RESUMEN
BACKGROUND: A subset of irritable bowel syndrome (IBS) patients, denoted post-infectious IBS (PI-IBS), develop symptoms after an enteric infection. Bacterial dysbiosis and mucosal inflammation have been proposed to be involved in the pathophysiology of this entity. AIM: To characterise the mucosal and faecal microbiota in PI-IBS, general IBS and healthy controls, and to investigate associations between the microbiota and the mucosal immune system. METHODS: Mucosal biopsies and faeces were collected from 13 PI-IBS patients, 19 general IBS patients and 16 healthy controls. Global bacterial composition was determined by generating 16S rRNA amplicons that were examined by phylogenetic microarray hybridisation, principal component and redundancy analysis. We correlated previously reported lymphocyte proportions with the microbiota. RESULTS: Faecal microbiota composition of PI-IBS patients differed significantly from both general IBS patients and healthy controls (P < 0.02). Both mucosal (P < 0.01) and faecal (P = 0.05) microbial diversity were reduced in PI-IBS compared to healthy controls. In the intraepithelial lymphocytes the previously published proportion of CD8(+) CD45RA(+) was negatively correlated with mucosal microbial diversity (P < 0.005). The previously published number of lamina propria lymphocytes was negatively correlated with mucosal microbial diversity (P < 0.05). Faecal microbial diversity was significantly negatively correlated with the Hospital Anxiety and Depression scale (P < 0.05). CONCLUSIONS: We present data that distinguishes the intestinal microbiota of PI-IBS patients from that of both general IBS patients and HC. The microbial composition is significantly associated with the HADs score and alterations in lymphocyte subsets proportions.
Asunto(s)
Heces/microbiología , Mucosa Intestinal/microbiología , Síndrome del Colon Irritable/microbiología , Adulto , Índice de Masa Corporal , Femenino , Gastritis/complicaciones , Humanos , Mucosa Intestinal/inmunología , Intestinos/patología , Síndrome del Colon Irritable/etiología , Síndrome del Colon Irritable/inmunología , Subgrupos Linfocitarios , Masculino , Microbiota/inmunología , Persona de Mediana Edad , Fenotipo , ARN Ribosómico 16SRESUMEN
One of the most widely used animal models for inflammatory bowel disease (IBD) is the dextran sulfate sodium (DSS)-induced colitis. We have previously reported that 5 days administration of DSS in C57Bl/6J mice induces a colonic inflammation that progresses into chronicity after DSS removal, whereas in BALB/cJ mice the inflammation resolves within 4 weeks post-DSS. Here we show that both thymic size and thymocyte numbers dramatically decreased in the acute phase of inflammation in C57Bl/6 mice, 7 days after DSS withdrawal. Mature, CD4(+) and CD8(+) single positive (SP) CD69(lo) CD62L(hi) thymocytes were enriched in these mice, accompanied by a major decrease in the number of immature double positive (DP) thymocytes. However, the different maturation stages within the DP thymocyte subset were unchanged between healthy and inflamed C57Bl/6J mice. Interestingly, as the inflammation progressed into the chronic phase, the thymus recovered and 2 weeks after the acute inflammatory phase all the thymic parameters investigated in this study were restored to normal. In contrast, BALB/cJ mice only develop mild thymic alterations. Nevertheless, we found that within the double negative (DN) thymocytes an increased frequency and also total numbers of CD44(+) CD25(-) (DN1) cells correlated with the severity of colitis, and that the frequency of CD44(-) CD25(-) (DN4) thymocytes decreased proportionally in the acute phase in BALB/cJ mice. Our observations suggest that the thymic effects are intimately connected to the intestinal inflammatory response in colitis regardless of the inflammatory stimuli.
Asunto(s)
Diferenciación Celular/inmunología , Colitis/patología , Sulfato de Dextran/administración & dosificación , Subgrupos Linfocitarios/patología , Subgrupos de Linfocitos T/patología , Timo/patología , Enfermedad Aguda , Animales , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/inmunología , Inmunofenotipificación , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Timo/efectos de los fármacos , Timo/inmunologíaRESUMEN
AIM: To study the serum concentration of IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-18 in Galphai2-deficient mice at the age of 6 (healthy), 12 (pre-colitic) and 24 wk (colitic) and in healthy control mice. METHODS: At the time of killing, serum samples were collected and IL-1beta, IL-1Ra and IL-18 levels were measured using enzyme-linked immunosorbent assays. RESULTS: Serum concentration of IL-1Ra was significantly increased in pre-colitic (median: 524 ng/L; P=0.02) and colitic (450 ng/L; P=0.01), but not in healthy (196 ng/L) Galphai2-deficient mice as compared with controls (217 ng/L). Serum concentrations of IL-1beta did not differ between Galphai2-deficient mice and their controls, irrespective of age, IL-18 was significantly increased in colitic, but not in pre-colitic mice compared with controls (510 ng/L vs 190 ng/L; P=0.05). CONCLUSION: The increased serum concentrations of IL-18 and IL-1Ra in established diseases are suggested as markers of ongoing colitis. Interestingly, the significantly increased serum concentration of IL-1Ra in pre-colitic mice is found to be an early marker of disease progression.
Asunto(s)
Colitis/sangre , Subunidad alfa de la Proteína de Unión al GTP Gi2/deficiencia , Interleucina-18/sangre , Sialoglicoproteínas/sangre , Animales , Biomarcadores , Colitis/diagnóstico , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/fisiología , Enfermedades Inflamatorias del Intestino/diagnóstico , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Bazo/patologíaRESUMEN
Mice deficient for the inhibitory G protein subunit alpha2 (Galphai2(-/-)) spontaneously develop a progressive inflammatory bowel disease resembling ulcerative colitis, and have a T helper 1 (Th1)-dominated immune response prior to onset of colitis, which is further augmented after the onset of disease. The present study was performed to investigate whether the Galphai2(-/-) mice were able to down-regulate the Th1-dominated inflammatory mucosal immune response and/or induce an anti-inflammatory Th2/T regulatory response and thereby diminish the severity of colitis following treatment with acellular Bordetella pertussis vaccine. The acellular vaccine against B. pertussis, the causative agent of whooping cough, has been demonstrated to induce a Th2-mediated response in both man and mice. We therefore treated Galphai2(-/-) mice intraperitoneally with a three-component acellular B. pertussis vaccine. The treated Galphai2(-/-) mice showed significantly increased interleukin (IL)-10 production in intestinal tissue, associated with significantly reduced colitis and decreased mortality, compared to untreated Galphai2(-/-) mice. The attenuation of colitis in Galphai2(-/-) mice was due, at least partly, to the B. pertussis surface antigen filamentous haemagglutinin (FHA), which almost completely inhibited proliferation of CD4(+) T cells and stimulated apoptosis of activated CD4(+) T helper 1 cells. In conclusion, the three-component acellular B. pertussis vaccine containing filamentous haemagglutinin increases the production of IL-10 in the intestinal mucosa, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2(-/-) mice.
Asunto(s)
Colitis Ulcerosa/terapia , Subunidades alfa de la Proteína de Unión al GTP/deficiencia , Interleucina-10/biosíntesis , Vacuna contra la Tos Ferina/inmunología , Células TH1/inmunología , Adhesinas Bacterianas/inmunología , Animales , Antígenos Bacterianos/inmunología , Apoptosis/inmunología , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Modelos Animales de Enfermedad , Hemaglutininas/inmunología , Inmunidad Mucosa , Inmunoglobulina G/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Vacuna contra la Tos Ferina/uso terapéutico , Tasa de Supervivencia , Factores de Virulencia de Bordetella/inmunologíaRESUMEN
We investigated dietary antigen-specific T-cell responses in mesenteric lymph nodes (MLN) and Peyer's patches (PP) in noncolitic control mice as well as in colitis-prone mice prior to onset of histological active colitis. T cells were restimulated in vitro with constituents isolated from the mouse diet. Interestingly, MLN T cells of littermate G(alpha)i2+/- control mice responded to soya with high production of interleukin (IL)-10, but did not produce proinflammatory T-helper 1 (Th1) cytokines. Recall dietary antigen stimulation of G(alpha)i2+/- PP T cells did not result in increased IL-10 production above the spontaneous production in the absence of antigenic stimulation. In strong contrast, MLN T cells from precolitic G(alpha)i2-/- mice produced high levels of interferon-gamma (IFN-gamma) upon restimulation with soya, which could be abolished using a major histocompatibility complex class II-blocking antibody. In conclusion, the present study demonstrates that MLN T lymphocytes in normal healthy mice respond with a significantly increased production of the regulatory cytokine IL-10 on re-encounter with dietary proteins in vitro. In marked contrast precolitic G(alpha)i2-/- mice respond to dietary antigens with a Th1-dominated cytokine response in the mucosa, prior to onset of colitis, with excessive IFN-gamma production. These results suggest that aberrant immune responses to dietary antigens could contribute as a potential pathogenic mechanism in the onset of colitis in G(alpha)i2-deficient mice.
Asunto(s)
Antígenos/administración & dosificación , Colitis/etiología , Colitis/inmunología , Interleucina-10/biosíntesis , Linfocitos T/inmunología , Alérgenos/administración & dosificación , Animales , Anticuerpos Bloqueadores/administración & dosificación , Citocinas/biosíntesis , Dieta , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Técnicas In Vitro , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglios Linfáticos Agregados/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Células TH1/inmunologíaRESUMEN
BACKGROUND: Mice with a targeted mutation in the G protein subunit G alpha i2 gene develop a colonic mucosal inflammation, with a highly activated B-cell response. We wanted to investigate whether this increased B-cell activity was directed against dietary antigens and/or various self tissues. METHODS: The level of antibodies specific for dietary (gliadin, soya and fish meal) antigens was measured by ELISA. Reactivity against self antigens was measured by immunohistochemistry on cryo-sectioned mouse and rat tissue. Sera and intestinal lavages were analysed from G alpha i2-/- mice before and after development of colitis and in age-matched wild type litter mates. RESULTS: Titres of antibodies against dietary antigens were significantly enhanced both in serum and in large intestinal lavages from G alpha i2-/- mice with ongoing colitis but not prior to disease, as compared to wild type mice. The autoreactivity to self tissues was significantly increased in G alpha i2-/- mice both before and after development of colitis as compared to litter mate control animals. Self tissue reactivity was directed not only against epithelial cells of the colon, small intestine and gastric glands, but also against smooth muscle cells, hepatocytes, bile duct cells, renal tubule and collecting tubule cells of the kidney. In analogy to human ulcerative colitis, autoantibodies against epithelial cells, bile duct epithelium and neutrophil granulocytes were found. CONCLUSIONS: Earlier increase in levels of autoantibodies (before onset of colitis) than of food antibodies (after onset of colitis) suggests the latter response to be a secondary phenomenon to e.g. a destroyed barrier function.
Asunto(s)
Autoanticuerpos/metabolismo , Linfocitos B/inmunología , Colitis/etiología , Proteínas en la Dieta/efectos adversos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/inmunología , Animales , Autoanticuerpos/sangre , Colitis/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Humanos , Mucosa Intestinal/inmunología , Ratones , Ratones SCID , Ratas , Ratas Sprague-DawleyRESUMEN
G-protein subunit Galphai2-deficient mice spontaneously develop an inflammatory bowel disease that clinically and histopathologically resembles ulcerative colitis in humans. The aim of this study was to determine whether immunological changes precede the development of colitis in Galphai2-deficient mice. Therefore, Galphai2-deficient mice with no clinical or histopathological signs of colitis were compared with Galphai2-deficient mice with established colitis and wild-type animals, concerning immunological parameters. Healthy Galphai2-deficient mice displayed an increased frequency of CD4+ T cells and a decreased frequency of CD19+ B lymphocytes in the intestinal mucosa compared with control mice. The CD4+ population was characterized by a memory phenotype, i.e. increased expression of CD44 and decreased expression of CD45RB and CD62L, as well as increased expression of the mucosal homing receptors integrins alpha4beta7 and alphaEbeta7. Production of pro-inflammatory cytokines, interleukin (IL)-1beta and interferon (IFN)-gamma, were increased in Galphai2-deficient mice before clinical signs of disease were evident. In addition, total immunoglobulin (Ig)G and IgA levels in large intestinal secretions were increased significantly compared with wild-type mice, and antibodies specific for the normal intestinal flora in large intestinal secretions were present in Galphai2-deficient mice several weeks before the onset of colitis. In contrast, antibodies against tropomyosin, a putative autoantigen in human ulcerative colitis, were not found in Galphai2-deficient mice before the onset of colitis, although they were present in animals with established disease. In conclusion, activation of the intestinal immune system precedes histopathological and clinical signs of inflammation in Galphai2-deficient mice, suggesting that immune abnormalities play an important role in the induction of colitis.