RESUMEN
BACKGROUND AND AIMS: Although the mechanisms underlying the formation of intestinal fibrostrictures in Crohn's disease [CD] are not fully understood, activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of such complications. Here, we investigated the role of cadherin-11 [CDH-11], a fibroblast-derived protein that induces collagen production in various organs, in intestinal fibrosis. METHODS: CDH-11 expression was evaluated in inflammatory [I] and fibrostricturing [FS] CD mucosal samples, ulcerative colitis [UC] mucosal samples, and ileal and colonic control samples, by real-time polymerase chain reaction, western blotting, and immunohistochemistry. CDH-11 expression was evaluated in normal and in CD intestinal fibroblasts stimulated with inflammatory/fibrogenic cytokines. FS CD fibroblasts were cultured either with a specific CDH-11 antisense oligonucleotide [AS], or activating CDH-11 fusion protein and activation of RhoA/ROCK, and TGF-ß pathways and collagen production were evaluated by western blotting. Finally, we assessed the susceptibility of CDH-11-knockout [KO] mice to colitis-induced intestinal fibrosis. RESULTS: CDH-11 RNA and protein expression were increased in both CD and UC as compared with controls. In CD, the greater expression of CDH-11 was seen in FS samples. Stimulation of fibroblasts with TNF-α, interleukin [IL]-6, IFN-γ, IL-13, and IL-1ß enhanced CDH-11 expression. Knockdown of CDH-11 in FS CD fibroblasts impaired RhoA/ROCK/TGF-ß signalling and reduced collagen synthesis, whereas activation of CDH-11 increased collagen secretion. CDH-11 KO mice were largely protected from intestinal fibrosis. CONCLUSIONS: Data show that CDH-11 expression is up-regulated in inflammatory bowel disease [IBD] and suggest a role for this protein in the control of intestinal fibrosis.
Asunto(s)
Cadherinas/metabolismo , Colitis Ulcerosa , Colágeno/biosíntesis , Enfermedad de Crohn , Mucosa Intestinal/metabolismo , Intestinos/patología , Animales , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colitis Ulcerosa/fisiopatología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Enfermedad de Crohn/fisiopatología , Citocinas/metabolismo , Progresión de la Enfermedad , Fibrosis/metabolismo , Humanos , Ratones , Ratones Noqueados , Transducción de Señal , Regulación hacia ArribaRESUMEN
In normal conditions gut homeostasis is maintained by the suppressive activity of regulatory T cells (Tregs), characterized by the expression of the transcription factor FoxP3. In human inflammatory bowel disease, which is believed to be the consequence of the loss of tolerance toward antigens normally contained in the gut lumen, Tregs have been found to be increased and functionally active, thus pointing against their possible role in the pathogenesis of this immune-mediated disease. Though, in inflammatory conditions, Tregs have been shown to upregulate the T helper (Th) type 1-related transcription factor Tbet and to express the pro-inflammatory cytokine IFNγ, thus suggesting that at a certain point of the inflammatory process, Tregs might contribute to inflammation rather than suppress it. Starting from the observation that Tregs isolated from the lamina propria of active but not inactive IBD patients or uninflamed controls express Tbet and IFNγ, we investigated the functional role of Th1-like Tregs in the dextran sulfate model of colitis. As observed in human IBD, Th1-like Tregs were upregulated in the inflamed lamina propria of treated mice and the expression of Tbet and IFNγ in Tregs preceded the accumulation of conventional Th1 cells. By using a Treg-specific Tbet conditional knockout, we demonstrated that Tbet expression in Tregs is required for the development of colitis. Indeed, Tbet knockout mice developed milder colitis and showed an impaired Th1 immune response. In these mice not only the Tbet deficient Tregs but also the Tbet proficient conventional T cells showed reduced IFNγ expression. However, Tbet deficiency did not affect the Tregs suppressive capacity in vitro and in vivo in the adoptive transfer model of colitis. In conclusion here we show that Tbet expression by Tregs sustains the early phase of the Th1-mediated inflammatory response in the gut.
Asunto(s)
Colitis/inmunología , Regulación de la Expresión Génica/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Proteínas de Dominio T Box/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Animales , Colitis/patología , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/patología , Masculino , Ratones , Linfocitos T Reguladores/patología , Células TH1/patologíaRESUMEN
In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-ß1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, ex vivo organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut.
Asunto(s)
Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Leucocitos Mononucleares/fisiología , Membrana Mucosa/inmunología , Sirtuina 1/metabolismo , Proteína smad7/metabolismo , Adolescente , Adulto , Animales , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Oligodesoxirribonucleótidos Antisentido/genética , ARN Interferente Pequeño/genética , Sirtuina 1/genética , Proteína smad7/genética , Ubiquitinación , Adulto JovenRESUMEN
Chronic inflammation drives colitis-associated colorectal cancer (CAC) in inflammatory bowel disease (IBD). FoxP3+ regulatory T cells (Treg) coexpressing the Th17-related transcription factor RORγt accumulate in the lamina propria of IBD patients, where they are thought to represent an intermediate stage of development toward a Th17 proinflammatory phenotype. However, the role of these cells in CAC is unknown. RORγt+FoxP3+ cells were investigated in human samples of CAC, and their phenotypic stability and function were investigated in an azoxymethane/dextran sulfate sodium model of CAC using Treg fate-mapping reporter and Treg-specific RORγt conditional knockout mice. Tumor development and the intratumoral inflammatory milieu were characterized in these mice. The functional role of CTLA-4 expressed by Tregs and FoxO3 in dendritic cells (DC) was studied in vitro and in vivo by siRNA-silencing experiments. RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice. Conditional RORγt knockout mice showed reduced tumor incidence, and dysplastic cells exhibited low Ki67 expression and STAT3 activation. Tumor-infiltrating DCs produced less IL6, a cytokine that triggers STAT3-dependent proliferative signals in neoplastic cells. RORγt-deficient Tregs isolated from tumors overexpressed CTLA-4 and induced DCs to have elevated expression of the transcription factor FoxO3, thus reducing IL6 expression. Finally, in vivo silencing of FoxO3 obtained by siRNA microinjection in the tumors of RORγt-deficient mice restored IL6 expression and tumor growth. These data demonstrate that RORγt expressed by tumor-infiltrating Tregs sustains tumor growth by leaving IL6 expression in DCs unchecked. Cancer Immunol Res; 6(9); 1082-92. ©2018 AACR.
Asunto(s)
Colitis/complicaciones , Neoplasias Colorrectales/inmunología , Células Dendríticas/inmunología , Interleucina-6/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Linfocitos T Reguladores/inmunología , Animales , Azoximetano , Antígeno CTLA-4/genética , Colitis/inducido químicamente , Neoplasias Colorrectales/etiología , Sulfato de Dextran , Proteína Forkhead Box O3/genética , Silenciador del Gen , Humanos , Inflamación , Interleucina-6/genética , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente PequeñoRESUMEN
Background: In Crohn's disease (CD), the pathogenic immune response is associated with high Smad7, an inhibitor of TGF-ß1 signaling. Smad7 knockdown with Mongersen, a specific antisense oligonucleotide-containing compound, restores TGF-ß1 activity leading to inhibition of inflammatory signals and associates with clinical benefit in CD patients. As TGF-ß1 is pro-fibrogenic, it remains unclear whether Mongersen-induced Smad7 inhibition increases the risk of intestinal fibrosis. We assessed the impact of Smad7 inhibition on the course of colitis-driven intestinal fibrosis in mice. Methods: BALB/c mice were rectally treated with increasing doses of trinitrobenzene sulfonic acid (TNBS) for 8 or 12 weeks. The effect of oral Smad7 antisense or control oligonucleotide, administered to mice starting from week 5 or week 8, respectively, on mucosal inflammation and colitis-associated colonic fibrosis was assessed. Mucosal samples were analyzed for Smad7 by immunoblotting and immunohistochemistry, TGF-ß1 by enzyme-linked immunosorbent assay, and collagen by immunohistochemistry. Results: TNBS-induced chronic colitis was associated with colonic deposition of collagen I and fibrosis, which were evident at week 8 and became more pronounced at week 12. TNBS treatment enhanced Smad7 in both colonic epithelial and lamina propria mononuclear cells. Colitic mice treated with Smad7 antisense oligonucleotide exhibited reduced signs of colitis, less collagen deposition, and diminished fibrosis. These findings were associated with diminished synthesis of TGF-ß1 and reduced p-Smad3 protein expression. Conclusion: Attenuation of colitis with Smad7 antisense oligonucleotide limits development of colonic fibrosis.
Asunto(s)
Colitis/genética , Oligonucleótidos Antisentido/farmacología , Proteína smad7/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Colitis/patología , Colágeno Tipo I/análisis , Colon/patología , Enfermedad de Crohn/terapia , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Fibrosis , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos/farmacología , Transducción de Señal , Proteína smad3/metabolismo , Ácido TrinitrobencenosulfónicoRESUMEN
Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc(min/+) mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.
Asunto(s)
Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Interleucinas/inmunología , Animales , Carcinogénesis , Procesos de Crecimiento Celular/inmunología , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/patología , Femenino , Células HT29 , Humanos , Inflamación/inmunología , Inflamación/patología , Interferón-alfa/inmunología , Interleucinas/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/inmunología , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT3/inmunología , Transducción de SeñalRESUMEN
Transforming growth factor-beta (TGF-ß) is deeply involved in colorectal cancer development and the disruption of the TGF-ß signaling in dysplastic cells is required for tumor to grow. Nevertheless, tumor cells express TGF-ß to escape the immune surveillance mediated by T cells. T-cell expression of Smad7, an intracellular inhibitor of the TGF-ß signaling, protects against colitis-associated colorectal cancer. However, whether Smad7 in T cells might influence colorectal cancer growth independently of chronic inflammation and which T-cell subset is involved in this process is unknown. To address this issue, T-cell-specific Smad7 transgenic mice and wild-type (WT) littermates were subcutaneously transplanted with syngenic MC38 colon carcinoma cells. Smad7Tg mice were resistant to tumor development compared with WT mice and protection was dependent on CD4(+) T cells. Smad7 expression in T cells increased the number of tumor-infiltrating Tbet/ROR-γ-t double-positive CD4 T cells characterized by the expression of tumor necrosis factor-alpha (TNF-α) and interferon-gamma but lower IL17A. The low expression of IL17A caused by the Smad7 expression in tumor-infiltrating CD4(+) T cells enabled the TNF-α-mediated killing of cancer cells both in vitro and in vivo, thus indicating that the Smad7-mediated plastic effect on T-cell phenotype induces protection against colorectal cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/patología , Interleucina-17/fisiología , Linfocitos Infiltrantes de Tumor/inmunología , Proteína smad7/fisiología , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Th17/patología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
UNLABELLED: Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. CONCLUSIONS: IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hepatocitos/patología , Interleucinas/uso terapéutico , Células Mieloides/patología , Linfocitos T/patología , Animales , Antígeno CD11b/metabolismo , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Técnicas de Cocultivo , Concanavalina A/efectos adversos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Galactosamina/efectos adversos , Hepatitis/metabolismo , Hepatitis/patología , Hepatocitos/metabolismo , Humanos , Interleucina-17/metabolismo , Interleucinas/metabolismo , Interleucinas/farmacología , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos BALB C , Células Mieloides/metabolismo , Receptores de Quimiocina/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
BACKGROUND & AIMS: Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice. METHODS: We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohn's disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-ß1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3-knockout (TIMP-3-KO), and transgenic (TIMP-3-Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3-KO T cells. RESULTS: Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-ß1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-ß1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3-KO mice developed severe colitis after administration of TNBS, whereas TIMP-3-Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3-KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells. CONCLUSIONS: TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-ß1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.
Asunto(s)
Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Mucosa Intestinal/metabolismo , ARN Mensajero/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Adulto , Anciano , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Citocinas/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Mucosa Intestinal/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Oligonucleótidos Antisentido/farmacología , Proteína smad7/genética , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Factor de Crecimiento Transformador beta/farmacología , Ácido TrinitrobencenosulfónicoRESUMEN
BACKGROUND: Interleukin (IL)-25, a Th2-related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL-25, however, remains unknown. Since Th2-cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL-25-mediated anticolitic effect. METHODS: AAM-related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis treated with IL-25 and/or neutralizing IL-4, IL-13, and transforming growth factor beta 1 (TGF-ß1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL-25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL-25 on the alternative activation of peritoneal F4/80+ cells. RESULTS: IL-25 enhanced the expression of AAM-related markers in F4/80(+) cells infiltrating the peritoneum and colon of naïve and colitic mice. Peritoneal F4/80(+) cells isolated from IL-25-treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL-25 did not directly induce AAM in vitro and in vivo in mice, IL-25 administration enhanced the expression of IL-4, IL-13, and TGF-ß1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL-25-driven AAM induction. Blockade of IL-4, IL-13, and TGF-ß1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL-25 on AAM gene expression. CONCLUSIONS: The IL-25-mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties.
Asunto(s)
Colitis/inmunología , Interleucina-17/inmunología , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Análisis de Varianza , Animales , Anticuerpos Neutralizantes/inmunología , Antígenos de Diferenciación/metabolismo , Arginasa/genética , Arginasa/metabolismo , Recuento de Células , Colitis/inducido químicamente , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-13/inmunología , Interleucina-17/farmacología , Interleucina-4/inmunología , Mucosa Intestinal/metabolismo , Lectinas/genética , Lectinas/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Estadísticas no Paramétricas , Factor de Crecimiento Transformador beta/inmunología , Ácido Trinitrobencenosulfónico , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Chronic inflammation is a major driving force in the development of cancer in many tissues, but the array of factors involved in this neoplastic transformation are not well understood. We have investigated the role of interleukin (IL)-21 in colitis-associated colon cancer (CAC), as this cytokine is overexpressed in the gut mucosa of patients with ulcerative colitis (UC), a chronic inflammatory disease associated with colon cancer. IL-21 was increased in the gut of patients with UC-associated colon cancer, and in mice with CAC induced by azoxymethane (AOM) and dextran sulfate sodium (DSS). After AOM+DSS treatment, IL-21 KO mice showed reduced mucosal damage, reduced infiltration of T cells, and diminished production of IL-6 and IL-17A. IL-21-deficient mice also developed fewer and smaller tumors compared with wild-type (WT) mice. Absence of IL-21 reduced signal transducer and activator of transcription 3 activation in tumor and stromal cells. Administration of a neutralizing IL-21 antibody to WT mice after the last DSS cycle decreased the colonic T cell infiltrate and the production of IL-6 and IL-17A and reduced the number of tumors. These observations indicate that IL-21 amplifies an inflammatory milieu that promotes CAC, and suggest that IL-21 blockade may be useful in reducing the risk of UC-associated colon cancer.
Asunto(s)
Colitis/complicaciones , Colitis/inmunología , Neoplasias del Colon/etiología , Neoplasias del Colon/inmunología , Interleucinas/inmunología , Animales , Azoximetano/efectos adversos , Linfocitos T CD4-Positivos/inmunología , Carcinógenos/farmacología , Línea Celular Tumoral , Colitis/inducido químicamente , Colitis/patología , Colon/citología , Colon/inmunología , Colon/patología , Neoplasias del Colon/patología , Sulfato de Dextran/efectos adversos , Factores de Transcripción Forkhead/inmunología , Humanos , Interleucina-17/inmunología , Interleucina-6/inmunología , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT3/metabolismoRESUMEN
Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to chronic inflammation. In IBD, chronic inflammation relies upon a TGFß signaling blockade, but its precise mechanistic relationship to colitis-associated colorectal cancer (CAC) remains unclear. In this study, we investigated the role of the TGFß signaling inhibitor Smad7 in CAC pathogenesis. In human colonic specimens, Smad7 was downregulated in CD4(+) T cells located in the lamina propria of patients with complicated IBD compared with uncomplicated IBD. Therefore, we assessed CAC susceptibility in a transgenic mouse model where Smad7 was overexpressed specifically in T cells. In this model, Smad7 overexpression increased colitis severity, but the mice nevertheless developed fewer tumors than nontransgenic mice. Protection was associated with increased expression of IFNγ and increased accumulation of cytotoxic CD8(+) and natural killer T cells in the tumors and peritumoral areas. Moreover, genetic deficiency in IFNγ abolished the Smad7-dependent protection against CAC. Taken together, our findings defined a novel and unexpected role for Smad7 in promoting a heightened inflammatory response that protects against CAC.
Asunto(s)
Colitis/metabolismo , Neoplasias Colorrectales/metabolismo , Proteína smad7/metabolismo , Linfocitos T/metabolismo , Adulto , Anciano , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Colitis/genética , Colitis/inmunología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Citotoxicidad Inmunológica/inmunología , Sulfato de Dextran/inmunología , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Proteína smad7/genética , Linfocitos T/inmunología , Linfocitos T/patología , Células TH1/inmunología , Células TH1/metabolismo , Adulto JovenRESUMEN
Chronic inflammation is thought to be the leading cause of many human cancers including colorectal cancer (CRC). Accordingly, epidemiologic and clinical studies indicate that patients affected by ulcerative colitis and Crohn's disease, the two major forms of inflammatory bowel disease, have an increased risk of developing CRC. In recent years, the role of immune cells and their products have been shown to be pivotal in initiation and progression of colitis-associated CRC. On the other hand, activation of the immune system has been shown to cause dysplastic cell elimination and cancer suppression in other settings. Clinical and experimental data herein reviewed, while confirming chronic inflammation as a risk factor for colon carcinogenesis, do not completely rule out the possibility that under certain conditions the chronic activation of the mucosal immune system might protect from colonic dysplasia.
Asunto(s)
Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/inmunología , Inflamación , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/inmunología , Intestinos/patología , Animales , Transformación Celular Neoplásica/inmunología , Quimiocinas/inmunología , Neoplasias Colorrectales/patología , Humanos , Sistema Inmunológico/fisiología , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/patología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-10/inmunología , Interleucina-6/inmunología , Intestinos/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is a crucial event in the control of tumor growth. However, many cancer cells, including colon cancer cells, are resistant to TRAIL-driven cell death. We have recently shown that 2-methoxy-5-amino-N-hydroxybenzamide (herein termed 2-14), a novel derivative of mesalamine, induces endoplasmic reticulum stress in colon cancer cells. Because endoplasmic reticulum stress-induced signals regulate the expression of molecules involved in TRAIL-driven apoptosis, we examined whether 2-14 makes colon cancer cells sensitive to TRAIL. Colon cancer cells were cultured with 2-14 and/or TRAIL. Death receptor (DR) 4/DR5 were analyzed by real-time PCR and flow cytometry. TRAIL pathway-associated proteins and extracellular signal-regulated kinase (ERK) were assessed by Western blotting. The in vivo capability of 2-14 to sensitize colon cancer cells to TRAIL-induced apoptosis was evaluated in a syngenic colon cancer model in which CT26-derived grafts were induced in mice. 2-14 promoted ERK-dependent induction of DR5, thereby enhancing TRAIL-mediated caspase-8 activation and apoptosis. Analysis of TRAIL-related pro- and antiapoptotic factors and functional studies revealed that survivin is involved in the protection of colon cancer cells against TRAIL-driven apoptosis. Notably, 2-14 enhanced ubiquitination and proteasome-mediated degradation of survivin. These data were confirmed in a murine model of TRAIL-resistant colon cancer in which 2-14 upregulated DR5, reduced survivin expression, and synergized with TRAIL in inhibiting tumor growth. Similarly, intraperitoneal administration of 2-14 to mice upregulated DR5 and downregulated survivin in a model of colitis-associated colon cancer. These findings indicate that 2-14 acts as a sensitizer for TRAIL-induced apoptosis and suggest that 2-14 can be useful in the therapy for TRAIL-resistant colon cancer.
Asunto(s)
Ácidos Aminosalicílicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ácidos Aminosalicílicos/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HCT116 , Células HT29 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Ratones , Ratones Endogámicos BALB C , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Survivin , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: The pathogenesis of inflammatory bowel disease (IBD) is believed to involve an altered balance between effector and regulatory T cells. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor that mediates the toxicity of dioxins, controls T-cell responses. We investigated the role of AhR in inflammation and pathogenesis of IBD in humans and mouse models. METHODS: AhR expression was evaluated in intestinal tissue samples from patients with IBD and controls by real-time polymerase chain reaction (PCR) and flow cytometry. Intestinal lamina propria mononuclear cells (LPMCs) were activated in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Colitis was induced in mice using trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS), or T-cell transfer. Mice were given injections of Ficz or the AhR antagonist 2-metyl-2H-pyrazole-3-carboxylic acid; some mice first received injections of a blocking antibody against interleukin (IL)-22. Cytokines were quantified by real-time PCR and flow cytometry. RESULTS: Intestine tissue from patients with IBD expressed significantly less AhR than controls. In LPMCs from patients with IBD, incubation with Ficz reduced levels of interferon gamma (IFN)-γ and up-regulated IL-22. Mice injected with Ficz were protected against TNBS-, DSS-, and T-cell transfer-induced colitis; they had marked down-regulation of inflammatory cytokines and induction of IL-22. Mice given AhR antagonist produced more inflammatory cytokines and less IL-22 and developed a severe colitis. Neutralization of endogenous IL-22 disrupted the protective effect of Ficz on TNBS-induced colitis. CONCLUSIONS: AhR is down-regulated in intestinal tissue of patients with IBD; AhR signaling, via IL-22, inhibits inflammation and colitis in the gastrointestinal tract of mice. AhR-related compounds might be developed to treat patients with IBDs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enfermedades Inflamatorias del Intestino/prevención & control , Interleucinas/metabolismo , Intestinos/inmunología , Receptores de Hidrocarburo de Aril/metabolismo , Adulto , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Biopsia , Carbazoles/farmacología , Estudios de Casos y Controles , Células Cultivadas , Sulfato de Dextran , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal , Linfocitos T/trasplante , Factores de Tiempo , Ácido Trinitrobencenosulfónico , Regulación hacia Arriba , Interleucina-22RESUMEN
Although interleukin-25 (IL-25) has been traditionally considered as a cytokine involved in T helper (Th) 2 cell-associated allergic diseases and host defence against helminthic parasites, recent studies have shown that IL-25 exerts negative effects on the initiation and progression of Th1/Th17-mediated pathologies. This later function of IL-25 is particularly evident at the gut level, where IL-25 could contribute to attenuate tissue-damaging immune responses. These new and exciting pre-clinical observations suggest that therapeutic interventions aimed at enhancing IL-25 activity could be useful in the management of patients with chronic gut inflammation.
Asunto(s)
Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Inmunoterapia , Interleucina-17/inmunología , Mucosa Intestinal/efectos de los fármacos , Animales , Colitis Ulcerosa/terapia , Enfermedad de Crohn/terapia , Retroalimentación Fisiológica , Humanos , Inmunidad Mucosa/efectos de los fármacos , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Interleucina-17/farmacología , Mucosa Intestinal/patología , Balance Th1 - Th2/efectos de los fármacos , Células Th17/efectos de los fármacos , Células Th17/inmunologíaRESUMEN
Interleukin-25 (IL-25), a T helper type 2 (Th2) -related factor, inhibits the production of inflammatory cytokines by monocytes/macrophages. Since Th2 cytokines antagonize classically activated monocytes/macrophages by inducing alternatively activated macrophages (AAMs), we here assessed the effect of IL-25 on the alternative activation of human monocytes/macrophages. The interleukins IL-25, IL-4 and IL-13 were effective in reducing the expression of inflammatory chemokines in monocytes. This effect was paralleled by induction of AAMs in cultures added with IL-4 or IL-13 but not with IL-25, regardless of whether cells were stimulated with lipopolysaccharide or interferon-γ. Moreover, pre-incubation of cells with IL-25 did not alter the ability of both IL-4 and IL-13 to induce AAMs. Both IL-4 and IL-13 activated signal transducer and activator of transcription 6 (STAT6), and silencing of this transcription factor markedly reduced the IL-4/IL-13-driven induction of AAMs. In contrast, IL-25 failed to trigger STAT6 activation. Among Th2 cytokines, only IL-25 and IL-10 were able to activate p38 mitogen-activated protein kinase. These results collectively indicate that IL-25 fails to induce AAMs and that Th2-type cytokines suppress inflammatory responses in human monocytes by activating different intracellular signalling pathways.
Asunto(s)
Interleucina-17 , Macrófagos/inmunología , Factor de Transcripción STAT6/inmunología , Células Cultivadas , Humanos , Interleucina-17/inmunología , Interleucina-17/metabolismo , Leucocitos Mononucleares/inmunología , Activación de MacrófagosRESUMEN
BACKGROUND & AIMS: During the pathogenesis of Crohn's disease (CD), interleukin (IL)-12, a cytokine produced by mucosal CD14+ monocyte-like cells, promotes tissue-damaging T helper cell (Th) 1-mediated inflammation through mechanisms that are not fully understood. IL-25 promotes Th2 cell responses by activating major histocompatibility complex class II-positive non-T and non-B cells. Because Th1 and Th2 cells, and the cytokines they release, are often mutually antagonistic, we examined whether IL-25 affects IL-12 production or Th1 cell-mediated inflammation in the gut. METHODS: Studies were performed using colonic samples from patients and mice with peptidoglycan (PGN)-, 2,4,6-trinitrobenzenesulphonic acid (TNBS)-, or oxazolone-induced colitis. IL-25 receptor (IL-25R) levels were evaluated in intestinal lamina propria mononuclear cells by flow cytometry, and IL-25 levels were measured by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. Mucosal CD14+ cells from patients with CD were incubated with IL-25 and/or lipopolysaccharide or PGN. Mice were injected with IL-25, and some mice first received injections of an IL-13 blocking antibody. Cytokines were quantified by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: CD14+ cells from the mucosa of CD patients expressed IL-25R and responded to IL-25 by decreasing the synthesis of IL-12 and IL-23. IL-25 prevented PGN-induced colitis in mice. IL-25 induced IL-13 production in the colon, but IL-13 was not required for suppression of PGN colitis. IL-25 ameliorated TNBS- and oxazolone-colitis. Patients with CD or ulcerative colitis produced significantly less IL-25 compared with controls. CONCLUSIONS: IL-25 inhibits CD14+ cell-derived cytokines and experimental colitis. IL-25 could be a useful treatment of CD and ulcerative colitis.
Asunto(s)
Citocinas/metabolismo , Interleucina-12/biosíntesis , Interleucina-17/metabolismo , Mucosa Intestinal/inmunología , Células TH1/inmunología , Animales , Western Blotting , Células Cultivadas , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Mediadores de Inflamación/análisis , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , Probabilidad , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Células TH1/fisiologíaRESUMEN
BACKGROUND & AIMS: Foxp3-expressing regulatory T cells (Tregs) play a key role in the maintenance of the gut immune homeostasis, and an intact transforming growth factor (TGF)-beta signaling is required for their function. In inflammatory bowel disease (IBD), the TGF-beta signaling is impaired because of high expression of the inhibitory molecule Smad7. Although no intrinsic defects in Tregs function have been shown in IBD, it is still unknown whether colitogenic T cells are susceptible to Treg-mediated suppression. In this study, we have investigated whether IBD mucosal CD4+ T cells are resistant to Tregs and whether Smad7 is involved in this process. METHODS: IBD lamina propria mononuclear cells (LPMC) were cultured with or without Tregs, and proliferation was assessed by flow cytometry. Proliferation of IBD LPMC was also evaluated after Smad7 antisense oligonuclotide treatment. Treg-mediated suppression of T-cell proliferation and proinflammatory cytokine expression was investigated in murine Smad7 transgenic cells. In vivo, the Smad7-dependent resistance of colitogenic naïve T cells to Tregs was studied in the adoptive transfer model of colitis. RESULTS: IBD LPMC were resistant to Treg-mediated suppression, and this phenomenon was reverted by Smad7 antisense treatment. Consistently, CD4+ T cells isolated from Smad7 transgenic mice showed high proliferation, produced considerable amount of inflammatory cytokines following activation, and induced a severe colitis when transferred in immunodeficient RAG1 knockout mice even in the presence of wild-type Tregs. CONCLUSIONS: Smad7 makes CD4+ T cells resistant to Tregs-mediated suppression thus fine-tuning their proinflammatory potential.
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Linfocitos T CD4-Positivos/patología , Comunicación Celular/fisiología , Colitis/metabolismo , Colitis/patología , Proteína smad7/metabolismo , Linfocitos T Reguladores/patología , Animales , Proliferación Celular , Células Cultivadas , Colon/metabolismo , Colon/patología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
IL-25, a member of the IL-17 cytokine family, is known to enhance Th2-like responses associated with increased serum levels of IgE, IgG1, IgA, blood eosinophilia, and eosinophilic infiltrates in various tissues. However, IL-25 also abrogates inflammatory responses driven by Th17 cells. However, the cell types that respond to IL-25 and the mechanisms by which IL-25 differentially regulates immune reactions are not well explored. To identify potential targets of IL-25, we initially examined IL-25 receptor (IL-25R) in human peripheral blood cells. IL-25R was predominantly expressed by CD14(+) cells. We next assessed the functional role of IL-25 in modulating the response of CD14(+) cells to various inflammatory signals. CD14(+) cells responded to IL-25 by down-regulating the synthesis of inflammatory cytokines induced by toll-like receptor (TLR) ligands and inflammatory cytokines. Inhibition of cytokine response by IL-25 occurred via a p38 Map kinase-driven Socs-3-dependent mechanism. In vivo, IL-25 inhibited monocyte-derived cytokines and protected against LPS-induced lethal endotoxemia in mice. These data indicate that IL-25 is a negative regulator of monocyte proinflammatory cytokine responses, which may have therapeutic implications.