RESUMO
Gαi2-deficient mice spontaneously develop colitis. Using xMAP technology and RT-PCR, we investigated cytokine/chemokine profiles during histologically defined phases of disease: (i) no/mild, (ii) moderate, (iii) severe colitis without dysplasia/cancer and (iv) severe colitis with dysplasia/cancer, compared with age-matched wild-type (WT) littermates. Colonic dysplasia was observed in 4/11 mice and cancer in 1/11 mice with severe colitis. The histology correlated with progressive increases in colon weight/cm and spleen weight, and decreased thymus weight, all more advanced in mice with dysplasia/cancer. IL-1ß, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 protein levels in colons, but not small intestines increased with colitis progression and were significantly increased in mice with moderate and severe colitis compared with WT mice, irrespective of the absence/presence of dysplasia/cancer. CCL5 did not change during colitis progression. Colonic IL-17 transcription increased 40- to 70-fold in all stages of colitis, whereas IFN-γ mRNA was gradually up-regulated 12- to 55-fold with colitis progression, and further to 62-fold in mice with dysplasia/cancer. IL-27 mRNA increased 4- to 15-fold during the course of colitis, and colonic IL-21 transcription increased 3-fold in mice with severe colitis, both irrespective of the absence/presence of dysplasia/cancer. FoxP3 transcription was significantly enhanced (3.5-fold) in mice with moderate and severe colitis, but not in mice with dysplasia/cancer, compared with WT mice. Constrained correspondence analysis demonstrated an association between increased protein levels of TNF-α, CCL2, IL-1ß, IL-6 and CXCL1 and dysplasia/cancer. In conclusion, colonic responses are dominated by a mixed T(h)1/T(h)17 phenotype, with increasing T(h)1 cytokine transcription with progression of colitis in Gαi2(-/-) mice.
Assuntos
Colite/imunologia , Colo/imunologia , Neoplasias do Colo/imunologia , Citocinas/imunologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/deficiência , Células Th1/imunologia , Células Th17/imunologia , Animais , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocinas/genética , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/imunologia , Regulação da Expressão Gênica/imunologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Índice de Gravidade de Doença , Transdução de Sinais , Baço/imunologia , Baço/patologia , Células Th1/metabolismo , Células Th1/patologia , Equilíbrio Th1-Th2 , Células Th17/metabolismo , Células Th17/patologia , Timo/imunologia , Timo/patologia , Transcrição Gênica/imunologiaRESUMO
BACKGROUND: Mice deficient in the inhibitory G protein subunit Gαi2 spontaneously develop a T helper 1 dominated colitis. We examined whether a defect in CD4(+)FoxP3(+) regulatory T cells (Treg) underpins the pathogenesis of colitis in the Gαi2(-/-) (Gαi2-deficient) colitis model. METHODOLOGY/PRINCIPAL FINDINGS: Using flow cytometry, we found that thymus and colonic lamina propria, but not spleen and mesenteric lymph nodes, of colitic Gαi2(-/-) mice contained increased frequencies of Treg, whereas FoxP3 expression intensity was similar in Gαi2(-/-) compared to Gαi2(+/-) or Gαi2(+/+) wild type (WT) mice. The frequency of CD4(+)FoxP3(+) T cells expressing CD103 was significantly increased in Gαi2(-/-) compared to WT mice. Treg in colons from WT mice clustered in the T cell areas of colonic lymphoid patches (CLP), with relatively few Treg in the lamina propria, as demonstrated by immunohistochemistry. In Gαi2(-/-) mice, CLP were not observed but lamina propria Treg were increased in number and frequency within the CD4(+) infiltrate, compared to WT mice. Using an in vitro co-culture system and flow cytometric analysis of cell division we could demonstrate that the in vitro suppressive function of WT and Gαi2(-/-) CD4(+)FoxP3(+) regulatory T cells (WT-Treg and KO-Treg) was indistinguishable, but that T effector cells (CD4(+)25(-) T cells) from Gαi2(-/-) mice were less readily suppressed than WT effectors (WT-Teff) by Treg from either source. However, neither WT nor Gαi2(-/-) Treg was able to suppress colitis induced by adoptive transfer of Gαi2(-/-) effector T cells (KO-Teff) to RAG2(-/-) recipients. The enhanced inflammatory activity of Gαi2(-/-) effectors was accompanied by increased expression of an effector/memory T cell phenotype and increased cytokine secretion, especially IL-4, IL-6 and IFN-γ. CONCLUSIONS: There is an increased frequency of Gαi2(-/-) Treg in the colon, and they demonstrate no endogenous functional defect. However, Gαi2(-/-) T effector cells are dramatically less susceptible to suppression in vitro, and in vivo, despite increased effective numbers of Treg, they cannot prevent disease.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Colite/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Linfócitos T Reguladores/metabolismo , Animais , Divisão Celular , Técnicas de Cocultura , Colite/patologia , Feminino , Citometria de Fluxo/métodos , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica , Receptores de Hialuronatos/biossíntese , Imuno-Histoquímica/métodos , Mucosa Intestinal/patologia , Selectina L/biossíntese , Masculino , Camundongos , Baço/metabolismo , Frações Subcelulares/metabolismoRESUMO
The aim of this study was to investigate the relevance of mouse ex vivo cultures as a first screening model for new therapeutic agents of Inflammatory Bowel Disease (IBD). Two murine models (dextran sodium sulphate (DSS)-induced colitis and Galphai2-deficient mice) and two anti-inflammatory agents (methyl-prednisolone and the proteasome inhibitor MG132) were evaluated. The in vivo effects of methyl-prednisolone were assessed in both models. Ex vivo colonic tissue from both mouse models were cultured in the presence or absence of the drugs and TaqMan Low-Density arrays were used to assess the regulation of inflammatory genes before and after drug treatment. Colitis induced a similar inflammatory gene profile in both mouse models in in vivo studies and in ex vivo cultures. The differences encountered reflected the different phases of colitis in the models, e.g. innate cytokine/chemokine profile in the DSS model and T cell related markers in Galphai2-deficient mice. After steroid treatment, a similar pattern of genes was suppressed in the two mouse models. We confirmed the suppression of inflammatory gene expression for IL-1beta, IL-6 and iNOS in ex vivo and in vivo colons from both mouse models by quantitative RT-PCR. Importantly, the inflammatory responses in the murine ex vivo culture system reflected the in vivo response in the inflamed colonic tissue as assessed by changes in inflammatory gene expression, suggesting that the murine culture system can be used for validation of future IBD therapies.