Augmenting Innate Immunity to Protect the Oral Mucosa

描述（由申请人提供）：在这个EUREKA项目（RFA-GM-10-009）中，我们将加速知识的发展，以临床方法阻止与牙周炎和念珠菌病相关的侵袭性口腔病原体。口腔角质形成细胞似乎使用两个区隔化的细胞内抗菌效应系统来保护和抑制侵袭性病原体：细胞质中的钙保护蛋白和内体中的LL-37。通过靶向这些效应物，我们计划使用两种方法来增强角质形成细胞对侵袭性病原体的细胞内抵抗力。我们假设，特定抗菌效应mrna（如S100A8和S100A9）或特定激动剂（如维生素D类似物上调LL-37）的瞬时递送可以增加角化细胞内对侵袭性微生物病原体的耐药性。为了避免使用转基因，我们将采用不寻常的方法将钙保护蛋白和/或LL-37 mrna引入角质形成细胞。为了比较，在一些实验中，我们将用维生素D类似物刺激细胞以上调LL-37。我们将了解是否可以选择性地增强内体和细胞质对入侵微生物的抗性。为了了解将这些特异性mrna或维生素A类似物应用于完整组织是否有效，并考虑细胞毒性、促炎免疫和致癌副作用，我们将同时在体外的人类扁桃体外植体中建立该系统的模型。总的来说，这些实验将表征维生素D类似物和抗菌效应mrna的转移，以增强口腔角化细胞对侵袭性口腔病原体的细胞内先天抗性的机制和有效性。我们的策略可能被证明是对大多数常见口腔感染的疫苗和抗生素的宝贵补充，这些感染不会危及生命，发病率相对较低。对于这些感染，疫苗具有不可接受的风险-效益比。抗生素增加了微生物耐药性和宿主过敏的风险。我们的方法可以规避这些担忧。

DESCRIPTION (provided by applicant): In this EUREKA project (RFA-GM-10-009), we will accelerate the development of knowledge towards a clinical approach to thwart invasive oral pathogens associated with periodontitis and candidiasis. Oral keratinocytes appear to protect against and suppress invasive pathogens using two compartmentalized, intracellular antimicrobial effector systems: calprotectin in the cytosol and LL-37 in endosomes. By targeting these effectors, we plan to use two approaches to augment keratinocyte intracellular resistance to invasive pathogens. We hypothesize that intra-keratinocyte resistance against invasive microbial pathogens can be increased by transient delivery of specific antimicrobial effector mRNAs (e.g., S100A8 and S100A9) or specific agonists (e.g., vitamin D analogues to upregulate LL-37). Avoiding the use of transgenes, we will use the unusual approach of introducing calprotectin and/or LL-37 mRNAs into the keratinocyte. For comparison, in some experiments we will stimulate cells with vitamin D analogues to upregulate LL-37. We will learn whether endosomal and cytoplasmic resistance against invasive microbes can be selectively augmented. To learn whether the application of these specific mRNAs or vitamin A analogues to intact tissues will prove efficacious, and to consider cytotoxic, proinflammatory immune, and carcinogenic side effects, we will concurrently model this system in human tonsil explants ex vivo. Collectively, the experiments will characterize vitamin D analogues and transfer of antimicrobial effector mRNAs for the mechanism and effectiveness in augmenting intracellular innate resistance to invasive oral pathogens in oral keratinocytes. Our strategy may prove to be a valuable compliment to vaccines and antibiotics for most common oral infections, which are not life threatening and have relatively low morbidity. For these infections, vaccines have an unacceptable risk-benefit ratio. Antibiotics promote the risk of microbial resistance and host allergy. Our approach would circumvent these concerns. PUBLIC HEALTH RELEVANCE: Antibiotics do not cure common oral infections such as periodontitis and candidiasis and vaccines are unavailable. The challenge is how to make the oral mucosa more resistant to invasive oral pathogens. To respond to this challenge, we will develop an alternative approach to gene therapy, which will increase production of normal antimicrobial effector molecules in oral mucosal epithelial cells.