The role of gut microbe-derived choline metabolites in driving the pro-inflammatory macrophage phenotype and restricting pancreatic cancer

项目摘要 胰腺导管腺癌（PDAC）的治疗仍然是一个主要障碍，5年生存率 率仅为9%。PDAC对化疗和免疫治疗的耐药性被认为主要是由于 免疫抑制性肿瘤微环境（TME）的特征是致密的纤维化基质和高密度的纤维化基质。 免疫抑制细胞浸润，包括肿瘤相关巨噬细胞（TAM）。TAMs阻滞效应器 T细胞功能和触发衰竭。TME中的免疫反应可能受多种免疫系统的控制。 机制，包括宿主内在因素，以及最近认识到的肠道微生物组的变化。 然而，肠道微生物组影响免疫的分子机制和途径 对PDAC TME的反应仍然知之甚少。我们新的初步研究表明， 微生物群可以通过进入血流的微生物代谢物影响免疫反应， 调节TAM活性。在对循环代谢物的非靶向代谢组学筛选中，我们发现了 最显着的变化是在三甲基胺N-氧化物（TMAO）水平急剧下降（>80倍）， 用抗生素甲硝唑治疗的携带PDAC的小鼠。氧化三甲胺的形成分两步：肠道微生物 TMA裂解酶将胆碱降解为三甲胺（TMA），然后在肝脏中进行氧化，形成 TMAO。生理相关量的TMAO腹膜内给药至携带PDAC的小鼠 显著抑制肿瘤生长。这伴随着肿瘤浸润的显著减少， 免疫抑制性骨髓细胞，促炎性TAM表型的增加，以及免疫抑制性骨髓细胞的显著增加。 PDAC TME中活化的CD 8 + T细胞浸润。这些和其他数据支持我们的假设， 饮食胆碱和肠道微生物TMA裂解酶是影响循环TMAO水平的关键因素， 促炎TAM表型。我们建议，补充TMAO或制定战略， 增加它将改善PDAC中对免疫疗法的应答。目的1检验膳食胆碱 和肠道微生物TMA途径有助于获得促炎性TAM表型， 诱导抗肿瘤免疫应答。我们将采用补充胆碱的饮食，耗尽TMA- 生产细菌，并抑制TMA裂解酶，以评估膳食前体之间的机械联系 TMAO，肠道微生物TMA途径，以及PDAC TME中的免疫反应。目标2测试 假设增加TMAO会使PDAC肿瘤对免疫治疗敏感。我们将直接评估 施用TMAO或富集产生TMA的肠道细菌如生孢梭菌， 与免疫检查点阻断的组合。这一目的也将评估的临床意义， 通过确定血浆TMAO水平与临床生存结局之间的相关性， 和PDAC中的疾病进展。从长远来看，这项工作可能会成为开发新的， 基于微生物组的治疗方法用于侵袭性和难以治疗的PDAC。

PROJECT SUMMARY The treatment of pancreatic ductal adenocarcinoma (PDAC) remains a major hurdle, with a 5-year survival rate of only 9%. Resistance of PDAC to chemotherapy and immunotherapy is thought to arise primarily due the immunosuppressive tumor microenvironment (TME), characterized by a dense fibrotic stroma and high infiltrates of immunosuppressive cells, including tumor associated macrophages (TAM). TAMs block effector T cell function and trigger exhaustion. Immune responses in the TME are likely controlled by multiple mechanisms, including host intrinsic factors and, more recently appreciated, changes in the gut microbiome. However, the molecular mechanisms and pathways by which the gut microbiome impact the immune responses in the PDAC TME remain poorly understood. Our new preliminary work indicates that the gut microbiota can influence the immune response through microbial metabolites that enter the blood stream to modulate TAM activity. In an untargeted metabolomic screen on circulating metabolites, we discovered the most significant change was a dramatic reduction (>80-fold) in trimethylamine N-oxide (TMAO) levels in PDAC-bearing mice treated with antibiotic metronidazole. TMAO is formed in two steps: the gut microbial TMA lyase degrades choline to trimethylamine (TMA), which then undergoes oxidation in the liver to form TMAO. Intraperitoneal administration of physiologically relevant amounts of TMAO to PDAC-bearing mice significantly suppressed tumor growth. This was accompanied by a significant decrease in tumor-infiltrating, immunosuppressive myeloid cells, an increase in a pro-inflammatory TAM phenotype, and a striking increase in infiltrates of activated CD8+ T cells in the PDAC TME. These and other data support our hypothesis that dietary choline and the gut microbial TMA lyase are key factors contributing to circulating TMAO levels and the pro-inflammatory TAM phenotype. We propose that supplementing TMAO or enacting strategies that increase it will improve response to immunotherapy in PDAC. Aim 1 tests the hypothesis that dietary choline and the gut microbial TMA pathway contribute to the acquisition of a pro-inflammatory TAM phenotype and induction of anti-tumor immune response. We will employ supplementation of choline diet, depletion of TMA- producing bacteria, and inhibition of TMA lyase to evaluate the mechanistic links between dietary precursors of TMAO, the gut microbial TMA pathway, and immune responses in the PDAC TME. Aim 2 tests the hypothesis that increasing TMAO sensitizes PDAC tumors to immunotherapy. We will evaluate direct administration of TMAO or enrichment of TMA-producing gut bacteria such as Clostridium sporogenes in combination with immune checkpoint blockade. This aim will also evaluate the clinical significance of the TMAO biology by determining the association between plasma TMAO levels and clinical outcome of survival and disease progression in PDAC. In the longer term, this work may form the basis for developing new, microbiome-based therapies for the aggressive and hard to treat PDAC.