Identifying the active factor of an anti-inflammatory chemopreventive bacterium

项目摘要/摘要 结直肠癌(CRC)仍然是一个重大的公共卫生问题，约有145,000个新病例和51,000个 仅在美国每年死于这种疾病的人数。肠道微生物区系组成的变化和 炎症升高是结直肠癌的既定危险因素。我们做了一个新奇的观察，正常的肠道 帕氏杆菌能抑制小鼠的结肠炎和肿瘤的形成。 重要的是，我们发现PD抑制了促炎症、促肿瘤的TLR4信号的激活 路径。这个应用程序的目的是确定是否需要阻断TLR4来抗肿瘤 帕金森病的作用，确定帕金森病的活性因子及其宿主靶点，最终确定帕金森病如何影响免疫 结肠中的细胞群。根据我们的可靠数据，我们假设帕金森病的化学预防作用是 通过抑制促炎TLR4信号和募集抗炎T细胞实现的 调节性(Treg)细胞进入结肠。本研究的具体目的是：1)确定丹参的抗炎因子 PD；1A)测试PD的抗TLR4功能是否是其化学预防作用所必需的；1B)识别 帕金森病抗炎分子的分子靶点和2)确定帕金森病如何影响免疫细胞 结肠粘膜中的种群。这项研究的目的将通过比较不同的 近缘种细菌体外抑制TLR4信号转导的比较基因组学鉴定 抑制物种共有的基因，但非抑制物种中没有同源基因。候选人的作用 将通过在E.coliNissle 1917(ECN)中表达它们并在PD中突变它们来进行评估。评估是否 PD的化学预防需要抑制TLR4，我们将比较PD和缺乏抗TLR4的PD的能力 表达PD抗TLR4基因的ECN和ECN抑制结肠癌发生的作用宿主靶蛋白 将通过与重组标记PD蛋白的免疫共沉淀进行鉴定。最后，PD的影响 将通过比较特定的癌症和微生物群相关的免疫细胞来评估对结肠免疫细胞的影响 PD小鼠和对照组小鼠之间的种群随着时间的推移而变化。理解帕金森病的作用机制和识别 它的活性分子和靶分子将为这种细菌或其活性成分在 预防CRC。我们的长期目标是鉴定具有化学预防特性的细菌，并了解 它们的作用机制。重要的是，帕金森病似乎与其他微生物疗法兼容，并确实 不会扰乱当地的微生物区系。这些研究是非常有价值的，因为尽管一些代理人表明 阿司匹林在临床前研究中有望成为化学预防药物(一种100多年前开发的药物) 仍然是我们有令人信服的有效性证据的唯一制剂，其有害影响可能 超过了这些好处。这些研究直接涉及NCI开发知识库的任务 这将减轻美国和世界各地的癌症负担。

Project Abstract/Summary Colorectal cancer (CRC) remains a major public health issue, with approximately 145,000 new cases and 51,000 deaths from this disease per year in the US alone. Alterations in the composition of the gut microbiota and elevated inflammation are established risk factors for CRC. We made the novel observation that the normal gut bacterium Parabacteroides distasonis (Pd) suppresses colorectal inflammation and tumor formation in mice. Importantly, we show that Pd suppresses the activation of the pro-inflammatory, pro-tumorigenic TLR4 signaling pathway. The objectives of this application are to determine whether blocking TLR4 is required for the anti-tumor effects of Pd, to identify the active factor of Pd and its host target and finally to define how Pd influences immune cell populations in the colon. Based on our robust data, we hypothesize that the chemopreventive effect of Pd is achieved by the suppression of pro-inflammatory TLR4 signaling and the recruitment of inflammation quelling T regulatory (Treg) cells to the colon. The specific aims of the study are to: 1) To identify anti-inflammatory factor of Pd; 1A) To test whether the anti-TLR4 function of Pd is required for its chemopreventive effect; 1B) To identify the molecular target of the Pd anti-inflammatory molecule and 2) To define how Pd influences immune cell populations in the colonic mucosa. The aims of this study will be addressed by comparing the ability of various closely-related bacterial species to inhibit TLR4 signaling in vitro followed by comparative genomics to identify genes common to inhibitory species but without homologs in non-inhibitory species. The function of candidates will be evaluated by expressing them in E.coli Nissle 1917 (EcN) and mutating them in Pd. To evaluate whether TLR4 inhibition is required for chemoprevention by Pd we will compare the ability of Pd and Pd lacking anti-TLR4 function to suppress colon tumorigenesis with EcN and EcN expressing Pd anti-TLR4 genes. Host target proteins will be identified by co-immunoprecipitation with the recombinant tagged Pd protein. Finally, the influence of Pd on colonic immune cells will be evaluated by comparing specific cancer and microbiota relevant immune cell populations between Pd fed and control mice over time. Understanding Pd’s mechanism of action and identifying its active and target molecules will pave the way for this bacterium, or its active component, to be utilized in the prevention of CRC. Our long-term goal is to identify bacteria with chemopreventive properties and to understand their mechanisms of action. Importantly, Pd appears to be compatible with other microbial therapies and does not disrupt the resident microbiota. These studies are highly worthwhile because although a few agents show promise as chemopreventive agents in pre-clinical studies, aspirin (a drug developed over 100 years ago) remains the only agent for which we have compelling evidence of efficacy and its detrimental effects may outweigh those benefits. These studies directly address the mission of the NCI to develop the knowledge base that will lessen the burden of cancer in the United States and around the world.