Decoding the Role of Bacterial Outer Membrane Vesicle and Small RNAs in Host Inflammation

项目摘要 慢性炎症与大多数非传染性疾病有关，这些疾病影响超过40%的人。 在美国的个人。虽然靶向抑制炎症效应通路的药物是可用的， 没有针对慢性炎症的解决方案，部分原因是缺乏对如何重建的了解。 免疫稳态结肠中的细菌在调节急性和慢性结肠炎之间的平衡方面起着关键作用。 慢性炎症，了解如何重建免疫稳态是至关重要的， 预防慢性炎症性疾病的发展。产肠毒素脆弱类杆菌（ETBF）是一种 代表一个物种，负责腹泻，结肠炎和结肠癌。然而， B的非致病性菌株。fragilis（NTBF）似乎可以预防炎症。我们的研究小组和其他人 已经证明了细菌的一个主要通讯工具是细胞外小RNA，它可以抑制 或者激活免疫系统然而，人们对sRNA种类在调控基因表达中的作用知之甚少。 微生物-宿主通讯最近，细菌小RNA（sRNA）物种被确定在外部 膜囊泡（OMV）是由细菌脱落。小RNA代表了一个关键的触发因素， 宿主细胞是否响应促炎或抗炎信号。有趣的是， B。fragilis和E. coli对化学诱导的小鼠结肠炎有预防作用，而致病性E.大肠杆菌诱导 全身性炎症我们实验室的初步证据表明，ETBF和NTBF的OMV 差异激活TLR途径，并且它们的OMV携带不同的sRNA货物。源自我们的 初步研究表明，OMV衍生的小RNA在来自 与NTBF相比，ETBF。我们还表明，来自ETBF的OMV特异性激活TLR 7， NTBF的OMV。在这项研究中，我们假设对OMV的不同炎症反应 从有毒和非致炎性脆弱拟杆菌中分离出来的细菌，部分是由sRNA与TLR结合控制的， 这种机制部分解释了慢性炎症的预防或发展。目的 这项建议的一个重要目的是确定来自致病性ETBF的OMV或OMV衍生的sRNA是否阻止了 或诱导慢性炎症。我们的具体目标是：（1） 阐明先天性免疫应答在肠源性和肠源性之间的差异激活 来自B的OMV。利用已建立的三重培养体系，2）确定免疫- 刺激从大肠杆菌相对于肠杆菌B的细胞内摄取和亚细胞定位。脆弱拟 OMV进入宿主细胞，和3）确定肠源性B与肠源性B。fragilis small RNA宿主 使用生物信息学工具在计算机上实现靶点，并使用三培养物和纳米脂质囊泡进行体外验证 转染这一假设的证实将解决该领域的一个悬而未决的差距，以解释如何 来自致病性菌株的OMV与致病性菌株的OMV诱导或预防炎症。

PROJECT SUMMARY Chronic inflammation is associated with most non-communicable diseases, which affect over 40% of individuals in the U.S.. While drugs are available that target suppression of inflammatory effector pathways, none target resolution of chronic inflammation, in part due to a lack of understanding of how to re-establish immune homeostasis. Bacteria in the colon play a key role in regulating the balance between acute and chronic inflammation, and understanding how to re-establish immune homeostasis is paramount in preventing chronic inflammatory disease development. Enterotoxigenic Bacteroides fragilis (ETBF) is representative of one species that is responsible for diarrhea, colitis and colon cancer. Yet, the commensal non-toxigenic strain of B. fragilis (NTBF) appears to prevent inflammation. Our research group and others have documented a major communication tool of bacteria are extracellular small RNAs, which can dampen or active the immune system. Little is known, however, about the contribution of sRNA species in governing microbe-host communication. Recently, bacterial small RNA (sRNA) species were identified within outer membrane vesicles (OMVs) that are shed by bacteria. Small RNAs represent a critical trigger governing whether host cells respond with pro- or anti-inflammatory signals. Intriguingly, OMVs from the commensal B. fragilis and E. coli prevent chemically induced murine colitis, while those from pathogenic E. coli induce systemic inflammation. Preliminary evidence from our lab indicate that OMVs from ETBF and NTBF differentially activate the TLR pathway, and their OMVs carry distinct sRNA cargo. Results from our preliminary research demonstrate that OMV-derived small RNAs are differentially abundant in OMVs from ETBF as compared to NTBF. We also show that OMVs from ETBF specifically activate TLR7 as compared to OMVs from NTBF. In this study, we hypothesize that the differential inflammatory response to OMVs from toxic and non-toxigenic Bacteroides fragilis is governed, in part, by sRNA engagement with TLRs, and that this mechanism partially explains the prevention or development of chronic inflammation. The purpose of this proposal is to determine if OMVs or OMV-derived sRNA from the pathogenic ETBF either prevent or induce chronic inflammation as compared to the commensal strain NTBF. Our specific aims will 1) Elucidate the differential activation of the innate immune response between commensal and enterotoxigenic OMVs from B. fragilis using an established tri-culture system, 2) Identify the mechanism of immune- stimulating intracellular uptake and sub-cellular localization from commensal vs. enterotoxigenic B. fragilis OMVs into host cells., and 3) Determine the commensal vs. enterotoxigenic B. fragilis small RNA host targets in silico using bioinformatics tools and validate in vitro using tri-culture and nanolipovesicle transfection. Confirmation of this hypothesis would resolve an outstanding gap in the field to explain how OMVs from commensal vs pathogenic strains induce or prevent inflammation.