IDO调控Kyn/AhR介导的T细胞分化及肿瘤细胞增殖在结肠癌化学预防中的作用及机制研究

Indoleamine 2,3-dioxygenase (IDO) is greatly involved in the development and progression of many cancer types. It was reported that IDO played a critical role in induction of immune escape. IDO activation was reported to correlate with poor clinical outcomes in patients with endometrial carcinoma, ovarian carcinoma and CRC. In the tumor microenvironment, kyn and other metabolites derived from tryptophan through IDO activation could suppress the proliferation of functional T cells and natural killer cells, but promote the differentiation and activation of Foxp3+ regulatory T cells. We demonstrated that kynurenine was required for IDO-mediated T cells function. Kyn promoted AhR nuclear translocation, and increased the transcription of Foxp3, through improving the interaction between AhR and Foxp3 promoter. Additionally, comparing to WT mice, IDO-/- mice treated with AOM/DSS exhibited fewer and smaller tumor burdens in colon, with fewer Tregs and more CD8+ T cells infiltration, while Kyn administration abolished this regulation. Rag1-/- mice, which lacked T cells, were sensitive to AOM/DSS-induced CRC, verifying that T cells-mediated adaptive immunity indeed played a critical role in CRC. We also demonstrated the chemopreventive effects of 1-L-MT on colitis-associated cancer, which were independent of adaptive immunity. IDO inhibition induced mitotic death, which was mediated by decreased Kyn production. The aim of this study is to investigate the role of IDO in the development of intestinal tumor induced by AOM/DSS, and illustrate the mechanisms of how IDO/Kyn/AhR pathway is involved in the progression of colorectal cancer. The result of this study would shed light on the prevention of the colorectal cancer.

IDO与结肠癌的恶性程度及不良预后密切相关。IDO介导的低色氨酸环境，或其代谢产物犬尿氨酸等可促进Tregs增殖，髓系抑制细胞招募增多，IL-10和TGF-β分泌增加，维持免疫耐受环境。本课题组前期研究发现：在小鼠结肠癌模型中，IDO的表达与活性显著升高，而IDO-/-小鼠的成瘤率明显低于野生型小鼠；给予IDO抑制剂后，结肠部位的免疫耐受微环境得以逆转，结肠癌增殖受阻；提示IDO既参与调控T细胞的不平衡分化，也可通过免疫非依赖的途径直接调控肿瘤细胞增殖，其确切机制有待进一步研究。目前国内外对IDO在结肠癌中的调控作用及其抑制剂用于结肠癌化学预防鲜有报道。本项目拟运用IDO、Rag1、AhR等敲除小鼠模型，结合肿瘤细胞-T细胞共培养体外模型，深入研究IDO对T细胞分化及对肿瘤细胞增殖的调控作用和确切机制，找寻免疫调控干预结肠癌进程的有效途径，为非可控性炎症恶性转化的防治提供新思路。

慢性炎症与结肠癌的风险密切相关。而炎症相关的T细胞浸润在结肠癌的发生发展中起着重要作用。IDO与结肠癌的恶性程度及不良预后密切相关。我们研究发现，犬尿氨酸对IDO介导的T细胞功能是不可或缺的。Kyn 通过促进AhR与Foxp3启动子结合而促进AhR入核，激活Foxp3转录，从而调节Tregs的功能。在小鼠结肠癌模型中，IDO的表达与活性显著升高，而IDO-/-小鼠的成瘤率明显低于野生型小鼠，伴随着更少的Treg 和更多的CD8 T细胞浸润。给予IDO抑制剂后，结肠部位的免疫耐受微环境得以逆转，结肠癌增殖受阻。Rag1-/-小鼠对AOM/DSS诱导的结肠癌形成更加敏感，提示T细胞介导的获得性免疫在炎症相关结肠癌发生发展中起着重要作用。通过抑制IDO可以抑制Kyn/AhR介导的Treg分化，可作为预防和治疗炎症相关结肠癌的靶点。.进一步研究发现，IDO抑制剂1-L-MT, 通过抑制有丝分裂死亡而抑制结肠癌细胞的增殖。抑制IDO可以抑制CDC20转录, 导致细胞G2/M周期阻滞。1-L-MT可诱导线粒体损伤而促进细胞凋亡。体内实验发现，1-L-MT 可减少AOM/DSS诱导的结肠癌模型中小鼠的死亡率，肿瘤数量和体积。IDO-/-小鼠中肿瘤负荷相较野生型小鼠减少，且伴随恶性上皮细胞增殖的减少，而1-L-MT在IDO-/-没有表现出进一步的保护作用，而1-L-MT在Rag1-/-小鼠亦可发挥保护作用，提示IDO抑制有不依赖于其调控获得性免疫的机制。综上，我们发现了IDO抑制剂1-L-MT 抑制结肠癌发生发展是通过调控有丝分裂灾难从而抑制肿瘤细胞增殖。IDO既参与调控T细胞的不平衡分化，也可通过免疫非依赖的途径直接调控肿瘤细胞增殖。我们研究发现了IDO对T细胞分化及对肿瘤细胞增殖的调控作用和确切机制，为非可控性炎症恶性转化的防治提供新思路。.我们结合数据库分析和文献检索筛选出多个可能与IDO1功能相关的趋化因子。结果显示抑制IDO1导致CXCL9和CXCL10 RNA表达和分泌蛋白表达均下降，因此推测在使用IDO1抑制剂时补回CXCL9或CXCL10可能可以促进IDO1抑制剂的抗肿瘤作用。实验证明，IDO1抑制剂联用CXCL10有协同作用，而联合CXCL9则没有。针对IDO1影响CXCL10表达的机制进行研究，发现其并不是通过犬尿氨酸实现的。我们将对相关机制进行深入研究。