RESUMO
Sodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phase-arrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxy-methylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested.
Assuntos
Proliferação de Células/efeitos dos fármacos , Desmetilação do DNA/efeitos dos fármacos , Decitabina/farmacologia , Fase G1/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Ácido Valproico/farmacologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
SCOPE: In the attempt to develop new therapeutic treatments for colitis, fractions containing phenolic compound isolate (Phi) and phenolic reduced-flaxseed protein hydrolysate (phr-FPH) from flaxseed are evaluated for their effects on the in vitro production of pro-inflammatory mediators and on the course of experimental colitis. METHODS AND RESULTS: The anti-inflammatory effects of Phi and phr-FPH from flaxseeds are studied in RAW264.7 cells and in trinitrobenzene sulphonic acid (TNBS) colitis model. It is observed that the incubation with Phi or phr-FPH result in lower levels of tumor necrosis factor α and nitric oxide in macrophages stimulated with bacterial lipopolysaccharide + interferon-γ. Prophylactic and therapeutic treatments with Phi and phr-FPH, respectively, greatly contribute to the prevention of weight loss and colon inflammation in colitic BALB/c mice. T cell proliferation, expansion of TH1 and TH17 cells, and pro-inflammatory cytokines are lower, whereas Treg cells are higher in spleen cell cultures from Phi-treated mice. In addition, therapeutic phr-FPH treatment is able to reduce the expansion of TH17 in splenic cell cultures. CONCLUSION: The consumption of phenolic and protein compounds extracted from flaxseeds has a protective effect on TNBS-induced colitis, and may be useful in the control of other inflammatory disorders.
Assuntos
Colite/tratamento farmacológico , Linho/química , Hidrolisados de Proteína/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colite/induzido quimicamente , Colite/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Fenóis/farmacologia , Células RAW 264.7 , Células Th17/efeitos dos fármacos , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
INTRODUCTION: In addition to conventional therapies, several new strategies have been proposed for modulating autoimmune diseases, including the adoptive transfer of immunological cells. In this context, dendritic cells (DCs) appear to be one of the most promising treatments for autoimmune disorders. The present study aimed to evaluate the effects of adoptive transfer of DCs obtained from both naïve and ovalbumin (OVA)-tolerant mice on the severity of TNBS induced colitis and analyze the eventual protective mechanisms. METHODS AND RESULTS: To induce oral tolerance, BALB/c mice were fed 4mg/mL OVA solution for seven consecutive days. Spleen DCs were isolated from tolerant (tDC) and naïve (nDC) mice, and then adoptively transferred to syngeneic mice. Three days later, colitis was induced in DC treated mice by intrarectal instillation of 100µg2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol. Control subjects received only intrarectal instillation of either TNBS solution or a vehicle. Five days later, mice from all groups were euthanized and examined for physiological and immunological parameters. Regarding the phenotype, we observed that the frequencies of CD11+ MHC II+ and CD11+ MHCII+ CD86+ cells were significantly lower in DCs isolated from tolerant mice than in those from naive mice. However, pretreatment with both types of DCs was able to significantly reduce clinical signs of colitis such as diarrhea, rectal prolapse, bleeding, and cachexia, although only treatment with tDCs was able to prevent weight loss from instillation of TNBS. In vitro proliferation of spleen cells from mice treated with either type of DCs was significantly lower than that observed in splenic cell cultures of naïve mice. Although no significant difference was observed in the frequencies of Treg cells in the experimental groups, the frequency of Th17+CD4+cellsand the secretion of IL-17 were more reduced in the cultures of spleen cells from mice treated with either type of DCs. The levels of IL-9 and IFN-γ were lower in supernatants of cells from mice treated with nDCs. CONCLUSION: The results allow us to conclude that the adoptive transfer of cells expressing CD11c is able to reduce the clinical and immunological signs of drug-induced colitis. Adoptive transfer of CD11c+DC isolated from both naive and tolerant mice altered the proliferative and T cell responses. To the best of our knowledge, there is no previously published data showing the protective effects of DCs from naïve or tolerant mice in the treatment of colitis.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Colite/terapia , Células Dendríticas/transplante , Tolerância Imunológica , Transferência Adotiva/métodos , Animais , Antígeno B7-2/imunologia , Antígeno CD11c/imunologia , Colite/induzido quimicamente , Colite/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Camundongos , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
INTRODUCTION: Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease. METHODS AND RESULTS: Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-γ. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-γ) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher. CONCLUSION: Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression.
Assuntos
Efeito Espectador/imunologia , Colite/imunologia , Tolerância Imunológica/imunologia , Ovalbumina/imunologia , Linfócitos T Reguladores/imunologia , Animais , Colite/induzido quimicamente , Feminino , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Owing to its high contents of fructooligosaccharides (FOSs), the yacon (Smallanthus sonchifolius) root is used in traditional Andean medicine as a substitute for cane sugar in diabetes and for obesity prevention. This study was designed to test the hypothesis that regular consumption of yacon works to improve the immune system. BALB/c mice were fed with the AIN-93 diet supplemented with 5% commercial FOS or either 3% or 5% yacon FOS for 30 consecutive days. Animals in the control group were fed with nonsupplemented ration. Food intake; weight gain; serum levels of IgA, IgM, and IgG; levels of fecal IgA, production of nitric oxide by peritoneal macrophages, frequencies of T and B lymphocytes in the spleen and peripheral blood, T-cell proliferation, and cytokine production were evaluated in all groups. No significant differences were observed in food intake and weight gain when the experimental and control groups were compared. Also, serum levels of IgA, IgM, and IgG; nitric oxide production in peritoneal macrophages; frequencies of T and B lymphocytes in the spleen and peripheral blood; T-cell proliferation; and production of interleukin (IL)-4, interferon-γ, IL-10, and tumor necrosis factor α did not differ in the different groups. The intake of FOS, however, led to a significant reduction of the proinflammatory cytokine IL-1ß in macrophage cultures and elevation of the levels of fecal IgA. Together, these results indicate that the daily consumption of yacon does not exert negative effects on the immune system, helps to preserve an anti-inflammatory state in phagocytic cells, and improves mucosal immunity, possibly preventing the risks associated with autoimmune and metabolic diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Asteraceae/química , Imunoglobulina A/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Oligossacarídeos/farmacologia , Extratos Vegetais/farmacologia , Animais , Suplementos Nutricionais , Fezes/química , Feminino , Frutose/farmacologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/químicaRESUMO
BACKGROUND: Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells. METHODOLOGY/PRINCIPAL FINDINGS: Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1-24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA. CONCLUSIONS/SIGNIFICANCE: The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death.
Assuntos
Anticonvulsivantes/farmacologia , Morte Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Ácido Valproico/farmacologia , Proliferação de Células/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HeLa , Humanos , Testes para Micronúcleos , Mitose/efeitos dos fármacos , Imagem MolecularRESUMO
Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account for 70% of leukocytes in early gestational uteri of humans and rodents. These cells make unique contributions to pregnancy, contributing to the success of embryo implantation and maintenance of decidual tissue that supports placental and fetal development. We postulated that uNK cells express molecules that are not shared by circulating NK (cNK) cells or other leukocytes and, therefore, would be immunogenic for male mice. We isolated viable uNK cells from gestation day 9 pregnant mice and inoculated them into syngeneic males. This induced antibodies reactive with mouse uNK cells but not with cNK cells or other lymphocytes. The antibodies reacted identically with uNK cells in tissue sections from five different mice strains from gestational day 7-12 and in pregnant rat uterus, suggesting that the recognized antigen should be a specific marker of uNK cell. Spleen cells from inoculated males were used subsequently to produce a monoclonal antibody reactive to a uNK cell surface antigen. These experiments confirm that uNK cells are a pregnancy-specific subset of NK cells expressing distinct surface antigen from those found in other tissues.
Assuntos
Células Matadoras Naturais/imunologia , Útero/imunologia , Animais , Anticorpos/análise , Antígeno CD56/análise , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Gravidez , Ratos , Ratos WistarRESUMO
BACKGROUND: The anti-inflammatory properties of some flavonoids have been attributed to their ability to inhibit the production of NO by activated macrophages. Soybean cotyledons accumulate certain flavonoids following elicitation with an extract of the fungal pathogen Diaporthe phaseolorum f. sp. meridionalis (Dpm). Sodium nitroprusside (SNP), a nitric oxide donor, can substitute for Dpm in inducing flavonoid production. In this study, we investigated the effect of flavonoid-containing diffusates obtained from Dpm- and SNP-elicited soybean cotyledons on NO production by lipopolysaccharide (LPS)- and LPS plus interferon-gamma (IFNgamma)-activated murine macrophages. RESULTS: Significant inhibition of NO production, measured as nitrite formation, was observed when macrophages were activated in the presence of soybean diffusates from Dpm- or SNP-elicited cotyledons. This inhibition was dependent on the duration of exposure to the elicitor. Daidzein, genistein, luteolin and apigenin, the main flavonoids present in diffusates of elicited cotyledons, suppressed the NO production by LPS + IFNgamma activated macrophages in a concentration-dependent manner, with IC50 values of 81.4 microM, 34.5 microM, 38.6 microM and 10.4 microM respectively. For macrophages activated with LPS alone, the IC50 values were 40.0 microM, 16.6 microM, 10.4 microM and 2.8 microM, respectively. Western blot analysis showed that iNOS expression was not affected by daidzein, was reduced by genistein, and was abolished by apigenin, luteolin and Dpm- and SNP-soybean diffusates at concentrations that significantly inhibited NO production by activated macrophages. CONCLUSIONS: These results suggest that the suppressive effect of flavonoids on iNOS expression could account for the potent inhibitory effect of Dpm- and SNP-diffusates on NO production by activated macrophages. Since the physiological concentration of flavonoids in plants is normally low, the treatment of soybean tissues with SNP may provide a simple method for substantially increasing the concentration of metabolites that are beneficial for the treatment of chronic inflammatory diseases associated with NO production.