RESUMO
Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.
Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Citocinas/biossíntese , Humanos , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Intestinos/patologia , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Fator de Transcrição STAT4/metabolismo , Proteínas com Domínio T/metabolismoRESUMO
An altered balance between effector and regulatory factors is supposed to sustain the tissue-damaging immune response in inflammatory bowel disease (IBD). We have recently shown that in IBD, there is a defective synthesis of the counter-regulatory cytokine, interleukin (IL)-25. In this study we investigated factors that control IL-25 production in the gut. IBD patients produced less IL-25 when compared with normal controls. Stimulation of normal intestinal explants with tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ) or IL-21, reduced IL-25 synthesis. Consistently, IL-25 production was enhanced by anti-TNF-α both in vitro and in vivo. Upregulation of IL-25 was also seen in normal colonic explants stimulated with transforming growth factor-ß1 (TGF-ß1). As in IBD, TGF-ß1 activity is abrogated by Smad7, we next assessed whether inhibition of Smad7 with an antisense oligonucleotide enhanced IL-25 expression. Knockdown of Smad7 was accompanied by an increase in IL-25 production. Data show that IL-25 production is differently regulated by TNF-α and TGF-ß1 in the human gut.
Assuntos
Regulação da Expressão Gênica , Interleucina-17/metabolismo , Mucosa Intestinal/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Doença Celíaca/imunologia , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Doenças Inflamatórias Intestinais/imunologia , Interleucina-17/genética , Proteína Smad7/genética , Proteína Smad7/metabolismoRESUMO
Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1ß and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1ß and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.
Assuntos
Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adolescente , Adulto , Western Blotting , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Imidazóis/farmacologia , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Niacinamida/farmacologia , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
BACKGROUND AND AIMS: Interleukin 17 (IL17) is now known to be involved in a number of chronic inflammatory disorders. However, the mechanisms regulating its production in inflammatory bowel disease (IBD) are still unclear. METHODS: Endoscopic biopsies or surgical specimens were taken from inflamed and uninflamed colonic mucosa of 72 patients with IBD (38 with Crohn's disease and 34 with ulcerative colitis), and normal colon of 38 control subjects. IL17 and interferon gamma (IFNgamma) were detected by ELISA in the supernatants of biopsies cultured ex vivo, and anti-CD3/CD28-stimulated lamina propria mononuclear cells (LPMCs) incubated with IL12, IL23, IL1beta plus IL6, transforming growth factor beta1 (TGFbeta1), or anti-IL21 neutralising antibody. Intracellular flow cytometry was performed to analyse mucosal Th17 and Th1/Th17 cells. RESULTS: IL17 production by organ culture biopsies was higher in IBD inflamed mucosa than IBD uninflamed mucosa and controls, and was equivalent in amount to IFNgamma. Anti-CD3/CD28-stimulated IBD LPMCs produced higher IL17 amounts compared to controls. The percentages of Th17 and Th1/Th17 cells were increased in patients with IBD. IL23 and IL1beta plus IL6 had no effect on IBD LPMC production of IL17; however, IL12 markedly increased IFNgamma production and decreased IL17 production. TGFbeta1 dose-dependently decreased IFNgamma, but had no significant inhibitory effect on IL17 production. Blocking IL21 significantly downregulated IL17 production. CONCLUSIONS: Our findings support a role for IL12, TGFbeta and IL21 in modulating IL17/IFNgamma production in IBD. The abundant IL17 in inflamed IBD mucosa may help explain the relative lack of efficacy of anti-IFNgamma antibodies in clinical trials of Crohn's disease.
Assuntos
Doenças Inflamatórias Intestinais/imunologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Adolescente , Adulto , Células Cultivadas , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Citocinas/imunologia , Relação Dose-Resposta Imunológica , Proteínas da Matriz Extracelular/imunologia , Humanos , Imunidade nas Mucosas , Mediadores da Inflamação/imunologia , Mucosa Intestinal/imunologia , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Fator de Crescimento Transformador beta/imunologia , Adulto JovemRESUMO
BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.
Assuntos
Doença de Crohn/metabolismo , Mucosa Intestinal/metabolismo , Metaloproteinases da Matriz/biossíntese , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Apoptose , Western Blotting/métodos , Estudos de Casos e Controles , Células Cultivadas , Senescência Celular , Colo/patologia , Doença de Crohn/patologia , Fibroblastos/metabolismo , Fibrose , Humanos , Mucosa Intestinal/patologia , Metaloproteinase 12 da Matriz/análise , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/análise , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinases da Matriz/análise , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína Smad2/análise , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/análise , Proteína Smad3/genética , Proteína Smad3/metabolismo , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/genética , Adulto JovemRESUMO
BACKGROUND AND AIMS: The role of transforming growth factor beta (TGFbeta) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFbeta inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFbeta blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon gamma (IFN gamma) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS: TGFbeta transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFbeta neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN gamma, tumour necrosis factor alpha (TNFalpha), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS: A higher number of TGFbeta transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFbeta antibody than in those cultured with control antibody. TGFbeta blockade downregulated T cell apoptosis, and induced a significant increase in IFN gamma, TNFalpha, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFbeta antibody. CONCLUSIONS: The findings support a crucial role for TGFbeta in dampening T cell-mediated tissue-damaging responses in the human gut.