Identifying the active factor of an anti-inflammatory chemopreventive bacterium

项目摘要/摘要 结直肠癌（CRC）仍然是一个主要的公共卫生问题，大约有145，000例新发病例和51，000例 仅在美国，每年就有500人死于这种疾病。肠道微生物群组成的改变以及 升高的炎症是CRC的既定危险因素。我们发现正常的肠道 Distasonis副拟杆菌（Parabacteroides distasonis，Pd）抑制小鼠结肠直肠炎症和肿瘤形成。 重要的是，我们发现Pd抑制了促炎、促肿瘤发生的TLR4信号传导的激活。 通路本申请的目的是确定阻断TLR4对于抗肿瘤治疗是否是必需的。 研究Pd对机体免疫功能的影响，确定Pd的活性因子及其宿主靶点，最终确定Pd对机体免疫功能的影响 结肠中的细胞群。基于我们的可靠数据，我们假设Pd的化学预防作用是 通过抑制促炎性TLR4信号传导和募集炎症抑制T细胞来实现。 调节性（Treg）细胞转移到结肠。本研究的具体目的是：1）鉴定抗炎症因子， 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）鉴定 Pd抗炎分子的分子靶点; 2）确定Pd如何影响免疫细胞 结肠粘膜中的细胞群。本研究的目的将通过比较不同的能力来解决 密切相关的细菌物种，以抑制TLR4信号在体外，然后比较基因组学，以确定 抑制性物种共有的基因，但在非抑制性物种中没有同源物。候选人的作用 将通过在大肠杆菌Nissle 1917（EcN）中表达它们并在Pd中突变它们来评估。评估是否 Pd的化学预防需要TLR4抑制，我们将比较Pd和缺乏抗TLR4的Pd的能力 用EcN和表达Pd抗TLR4基因的EcN抑制结肠肿瘤发生。宿主靶蛋白 将通过与重组标记的Pd蛋白的免疫共沉淀来鉴定。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结肠免疫细胞 随着时间的推移，Pd喂养小鼠和对照小鼠之间的种群数量。了解Pd的作用机制并识别 它的活性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， 他们的行动机制。重要的是，Pd似乎与其他微生物疗法相容， 而不是破坏体内的微生物群这些研究是非常值得的，因为虽然一些代理人显示， 在临床前研究中，阿司匹林（一种100多年前开发的药物）有望成为化学预防剂。 仍然是唯一的代理，我们有令人信服的证据，其有效性和有害影响， 超过这些好处。这些研究直接涉及NCI发展知识库的使命 这将减轻美国和全世界的癌症负担。

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 ant-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.