Immunoregulatory Defects In Inflammatory Bowel Disease

项目1：明确的Th1反应导致肠道炎症，如在患有TNBS-结肠炎的SJL/J小鼠中发生的反应，为确定导致结肠炎的遗传位点(并最终确定的基因)提供了一个极好的机会。由于对TNBS-结肠炎的易感性存在巨大的小鼠品系差异，这一事实促进了这一方法：SJL/J小鼠和C57BL/10小鼠高度敏感，而C25BL/6小鼠高度耐药。因此，我们在SJL/J和C57BL/6小鼠的F2代中进行了广泛的全基因组连锁分析，方法是识别易感和抗性后代，然后将其与与这两个品系相关的多个染色体标记相关联。这一分析导致了两个易感基因座的鉴定，一个在9号染色体上，一个在11号染色体上，分别称为TNBS1和TNBS2。此外，虽然在雄性小鼠中，TNBS与疾病有很强的相关性，但在雌性小鼠中，它与疾病的关联相对较弱。相反，尽管TNBS与雌性小鼠的疾病密切相关，但它与雄性小鼠的疾病无关。然而，对TNBS1阴性的抗性雄性小鼠的进一步分析发现与TNBS2有关；因此，雄性和雌性小鼠都在11号染色体上有一个易感基因。认识到11号染色体上的该基因包含IL-12基因，我们进行了进一步的研究，以确定IL-12反应异常也可能映射到该基因。因此，我们给SJL/J和C57BL/6小鼠ip内毒素，结果表明，虽然两个品系的内毒素诱导的IL-12p40的产生是相同的，但SJL/J品系的IL-12p70的量远远高于C57BL/6品系。然后，我们进行了进一步的基因组分析，以定位这种反应的差异，并发现安装高IL-12p70反应的能力被映射到SJL/J染色体11等位基因。因此，这些数据强烈暗示，涉及IL-12基因的遗传异常在一定程度上与TNBS-结肠炎的易感性有关。这一结论也得到了以下事实的支持：雄性C57BL/10小鼠的基因组与C57BL/6小鼠的基因组有99%的同源性，在11号染色体易感区域有很大差异，也对TNBS-结肠炎易感，并对内毒素诱导的IL-12p70产生高反应。最后，值得注意的是，人类染色体5(5q33-34)上与11号染色体上的TNBS2同线的区域与人类克罗恩-S病有关：在此基础上，TNBS2可能与人类疾病有关。

Project 1: Defined Th1 responses causing intestinal inflammation such as that occurring in SJL/J mice with TNBS-colitis offer an excellent opportunity to identify genetic loci (and ultimately defined genes) that are responsible for the colitis. This approach is facilitated by the fact that there is enormous mouse strain variability in susceptibility to TNBS-colitis: SJL/J mice and C57BL/10 mice are highly susceptible whereas C25BL/6 mice are highly resistant. Accordingly, we conducted an extensive genome-wide linkage analysis in F2 progeny of SJL/J and C57BL/6 mice by identifying susceptible and resistant progeny and then correlating that with multiple chromosomal markers associated with the two strains. This analysis led to the identification of two susceptibility loci, one on chromosome 9 and one on chromosome 11 called TNBS1 and TNBS2 respectively. Furthermore, while TNBS was strongly associated with disease in male mice, it was relatively weakly associated with disease in females. Conversely, while TNBS was strongly associated with disease in female mice, it was not associated with disease in male mice. However, further analysis of resistant male mice negative for TNBS1 revealed an association with TNBS2; thus, both male and female mice have a susceptibility locus on chromosome 11. Recognizing that locus on chromosome 11 contains the IL-12 gene, we conducted futher studies to identify IL-12 response abnormalities that might also map to this locus. Accordingly, we administered IP LPS to SJL/J and C57BL/6 mice and showed that whereas IL-12 p40 production induced by the LPS was equivalent in the two strains, the amount of IL-12 p70 was vastly greater in the SJL/J strain than in the C57BL/6 strain. We then conducted further genome-analysis to map this difference in responses and found that the ability to mount a high IL-12 p70 response mapped to the SJL/J chromosome 11 allele. Thus, these data were strongly suggestive that a genetic abnormality involving the IL-12 gene is partly responsible for susceptibility to TNBS-colitis. This conclusion was also supported by the fact that male C57BL/10 mice whose genome is 99% identical to that of C57BL/6 mice and differs largely at the chromosome 11 susceptibility region, is also susceptible to TNBS-colitis and mounts a high LPS-induced IL-12 p70 response. Finally, it is important to note that a human chromosome region on human chromosome 5 (5q33-34) syntenic to the TNBS2 on chromosome 11 has been linked to human Crohn?s disease: on this basis, TNBS2 may have relevance to the human disease.