Augmenting Innate Immunity to Protect the Oral Mucosa

描述(由申请人提供)：在这个尤里卡项目(RFA-GM-10-009)中，我们将加快知识的发展，朝着临床方法发展，以挫败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通过两个分隔的、细胞内的抗微生物效应系统来保护和抑制侵袭性病原体：胞浆中的钙保护素和内体中的LL-37。通过针对这些效应器，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。我们假设角质形成细胞对侵袭性微生物病原体的抵抗力可以通过瞬时传递特定的抗微生物效应物(例如，S100A8和S100A9)或特定的激动剂(例如，上调LL-37的维生素D类似物)来增强。为了避免转基因的使用，我们将使用一种不同寻常的方法，将钙保护素和/或LL-37mRNAs引入角质形成细胞。作为比较，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的表达。我们将了解是否可以选择性地增强内体和细胞质对侵袭性微生物的抵抗力。为了了解将这些特定的mRNAs或维生素A类似物应用于完整组织是否有效，并考虑到细胞毒性、促炎免疫和致癌副作用，我们将在体外对这一系统进行模拟。总的来说，这些实验将表征维生素D类似物和抗微生物效应物mRNAs的转移，以了解在增强口腔角质形成细胞对侵袭性口腔病原体的天然抵抗力方面的机制和有效性。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有价值的补充，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-收益比。抗生素会增加微生物耐药性和宿主过敏的风险。我们的做法将绕过这些担忧。 公共卫生相关性：抗生素不能治愈牙周炎和念珠菌病等常见的口腔感染，也无法获得疫苗。挑战是如何使口腔粘膜对侵袭性口腔病原体更具抵抗力。为了应对这一挑战，我们将开发一种替代基因治疗的方法，该方法将增加口腔粘膜上皮细胞产生正常的抗微生物效应分子。

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.