【作者】 李平；

【导师】 温海；

【作者基本信息】 第二军医大学 ， 皮肤病与性病学（专业学位）， 2013， 博士

【摘要】 隐球菌系环境腐生菌，常引起免疫功能低下人群的感染，隐球菌性脑膜炎/脑膜脑炎是其高病死率的最主要的原因。隐球菌感染一般有两个结局，一是宿主免疫反应清除隐球菌，另一个就是隐球菌成功逃逸机体的免疫应答，从而造成潜伏感染或播撒。目前认为隐球菌逃逸机体的免疫杀伤有以下几个途径：1、“吞噬体挤出”与细胞内复制；2、溶解巨噬细胞或诱导巨噬细胞凋亡；3、避免被巨噬细胞吞噬；4、遮盖病原体相关分子模式（PAMP），避免宿主免疫识别；5、诱导机体免疫抑制。甘露糖受体（Mannose receptor，MR）也叫CD206，主要由巨噬细胞及树突状细胞表达，属于C型凝集素样受体，是一种模式识别受体，不仅可以识别并结合病原生物的多种糖蛋白，介导固有免疫应答，而且通过其抗原提呈等功能，可以促进T细胞活化和细胞因子释放，启动适应性免疫应答。新生隐球菌甘露糖蛋白（Mannoprotein，MP）是MR的配体，MP可有效激活机体T细胞免疫应答，增强小鼠的抗隐球菌感染免疫，表现在MP免疫小鼠可以延长其新生隐球菌感染后的生存时间和降低器官的真菌荷载，且DCs可以通过MR和FC受体吞噬隐球菌，进而递呈抗原给T细胞，诱发机体的抗隐球菌免疫应答。目前有关隐球菌逃逸机体免疫应答的研究主要集中在隐球菌荚膜相关基因及隐球菌巨噬细胞内复制，而对于隐球菌是否通过影响某些重要受体的表达从而抑制免疫细胞的功能尚未见相关报道。鉴于MR在抗隐球菌感染免疫的重要作用，我们观察了隐球菌对MR的表达调节及其调节机制，并对其病理生理意义进行了研究。隐球菌困扰人类的另一大问题就是其嗜中枢性，常表现为致命性的脑膜炎/脑膜脑炎。目前认为隐球菌嗜中枢性与以下因素有关：1、依赖于单核细胞的木马机制；2、活化Rho GTPases；3、活化尿激酶纤溶酶原系统；4、新生隐球菌CPS1基因透明质酸CD44分子；5、自身的尿素酶。我们前期研究发现隐球菌可以上调人脐静脉内皮细胞S100A10的表达，且抑制S100A10表达降低了脑微血管内皮细胞吞噬隐球菌的能力，被吞噬的隐球菌出芽率降低，荚膜增厚，表明沉默S100A10蛋白的表达降低了隐球菌在脑微血管内皮细胞内的生存能力，提示S100A10可能与隐球菌入侵脑微血管内皮细胞有关。因为尿激酶活化的纤溶酶系统有助于隐球菌通过血脑屏障，而纤溶酶系统的活化需要内皮细胞S100A10的参与，因此，我们对隐球菌上调脑微血管内皮细胞S100A10表达的病理生理意义进行了深入的研究，拟明确隐球菌上调S100A10的表达与其通过血脑屏障的关系。一、隐球菌调节MR表达及其相关机制研究首先我们观察了隐球菌对免疫细胞MR的表达的调节，我们发现隐球菌可以下调DCs及巨噬细胞MR基因水平及蛋白水平的表达，MR表达的下调并不是由于膜MR脱落成可溶性SMR所致，而是基因水平的下降。有趣的是，使用Transwell小室将菌体与细胞隔开并没有观察到这种变化，说明隐球菌下调MR的表达依赖于菌体与细胞的直接接触，但与菌的活力无关。进一步研究发现，MR的配体MP可以下调MR的表达，这一现象类似于LPS下调TLR4的表达，而后者被认为是导致内毒素耐受的机制之一，这可能是隐球菌逃逸机体免疫的一种手段。二、MR信号转导通路的研究因MR胞内段缺乏ITAM模序，因此我们推测MP作用于MR启动的信号通路的活化需要其他受体的参与。首先我们观察了MP刺激后巨噬细胞MAPK、NF κB的活化情况，我们发现，MP可以促进巨噬细胞p38、ERK、JNK、p65的磷酸化，敲除TLR2后上述信号通路的磷酸化均明显受到影响，说明TLR2参与了MR的信号转导。进一步研究发现，敲除TLR2明显降低了MR促进的巨噬细胞IL6、TNF α、IL1β等促炎细胞因子的分泌，并且降低了巨噬细胞杀伤隐球菌的能力，说明TLR2在巨噬细胞抗隐球菌感染免疫中具有重要的作用。应用p38抑制剂我们发现，隐球菌和MP下调MR的作用被抑制，因此，隐球菌下调MR的表达是通过活化p38MAPK实现的。三、隐球菌下调MR表达的病理生理意义研究由于观察到隐球菌可以下调MR的表达，因此我们探索了该现象的病理生理意义。我们发现过表达MR的DCs抗原递呈MP的能力明显增强，活化T细胞的能力也增强，说明DCs的抗隐球菌免疫能力与MR的表达水平呈正相关。接下来我们观察了降低MR表达对巨噬细胞功能的影响，我们发现，抑制MR的表达降低了巨噬细胞吞噬和杀伤隐球菌的能力，说明巨噬细胞抗隐球菌感染免疫的能力也与MR的表达水平呈正相关。因此，我们认为，隐球菌下调MR的表达抑制了机体的免疫应答，从而逃避机体的抗隐球菌免疫反应，这可能是隐球菌免疫逃逸的一个新的机制。四、S100A10与隐球菌嗜中枢性的机制研究我们的前期研究发现，隐球菌可以上调人脐静脉内皮细胞S100A10的表达，抑制S100A10的表达明显降低了内皮细胞吞噬隐球菌的能力,并且被吞噬的隐球菌出芽率降低、荚膜变厚，说明S100A10可能在隐球菌侵袭血管内皮细胞的过程中具有重要作用。我们的研究发现，隐球菌可以上调小鼠脑微血管内皮细胞S100A10的表达，通过干扰慢病毒载体沉默S100A10的表达降低了依赖于纤溶酶的隐球菌通过血脑屏障的能力，但对未包被纤溶酶原的隐球菌通过血脑屏障的能力影响不大，说明S100A10影响隐球菌通过血脑屏障依赖于纤溶酶原的存在。进一步的研究发现，沉默S100A10的表达降低了尿激酶及纤溶酶原的活化，而升高的尿激酶纤溶酶的活性被认为与隐球菌侵袭血脑屏障有关，因此，隐球菌促进S100A10尿激酶纤溶酶系统的活化可能与其嗜中枢性有关。综上所述，我们发现了隐球菌可以下调MR的表达，MR表达的降低可能是隐球菌诱导免疫细胞的功能抑制的一个新的途径，而隐球菌上调S100A10的表达与其嗜中枢性有关，下一步研究可通过构建MR转基因小鼠和S100A10基因敲除小鼠进一步从体内予以证实。更多还原

【Abstract】 Cryptococcus neoformans(C.neoformans) is an opportunistic fungal pathogen thatcauses central nervous system and pulmonary disease among immunocompromisedpatients. Despite the availability of antifungal agents with anticryptococcal activity, themortality and treatment failure rates associated with cryptococcosis remain unacceptablyhigh. In cryptococcal infection, one of two things happens: either the host immuneresponse clears the infection, or C.neoformans successfully evades this response and,without treatment, will disseminate with potentially fatal consequences. An additionalpossible outcome is that cryptococcal infection may be contained but not cleared andbecomes latent. There are several ways for C.neoformans evade the body’s immuneresponse:1,"Phagosome extrusion" and intracellular replication;2, Lysis of macrophagesor inducing macrophage apoptosis;3, Avoiding being engulfed by macrophages;4,Avoiding immune recognition: shielding PAMPs and physical barriers;5,Inducing immunesuppression.Mannose receptor, also known as CD206, mainly expressed by macrophages anddendritic cells, are the C type lectin like receptor and pattern recognition receptors.MR cannot only recognize and bind to glycoprotein of pathogens and induce innate immuneresponse, but also can promote T cell activation and cytokine release, starting the adaptiveimmune response. C.neoformans mannoprotein(MP) is the ligand of MR, MP caneffectively activate the T cell immune response and enhance the immunity againstcryptococcal infection. MP immunized animals exhibited prolonged survival and reducedbrain and kidney fungal loads following intravenous challenge with C. neoformans, anddendritic cells can phagocytize C. neoformans via mannose receptors and Fc receptor IIand present antigens to T lymphocytes, inducing the antifungal immune response.Researchs on C.neoformans evading the immune response are mainly concentrated oncryptococcal capsular genes and intracellular replication, yet there is no report aboutC.neoformans evading immune response by regulating some important receptors expression.Because of the importance of MR in anti cryptococcal infection, we observed howC.neoformans regulate the expression of MR and its pathophysiological significance.C. neoformans is encapsulated yeast responsible for life threatening central nervoussystem (CNS) infections primarily in immunocompromised patients. How Cryptococcus addict to the central nervous system may involve the following factors:1, Trojanmechanism which depends on the the monocytes;2, Activating Rho GTPases;3,Activating urokinase plasminogen system;4, C. neoformans CPS1gene hyaluronicacid CD44;5, Urease. Our previous studies found that Cryptococcus can up regulate theexpression of S100A10in human umbilical vein endothelial cells, and suppression ofS100A10expression was associated with significantly reduced rates of phagocytosis andbudding of C. neoformans, the capsule of intracellular C. neoformans also became thickerafter S100A10was knocked down, indicating that S100A10may involved in C.neoformans invading brain microvascular endothelial cells. C. neoformans can invade theblood brain barrier though plasminogen system which was activated by UPA, and UPAactivating the plasminogen needs the expression of S100A10on endothelial cells,therefore, we intend to clarify the relationship between Cryptococcus upregulating theexpression of S100A10and its traversal across the blood brain barrier.1、The mechanisms of Cryptococcus regulating the expression of MRFirst, we found that Cryptococcus can down regulate the expression of MR in DCsand macrophages at both gene level and protein level. The downregulation of MR was notdue to its sheding into soluble MR(SMR). Interestingly, when using the Transwellchamber to separate the fungus and cells we did not observe such a change, indicating thatCryptococcus down regulate MR expression depending on the direct contact between thefungus and cells, and regardless of the vitality of the fungus. Further studies showed thatthe ligand MP can down regulate MR expression,it was similar to LPS down regulatingTLR4expression which was considered to be responsible for endotoxin tolerance. Somaybe this is the new mechanism used by Cryptococcus to evade the immune response.2、 The signal pathway of MRBecause the MR cytoplasmic tail does not contain intracellular ITAM signaling motifs, wehypothesized that direct interaction of other receptors and MR may happen,these receptorsmay form a functional complex, whereby MR may capture MP at the cell membrane,andthe other receptors signaling motif of the associated receptors may provide the necessaryintracellular signal transduction via NF κB and MAPKs to promote cytokine release. First,we observed that MP can activate MAPK and NF κB pathway, it’s shown that the MP canpromote p38, ERK, JNK and p65phosphorylation, knockout of TLR2significantlyaffected the phosphorylation of MAPK and NF κB, demonstrating that MR signaltransduction need the expression of TLR2. And it’s shown that knockout of TLR2 significantly reduced the secretion of pro inflammatory cytokines such as IL6,TNF α andIL1β and affected the ability of macrophages in killing Cryptococcus.Finally,we foundthat p38inhibitors can suppress the down regulation of MR induced by Cryptococcus andMP,therefore, Cryptococcus and MP down regulate the expression of MR depends on theactivation of p38MAPK.3、The significance of down regulation of MR induced by CryptococcusFirst,we found that overexpression of MR enhanced the ability of DCs inpresenting MP antigen and significantly enhanced the activation of T cells, demonstratingthat the antifungal immunity of DCs was positively correlated with the level of MR. Next,we observed that silencing MR reduced the ability of macrophage in phagocytizing andkilling C. neoformans, indicating that the antifungal immunity of macrophages was alsopositively correlated with the level of MR. Therefore, we supposed that thedown regulation of MR expression induced by Cryptococcus suppressed the antifungalimmunity,which may provided new mechanism for Cryptococcus immune evasion.4、S100A10with cryptococcal addicted central MechanismOur previous studies showed that Cryptococcus can up regulate the expression ofS100A10in human umbilical vein endothelial cells,and silencing S100A10significantlyreduced the ability of endothelial cells in phagocytizing Cryptococcus. Additionally,suppression of S100A10expression was associated with significantly reduced rate ofphagocytosis and budding of C. neoformans. The capsule of intracellular C. neoformansalso became thicker after S100A10was knocked down, indicating that S100A10may playan important role in transpenetration of C. neoformans across the vascular endothelia.Inthis research,we found that C. neoformans can up regulate the expression of S100A10inmouse brain microvascular endothelial cells, silencing S100A10reduced the ability ofplasminogen coated C. neoformans in invading the blood brain barrier, but it didn’t affectthe ability of C. neoformans not coated with plasminogen in crossing the blood brainbarrier. Further we found that silencing S100A10reduced the activation of urokinase andplasminogen, therefore, the activated S100A10urokinase plasminogen system may beinvolved in C. neoformans addicting to central nervous system.In summary, we found that C. neoformans can downregulate the expression of MRand the reduced MR expression may be responsible for C. neoformans immuneevasion.The upregulation of S100A10may be involved in C. neoformans addicting tocentral nervous system.更多还原