RESUMEN
The intracellular signaling molecule TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). We now report that DC-specific deletion of TRAF6 (TRAF6ΔDC) resulted, unexpectedly, in loss of mucosal tolerance, characterized by spontaneous development of T helper 2 (Th2) cells in the lamina propria and eosinophilic enteritis and fibrosis in the small intestine. Loss of tolerance required the presence of gut commensal microbiota but was independent of DC-expressed MyD88. Further, TRAF6ΔDC mice exhibited decreased regulatory T (Treg) cell numbers in the small intestine and diminished induction of iTreg cells in response to model antigen. Evidence suggested that this defect was associated with diminished DC expression of interleukin-2 (IL-2). Finally, we demonstrate that aberrant Th2 cell-associated responses in TRAF6ΔDC mice could be mitigated via restoration of Treg cell activity. Collectively, our findings reveal a role for TRAF6 in directing DC maintenance of intestinal immune tolerance through balanced induction of Treg versus Th2 cell immunity.
Asunto(s)
Células Dendríticas/inmunología , Enteritis/inmunología , Eosinofilia/inmunología , Eosinófilos/inmunología , Gastritis/inmunología , Intestinos/inmunología , Linfocitos T Reguladores/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Células Th2/inmunología , Animales , Células Cultivadas , Células Dendríticas/microbiología , Enteritis/genética , Eosinofilia/genética , Gastritis/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica/genética , Interleucina-2/genética , Interleucina-2/metabolismo , Intestinos/microbiología , Intestinos/patología , Activación de Linfocitos/genética , Metagenoma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/genética , Linfocitos T Reguladores/microbiología , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/inmunología , Células Th2/microbiologíaRESUMEN
PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Células Epiteliales/fisiología , Simportadores/metabolismo , Animales , Células CACO-2 , Regulación de la Expresión Génica , Células HT29 , Humanos , Concentración de Iones de Hidrógeno , Inflamación/metabolismo , Mucosa Intestinal/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Transportador de Péptidos 1RESUMEN
Inflammatory bowel disease (IBD) is thought to result from commensal flora, aberrant cellular stress, and genetic factors. Here we show that the expression of colonic Ste20-like proline-/alanine-rich kinase (SPAK) that lacks a PAPA box and an F-alpha helix loop is increased in patients with IBD. The same effects were observed in a mouse model of dextran sodium sulfate-induced colitis and in Caco2-BBE cells treated with the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha. The 5'-flanking region of the SPAK gene contains two transcriptional start sites, three transcription factor Sp1-binding sites, and one transcription factor nuclear factor (NF)-kappaB-binding site, but no TATA elements. The NF-kappaB-binding site was essential for stimulated SPAK promoter activity by TNF-alpha, whereas the Sp1-binding sites were important for basal promoter activity. siRNA-induced knockdown of NF-kappaB, but not of Sp1, reduced TNF-alpha-induced SPAK expression. Nuclear run-on and mRNA decay assays demonstrated that TNF-alpha directly increased SPAK mRNA transcription without affecting SPAK mRNA stability. Furthermore, up-regulation of NF-kappaB expression and demethylation of the CpG islands induced by TNF-alpha also played roles in the up-regulation of SPAK expression. In conclusion, our data indicate that during inflammatory conditions, TNF-alpha is a key regulator of SPAK expression. The development of compounds that can either modulate or disrupt the activity of SPAK-mediated pathways is therefore important for the control and attenuation of downstream pathological responses, particularly in IBD.
Asunto(s)
Colitis/enzimología , Colitis/patología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Sitios de Unión , Células CACO-2 , Clonación Molecular , Colitis/inducido químicamente , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Sulfato de Dextran , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Estabilidad del ARN/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. RESULTS: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. CONCLUSIONS: Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.
Asunto(s)
Ácido Butírico/farmacología , Simportadores/genética , Transcripción Genética/efectos de los fármacos , Acetilación , Sitios de Unión , Western Blotting , Factor de Transcripción CDX2 , Células CACO-2 , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Transportador de Péptidos 1 , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores/metabolismoRESUMEN
Cell-cell and cell-matrix interactions are of utmost importance in the pathogenesis of inflammatory diseases. For example, cell-cell and cell-matrix interactions are crucial for leukocyte homing and recruitment to inflammatory sites. The discovery of the disintegrin and metalloprotease (ADAM) proteins, which have both adhesive and proteolytic activities, raised the question of their involvement in inflammatory processes. More interestingly, the presence of the RGD integrin-binding sequence in the disintegrin domain of ADAM-15 (MDC-15; metargidin) highlighted ADAM-15 as a protein particularly involved in cell-cell interactions. These findings therefore prompted authors to investigate the roles of ADAM-15 in inflammatory diseases. Because of the early description of ADAM-15 expression in endothelial cells, work first focused on the roles of ADAM-15 in vascular diseases, and ADAM-15 was found to be associated with atherosclerosis. Other studies also pointed at ADAM-15 as a mediator of rheumatoid arthritis and intestinal inflammation as well as inherent angiogenesis. The roles of ADAM-15 in these diseases appear to involve mechanisms as different as cell-cell interactions, cell-extracellular matrix (ECM) interactions, and shedding activity. Here we review and discuss these recent discoveries pointing to ADAM-15 as a mediator of mechanisms underlying inflammation and as a possible therapeutic target for prevention of inflammatory diseases.
Asunto(s)
Proteínas ADAM/metabolismo , Artritis Reumatoide/inmunología , Aterosclerosis/inmunología , Inflamación/inmunología , Síndrome del Colon Irritable/inmunología , Proteínas de la Membrana/metabolismo , Humanos , Neovascularización Patológica/inmunologíaRESUMEN
BACKGROUND & AIMS: KPV is a tripeptide (Lys-Pro-Val), which possesses anti-inflammatory properties; however, its mechanisms of action still remain unknown. PepT1 is a di/tripeptide transporter normally expressed in the small intestine and induced in colon during inflammatory bowel disease (IBD). The aim of this study was to 1) investigate whether the KPV anti-inflammatory effect is PepT1-mediated in intestinal epithelian and immune cells, and 2) examine the anti-inflammatory effects in two models of mice colitis. METHODS: Human intestinal epithelial cells Caco2-BBE, HT29-Cl.19A, and human T cells (Jurkat) were stimulated with pro-inflammatory cytokines in the present or absence of KPV. KPV anti-inflammatory effect was assessed using a NF-kappaB luciferase gene reporter, Western blot, real-time RT-PCR and ELISA. Uptake experiments were performed using cold KPV as a competitor for PepT1 radiolabelled substrate or using [(3)H]KPV to determine kinetic characteristics of KPV uptake. Anti-inflammatory effect of KPV was also investigated in DSS- and TNBS-induced colitis in mice. KPV was added to drinking water and inflammation was assessed at the histologic level and by proinflammatory cytokine mRNA expression. RESULTS: Nanomolar concentrations of KPV inhibit the activation of NF-kappaB and MAP kinase inflammatory signaling pathways, and reduce pro-inflammatory cytokine secretion. We found that KPV acts via PepT1 expressed in immune and intestinal epithelial cells. Furthermore, oral administration of KPV reduces the incidence of DSS- and TNBS-induced colitis indicated by a decrease in pro-inflammatory cytokine expression. CONCLUSIONS: This study indicates tht KPV is transported into cells by PepT1 and might be a new therapeutic agent for IBD.
Asunto(s)
Colitis/metabolismo , Colitis/prevención & control , Hormonas Estimuladoras de los Melanocitos/farmacocinética , Hormonas Estimuladoras de los Melanocitos/uso terapéutico , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/uso terapéutico , Simportadores/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Quimiocinas/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Transportador de Péptidos 1RESUMEN
The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-beta-cyclodextrin (MbetaCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MbetaCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.