TLR2激动剂预处理对急慢性耐甲氧西林金葡菌感染的作用研究

Due to the increasing the emergence of methicillin-resistant S.arueus (MRSA),the immunotherapy and prophylaxis have been attracted great attention. The previously described strategies seek to alter the balance of host immunity and pathogen virulence to favor bacterial clearance. However, bacterial clearance alone frequently is insufficient to optimize amelioration of infectious pathology. For example, it is well appreciated that in the case of severe pneumonia or sepsis, patients can sustain prolonged immunopathology long after the clearance of bacteria from the tissues, and overly exuberant host immunity in these cases is ultimately the major cause of morbidity and mortality. So it essential to effectively control inflammation during clearance of bacteria. TLRs(toll like receptors) are one of two types of PRRs expressed on the surface of cell and endosomal membranes, and show variable contribution to host defense in different infection models. For S.aureus, TLR2 shown to be an essential component of host innate defense against in murine brain abscess, cutaneous, and systemic infection models, which activates a broad array of pro-inflammatory pathways principally through activation of the NF-κB pathway, and regulates neutrophil recruitment and bactericidal activities. In this project, we will attempt to evaluate the protective effect and mechanism of acute or chronic murine infection models challenge by MRSA with pretreatment with Pam3CSK4(TLR2 agonist),and to understand innate and acquired immunity against MRSA.Such as secondary humural immune response,especially T cell mediated immune response will be studied. In our previous work, found that mice(Balb/c)after pretreatment with the Pam3CSK4(TLR2 agonist) improves bacterial clearance and decrease inflammation and mortality challenged with MRSA（ATCC43300）, in addition, the number of renal abscess decreased in murine pretreated with Pam3CSK4. The significance of this project are as follows ①this prophylaxis is the new attempt against the infection by MRSA that resists multi-antibiotics and increases the emergence in clinical medicine, which will also reduce the indiscriminate usage of antibiotics against MRSA. ② it essential to effectively control inflammation during clearance of bacteria. Our previous works found that Pretreatment with the Pam3CSK4(TLR2 agonist) improved bacterial clearance ，and decreases inflammation and mortality in mice challenged with methicillin-resistant S.arueus. ③ To reveal and change the regularity of the innate and acquired response against MRSA in murine pretreatment with the Pam3CSK4 .

耐甲氧西林金葡菌（MRSA）感染日越严重，免疫学防治被寄予希望。有效的免疫防治既要有效清除病原体又要抑制免疫病理反应和改变机体对金葡菌感染的无免疫记忆状态；TLR2是对抗金葡菌感染的关键受体，干预TLR2的状态能影响机体对金葡菌的应答能力。本项目拟观察TLR2配基Pam3CSK4对小鼠预处理后MRSA急慢性感染的保护性防御机制；以期既增强感染小鼠清除MRSA能力的同时抑制其过度炎症反应，同时期望干预TLR2后改变机体对MRSA的获得性免疫应答规律；我们初步证实Pam3CSK4预处理对MRSA感染小鼠具保护作用，同时降低了血清中TNFα、IL6水平，目前尚未见国内外同类报导。项目意义①本项目可望为MRSA感染提供新的防治思路与手段，实现增强抗菌免疫反应的同时也降低炎症损伤,以减少抗菌药物的使用及滥用；②揭示以天然免疫模式识别受体TLR2为切入点，改变机体对MRSA的免疫应答的特性与规律。

临床上由耐甲氧西林金葡菌（MRSA）导致的感染，是临床用药面临的较为棘手难题之一；本研究选择TLR2配基Pam3CSK4预处理，观察小鼠对MRSA的免疫应答。本项目的主要结论如下：.一、Pam3CSK4预处理增强小鼠腹腔巨噬细胞对MRSA的杀菌作用：在蛋白和基因水平Pam3CSK4预处理减弱MRSA刺激腹腔巨噬细胞释放前炎症介质TNF-α、IL6和IL-1能力；增强巨噬细胞对金葡菌杀菌和吞噬能力可能与增强巨噬细胞吞噬相关受体、iNOS和LL37的表达有关。.二、Pam3CSK4预处理增强中性粒细胞对MRSA杀菌作用：Pam3CSK4增强骨髓来源中性粒细胞对MRSA的杀菌和吞噬能力；促进中性粒细胞的呼吸爆发、乳铁蛋白和细胞因子的释放，其机制可能与激活MAPK 和PI3K通路有关。Pam3CSK4预处理中性粒细胞过继免疫对MRSA攻击小鼠有保护作用。.三、Pam3CSK4预处理增强对耐甲氧西林金葡菌小鼠肺炎模型和脓毒症模型的保护性：Pam3CSK4预处理增强MRSA小鼠肺炎模型和脓毒症模型的生存率，肺、支气管及肝脾肾等组织细菌清除，降低肺趋化因子表达、中性粒细胞的浸润，以及肺炎及脓毒症模型过度炎症反应，其机制可能与降低NF－kB 、MyD88、 IRAK1/4的磷酸化和增强负调控因子蛋白IRAK-M 和Tollip的表达有关。Pam3CSK4预处理增加肺组织和脾脏吞噬相关受体Fc和补体受体的表达，PAM3Csk4预处理也显著降低小鼠肾脓肿的形成。.四、pam3CSK4预处理增强DC细胞的免疫递呈功能。Pam3CSK4显著增强骨髓来源DC对MRSA摄取作用，增强骨髓来源DC表面TLR2的表达并随时间增加而增强，降低骨DC表面CD40/86表达；显著增加DC对初始T-cell的活化和CD4 /CD8+T增值。.本项目用低剂量Pam3CSK4体内预处理增强MRSA感染动物清除细菌同时降低炎症反应，并阐明其作用机制，国内外尚未见同类报道。本项目的实施对MRSA等多重耐药菌的免疫学防治，包括治疗性疫苗设计、新药研发及开创新疗法提供新的思路与实验依据。