Role of microbiome in cancer and inflammation

微生物组在癌症和炎症中的作用

• 批准号: 10014549
• 负责人: GIORGIO TRINCHIERI
• 金额: $ 159.64万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014549
• 关键词: 4T1; Address; Adipose tissue; Adverse reactions; Affect; Anorexia; Antibiotic Therapy; Antibiotics; Attenuated; Bacteria; Bioinformatics; Birth; Body Weight decreased; Brown Fat; CCR; CPG-oligonucleotide; CSF3 gene; Cachexia; Cancer Control; Cancer Patient; Cell Line; Cell Proliferation; Cell Respiration; Cell physiology; Cells; Characteristics; Chemotherapy-Oncologic Procedure; Chronic; Cisplatin; Clinical; Colitis; Collaborations; Colorectal Cancer; Comorbidity; Crohn' s disease; Cutaneous; Cysteine; Dependence; Development; Diet; Disease; Disease Outcome; Distal; Distant; Epithelial; Eukaryota; Experimental Models; Exposure to; Failure; Fluorescent in Situ Hybridization; Genes; Genetic; Germ-Free; Glucose; Gnotobiotic; Health; Homeostasis; Housing; Human; Human body; Immune; Immune response; Immune system; Immunity; Immunotherapy; Impairment; Individual; Infiltration; Inflammation; Interleukin-1 Receptors; Interleukin-13; Interleukin-18; Interleukin-6; Intestinal Mucosa; Intestines; Inulin; Invaded; Label; Lewis Lung Carcinoma; Lipolysis; Lung; Lymphocyte Function; Lymphoid; Maintenance; Malignant Neoplasms; Mediating; Metabolic Control; Metabolism; Metagenomics; Methods; Microbe; Microbiology; Mitochondria; Modeling; Molecular; Mouse Cell Line; Mucous body substance; Mus; Muscle; Mutation; Myelogenous; Myeloid Cells; NADPH Oxidase; Necrosis; Neoplasm Transplantation; Neutrophil Infiltration; Obesity; Pancreas; Patients; Physiological; Physiology; Platinum; Play; Predisposition; Production; Prokaryotic Cells; Proto-Oncogene Protein c-kit; RNA; Reactive Oxygen Species; Regulation; Resistance to infection; Role; SW480; Science; Signal Transduction; Site; Skin; Spleen; Sterility; T-Lymphocyte; TNF gene; TP53 gene; Testing; Time; Tissues; Toxic effect; Transforming Growth Factor beta; Universities; Weight; base; cancer cachexia; cancer immunotherapy; cancer therapy; carcinogenesis; chemical carcinogenesis; chemotherapy; colitis associated cancer; colon cancer patients; commensal bacteria; commensal microbes; cytokine; dysbiosis; eosinophil; experimental study; fatty acid oxidation; fecal transplantation; fortification; genotoxicity; gut microbiome; gut microbiota; immature animal; immune activation; improved; inflammatory milieu; insight; irradiation; lipid biosynthesis; metabolic abnormality assessment; metatranscriptomics; microbial; microbial host; microbiome; microbiota; microorganism; mitochondrial metabolism; monocyte; mouse model; mutualism; nephrotoxicity; neutrophil; pathogen; pathogenic microbe; prevent; reconstitution; resident commensals; response; skin microbiota; soluble fiber; stem; subcutaneous; symbiont; targeted treatment; tumor; tumor microenvironment

We extensively use mice deficient for immune or inflammation-related genes and it is always difficult to distinguish a direct effect of those genes on the colitis or cancer, or an indirect one through the regulation of the intestinal microbiota. Overall these studies will greatly benefit by the access to a germ free facility that we are contributed to establish in Frederick and particularly by the availability of committed expertise in gut microbiology based on state of the art sequencing and bioinformatics, expertise that is provided by the microbiome core that we have established in Bethesda. We have established methods for the determination of mouse microbioma using 454 sequencing or MiSeq sequencingof 16 RNA, metagenomic analysis using NextSeq sequencing, and cytofluorimetric analysis of FISH labeling of specific bacterial types. We also initiated studies with germ free mice, gnotobiotic mice with defined intestinal flora, and mice reconstitute after antibiotic treatment. Initially we studied the role of the intestinal microbiota in experimental models of colitis and colitis-associated cancer using mice genetically deficient for inflammation-controlling genes such as MyD88, IL-18, TNF, TLRs, and others. In these mice the genetic defects induce a dysbiosis that can be transferred to normal mice by co-housing or fecal transplant and enhance susceptibility to chemical carcinogenesis. The bacterial species responsible of this increased susceptibility to carcinogenesis and their mechanism of action are being investigated. The role of commensal microbiota in energetic alteration associated with cancer (i.e. obesity, cachexia, anorexia, cancer treatment, irradiation) has been initiated in murine experimental models and in observational clinical experimentation. Compartmentalized control of skin immunity by resident commensals (Science. 2012;337:1115-9). Intestinal commensal bacteria induce protective and regulatory responses that maintain host-microbial mutualism. However, the contribution of tissue-resident commensals to immunity and inflammation at other barrier sites has not been addressed. We found that in mice, the skin microbiota has an autonomous role in controlling the local inflammatory milieu and tuning resident T lymphocyte function. Protective immunity to a cutaneous pathogen was found to be critically dependent on the skin microbiota but not the gut microbiota. Furthermore, skin commensals tuned the function of local T cells in a manner dependent on signaling downstream of the interleukin-1 receptor. These findings underscore the importance of the microbiota as a distinctive feature of tissue compartmentalization, and provide insight into mechanisms of immune system regulation by resident commensal niches in health and disease. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment (Science 342:967-970). The gut microbiome influences both local and systemic inflammation. Although the role of inflammation in cancer is well documented, whether commensal bacteria can exert distant effects on the inflammation in the sterile tumor microenvironment remains unclear. Here we show that microbiota perturbation impairs the response of subcutaneous cancers to CpG-oligonucleotide-immunotherapy or platinum chemotherapy. In antibiotic-treated or germ-free mice, decreased cytokine production from tumor-infiltrating monocyte-derived cells following CpG-ODN treatment reduced tumor necrosis, whereas deficient chemotherapy-induced production of reactive oxygen species by myeloid cells impaired genotoxicity and tumor destruction. Thus, optimal response to cancer immunotherapy and chemotherapy requires an intact commensal microbiota that acts distantly by modulating myeloid-derived cell function in the tumor microenvironment. These findings underscore the importance of the microbiota in the outcome of disease treatment. The toxicity mediated by cisplatin (intestinal mucosa damage, nephrotoxicity, decrease of adipose and muscular tissues (cachexia)) require the presence of gut microbiota. The participation of different microbial species and the mechanisms by which they allow the cisplatin toxicity are being investigated. We also study cancer associated cachexia in mice and humans to investigate whether the microbiota regulates the establishment of this devastating cancer comorbidity and could be targeted therapeutically. In mice we focused initially on a model of cachexia induced by the Lewis Lung Carcinoma (LLC) tumor that in 18-21 days induces weight loss associated with loss of adipose and muscle tissue. LLC-induced cachexia is due to increased lipolysis in the white adipose tissue (WAT). Unlike what we observed in cisplatin induced cachexia, beiging (switch from WAT to brown adipose tissue) is not an important component of LLC induced cachexia. LLC induces cachexia by inducing WAT infiltration of c-Kit expressing immature oxidative neutrophils that induce lipolysis by ROS production. Deletion of neutrophils or treatment of mice with N-acetyl cysteine prevent cachexia. Cachexia is accelerated in germ free mice due to increased lipolysis and failure to upregulate compensatory mechanisms, adipogenesis and lipogenesis. The microbiota delays cancer cachexia at least in part by producing SCFA that regulate lipolysis, lipogenesis and adipogenesis. The results obtained in the LLC model showing absence of beiging, neutrophil infiltration and lack of dependence from IL-6 differ in part from those described in other experimental models. Because different tumor types may induce cachexia through different mechanisms in mice as well as in patients, we will compare the mechanism involved and the role of the microbiota in different models (SW480, 4T1 and C26 transplantable tumors; Kras/P53 pancreatic GEMM and cell lines). Kras/P53 mice and cell lines will be studied in collaboration with Perwez Hussein, CCR. The group of Dan McVicar, CIP, has shown in the spleen of 4T1 tumor bearing animals that immature neutrophils, defined by expression of c-Kit and dependent on c-Kit signaling, possess the capacity for oxidative mitochondrial metabolism and in limited glucose use their mitochondria to support NADPH-oxidase dependent ROS production via fatty acid oxidation. The characteristics of these splenic neutrophils are like those that we observed in the cachectic adipose tissue and that induce lipolysis through ROS, thus we plan to collaborate with Dan McVicar in studying the metabolism of neutrophil and adipose tissue during cachexia. The role of the microbiota will be studied by modifying the microbiota by diet (e.g. diet supplemented with soluble fibers such as inulin) or other perturbations (followed by metagenomic and metatranscriptomic analysis) or by targeted gnotobiotic experiments, focusing initially on the production of SCFA. Cancer patients' neutrophils display immaturity and oxidative metabolism, thus, the mechanism of cachexia observed in mice may extend to humans. We collaborate with Marilia Seelaender, University of Sao Paulo, testing the hypothesis that in cachectic patients gut barrier disruption associated with altered microbiota composition may elicit persistent immune activation in the host. We have analyzed 7 cachectic patients with colorectal cancer (CC) and 14 weight stable patients (WSC) by 16S analysis. We will extend this analysis to a larger number of patients by metagenomic and metatranscriptomic analysis. The initial studies have shown an increased number of lymphoid aggregates in CC patients associated with degradation of mucus layer, infiltration with eosinophils, increased G-CSF, IL-13 and TGF-beta thus showing analogy with the mouse results.

我们广泛使用缺乏免疫或炎症相关基因的小鼠，但总是很难区分这些基因对结肠炎或癌症的直接影响，还是通过肠道微生物群调节产生的间接影响。总的来说，这些研究将受益于我们在弗雷德里克贡献的无菌设施，特别是基于最先进的测序和生物信息学的肠道微生物学专业知识，以及我们在贝塞斯达建立的微生物组核心提供的专业知识。我们建立了使用 16 RNA 的 454 测序或 MiSeq 测序、使用 NextSeq 测序进行宏基因组分析以及特定细菌类型的 FISH 标记的细胞荧光分析来确定小鼠微生物瘤的方法。我们还启动了对无菌小鼠、具有明确肠道菌群的无菌小鼠以及抗生素治疗后恢复正常的小鼠的研究。最初，我们使用 MyD88、IL-18、TNF、TLR 等炎症控制基因遗传缺陷的小鼠，研究了肠道微生物群在结肠炎和结肠炎相关癌症实验模型中的作用。在这些小鼠中，遗传缺陷会导致生态失调，这种失调可以通过共同饲养或粪便移植转移到正常小鼠身上，并增加对化学致癌的易感性。导致致癌易感性增加的细菌种类及其作用机制正在研究中。共生微生物群在与癌症相关的能量改变（即肥胖、恶病质、厌食、癌症治疗、辐射）中的作用已在小鼠实验模型和观察性临床实验中启动。通过常驻共生体对皮肤免疫进行分区控制（Science.2012；337：1115-9）。肠道共生细菌诱导保护和调节反应，维持宿主-微生物的互利共生。然而，组织驻留共生体对其他屏障位点的免疫和炎症的贡献尚未得到解决。我们发现，在小鼠中，皮肤微生物群在控制局部炎症环境和调节常驻 T 淋巴细胞功能方面具有自主作用。研究发现，对皮肤病原体的保护性免疫力严重依赖于皮肤微生物群，而不是肠道微生物群。此外，皮肤共生体以依赖于 IL-1 受体下游信号传导的方式调节局部 T 细胞的功能。这些发现强调了微生物群作为组织区室化的独特特征的重要性，并提供了对健康和疾病中常驻共生生态位的免疫系统调节机制的见解。共生细菌通过调节肿瘤微环境来控制癌症对治疗的反应 (Science 342:967-970)。肠道微生物组影响局部和全身炎症。尽管炎症在癌症中的作用已得到充分证明，但共生细菌是否可以对无菌肿瘤微环境中的炎症产生遥远的影响仍不清楚。在这里，我们发现微生物群扰动会损害皮下癌对 CpG 寡核苷酸免疫疗法或铂类化疗的反应。在接受抗生素治疗或无菌小鼠中，CpG-ODN 治疗后肿瘤浸润单核细胞衍生细胞产生的细胞因子减少，从而减少了肿瘤坏死，而化疗引起的髓系细胞活性氧产生不足会损害基因毒性和肿瘤破坏。因此，对癌症免疫疗法和化疗的最佳反应需要完整的共生微生物群，该微生物群通过调节肿瘤微环境中的骨髓源性细胞功能来发挥远程作用。这些发现强调了微生物群在疾病治疗结果中的重要性。顺铂介导的毒性（肠粘膜损伤、肾毒性、脂肪和肌肉组织减少（恶病质））需要肠道微生物群的存在。不同微生物物种的参与及其导致顺铂毒性的机制正在研究中。我们还研究了小鼠和人类与癌症相关的恶病质，以研究微生物群是否调节这种破坏性癌症合并症的形成，并可以作为治疗的目标。在小鼠中，我们最初关注的是由刘易斯肺癌 (LLC) 肿瘤引起的恶病质模型，该肿瘤在 18-21 天内导致与脂肪和肌肉组织损失相关的体重减轻。 LLC 引起的恶病质是由于白色脂肪组织 (WAT) 中的脂肪分解增加所致。与我们在顺铂引起的恶病质中观察到的情况不同，beiging（从 WAT 转变为棕色脂肪组织）并不是 LLC 引起的恶病质的重要组成部分。 LLC 通过诱导表达未成熟氧化性中性粒细胞的 WAT 浸润来诱导恶病质，这些细胞通过 ROS 产生诱导脂肪分解。删除中性粒细胞或用 N-乙酰半胱氨酸治疗小鼠可预防恶病质。由于脂肪分解增加以及无法上调代偿机制、脂肪生成和脂肪生成，无菌小鼠中恶病质加速。微生物群至少部分通过产生调节脂解、脂肪生成和脂肪生成的 SCFA 来延迟癌症恶病质。 LLC 模型中获得的结果显示不存在米色、中性粒细胞浸润并且缺乏对 IL-6 的依赖性，这与其他实验模型中描述的结果部分不同。由于不同的肿瘤类型可能通过不同的机制在小鼠和患者中诱发恶病质，因此我们将比较不同模型（SW480、4T1 和 C26 可移植肿瘤；Kras/P53 胰腺 GEMM 和细胞系）中所涉及的机制和微生物群的作用。 Kras/P53 小鼠和细胞系将与 CCR 的 Perwez Hussein 合作进行研究。 Dan McVicar (CIP) 小组在 4T1 荷瘤动物的脾脏中发现，未成熟的中性粒细胞（由 c-Kit 表达定义并依赖于 c-Kit 信号传导）具有氧化线粒体代谢的能力，并且在有限的葡萄糖条件下，利用线粒体通过脂肪酸氧化支持 NADPH 氧化酶依赖性 ROS 产生。这些脾脏中性粒细胞的特征与我们在恶病质脂肪组织中观察到的特征相似，并且通过ROS诱导脂肪分解，因此我们计划与Dan McVicar合作研究恶病质期间中性粒细胞和脂肪组织的代谢。微生物群的作用将通过饮食（例如补充可溶性纤维如菊粉的饮食）或其他扰动（随后进行宏基因组和宏转录组分析）或通过有针对性的无菌实验来改变微生物群来研究，最初侧重于 SCFA 的生产。癌症患者的中性粒细胞表现出不成熟和氧化代谢，因此，在小鼠中观察到的恶病质机制可能会延伸到人类。我们与圣保罗大学的 Marilia Seelaender 合作，测试了以下假设：在恶病质患者中，与微生物群组成改变相关的肠道屏障破坏可能会引发宿主持续的免疫激活。我们通过 16S 分析分析了 7 名恶病质结直肠癌 (CC) 患者和 14 名体重稳定患者 (WSC)。我们将通过宏基因组和宏转录组分析将该分析扩展到更多的患者。初步研究表明，CC 患者淋巴聚集数量增加，与粘液层降解、嗜酸性粒细胞浸润、G-CSF、IL-13 和 TGF-β 增加相关，因此与小鼠结果类似。