CELLULAR AND MOLECULAR BIOLOGY OF CHOLESTEATOMA

描述(申请人提供)：耳胆脂瘤是一种上皮性囊状结构，扩张到邻近的骨骼，导致慢性感染和听力丧失。本研究的主要目的是探讨铜绿假单胞菌(PA)感染胆脂瘤的致病机制和致病机制。这些细菌在胆脂瘤内形成生物膜，导致慢性感染，而这种感染对抗生素和宿主防御是顽固的。感染胆脂瘤的病理生理学是复杂的，涉及细菌因子、下层上皮和邻近骨骼之间的相互作用。在具体目标I中，我们将使用实验性胆脂瘤沙土鼠模型来评估从人胆脂瘤分离的耳源性PA分离株的发病机制。我们选择了一株具有代表性的菌株OPPA8进行测序和分析。对定点缺失突变体的评估将提供对胆脂瘤发生和增强侵袭性所必需的基因的洞察。我们还计划通过有针对性地删除关键毒力基因(Galu、algC、algD、LASI、LasR、flic、exsA)来评估沙土鼠模型中的一些已知毒力因子。在特定的目标II中，我们的目标是阐明PA内毒素在宿主损伤和反应中的机制，特别是解除对角质形成细胞增殖和骨吸收的调控。我们将使用永生化细胞系、原代人和原代小鼠角质形成细胞来检测脂多糖在体外对角质形成细胞增殖和分化的影响。我们将通过评估参与PA内毒素诱导的增殖和分化的下游效应分子，进一步探讨PA内毒素诱导体外增殖的机制。通过比较PA脂多糖刺激的TLR2-/-、TLR4-/-和MyD88-/-基因敲除小鼠的角质形成细胞的细胞因子谱，我们希望描述这一过程发生的机制。此外，我们还将使用小鼠局部骨吸收模型来评估PA-内毒素诱导的体内骨吸收的机制。我们实验室以前的研究表明，PA-LPS在体外通过TLR4、MyD88依赖的途径诱导骨吸收，但初步的体内数据也表明TLR2也有作用。了解PA在胆脂瘤中的致病性和毒性可能会导致药物治疗或治疗方式的新靶点，从而预防或改善与这种疾病相关的听力损失。 与公共卫生相关：耳胆脂瘤是一种发生在中耳的上皮性囊样结构，是中耳炎的并发症，感染后具有侵袭性、复发性和顽固性。铜绿假单胞菌是从人和沙土鼠胆脂瘤中分离到的最常见的革兰氏阴性菌。更深入地了解PA的发病机制和细菌毒力因子在组织破坏(角质形成细胞增殖和骨吸收)中的作用，可能会导致新的治疗方法，以降低与该疾病相关的发病率、慢性化和听力损失的发生率。

DESCRIPTION (provided by applicant): Aural cholesteatomas are epithelial cyst-like structures that expand into adjacent bone, resulting in chronic infection and hearing loss. The overall goal of this proposal is to investigate the mechanisms of bacterial-host interactions in the pathogenesis and virulence of cholesteatomas infected with Pseudomonas aeruginosa (PA). These bacteria form biofilms within the cholesteatoma resulting in chronic infection which is recalcitrant to antibiotics and host defenses. The pathophysiology of infected cholesteatomas is complex involving interactions among bacterial factors, the underlying epithelium and adjacent bone. In Specific Aim I, we will use a gerbil model of experimental cholesteatoma to evaluate the pathogenesis of otopathogenic PA isolates isolated from human cholesteatomas. We have chosen a representative strain, OPPA8 for sequencing and analysis. Evaluation of site directed deletion mutants will provide insight into the genes necessary for cholesteatoma development and enhanced aggressiveness. We also plan to evaluate a number of known virulence factors in the gerbil model by using targeted deletions of key virulence genes (galU, algC, algD, lasI, lasR, fliC, exsA). In Specific Aim II, our goal is to elucidate the mechanisms of PA LPS in host injury and response, specifically deregulated keratinocyte proliferation and bone resorption. We will examine the effect of lipopolysaccharide in the proliferation and differentiation of keratinocytes in vitro using an immortalized cell line, primary human and primary mouse keratinocytes. We will further examine the mechanism of PA LPS induced proliferation in vitro by evaluating downstream effectors involved in PA LPS mediated proliferation and differentiation. By comparing cytokine profiles from PA LPS stimulated keratinocytes derived from TLR2-/-, TLR4-/- and MyD88-/- knockout mice we hope to delineate the mechanism by which this process occurs. Additionally, we will evaluate the mechanisms of PA LPS induced bone resorption in vivo using a murine model of localized bone resorption. Previous studies in our laboratory demonstrated PA LPS induces bone resorption in vitro through a TLR4, MyD88 dependent pathway but preliminary in vivo data also suggests a role for TLR2. An understanding of the pathogenicity and virulence of PA in cholesteatoma may lead to new targets of drug therapy or treatment modalities that will prevent or ameliorate the hearing loss associated with this disease. PUBLIC HEALTH RELEVANCE: Aural cholesteatomas are epithelial cyst-like structures occurring in the middle ear as a complication of otitis media and when infected they are aggressive, recurrent and recalcitrant to treatment. Pseudomonas aeruginosa is the most common gram negative organism isolated from both human and gerbil cholesteatomas. A greater understanding of the pathogenesis of PA and the role of bacterial virulence factors in tissue destruction (keratinocyte proliferation and bone resorption) may lead to new therapeutic modalities to reduce the morbidity, chronicity and incidence of hearing loss associated with this disease.