Analyses of Host-Biofilm Interactions: A Novel Polymicrobial Model

描述(申请人提供)：牙周炎是人类分布最广泛的多菌疾病。导致牙周软组织和牙槽骨功能丧失的组织破坏过程是由于局部组织中宿主炎症、先天免疫和获得性免疫反应的慢性多菌生物膜激活所致。多菌生物膜的形成是一个连续的过程，从宿主和细菌糖蛋白沉积到清洁的牙齿表面开始。其次是口腔微生物的初始定植，口腔链球菌是主要的早期定殖者。链球菌最初的定植之后是放线菌、韦洛氏菌和梭杆菌的比例增加。(中间定殖者)，随后的定殖者主要是革兰氏阴性厌氧细菌(晚期定殖者)。这种从革兰氏阳性兼性微生物为主到以革兰氏阴性厌氧菌为主的微生物区系的转变在临床上表现为宿主对这种微生物挑战做出反应时牙周组织中的炎症变化。有人提出，与浮游细菌感染挑战相比，这种多细胞细菌-宿主相互作用具有独特的特征；然而，几乎没有客观证据支持这一论点。这项R21探索性/开发赠款将特别侧重于使用一种新的多微生物生物膜模型来开发这一证据。要检验的一般假设是：(1)生物膜中的口腔细菌将引起牙龈上皮细胞的不同模式的宿主反应；(2)宿主细胞对以单一物种和多物种生物膜存在的细菌物种的反应不同。我们开发了一种新型的体外生物膜-宿主细胞模型系统，该系统使用了一种刚性的、透气的隐形眼镜材料。在具体目标1中，我们将确定在该材料上形成的单物种和多物种生物膜的特征。在这个模型系统中开发的生物膜然后将用于特定的目标2来挑战口腔上皮细胞，使我们能够表征这些宿主细胞中由细菌挑战引起的反应的模式。为了实现这些目标，我们将开发一种新的方法来评估宿主对浮游生物膜、单物种生物膜和多微生物生物膜挑战的反应模式。所获得的结果将使我们能够提供开创性的数据，使进一步探索宿主对不同组成和复杂的生物膜的反应。在牙菌斑生物膜中发现的细菌与支持牙齿的组织相互作用，启动炎症反应。与这种炎症反应相关的临床疾病是牙周炎和/或牙周炎。然而，这种相互作用引起的确切宿主反应尚未确定。我们正在开发这种相互作用的体外模型，以更好地了解牙周炎和牙周炎的病理机制。这种理解对于开发和评估控制这些慢性炎症性疾病的破坏性特征的新策略是必要的。

DESCRIPTION (provided by applicant): Periodontitis represents the most broadly distributed polymicrobial disease of mankind. The tissue destructive processes that lead to loss of function of periodontal soft tissues and alveolar bone result from a chronic polymicrobial biofilm activation of host inflammatory, innate immune, and adaptive immune responses in the local tissues. Development of the polymicrobial biofilm is a sequential process that begins with deposition of host and bacterial glycoproteins onto the clean tooth surface. Initial colonization by oral microorganisms occurs next, with oral streptococci being the primary early colonizers. This initial colonization by streptococci is followed by increasing proportions of Actinomyces, Veillonella, and Fusobacterium spp. (middle colonizers) with subsequent colonization predominated by Gram-negative, anaerobic bacteria (late colonizers). This shift from a predominance of Gram-positive, facultative microorganisms to a microbiota dominated by Gram- negative, anaerobic species is manifest clinically by inflammatory changes in the periodontal tissues as the host responds to this microbial challenge. It has been proposed that this multicellular bacterial-host interaction possesses unique features compared to planktonic bacterial infectious challenges; however, there is little objective evidence to support this contention. This R21 exploratory/development grant will specifically focus on developing this evidence using a novel polymicrobial biofilm model. The General Hypotheses to be tested are: (1) Oral bacteria in biofilms will elicit different patterns of host responses from gingival epithelial cells; and (2) Host cells will respond differently to bacterial species present as monospecies versus multispecies biofilms. We have developed a novel in vitro biofilm-host cell model system using a rigid, gas permeable contact lens material. In Specific Aim 1 we will determine the characteristics of single species and multispecies biofilms created on this material. The biofilms developed in this model system will then be used in Specific Aim 2 to challenge oral epithelial cells, allowing us to characterize the patterns of responses elicited in these host cells by the bacterial challenge. In accomplishing these aims, we will have developed a novel approach for evaluating host response patterns to planktonic, single species biofilm and polymicrobial biofilm challenges. The results obtained will enable us to provide seminal data to enable a further exploration of host responses to biofilms of various compositions and complexities. The bacteria found in dental plaque biofilms interact with tissues supporting the teeth to initiate an inflammatory response. The clinical diseases associated with this inflammatory response are gingivitis and/or periodontitis. However, the exact host responses elicited by this interaction have not been determined. We are developing an in vitro model of this interaction to gain a better understanding of the pathologenesis of gingivitis and periodontitis. This understanding is necessary to develop and evaluate new strategies for controlling the destructive features of these chronic inflammatory diseases.