Role of microbiome in cancer and inflammation

我们广泛使用缺乏免疫或炎症相关基因的小鼠，总是很难区分这些基因对结肠炎或癌症的直接影响，还是通过调节肠道微生物群的间接影响。总的来说，这些研究将大大受益于我们在弗雷德里克建立的无菌设施，特别是得益于我们在贝塞斯达建立的微生物组核心提供的基于最先进测序和生物信息学的肠道微生物学专业知识的可用性。我们建立了检测小鼠微生物瘤的方法，使用454测序或16 RNA的MiSeq测序，使用NextSeq测序进行宏基因组分析，以及对特定细菌类型进行FISH标记的细胞荧光分析。我们还启动了无菌小鼠、肠道菌群确定的非生菌小鼠和抗生素治疗后重建的小鼠的研究。最初，我们使用MyD88、IL-18、TNF、TLRs等炎症控制基因基因缺陷的小鼠，研究了肠道微生物群在结肠炎和结肠炎相关癌症的实验模型中的作用。在这些小鼠中，遗传缺陷会引起生态失调，这种失调可以通过同居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。目前正在研究导致这种致癌性增加的细菌种类及其作用机制。在小鼠实验模型和观察性临床实验中，已经开始研究共生微生物群在与癌症相关的能量改变（即肥胖、恶病质、厌食症、癌症治疗、辐射）中的作用。寄生生物对皮肤免疫的分区控制（Science. 2012;337:1115-9）。肠道共生菌诱导保护和调节反应，维持宿主-微生物共生。然而，组织常驻共生体对其他屏障部位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。发现对皮肤病原体的保护性免疫严重依赖于皮肤微生物群，而不是肠道微生物群。此外，皮肤共生体以一种依赖于白细胞介素-1受体下游信号的方式调节局部T细胞的功能。这些发现强调了微生物群作为组织区隔化的独特特征的重要性，并为健康和疾病中常驻共生生态位调节免疫系统的机制提供了见解。共生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被充分证明，但共生菌是否能在无菌肿瘤微环境中对炎症产生远端影响尚不清楚。在这里，我们表明微生物群的扰动削弱了皮下肿瘤对cpg -寡核苷酸免疫治疗或铂化疗的反应。在抗生素治疗或无菌小鼠中，CpG-ODN治疗后，肿瘤浸润性单核细胞来源细胞因子产生的减少减少了肿瘤坏死，而化疗诱导的髓细胞活性氧产生不足则损害了遗传毒性和肿瘤破坏。因此，对癌症免疫治疗和化疗的最佳反应需要一个完整的共生微生物群，通过调节肿瘤微环境中髓源性细胞的功能来远距离起作用。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（肠黏膜损伤、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。目前正在研究不同微生物种类的参与以及它们允许顺铂毒性的机制。我们还研究了小鼠和人类中与癌症相关的恶病质，以研究微生物群是否调节这种毁灭性癌症合并症的建立，以及是否可以靶向治疗。在小鼠中，我们最初关注的是由Lewis肺癌（LLC）肿瘤诱导的恶病质模型，该模型在18-21天内诱导体重减轻，并伴有脂肪和肌肉组织的损失。llc诱导的恶病质是由于白色脂肪组织（WAT）中脂肪分解增加所致。与我们在顺铂诱导的恶病质中观察到的不同，北京（从WAT到棕色脂肪组织的转换）并不是LLC诱导的恶病质的重要组成部分。LLC通过诱导WAT浸润表达未成熟氧化中性粒细胞的c-Kit诱导恶病质，通过产生ROS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，无法上调代偿机制、脂肪生成和脂肪生成，无菌小鼠的恶病质加速。微生物群至少在一定程度上通过产生调节脂肪分解、脂肪生成和脂肪生成的短链脂肪酸来延缓癌症恶病质。在LLC模型中获得的结果显示没有北京，中性粒细胞浸润和缺乏对IL-6的依赖性与其他实验模型中描述的部分不同。由于不同的肿瘤类型在小鼠和患者中可能通过不同的机制诱导恶病质，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）中微生物群的机制和作用。Kras/P53小鼠和细胞系将与CCR Perwez Hussein合作进行研究。Dan McVicar， CIP的研究小组在4T1肿瘤动物的脾脏中发现，由c-Kit表达定义并依赖于c-Kit信号传导的未成熟中性粒细胞具有线粒体氧化代谢的能力，在有限的葡萄糖条件下，它们的线粒体通过脂肪酸氧化来支持nadph氧化酶依赖的ROS生成。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相似，并通过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通过饮食（例如在饮食中添加可溶性纤维，如菊粉）或其他扰动（随后进行宏基因组学和元转录组学分析）来改变微生物群，或通过有针对性的生物实验来研究，最初的重点是SCFA的产生。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延伸到人类。我们与圣保罗大学的Marilia Seelaender合作，验证了一种假设，即在病毒症患者中，肠道屏障破坏与微生物群组成改变相关，可能会引发宿主体内持续的免疫激活。本文采用16S分析法分析了7例结直肠癌（CC）和14例体重稳定（WSC）患者。我们将通过宏基因组和亚转录组分析将这一分析扩展到更多的患者。初步研究表明，CC患者中淋巴样聚集体数量增加与黏液层降解、嗜酸性粒细胞浸润、G-CSF、IL-13和tgf - β增加有关，因此与小鼠结果相似。

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.