Modulation of innate epithelial cell responses by oral commensal bacteria

 描述（由申请人提供）：口腔上皮细胞（OEC）持续识别口腔粘膜细菌减轻口腔粘膜不受控制的炎症反应的机制仍然未知。有证据表明，上皮细胞可以通过反应的差异激活，特别是细胞因子/趋化因子模式，最终影响免疫反应的先天性和适应性臂来区分细菌和病原菌。我们实验室最近的工作是确定OECs在口腔细菌和致病性口腔细菌挑战后的反应模式。结果表明，在响应口服的S.p.S.的情况下，趋化因子的转录活性显著更强（质量和数量）。gordonii（Sg）感染的结果与用口腔病原体F.具核胞菌（Fn）和牙龈卟啉单胞菌。随后对所选趋化因子的蛋白质分析显示，与Fn的作用相比，在Sg诱导的趋化因子转录激活与OEC提取物和上清液中发现的降低和有限的蛋白质水平之间存在明显的脱节，尽管Fn诱导较低的mRNA趋化因子水平（低2至4倍），但如先前所示增强了稳健的蛋白质趋化因子产生。因此，我们假设Sg和其他口腔黏膜细菌物种有能力有效地激活趋化因子转录和翻译的调节机制，以最大限度地减少粘膜组织中OEC驱动的病理性炎症反应。一致地，初步分析显示，Sg具有显著上调（>2倍）OEC中115/2578个miRNA的表达的能力，由此miR-663 a、miR-4516、miR 492和miR 193 a-5 p已经验证了参与TLR和精氨酸诱导的趋化因子转录和翻译的基因靶标。为了验证这一假设，我们提出了以下两个具体目标：（i）确定OECs对口腔细菌的趋化因子转录和翻译反应，以及（ii）确定特定miRNA在调节口腔细菌诱导的OECs中趋化因子产生中的作用。为了解决这些知识缺口，我们将使用OEC培养物来：（a）确定OEC中趋化因子的转录和翻译之间的断开是否是口腔粘膜细菌物种的共同特征，以及（B）确定口腔粘膜对作为这些炎症分子的调节剂的特定miRNA表达的影响。这项研究的贡献预计是鉴定特定miRNA对OECs炎症反应的主动调节，作为口腔粘膜细菌与宿主保持共生的潜在“致耐受性机制”。重要的是，将被口腔粘膜细菌特异性激活的miRNA的减少（例如，在生态失调期间）可能与疾病中观察到的病理性炎症变化有关。该项目的意义将使我们能够更好地了解上皮耐受性和病理反应之间的“临界点”的细胞和分子机制。预计这些结果将为未来旨在针对这些机制的研究奠定坚实的循证基础，并确定与口腔细菌调节的上皮趋化因子产生相关的新分子靶点，并最终为开发创新方法提供新的机会预防/治疗牙周炎。

 DESCRIPTION (provided by applicant): The mechanisms through which a persistent recognition of oral commensal bacteria by oral epithelial cells (OECs) mitigates an uncontrolled inflammatory response of the oral mucosa remain unknown. Evidence suggests that epithelial cells may discriminate between commensals and pathogenic bacteria through differential activation of responses, particularly cytokine/chemokine patterns, which ultimately influence both innate and adaptive arms of the immune response. Recent work in our laboratory has been identifying patterns of OECs responses following challenge with commensal and pathogenic oral bacteria. The results showed a significantly more robust (quality and quantity) chemokine transcriptional activity in response to the oral commensal S. gordonii (Sg) compared to the outcomes following challenge with the oral pathogens F. nucleatum (Fn) and P. gingivalis. Subsequent protein analysis of selected chemokines showed that there was a clear disconnect between the Sg-induced chemokine transcriptional activation and the reduced and limited protein levels found in OEC extracts and supernatants in comparison with the effect of Fn, which despite inducing lower mRNA chemokines levels (2 to 4-fold less), enhanced a robust protein chemokine production as previously shown. Therefore, we hypothesized that Sg and perhaps other oral commensal bacterial species have the ability to efficiently activate regulatory mechanisms for chemokine transcription and translation in order to minimize pathological inflammatory responses driven by OECs in mucosal tissues. Consistently, preliminary analysis showed that Sg has the ability to significantly up-regulate (>2- fold) the expression of 115/2578 miRNAs in OECs, whereby miR-663a, miR-4516, miR492, and miR193a-5p have validated gene targets involved in TLR- and cytokine-induced chemokine transcription and translation. To test this hypothesis we propose the following two specific aims: (i) To determine the chemokine transcriptional and translational responses of OECs to oral commensal bacteria, and (ii) To determine the role of specific miRNAs in regulating the chemokine production induced by oral commensal bacteria in OECs. To address these knowledge gaps, we will use OEC cultures to: (a) determine whether the disconnect between transcription and translation of chemokines in OECs is common feature of oral commensal bacterial species, and (b) determine the effect of oral commensals on the expression of specific miRNAs as regulators of these inflammatory molecules. The contribution of this investigation is expected to be the identification of the activ regulation of inflammatory responses in OECs by specific miRNAs as a potential "tolerogenic mechanism" by which oral commensal bacteria maintain symbiosis with the host. Importantly, diminutions of miRNAs that would be activated specifically by oral commensal bacteria (e.g., during dysbiosis) could be related to pathologic inflammatory changes observed in disease. This project's significance will enable a better understanding of the cellular and molecular mechanisms involved in the "tipping point" between epithelial tolerogenic and pathologic responses to oral bacteria. The results are expected to contribute to a strong evidence-based foundation for future studies designed to target these mechanisms and identify new molecular target(s) associated with oral bacteria-regulated epithelial chemokine production, and will ultimately provide new opportunities for the development of innovative approaches to prevent/treat periodontitis.