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

项目总结 胰腺导管腺癌(Pdac)的治疗仍然是一个主要障碍，其存活期为5年。 利率只有9%。PDAC对化疗和免疫治疗的耐药性被认为主要是由于 免疫抑制肿瘤微环境(TME)，以致密的纤维间质和高密度的 免疫抑制细胞的渗出，包括肿瘤相关巨噬细胞()。TAMS块效应器 T细胞功能和触发衰竭。TME中的免疫反应可能受多个 机制，包括宿主内在因素，以及最近认识到的肠道微生物群的变化。 然而，肠道微生物群影响免疫的分子机制和途径 PDAC TME中的反应仍然知之甚少。我们新的初步工作表明，内脏 微生物区系可以通过进入血流的微生物代谢产物来影响免疫反应 调节的活动。在对循环代谢物的非靶向代谢组筛查中，我们发现 最重要的变化是三甲胺氮氧化物(TMAO)水平显著降低(&gt；80倍)。 用抗生素甲硝唑治疗PDAC荷瘤小鼠。氧化三甲胺的形成分两步进行：肠道微生物 TMA裂解酶将胆碱降解为三甲胺(TMA)，然后在肝脏中氧化形成 TMAO。生理剂量的TMAO对荷PDAC小鼠的腹腔注射 显著抑制肿瘤生长。与此同时，肿瘤浸润率显著下降， 免疫抑制的髓系细胞，促炎的表型增加，并且显著增加 在PDAC TME中活化的CD8+T细胞的浸润液中。这些数据和其他数据支持我们的假设 膳食胆碱和肠道微生物TMA裂解酶是导致循环TMAO水平和 促炎的表型。我们建议补充TMAO或制定战略， 增加它将改善PDAC对免疫治疗的反应。Aim 1测试饮食中胆碱的假设 肠道微生物的TMA途径有助于获得促炎的表型和 诱导抗肿瘤免疫反应。我们将采用补充胆碱饮食，消耗TMA- 产生细菌和抑制TMA裂解酶以评估饮食前体之间的机制联系 肠道微生物TMA途径和PDAC TME中的免疫反应。AIM 2测试 假设增加TMAO使PDAC肿瘤对免疫治疗敏感。我们将直接进行评估 TMAO给药或产TMA肠道细菌如生孢梭菌的培养 与免疫检查站封锁相结合。这一目的也将评估临床意义的 通过确定血浆TMAO水平与临床生存结果之间的关系来研究TMAO生物学 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、 以微生物群为基础的治疗侵袭性和难治性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.