonucleotídeos/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Receptores de Superfície Celular/imunologia , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Immunostimulatory CpG motifs can preferentially induce Th1 immune responses and have been applied to treat Th2-dominant disease. In this study, we investigated whether a plasmid with the addition of 20 copies of an immunostimulatory CpG motif (pB-CpG20) might prevent the development of scleroderma-like syndrome in tight-skin (Tsk/+) mice. Administration of pB-CpG20 to Tsk/+mice every 3 weeks starting at the age of 1 week reduced skin thickness and collagen content compared to that of pB or saline. The reduction was long lasting even after halting the treatment. Furthermore, this treatment partially reduced the production of anti-nuclear antibodies although it did not decrease the incidence of lung emphysema. pB-CpG20 increased the number of spleen cells secreting IFN-gamma and reduced that of the cells secreting IL-4 in vivo and in vitro compared to saline. These results suggest that repeated administration of a CpG-enriched plasmid can ameliorate scleroderma-like syndrome by biasing Th1 immunity in young Tsk/+mice.
Assuntos
Sequência Rica em GC/imunologia , Imunidade Celular/efeitos dos fármacos , Plasmídeos/administração & dosagem , Escleroderma Sistêmico/tratamento farmacológico , Células Th1/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , Plasmídeos/imunologia , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Pele/imunologia , Pele/patologia , Células Th2/imunologiaRESUMO
Different DNA motifs are required for optimal stimulation of mouse and human immune cells by CpG oligodeoxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CpG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 MAPK than optimal motifs. When the CpG dinucleotide was inverted to GC in each ODN, some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may be responsible for mediating many published CpG-independent responses to PS-ODN.
Assuntos
Adjuvantes Imunológicos/metabolismo , Ilhas de CpG/imunologia , Proteínas de Ligação a DNA/fisiologia , Oligodesoxirribonucleotídeos/metabolismo , Receptores de Superfície Celular/fisiologia , Tionucleotídeos/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/imunologia , Sequência Rica em GC/imunologia , Humanos , Camundongos , Oligodesoxirribonucleotídeos/imunologia , Ligação Proteica/imunologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/imunologia , Especificidade da Espécie , Tionucleotídeos/imunologia , Receptor Toll-Like 9RESUMO
Bacterial DNA and immunostimulatory CpG oligodeoxynucleotides (ODNs) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG motifs are currently used as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. In this study, we show that the application of an immunomodulatory GpG ODN, with a single base switch from CpG to GpG, can effectively inhibit the activation of Th1 T cells associated with autoimmune disease. Moreover, this immunomodulatory GpG ODN suppresses the severity of experimental autoimmune encephalomyelitis in mice, a prototypic Th1-mediated animal disease model for multiple sclerosis.
Assuntos
Adjuvantes Imunológicos/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fosfatos de Dinucleosídeos/farmacologia , Sequência Rica em GC/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1/biossíntese , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Fosfatos de Dinucleosídeos/administração & dosagem , Fosfatos de Dinucleosídeos/imunologia , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Sequência Rica em GC/genética , Glicoproteínas/biossíntese , Inibidores do Crescimento/farmacologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Imunidade Inata/genética , Imunossupressores/administração & dosagem , Imunossupressores/farmacologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Básica da Mielina/antagonistas & inibidores , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteína Proteolipídica de Mielina/antagonistas & inibidores , Proteína Proteolipídica de Mielina/fisiologia , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosforilação , Serina/metabolismo , Baço/citologia , Baço/imunologia , Células Th1/imunologiaRESUMO
DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.