NLRP3炎性小体活化介导的MDSCs异常分化在奥沙利铂药物抵抗中的作用及分子机制

The components of tumor microenvironment (TME) could be altered by anticancer chemotherapeutics, which is the main cause of chemotherapeutics resistance. To overcome chemotherapeutics resistance, we should focus on both the elimination of cancer cells and the alteration of tumor microenvironment. As important immune cells in tumor microenvironment, myeloid-derived suppressor cells (MDSCs) could differentiate into tumoricical M1 macrophage or protumoral M2 macrophage in tumor microenvironment. In our previous studies, the frequency of MDSCs and M1/M2 ratio decreased significantly in tumor- bearing mice with the treatment of oxaliplatin. In vitro, oxalipaltin promoted MDSCs to differentiate into M2 macrophage. Our data suggested that oxaliplatin could be immunosuppressive, but the underlying mechanism was unclear. Recent studies showed that NLRP3 inflammasome was the “switch” of myeloid cells differentiation. We found the activity of inflammasome increased in oxaliplatin-treatment group and oxaliplatin-medicated MDSCs differentiation could be inhibited by suppressing the activity of inflammasome in MDSCs. By bioinformatics analysis, we found there was a predict cleavage site in IRF5 protein sequence by caspase-1. So, we hypothesized that inflammasome was involved in oxaliplatin-induced MDSCs differentiation, which could be a new mechanism of chemotherapeutics resistance. In the present studies, we further investigate the effects of oxaliplatin on the differentiation of MDSCs and the underlying mechanism in vitro and in vivo. We focus on the NLRP3/caspase-1 - dependent IRF5 transcription regulation. Our research will provide a new insight on developing new antitumor methods with less side-effects and more effective.

化疗药物导致的肿瘤微环境改变是产生化疗药物抵抗的重要原因，只有在杀灭肿瘤细胞的同时彻底改变其赖以生存的微环境，才能彻底解决化疗药物抵抗问题。髓源抑制性细胞（MDSCs）是微环境中重要的免疫细胞，我们观察到奥沙利铂有促进MDSCs向具有促瘤活性的M2型巨噬细胞分化的效应，提示其具有抑制肿瘤免疫的副作用，但机制未明。研究证实NLRP3炎性小体是调节髓系细胞分化的关键节点，我们发现奥沙利铂可激活MDSCs内炎性小体活性，降低转录因子IRF5（M1标志分子）表达，抑制炎性小体活性可逆转前述M2分化效应。因此，我们猜想“炎性小体活化介导的MDSCs异常分化”是奥沙利铂药物抵抗机制之一。本课题拟继续探讨肠癌化疗过程中奥沙利铂对MDSCs分化的影响，明确炎性小体活化对MDSCs分化的作用及分子机制，尤其是NLRP3/caspase-1轴介导的IRF5转录调控，为克服肿瘤化疗药物抵抗提供实验数据及新思路。

奥沙利铂耐药是影响临床化疗方案疗效的重要原因，其耐药机制被普遍认为与细胞转运，DNA损伤修复，细胞凋亡，表观遗传学改变等机制有关。然而其对免疫细胞的潜在调节机制至今尚不明确。髓源抑制性细胞（MDSCs）以及由其分化而来的肿瘤相关巨噬细胞（TAMs）作为肿瘤微环境中重要的免疫抑制细胞，在化疗耐药中起到重要作用。本项目通过流式细胞术、免疫组化、免疫荧光、qPCR等手段分别在结直肠癌患者、体内、体外实验中证实了奥沙利铂可以显著抑制MDSCs向M1型巨噬细胞分化。此外，M1型巨噬细胞上清可以通过增强奥沙利铂对肿瘤细胞诱导凋亡能力及对肿瘤细胞侵袭迁移的抑制能力，而且这种效应呈剂量依赖性。这表明奥沙利铂可能通过抑制MDSCs向M1型巨噬细胞分化产生化疗耐药。研究进一步探讨奥沙利铂对MDSCs异常分化调节的分子机制，MDSCs经过奥沙利铂处理后，Caspase-1活性显著增高，而IRF-5的表达则明显下降，而通过WB实验进一步验证经过Caspase-1抑制剂Z-YVAD-FMK处理后IRF-5表达量显著提高，并且流式结果显示MDSCs向M1型巨噬细胞分化比例明显增加，这提示Caspase-1可能是IRF-5上游信号分子。我们进一步使用CO-IP技术证明Caspase-1与IRF-5之间可能存在结合位点。体内实验也证明敲除Caspase-1促进MDSCs向M1型巨噬细胞分化最终提高化疗疗效，而敲除IRF-5则起相反作用。本项目揭示了奥沙利铂耐药新的免疫学机制，为临床奥沙利铂耐药提供一种新的解决方案。目前课题进展顺利，可以按期结题。课题前期工作发表SCI收录论文3篇，1篇论文摘要被“美国消化病周”（DDW）接受为壁报展示，1篇英文论文正在审稿，另培养硕士研究生3名，其中毕业2人，1在读。