Role of microbiome in cancer and inflammation

在我们的研究中，我们广泛使用免疫或炎症相关基因缺失的小鼠，很难区分这些基因对结肠炎、癌症和治疗反应的直接影响，还是通过调节肠道微生物群的间接影响。我们已经建立了测定小鼠微生物瘤的方法，使用454测序或16 RNA的MiSeq测序，使用NextSeq测序的宏基因组分析，以及特定细菌类型的FISH标记的细胞荧光分析，我们已经建立了一个微生物组核心，为NIH社区提供这些技术服务。我们对无菌小鼠、具有明确肠道菌群的非生物小鼠和抗生素治疗后的小鼠进行了广泛的研究。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。我们是第一个证明共生菌通过调节肿瘤微环境来控制癌症对治疗的反应的人（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。许多实验室已经将我们的结果扩展到患者的癌症免疫治疗，并表明微生物组组成决定了黑色素瘤和其他类型癌症患者对抗pd1治疗的反应能力。然而，不同的研究已经确定了造成这种效果的不同类型的细菌，其机制尚不清楚。我们已经将分析扩展到匹兹堡大学的一大群黑色素瘤患者，我们开始阐明不同组中结果不一致的原因，并确定共同的机制。在与MD安德森癌症中心的合作中，我们已经确定，高纤维饮食的患者对抗pd1治疗的反应更好，我们已经在小鼠研究中研究了这种作用的机制。通过与匹兹堡大学癌症中心的合作，我们已经治疗了14例抗pd1难治性黑色素瘤患者，患者的粪便微生物群移植来自pd - 1反应性患者，在几乎一半的患者中，快速进展的肿瘤稳定或对持续的抗pd1治疗有显著反应。这种作用的机制正在通过微生物组、细胞因子和代谢组学研究进行研究。

In our studies, 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, cancer and response to therapy , or an indirect one through the regulation of the intestinal microbiota. We have established methods for the determination of mouse microbioma using 454 sequencing or MiSeq sequencing of 16 RNA, metagenomic analysis using NextSeq sequencing, and cytofluorimetric analysis of FISH labeling of specific bacterial types and we have established a Microbiome core for providing these technologies as a service to the NIH community. We work extensively 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. We were among the very first to show that 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. Many laboratories have extended our results to cancer immunotherapy in patients and suggested that the microbiome composition determine the ability of patients with melanoma and other type of cancer to respond to anti-PD1 therapy. However, the different studies have identified different types of bacteria has been responsible for this effect and the mechanisms remain unclear. We have extended the analysis to a large cohort of melanoma patients at the University of Pittsburgh and we are starting to elucidate the reason of the discordant results in the various group and to identify common mechanisms. In collaboration with MD Anderson cancer center we have established that patients with a high fiber diet respond better to anti-PD1 therapy and we have studied the mechanisms underlying this effect in mouse studies. In collaboration with the University of Pittsburgh Cancer Center we have treated 14 anti-PD1 refractory melanoma patients with a fecal microbiota transplant from PD1-responsive patients and in almost half of the patients the rapidly progressive tumors stabilized or significantly responded to the continued anti-PD1 therapy. The mechanisms underlying this effect are being studied with microbiome, cytokine and metabolomic studies.