Immunoregulatory Defects in Inflammatory Bowel Disease

项目1：对小鼠肠道炎症模型的研究为人类克罗恩病的发病机制和治疗提供了丰富的新见解。本实验室广泛研究的一种这样的模型是TNBS结肠炎模型，这是一种半抗原诱导的结肠炎，原因是SJL/J小鼠过度的IL-12驱动的Th1 T细胞介导的免疫反应。在本研究中，我们探索了重组霍乱毒素B链(RCT-B)作为治疗TNBS结肠炎的药物。在最初的研究中，我们发现，在通过直肠内滴注TNBS诱导TNBS结肠炎时口服RCT-B，可以防止结肠炎的发展，或者在TNBS结肠炎确诊后，带来结肠炎的缓解。在进一步的研究中，我们发现，这种CT-B给药伴随着阻止或逆转干扰素-γ的分泌(Th1反应的标志)，而对IL-4的分泌没有伴随的影响。这种对干扰素-γ分泌的影响不是由于对反调节细胞因子IL-10或转化生长因子-β的分泌的影响，而是由于对IL-12分泌的显著抑制。最后，我们发现RCT-B给药导致固有层细胞的凋亡增加，这一效应以前被证明是IL-12剥夺的迹象。从这些研究中，RCT-B成为一种强大的Th1 T细胞驱动的炎症抑制剂，可能应用于克罗恩病的治疗。项目2：在对TNBS-结肠炎的进一步研究中，我们探索了通过DNA质粒传递的转化生长因子-β抑制Th1 T细胞介导的炎症的能力。在这些研究中，我们最初表明，单次鼻腔注射编码活性转化生长因子-β1(pCMV-转化生长因子-β1)的质粒可防止TNBS-结肠炎的发生。此外，在TNBS-结肠炎建立后，这样的质粒注射可以消除它，而相反，I.P.给予rTGF-beta1蛋白则不具有这种作用。在进一步研究转化生长因子-βDNA的作用机制时，我们首先发现，鼻腔注射pCMV-转化生长因子-β1可导致肠道固有层和脾组织中转化生长因子-β1基因的表达，并在这些组织中出现转化生长因子-β1产生的T细胞和巨噬细胞；此外，它与组织纤维化的发生无关。我们随后发现，产生转化生长因子-β1的细胞可显著抑制IL-12和干扰素-γ的产生，促进IL-10的产生；此外，它们还抑制IL-12Rβ2链的表达。最后，我们发现在pCMV-转化生长因子-β1注射时联合应用抗IL-10可以阻止IL-10产生的增强，并逆转对IL-12的抑制，但不能逆转干扰素-γ的分泌；然而，抗IL-10会导致肿瘤坏死因子-α的产生增加，特别是在已建立的结肠炎中。综上所述，这些研究表明，转化生长因子-β1抑制Th1介导的结肠炎是由于：1)通过IL-10诱导抑制IL-12的分泌；2)通过下调IL-12Rβ2链的表达抑制IL-12信号转导；此外，转化生长因子-β1也可能对干扰素-γ的转录有抑制作用。

Project 1: The study of murine models of intestinal inflammation has been a rich source of new insights concerning the pathogenesis and treatment of human Crohn's disease. One such model that has been extensively studied in this laboratory is the model known as TNBS-colitis, a haptene-induced colitis due to an excessive IL-12-driven, Th1 T cell-mediated immune response in SJL/J mice. In the present study, we explored the use of recombinant B chain of cholera toxin (rCT-B) as an agent for the treatment of TNBS-colitis. In initial studies we showed that oral administration of rCT-B at the time of TNBS-colitis induction by intra-rectal TNBS instillation, prevents the development of colitis or, at later time when TNBS-colitis is well established, brings about resolution of the colitis. In further studies we showed that such CT-B administration is accompanied by prevention or reversal of IFN-gamma secretion (the hallmark of a Th1 response) without a concomitant effect on IL-4 secretion. This effect on IFN-gamma secretion is not due to an effect on the secretion of the counter-regulatory cytokines IL-10 or TGF-beta but, instead, is due to marked inhibition of IL-12 secretion. Finally, we showed that rCT-B administration results in increased apoptosis of lamina propria cells, an effect previously shown to be indicative of IL-12 deprivation. From these studies, rCT-B emerges as a powerful inhibitor of Th1 T cell-driven inflammation that can potentially be applied to the treatment of Crohn's disease. Project 2: In further studies of TNBS-colitis we explored the capacity of TGF-beta delivered via a DNA plasmid to inhibit the Th1 T cell-mediated inflammation. In these studies we initially showed that a single intranasal dose of a plasmid encoding active TGF-beta1 (pCMV-TGF-beta1) prevents the development of TNBS-colitis. In addition, such plasmid administration abrogates TNBS-colitis after it has been established, whereas in contrast, i.p. administration of rTGF-beta1 protein does not have this effect. In further studies addressing the mechanism of the effect of the TGF-beta DNA plasmid we showed first that intranasal pCMV-TGF-beta1 administration leads to the expression of TGF-beta1 mRNA in the intestinal lamina propria and spleen for two weeks, as well as the appearance of TGF-beta1 producing T cells and macrophages in these tissues; in addition, it is not associated with the occurrence of tissue fibrosis. We then showed that TGF-beta1-producing cells cause marked suppression of IL-12 and IFN-gamma production and enhancement of IL-10 production; in addition, they inhibit IL-12R beta2 chain expression. Finally, we showed that co-administration of anti-IL-10 at the time of pCMV-TGF-beta1 administration prevents the enhancement of IL-10 production and reverses the suppression of IL-12, but not IFN-gamma secretion; however, anti-IL-10 leads to increased TNF-alpha production, especially in established colitis. Taken together, these studies demonstrate that TGF-beta1 inhibition of a Th1-mediated colitis is due to: 1) suppression of IL-12 secretion by IL-10 induction; and 2) inhibition of IL-12 signaling via down-regulation of IL-12R beta2 chain expression; in addition, TGF-beta1 may also have an inhibitory effect on IFN-gammatranscription.