尘螨变应原舌下免疫治疗对变应性鼻炎TSLP-OX40L信号通路介导的TH2型免疫反应的影响及其临床疗效观察
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摘要
研究背景
WHO指出“变应性疾病是二十一世纪人类面临的三大健康问题之一”。变应性鼻炎又称过敏性鼻炎,是以鼻腔粘膜为主要的效应部位,以机体免疫系统异常为主要的病理基础,在特异性体质的患者接触变应原后引起的,以IgE介导的,嗜酸性粒细胞反应为主的,由多种炎症细胞及细胞因子共同参与的Ⅰ型变态反应性疾病。其共同特征即所谓的“TH2型免疫反应”。
目前认为,有多种细胞共同参与到TH2(Ⅱ型辅助性T细胞,T help cell2)型免疫反应过程中,形成了一个复杂的免疫反应的网路系统。其中涉及了T细胞、B细胞、嗜酸细胞、嗜碱细胞、抗原递呈细胞以及最新发现的先天辅助细胞群等。TH2型免疫应答的特点是诱导CD4+T细胞(分化群,cluste of differentiation)分化,其分泌的细胞因子包括：IL-4、EL-5、IL-9、IL-13(白细胞介素,interleukin)。TH2细胞通过分泌IL-4促进B细胞反应和IgE的分泌。当IgE免疫复合物结合到位于嗜碱细胞和肥大细胞表面的高亲和力的IgE受体(FceRl),导致上述细胞激活并释放多种细胞因子和炎症介质,包括：组织胺、肝素和血清素。这些细胞因子介导了一系列的以TH2型免疫反应为特点的效应功能。包括有选择的募集和活化巨噬细胞和粒细胞,引起平滑肌的收缩和粘液过量分泌。对这个复杂的免疫反应网路系统进行纵向的分析可见,整个TH2型免疫反应的过程大致包括4部分：机体免疫系统对变应原的识别、机体致敏、机体再次接触变应原后产生的TH2型免疫反应和机体免疫系统对TH2型免疫反应的记忆和免疫维持。而对这个免疫反应网路系统进行横向的比较就会发现,TH2型免疫反应主要涉及机体的粘膜免疫和系统免疫这两大免疫系统。同时还应看到,TH2型免疫反应本身也存在多样性,表现在启动TH2型免疫反应的刺激形式多样性、先天免疫系统感受刺激的机制多样性、调控免疫反应的细胞和分子通路多样性,甚至在Th2细胞免疫反应中应答的细胞因子也存在多样性。总之,虽然目前对TH2型免疫反应已经进行了大量的临床和实验研究,但是人们对TH2型反应如何启动、反应、维持及调控仍知之甚少。
变应原特异性免疫治疗(allergen specific immunotherapy,SIT)的临床应用已经有一百多年的历史,它是目前唯一被认可的能够改变变应性疾病自然进程,针对病因的治疗方案。根据ARIA指南推荐,适用于持续性中重度变应性鼻炎患者。目前临床常用的免疫治疗包括：变应原皮下免疫治疗(subcutaneous immunotherapy,SCIT)和变应原舌下免疫治疗(sublingual immunotherapy,SLIT)。虽然已有大样本的随机双盲安慰剂对照的临床研究证实,SIT最终可明显改善变应性鼻炎患者相关的临床症状,减少对药物的依赖,但是SIT的作用机制仍存在许多未知。目前有关SIT作用机制的结论多是基于SCIT的研究结果。尘螨变应原SLIT应用于变应性鼻炎的临床治疗始于1986年,直到目前为止,人们对尘螨变应原SLIT涉及的许多问题仍然存在争议。这其中主要包括：临床疗效如何、作用机制是什么、尘螨变应原SLIT对机体免疫系统的影响是什么、与花粉变应原SLIT相比,尘螨SLIT的特点和规律是什么、免疫治疗的疗程需要多久、免疫维持所需的变应原的浓度是多少等诸多问题都仍然未知。
目前,SLIT主要在欧洲地区的国家得到认可和较广泛的临床应用,其适用对象主要是间歇性变异性鼻炎的患者,即花粉症的患者。在已经发表的超过60项随机双盲安慰剂对照的SLIT中,花粉症患者约占SLIT总数的30%以上。因此目前SLIT相关的结论多是基于欧洲团队针对花粉症的研究结果。但是近年来随着对SLIT基础研究及临床应用研究的进展,SLIT凭借其所特有优势正逐步得到越来越多关注。近几年尘螨变应原SLIT的相关研究也逐步增多,多项最新的研究初步证实了尘螨变应原SLIT的临床疗效及安全性,临床疗效方面显示尘螨变应原SLIT能有效的缓解变应性鼻炎及哮喘的临床症状,减轻对药物的依赖,降低患者产生新的过敏原的危险。安全性方面,SLIT相对于SCIT更具有明显的优势,但是其中的很多细节仍不明确,而且仍有相反结论的研究报道。而有关尘螨变应原SLIT作用机制的最新研究认为：SLIT与SCIT的作用机制可能存在部分的重叠或者有相似的作用机制,由于作用途径及使用剂量的不同,因而存在的差异部分可能主要在对粘膜免疫的影响,而SLIT是否也能引起系统免疫显著的变化也没有明确的答案。
有关变应性鼻炎免疫机制的最新研究发现了一种新的细胞类型,被称为“先天固有免疫细胞”(先天辅助细胞,innate helper cell)。研究发现它们能通过直接产生TH2型免疫反应的细胞因子的方式介导TH2型免疫反应。其中TSLP-OX40L信号通路在TH2型免疫反应的启动及免疫维持发挥的重要作用正逐步受到重视。
胸腺间质淋巴细胞生成素(Thymic stromal lymphopoietin,TSLP)是一种IL-7样的Ⅰ型细胞因子,属于IL-2细胞因子家族成员之一,主要由作为粘膜屏障的上皮细胞在外界环境刺激下产生。有研究资料显示TSLP是TH2型免疫反应重要的启动因素,当作为机体屏障的粘膜部位受到变应原的刺激,粘膜内的上皮细胞产生大量的TSLP,TSLP能够通过极化人树突状细胞诱导naive T细胞向TH2细胞分化,从而启动TH2型免疫反应。此外,TSLP能够诱导强烈的人类TH2记忆细胞的扩增,从而维持中央记忆细胞的表型和TH2细胞反应状态。OX40L为O型跨膜糖蛋白,属TNF家庭成员。OX40L由抗原递呈细胞(allergen present cell,APC)如B细胞、树突状细胞(dendritic cells,DCs)诱导表达,多种促进APC成熟的因素都能够促进OX40L的表达。在TSLP介导的TH2型免疫反应中,OX40L通过于OX40相互作用对于维持和重新激活TH2记忆细胞反应是非常重要的。TSLP通过极化人树突状细胞实现诱导naive T细胞向TH2细胞分化,而这种功能又是通过介导树突状细胞表面的OX40L的表达的途径实现的。研究发现阻断OX40/OX40L的相互作用,能实现对TSLP诱导的炎症性TH2细胞或抗原特异性TH2记忆细胞的靶向抑制,为变应性疾病的临床治疗提供新的思路和方法。有关TSLP-OX40L介导的信号通路对DCs的作用和对T细胞的功能的影响已经在变应性疾病的研究中有大量的报道。最新的研究发现,记忆性TH2细胞是维持慢性变应性炎症反应和变应原再次暴露后引起的过敏性炎症反应再发的主细胞群,在特异性皮炎患者病变部位角蛋白细胞TSLP表达升高。这些都提示在变应性性疾病的病理生理过程中,TSLP和记忆性TH2细胞之间关系的重要性。上皮细胞表达的TSLP对启动和维持TH2细胞免疫反应的作用与其所具有的独特的DCs激活能力有密切的关系。此外研究发现,由TSLP激活的DCs表达的OX40L是TH2型免疫反应的重要的信号反应通路。TSLP-OX40L信号通路有可能成为应对变应性疾病的潜在的分子靶点。
总之,对TSLP-OX40L信号通路进行阻断,从而实现对变应性疾病的治疗已经成为目前研究的热点。针对变应性疾病的传统药物治疗重要局限在TH12型免疫反应的效应阶段,包括激活的T细胞、肥大细胞和嗜酸细胞。在变应性免疫反应的启动阶段,以TSLP和或OX40L为治疗靶点的新概念的出现,为变应性疾病的治疗开辟了新的研究方向。由于SLIT具有借助粘膜途径对机体TH2型免疫反应进行调控的特点,而TSLP-OX40L信号通路介导的TH2型免疫反应也是以粘膜的应激反应为启动阶段,进而引起包括粘膜免疫和系统免疫在内的一系列的免疫级联反应,因此尘螨变应原SLIT与TSLP-OX40L介导的TH2型免疫反应之间是否存在相互影响及作用关系值得期待,这也是本研究拟探讨的主要问题,而目前尚无相关报道。
研究目的
探讨尘螨变应原SLIT治疗12个月对持续性中重度变应性鼻炎患者机体TSLP-OX40L信号通路介导的TH2免疫反应占优势的免疫状态的影响,包括鼻腔局部粘膜免疫反应的变化和机体系统免疫的变化。同时结合对患者临床症状的动态观察和评估,进一步分析尘螨变应原SLIT对机体TH2型免疫反应的影响和作用机制,为尘螨SLIT临床治疗和临床疗效评估及作用机制研究提供指导和帮助依据。
研究方法’
本研究以中国华南地区对尘螨过敏的持续性中重度变应性鼻炎患者为研究对象,以尘螨变应原舌下特异性免疫治疗为干预因素,以免疫治疗12个月为研究的时间截点,采取病例对照的研究方法,分别在免疫治疗开始前及免疫治疗12个月对机体TH2型免疫反应相关客观指标进行检测和分析,在此期间,同时对患者变应性鼻炎相关临床症状进行动态的观察和分析。首先,本研究项目总结了尘螨变应原SLIT治疗12个月内相关临床症状变化特点及规律；在此基础上,以TH2型免疫反应及变应原舌下特异性免疫治疗方面的最新的研究进展为依托,选取TSLP-OX40L信号通路为切入点,进一步分析患者鼻粘膜免疫和机体系统免疫相关免疫指标的变化情况。其中包括：酶联免疫吸附测定(Enzyme-linked immuno sorbent assay,ELISA)法检测鼻腔灌洗液中IL-4、IL-13、干扰素-γ(Interferon-gama, INF-γ)及TSLP的表达；实时荧光定量PCR(Real-time Quantitative PCR,RT-PCR)检测外周血中转录因子T-bet mRNA(transcription factor T-bet mRNA,T-bet mRNA)、转录因子GATA-3mRNA(transcription factor GATA-3mRNA,GATA-3mRNA).及OX40L mRNA的表达；流式细胞检测外周血单个核细胞(peripheral blood monouclear cells,PBMC)中OX40L的表达和PBMC来源的DCs(CD11c+CD86+)表面OX40L的表达；免疫组化检测部分患者鼻粘膜TSLP的表达。从上述各项客观指标的变化情况反应TSLP-OX40L介导的TH2型免疫反应是否发生改变,包括反应局部鼻粘膜免疫的指标和反应机体系统免疫的指标。最后将临床观测结果与实验室检查的客观结果进行分析汇总,解释临床症状变化的理论依据,探讨尘螨变应原SLIT治疗持续性中重度变应性鼻炎的作用机制。
研究结果
(一)临床部分(第一章)：
经过12个月的尘螨变应原SLIT,对46例持续性中重度变应性性鼻炎患者临床症状评分(包括鼻腔总体症状评分和鼻腔各个单项症状评分)和药物使用评分进行整体比较,结果都呈现明显下降,差异有统计学意义(P<0.001)。其中鼻腔总体症状评分由免疫治疗前9.065±1.162降至4.413±0.717(X2=82.667,P<0.001)；在鼻腔单项症状评分之中,鼻痒症状评分由免疫治疗前2.391±0.493降至1.022±0.257(X2=86.681,P<0.001)；鼻塞症状评分由免疫治疗前2.000±0.633降至1.196±0.401(X2=60.057,P<0.001)；打喷嚏症状评分由免疫治疗前2.457±0.504降至1.109±0.315(X2=74.146,P<0.001)；流鼻涕症状评分由免疫治疗前2.217±0.417降至1.087±0.354(X2=83.532,P<0.001)。药物使用评分由治疗前2.870±0.341降至1.109±0.315(X2=79.590,P<0.001)。结果显示尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者临床症状显著改善,患者对药物的依赖明显降低。治疗期间无严重的不良事件的发生,无症状加重的病例报告。
动态观察临床症状变化发现,鼻腔总体症状评分在尘螨变应原SLIT治疗12个月内总体呈现逐步的下降趋势。其中免疫治疗1个月,鼻腔总体症状的评分由免疫治疗前9.065±1.162降至7.132±1.554,差异有统计学意义(Z=-5.264,P<0.001)。在免疫治疗前6个月,患者鼻腔总体症状评分的降幅较大,下降趋势明显,呈陡降型变化曲线,由免疫治疗前9.065±1.162降至4.935±1.063,差异有统计学意义(Z=-5.962,P<0.001)。而从免疫治疗6个月至免疫治疗12个月期间,鼻腔总体症状评分降幅不明显,呈现平台型变化曲线,变化差异无统计学意义(Z=-2.449,P=0.014>0.0125)(此处需调整检验水准,以P<0.0125为差异有统计学意义)。至免疫治疗12个月,患者鼻腔总体评分大约为治疗前的50%左右。鼻腔单项症状评分的变化趋势与鼻腔总体症状评分的变化趋势基本一致。只有鼻塞症状评分和药物使用评分在免疫治疗6个月与免疫治疗12个月比较时显示有统计学差异(P<0.0125)(检验水准需调整,以P<0.0125为差异有统计学意义)。
此外在研究中还发现,部分患者(13.04%,6/46)在鼻腔总体症状评分逐步下降的过程中出现评分的一过性反弹,呈现波浪状的下降趋势,但其症状反弹时的鼻腔总体症状评分始终低于治疗前的评分,反弹发生的时间在免疫治疗后3-7个月内。
(二)实验部分(第二章、第三章)：
HE染色(第二章)：采用HE染色对鼻分泌物涂片中嗜酸性粒细胞计数在尘螨变应原SLIT开始前、免疫治疗6个月和免疫治疗12个月三个时间点进行整体比较,结果显示差异有统计学意义(Cochran's Q=23.330,P<0.001).其后分别将三个时间点鼻分泌物嗜酸性粒细胞计数分别进行两两比较发现(此处需调整检验水准,以P<0.0125为差异有统计学意义)：免疫治疗后鼻分泌物嗜酸性粒细胞计数较免疫治疗前显著下降,差异有统计学意义(免疫治疗6个月：Z=-3.162,P<0.0125；免疫治疗12个月：Z=-3.873,P<0.001)；免疫治疗6个月与免疫治疗12个月相比,差异无统计学意义(Z=-2.236,P>0.0125)。
ELISA检测(第二章、第三章)：鼻腔灌洗液中的细胞因子IL-4、IL-13、INF-y及TSLP在尘螨SLIT治疗12个月后都显著下降,差异有统计学意义(P<0.05)。其中IL-4由76.010±9.394(pg/ml)降至32.910±8.752(pg/ml)(t=27.010,P<0.05); IL-13由55.680±12.860(pg/ml)降至30.000±9.453(pg/ml)(t=14.050,P<0.05); INF-γ由30.600±6.755(pg/ml)降至21.710±5.457(pg/ml)(1=8.087,P<0.05); TSLP由36.390±6.610(pg/ml)降至21.780±4.231(pg/ml)(t=11.110,P<0.05)。此外,IL-4/INF-γ比值也显著下降,由治疗前2.584±0.581降至治疗后1.611±0.593,差异有统计学意义(t=8.987,P<0.05)。
血清TIgE和sIgE(第二章)：在尘螨SLIT治疗12个月前后的表达分别为：TIgE治疗前为423.900±29.990(kU/L),治疗后为420.200±39.110(kU/L);sIgE治疗前为54.920±5.217(kU/L),治疗后56.790±6.750(kU/L),差异无统计学意义(P>0.05)。
QRT-PCR检测(第三章)：外周血中,T-bet mRNA表达在SLIT治疗12个月后显著升高,由治疗前3.458±0.620,提高至治疗后5.389±1.228差异有统计学意义(t=12.230,P<0.05);GATA-3mRNA表达显著降低,治疗前为12.440±2.536,治疗后为7.246±1.953,差异有统计学意义(t=11.920,P<0.05);T-bet mRNA/GATA-3mRNA的比值显著升高,治疗前为0.288±0.071,治疗后为0.785±0.251差异有统计学意义(t=13.890, P<0.05);OX40LmRNA的表达在SLIT治疗12个月后显著下降,由治疗前1.401±0.361,下降至治疗后0.772±0.301,差异有统计学意义(t=12.300,P<0.05)；外周血OX40L mRNA表达水平与T-bet mRNA/GATA-3mRNA比率相关性分析显示,免疫治疗12个月前后,OX40L mRNA表达水平与T-bet mRNA/GATA-3mRNA的比率都呈负相关,差异有统计学意义(免疫治疗前：r=-0.440,P<0.05；免疫治疗12个月后：r=-0.565,P<0.05)；外周血OX40L mRNA表达水平与鼻腔灌洗液TSLP表达水平相关性分析显示,免疫治疗12个月前后,OX40L mRNA表达水平与TSLP表达水平呈正相关(免疫治疗前：r=0.491,P<0.05；免疫治疗12个月后：r=0.548,P<0.05),差异有统计学意义。
流式细胞检测(第三章)：外周血单个核细胞表面OX40L的表达在SLIT治疗12个月后显著下降,由免疫治疗前(2.712±0.511)%,下降至免疫治疗12个月后(1.630±0.471)%,差异有统计学意义(t=13.100,P<0.05)；外周血单个核细胞来源的DCs(CD11c+CD86+)表面OX40L的表达也显著下降,由免疫治疗前(5.839±2.841)%,下降至免疫治疗12个月后(2.098±0.870)%,差异有统计学意义(t=9.946,P<0.05)。
免疫组化检测(第三章)：本研究中有6例患者在免疫治疗前和免疫治疗12个月后接受了鼻粘膜HE染色和TSLP免疫组化染色。研究发现在SLIT治疗前鼻粘膜HE染色见：鼻粘膜上皮细胞之间的间隙宽,部分上皮细胞层变薄,上皮层下大量嗜酸性粒细胞浸润；TSLP染色见：上皮细胞TSLP表达阳性染色比例为83.33%,镜下见多数上皮细胞胞浆内大量粗大的棕黄色颗粒染色。SLIT治疗12个月后,鼻粘膜HE染色：上皮细胞间排列较前紧密,上皮层下嗜酸性粒细胞浸润减少,部分上皮层变薄未见明显改善；TSLP染色见：上皮细胞TSLP表达阳性33.33%,上皮细胞胞浆内棕黄色染色颗粒变小,颜色变浅。
研究结论
1.尘螨变应原SLIT治疗12个月,对于持续性中重度变应性鼻炎具有显著的临床疗效和安全性。其鼻腔总体症状约为免疫治疗前的50%左右。
2.尘螨变应原SLIT治疗12个月期间,患者临床症状的缓解呈现逐步改善的趋势。其中免疫治疗前6个月,临床症状改善幅度大,呈现陡降型临床症状变化曲线；免疫治疗后6个月,临床症状相对稳定,变化不大呈现平台型临床症状变化曲线。部分患者(13.04%)在免疫治疗的前6个月左右,临床症状会出现一定程度的反弹,总体呈现为波浪状下降的临床症状变化曲线。
3.尘螨变应原SLIT治疗早期(免疫治疗开始后1个月)即可产生显著的临床疗效,该结果早于本研究对临床疗效起效时间的预期。
4.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者鼻粘膜免疫的TH2型免疫反应占优势的免疫状态得到下调,表现为鼻粘膜局部TH2型免疫反应强度的下调,TH1/TH2免疫反应之间的平衡状态向TH1反向偏倚。
5.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者机体的系统免疫中TH2型免疫反应占优势的免疫状态实现下调,表现为调控TH2型免疫维持或免疫记忆功能的OX40L表达水平的下调,TH1/TH2免疫反应之间的平衡状态向TH1反向偏倚。
6.尘螨变应原SLIT治疗12个月,能实现对持续性中重度变应性鼻炎患者粘膜免疫和系统免疫中TH2型免疫反应占优势的免疫状态的下调。
7.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者临床症状显著改善,机体OX4OL和粘膜TSLP表达水平的下调,结合TH2型免疫反应在粘膜免疫和系统免疫的下调和TH1/TH2免疫平衡向TH1方向偏倚,提示着患者机体TSLP-OX40L信号通路介导的TH2型免疫反应的下调。
8.尘螨变应原SLIT的临床疗效能在免疫治疗早期的实现,来源于鼻腔粘膜免疫中TH2型免疫反应下调的结果；而免疫治疗后期,临床疗效的相对稳定源于系统免疫中维持TH2型免疫记忆的OX40L下调的结果。
9.对于持续性中重度变应性鼻炎患者,尘螨变应原SLIT治疗后鼻粘膜TSLP表达下调可以作为预测患者系统免疫中TH2型免疫反应下调的指标。
Background
WHO had pointed "allergic disease would be one of the biggest challenges for human health in21-century". Allergic rhinitis belongs to allergic disease. The pathophysiology of allergic rhinitis is occurred in nasal mucosa and originated from systemic immune abnormalities. After in contact with allergens, TH2type immune responses were started by IgE mediated eosinophil reaction and dominated by a variety of inflammatory cells and cytokines involved in. The common features of allergic disease were so-called TH2type immune response.
In our knowledge of TH2type immune responds, many kinds of cells take part in this sophisticated network of immune responds. There are included T cell, B cell, Eosinophils, Basophil, APC, innate helper cell and so on. Type2responses are characterized by the induction of CD4+T helper (TH)2cells, which secrete cytokines such as interleukin-4(IL-4), IL-5, IL-9, and IL-13. TH2cells promote B cell responses and immunoglobulin E (IgE) secretion through their production of IL-4. IgE immune complexes bind to high-affinity IgE receptors (FceRl) on basophils and mast cells, leading to their activation and secretion of several cytokines and inflammatory mediators such as histamine, heparin, and serotonin. These factors mediate a range of effector functions characteristic of type2inflammation, including recruitment of alternatively activated macrophages and granulocytes, smooth muscle contractility, and mucus hypersecretion. There are four stages in the courses of the formation of TH2type immune responses in allergic rhinitis. There are so-called immune recognition, sensitization, immune responses and immune maintenance. At the same time, mucosa immune and system immune are all involved in TH2type immune responses. A marked feature of type2responses is their diversity. There is diversity in terms of the array of stimuli that trigger type2responses, the mechanisms by which the innate immune system senses such stimuli, and the cellular and molecular pathways that orchestrate the response. Indeed, there even appears to be diversity in the cytokine profiles of responding cells in a TH2cell response. In summary, although many attentions have been pay in the field of TH2type immune responses, but the mechanisms that initiate and control type2responses remain enigmatic.
Allergen-specific immunotherapy has been used more than100years as a desensitizing therapy for allergic diseases and represents the potentially curative and specific method of treatment. Specific immunotherapy is the only treatment modality with capacity of change the natural course of allergic diseases. According to the guideline of ARIA, allergen specific immunotherapy is referred to consistent moderate-severe allergic rhinitis. Now days, specific immunotherapy are applied in clinic treatment for allergic rhinitis with two ways, subcutaneous immunotherapy and sublingual immunotherapy. Although many big samples randomized double-blind placebo-controlled trails have confirmed the clinic efficiency of SIT in patients with allergic rhinitis. But the mechanism of SIT is unclear. Many conclusions about SIT were originated from SCIT. SLIT was first applied in clinic treatment in1986. Compared with the consensus about SCIT, many disputations are still existed about SLIT. The mainly parts are clinic efficiency and mechanism. Allergen specific sublingual immunotherapy has recently received much attention around the world as a treatment for allergic rhinitis and is now widely used to replace the subcutaneous route. To date, many randomized double-blind placebo-controlled trials have confirmed the clinic efficiency of SLIT for patients with allergic rhinitis.
The knowledge of SLIT is mainly originated from Europe study team and the patients received SLIT are mainly with seasonal allergic rhinitis sensitive to grass or pollen allergen. Although clinic efficiency of SLIT have already been confirmed in seasonal allergic rhinitis. But for consistent moderate-severe allergic rhinitis, whether house dust mite sublingual immunotherapy would possess the same result or not were uncertain. On account of new progressions of SLIT and the finds of innate immune system, increasing interest in SLIT has immerged in recent years.
The recent identification of previously unrecognized innate cell populations, termed innate helper cell. Studies show that innate helper cell has ability to induce TH2type immune responses by directly secreting TH2type cytokine. Thymic stromal lymphopoietin (TSLP), a distant paralog of IL-7, is a type I cytokine that is part of the IL-2cytokine family. TSLP are produced primarily by epithelial cell in airway during airway's allergic inflammation. Recently study shows that TSLP can induce a robust expansion of human TH2memory cells while maintaining their central memory phenotype and TH2commitments. OX40L and its receptor OX40are members of the TNF and TNF receptor super families, respectively. OX40L is expressed by APCs, endothelial cells and T cells, whereas OX40is found mainly on activated T cells. The expression of OX40L on dendritic cells is mainly induced by thymic stromal lymphopoietin. Many factors of promoting mature of APC could improve the expression of OX40L. TSLP has ability to induce naive T cell difference into TH2cell by polarizing dendritic cells and this role is accomplice by inducing the expression of OX40L on the surface of dendritic cells. New results demonstrated the critical roles of OX40/OX40L interaction on the maintenance and re-activation of TH2memory responses during TSLP-mediated allergic responses. Targeting the TSLP-induced inflammatory TH2cells or allergen-specific TH2memory cells by blocking OX40/OX40L interaction may be one of the new therapeutic approaches for the prevention and treatment of human-allergic diseases. The important roles of TSLP-OX40L signal in TH2type immune responses have been reported in many allergic disease trials. The previous studies had demonstrated that TSLP-OX40L signal play a crucial role in pathogenesis of allergic disease. Until now, there is no research to concern the influence of house dust mite sublingual immunotherapy in TSLP-OX40L mediated TH2type Immanuel responds in patient with allergic rhinitis. Memory TH2cell is main cell mass that could maintain chronic inflammation stage in allergic responses and insure allergic responses reappearance when allergen was exposure again. The expression of TSLP can conduce to maintain the function of memory TH2cell. So TSLP-OX40L signal would become a potential molecular target in treatment of allergic diseases.
In conclusion, TSLP-OX40L signal has become a hot spot in treatment of allergic diseases. The traditional medicine treatment strategy is mainly focused on the effector phase of allergic responses. Its role is mainly on been activated cells, such as T cell, Eosinophils and Basophil. Since the ability of SLIT to regulate TH2-type immune response is characterized by mucosal route, and TH2-type immune response mediated by TSLP-OX40L signaling pathway is also in the mucosal start-up phase, thereby causing the body's immune cascade series. Whether there exist interaction between house dust mite allergen SLIT and TH2type immune response mediated by TSLP-OX40L signal or not, is worth the wait. This is the main purpose of this study to investigate. But until now, no related reports were published.
Objective
To investigate the impact of house dust mite allergen sublingual immunotherapy on TSLP-OX40L mediated TH2type immune responds in patient with moderate to severe allergic rhinitis, including changes in local mucosal immune of nasal mucosa and the system immune. Combined with changes in the patients clinic symptoms of observation and assessment, further analysis of the impact of dust mite allergen SLIT on TH2-type immune response in patients with moderated to severe allergic rhinitis and mechanism were carried out. We look forward to provide guidance and assistance for the clinical treatment of dust mite SLIT and the research of mechanism of SLIT.
Methods
In this study, according to the guideline of ARIA,46cases of allergic rhinitis patients in southern China were enrolled in our study. All of the patients had moderate to severe persistent allergic rhinitis with or without asthma. All patients received house dust mite allergen specific sublingual immunotherapy. The cut-off point of trials is12months immunotherapy. The case-control study is employed in trials. Each patient was treated for a12-month period. Mixture extracts of D.f. and D.p.(Zhejiang Wolwo Bio-Pharmaceutical Co., Ltd., China) were used in our study. Changes of TH2-type immune response and clinical symptoms in patients with allergic rhinitis in our trials were dynamic observed and analyses respectively at the times before the start of immune therapy and immunotherapy for12months. First, the characteristics and rules of changes related to clinical symptoms were summarized within12months'dust mite allergen SLIT; On the basis of the progress in TH2-type immune response and sublingual allergen-specific immunotherapy, TSLP-OX40L signaling pathway was selected as a starting point for further analysis of the changes both occurred in the nasal mucosa immune and system immune by impact of SLIT. There are included the expressions of IL-4, IL-13, INF-γ and TSLP in nasal lavage detected by ELISA; The expressions of T-bet mRNA, GATA-3mRNA and OX40L mRNA in blood detected by QRT-PCR; The expressions of OX40L in PBMC detected by flow cytometric analysis; The expression of TSLP detected by immunohistochemistry and the counts of EOS in nasal secretion smear detected by HE staining. From changes in the above objective indicators, we can deduce whether TH2-type immune response mediated by TSLP-OX40L signaling pathway would be changed or not, including the mucosa immune response and system immune response. Finally, the results of clinical observations and laboratory tests are analyzed.
Results
Outcomes of syndrome score
After12-month treatment, TNSS, medication score and all INSS (nasal rhinorrhea, itching, sneezing and nasal congestion) significantly decreased compared with baseline value by overall evaluation (P<0.001). TNSS significantly decreased after12-month SLIT compared with the baseline value from9.065±1.162to4.413±0.717(X2=82.667, P<0.001); Medication score decreased from2.870±0.341to1.109±0.315(X2=79.590, P<0.001). Among all INSS, itching score decreased from2.391±0.493to1.022±0.257(X2=86.681, P<0.001); nasal congestion score decreased from2.000±0.633to1.196±0.401(X2=60.057,P<0.001); sneezing score decreased from2.457±0.504to1.109±0.315(X2=74.146,P<0.001);nasal rhinorrhea score decreased from2.217±0.417to1.087±0.354(X2=83.532, P<0.001). All show significant difference. The results showed that a significant improvement of clinical symptoms for patients with moderate to severe persistent allergic rhinitis had achieved after12-month dust mite allergen SLIT treatment. The dependence on drugs for all patients was reduced significantly. No serious adverse events and no symptoms aggravated case were reported during12-month SLIT treatment, symptoms aggravated case report.
Additionally, by dynamic observation of clinic syndromes, the changes of TNSS showed a gradual decline trend during12-month dust mite allergen SLIT. Among this trend, TNSS decreased significantly after1-month dust mite allergen SLIT from9.065±1.162to7.132±1.554(Z=-5.264, P<0.001). During the first6-month dust mite allergen SLIT, TNSS declined obviously and showed a steep drop trend from9.065±1.162to4.935±1.063. The difference was statistically significant(Z=-5.962, P <0.001). But during the second6-month SLIT, the changes of TNSS were relatively stable and the trend of TNSS presented a platform-type curve. The difference was not statistically significant(Z=-2.449, P=0.014>0.0125)(The significance level in here needed to be adjusted to P<0.0125for the statistical significant difference). Until to the endpoint of12-month SLIT, TNSS had been declined50%compared with baseline. The changes of INSS during12-month dust mite allergen SLIT were basically as same as TNSS. Only the differences of nasal congestion score and medicine score compared at the time of6-month SLIT and12-month SLIT were statistical significant(P<0.0125)(significance level was adjusted). The study proved the safety and efficiency of dust mite allergen SLIT.
The results also showed that the trend of syndromes improvement was gradual and wavy during12-month dust mite allergen SLIT period. The wavy or the transient rebound of syndromes presented mainly mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline.
Outcomes of laboratory parameter
HE staining:The counts of EOS in nasal secretion smear were decreased significantly by overall evaluation(Cochran's Q=23.330, P<0.001) after12-month dust mite allergen SLIT. After that, pair-wise comparisons of the counts of EOS in nasal secretion smear were carried respectively at the three time points (baseline,6-month and12-month) to evaluate the differences. The results showed that the counts of EOS in nasal secretion smear at the times of6-month SLIT and12-month SLIT decreased significantly compared with baseline(6-month SLIT: Z=-3.162, P<0.0125;12-month SLIT:Z=-3.873, P<0.001). But there were no statistical difference of the counts of EOS in nasal secretion smear between6-month SLIT with12-month SLIT(Z=-2.236,P>0.0125).
ELISA:The levels of IL-4, IL-13, INF-y and TSLP in nasal lavage were decreased significantly after12-month dust mite allergen SLIT (P<0.05). The level of IL-4decreased from76.010±9.394(pg/ml) to32.910±8.752(pg/ml)(t=27.010, P <0.05); The level of IL-13decreased from55.68±12.86(pg/ml) to30.000±9.453(pg/ml)(t=14.050, P<0.05); The level of INF-y decreased from30.600±6.755(pg/ml) to21.710±5.457(pg/ml)(t=8.087, P<0.05); The level of TSLP decreased from36.390±6.610(pg/ml) to21.780±4.231(pg/ml)(t=11.110, P<0.05). The ratio of IL-4/INF-γ decreased from2.584±0.581tol.611±0.593. The difference was statistically significant (t=8.987, P<0.05).
The expressions of TIgE and sIgE in serum didn't showed significant changes compared with baseline (TIgE:423.900±29.990(kU/L) vs420.200±39.110(kU/L); slgE:54.920±5.217(kU/L)vs56.790±6.750(kU/L)(P>0.05).
QRT-PCR:The expressions of T-bet mRNA in blood significantly increased from3.458±0.6202to5.389±1.228fold after12-month SLIT compared with baseline (t=12.230, P<0.05). The expressions of GATA-3mRNA in blood significantly decreased from12.440±2.536fold to7.246±1.953fold after12-month SLIT compared with baseline (t=11.920, P<0.05). The ratio of T-bet mRNA/GATA-3mRNA increased significantly from0.288±0.071to0.785±0.251(t=13.890, P<0.05). The level of OX40L mRNA in blood decreased significantly after12-month SLIT from1.401±0.361fold to0.772±0.301fold compared with baseline (t=12.300, P<0.05). The negative associations of the levels of OX40L mRNA and the ratios of T-bet mRNA/GATA-3mRNA in blood were observed before and after12-month SLIT(before SLIT:r=-0.440, P<0.05;after12-month SLIT:r=-0.565, P<0.05). The difference was statistically significant. The positive associations of the levels of OX40L mRNA in blood and the levels of TSLP in nasal lavage fluid were observed before and after12-month SLIT(before SLIT:r=0.491, P<0.05; after12-month SLIT:r=0.548, P<0.05). The difference was statistically significant.
Flow cytometry:The expressions of OX40L on the surface of peripheral blood mononuclear cells after12-month SLIT were decreased significantly (t=13.100, P<0.05), from before treatment (2.712±0.511)%, down to12-month treatment (1.630±0.471)%. The expressions of OX40L on the surface of CDllc+CD86+cells in peripheral blood mononuclear cells also significantly decreased after12-month SLIT from the former (5.839±2.841)%, reduced to (2.098±0.870)%. The difference was statistically significant (t=9.946, P<0.05).
Immunohistochemical staining:There were6cases of nasal mucosa biopsies be obtained to study the HE staining and the expression of TSLP before and after12-month SLIT. The results showed that positive staining of TSLP in nasal mucosa was weakened after12-month SLIT compared with baseline from83.33%to33.33%. From the HE staining, the results showed that epithelial cells arranged closely and eosinophil infiltration under the epithelium decreased after12-month SLIT.
Conclusion
1. Dust mite allergen SLIT treatment for patients with moderate to severe persistent allergic rhinitis has significant clinical efficacy and safety. After12-month dust mite allergen SLIT, the TNSS was about to decrease50%compared with before immunotherapy.
2. During12-month dust mite allergen SLIT, the remission of clinical symptoms showed a gradual improvement trend. An obviously clinic improvement was found in the first6-month dust mite allergen SLIT. The changes of clinic symptoms showed a steep drop trend in the first six months. During the second six months, the changes of clinic symptoms were relatively stable and showed a platform-type curve. The transient rebounds of syndromes were presented in some patients mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline. Overall clinic symptoms showed a waving decline trend.
3. The significant clinical efficacy could be achieved in early stage of dust mite allergen SLIT (one month), earlier than the onset time being expected.
4. TH2type immune responses in nasal mucosa immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the intensity of TH2type immune responses was weakened and the balance of TH1/TH2immune responses was shifted to TH1.
5. TH2type immune responses in system immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the level of OX40L which was in charge of immune memory of TH2type immune responses was down-regulated and the balance of TH1/TH2immune responses was shifted to TH1.
6. The down-regulation of OX40L in system immune, TSLP in mucosa immune and TH2type immune responses in both of system and mucosa system implied the signaling pathway conducted by TSLP-OX40L in TH2type immune responses was down-regulated after12-month dust mite allergen SLIT.
7. TH2type immune responses both in mucosa immune and in system immune were down-regulated by12-month dust mite allergen SLIL in patients with moderate to severe persistent allergic rhinitis.
8. The achievement of clinic efficiency in early stages of dust mite allergen SLIT was derived from the down-regulation of TH2immune responses in nasal mucosa immune. While the maintaining the clinical efficacy was derivered from the down-regulation of OX40L in system immune.
9. The expressions of TSLP could be a predictor of the down-regulation of TH2type immune responses in system immune responses for patients with moderate to severe persistent allergic rhinitis after being received house dust mite SLIT.
WHO指出“变应性疾病是二十一世纪人类面临的三大健康问题之一”。变应性鼻炎又称过敏性鼻炎,是以鼻腔粘膜为主要的效应部位,以机体免疫系统异常为主要的病理基础,在特异性体质的患者接触变应原后引起的,以IgE介导的,嗜酸性粒细胞反应为主的,由多种炎症细胞及细胞因子共同参与的Ⅰ型变态反应性疾病。其共同特征即所谓的“TH2型免疫反应”。
目前认为,有多种细胞共同参与到TH2(Ⅱ型辅助性T细胞,T help cell2)型免疫反应过程中,形成了一个复杂的免疫反应的网路系统。其中涉及了T细胞、B细胞、嗜酸细胞、嗜碱细胞、抗原递呈细胞以及最新发现的先天辅助细胞群等。TH2型免疫应答的特点是诱导CD4+T细胞(分化群,cluste of differentiation)分化,其分泌的细胞因子包括：IL-4、EL-5、IL-9、IL-13(白细胞介素,interleukin)。TH2细胞通过分泌IL-4促进B细胞反应和IgE的分泌。当IgE免疫复合物结合到位于嗜碱细胞和肥大细胞表面的高亲和力的IgE受体(FceRl),导致上述细胞激活并释放多种细胞因子和炎症介质,包括：组织胺、肝素和血清素。这些细胞因子介导了一系列的以TH2型免疫反应为特点的效应功能。包括有选择的募集和活化巨噬细胞和粒细胞,引起平滑肌的收缩和粘液过量分泌。对这个复杂的免疫反应网路系统进行纵向的分析可见,整个TH2型免疫反应的过程大致包括4部分：机体免疫系统对变应原的识别、机体致敏、机体再次接触变应原后产生的TH2型免疫反应和机体免疫系统对TH2型免疫反应的记忆和免疫维持。而对这个免疫反应网路系统进行横向的比较就会发现,TH2型免疫反应主要涉及机体的粘膜免疫和系统免疫这两大免疫系统。同时还应看到,TH2型免疫反应本身也存在多样性,表现在启动TH2型免疫反应的刺激形式多样性、先天免疫系统感受刺激的机制多样性、调控免疫反应的细胞和分子通路多样性,甚至在Th2细胞免疫反应中应答的细胞因子也存在多样性。总之,虽然目前对TH2型免疫反应已经进行了大量的临床和实验研究,但是人们对TH2型反应如何启动、反应、维持及调控仍知之甚少。
变应原特异性免疫治疗(allergen specific immunotherapy,SIT)的临床应用已经有一百多年的历史,它是目前唯一被认可的能够改变变应性疾病自然进程,针对病因的治疗方案。根据ARIA指南推荐,适用于持续性中重度变应性鼻炎患者。目前临床常用的免疫治疗包括：变应原皮下免疫治疗(subcutaneous immunotherapy,SCIT)和变应原舌下免疫治疗(sublingual immunotherapy,SLIT)。虽然已有大样本的随机双盲安慰剂对照的临床研究证实,SIT最终可明显改善变应性鼻炎患者相关的临床症状,减少对药物的依赖,但是SIT的作用机制仍存在许多未知。目前有关SIT作用机制的结论多是基于SCIT的研究结果。尘螨变应原SLIT应用于变应性鼻炎的临床治疗始于1986年,直到目前为止,人们对尘螨变应原SLIT涉及的许多问题仍然存在争议。这其中主要包括：临床疗效如何、作用机制是什么、尘螨变应原SLIT对机体免疫系统的影响是什么、与花粉变应原SLIT相比,尘螨SLIT的特点和规律是什么、免疫治疗的疗程需要多久、免疫维持所需的变应原的浓度是多少等诸多问题都仍然未知。
目前,SLIT主要在欧洲地区的国家得到认可和较广泛的临床应用,其适用对象主要是间歇性变异性鼻炎的患者,即花粉症的患者。在已经发表的超过60项随机双盲安慰剂对照的SLIT中,花粉症患者约占SLIT总数的30%以上。因此目前SLIT相关的结论多是基于欧洲团队针对花粉症的研究结果。但是近年来随着对SLIT基础研究及临床应用研究的进展,SLIT凭借其所特有优势正逐步得到越来越多关注。近几年尘螨变应原SLIT的相关研究也逐步增多,多项最新的研究初步证实了尘螨变应原SLIT的临床疗效及安全性,临床疗效方面显示尘螨变应原SLIT能有效的缓解变应性鼻炎及哮喘的临床症状,减轻对药物的依赖,降低患者产生新的过敏原的危险。安全性方面,SLIT相对于SCIT更具有明显的优势,但是其中的很多细节仍不明确,而且仍有相反结论的研究报道。而有关尘螨变应原SLIT作用机制的最新研究认为：SLIT与SCIT的作用机制可能存在部分的重叠或者有相似的作用机制,由于作用途径及使用剂量的不同,因而存在的差异部分可能主要在对粘膜免疫的影响,而SLIT是否也能引起系统免疫显著的变化也没有明确的答案。
有关变应性鼻炎免疫机制的最新研究发现了一种新的细胞类型,被称为“先天固有免疫细胞”(先天辅助细胞,innate helper cell)。研究发现它们能通过直接产生TH2型免疫反应的细胞因子的方式介导TH2型免疫反应。其中TSLP-OX40L信号通路在TH2型免疫反应的启动及免疫维持发挥的重要作用正逐步受到重视。
胸腺间质淋巴细胞生成素(Thymic stromal lymphopoietin,TSLP)是一种IL-7样的Ⅰ型细胞因子,属于IL-2细胞因子家族成员之一,主要由作为粘膜屏障的上皮细胞在外界环境刺激下产生。有研究资料显示TSLP是TH2型免疫反应重要的启动因素,当作为机体屏障的粘膜部位受到变应原的刺激,粘膜内的上皮细胞产生大量的TSLP,TSLP能够通过极化人树突状细胞诱导naive T细胞向TH2细胞分化,从而启动TH2型免疫反应。此外,TSLP能够诱导强烈的人类TH2记忆细胞的扩增,从而维持中央记忆细胞的表型和TH2细胞反应状态。OX40L为O型跨膜糖蛋白,属TNF家庭成员。OX40L由抗原递呈细胞(allergen present cell,APC)如B细胞、树突状细胞(dendritic cells,DCs)诱导表达,多种促进APC成熟的因素都能够促进OX40L的表达。在TSLP介导的TH2型免疫反应中,OX40L通过于OX40相互作用对于维持和重新激活TH2记忆细胞反应是非常重要的。TSLP通过极化人树突状细胞实现诱导naive T细胞向TH2细胞分化,而这种功能又是通过介导树突状细胞表面的OX40L的表达的途径实现的。研究发现阻断OX40/OX40L的相互作用,能实现对TSLP诱导的炎症性TH2细胞或抗原特异性TH2记忆细胞的靶向抑制,为变应性疾病的临床治疗提供新的思路和方法。有关TSLP-OX40L介导的信号通路对DCs的作用和对T细胞的功能的影响已经在变应性疾病的研究中有大量的报道。最新的研究发现,记忆性TH2细胞是维持慢性变应性炎症反应和变应原再次暴露后引起的过敏性炎症反应再发的主细胞群,在特异性皮炎患者病变部位角蛋白细胞TSLP表达升高。这些都提示在变应性性疾病的病理生理过程中,TSLP和记忆性TH2细胞之间关系的重要性。上皮细胞表达的TSLP对启动和维持TH2细胞免疫反应的作用与其所具有的独特的DCs激活能力有密切的关系。此外研究发现,由TSLP激活的DCs表达的OX40L是TH2型免疫反应的重要的信号反应通路。TSLP-OX40L信号通路有可能成为应对变应性疾病的潜在的分子靶点。
总之,对TSLP-OX40L信号通路进行阻断,从而实现对变应性疾病的治疗已经成为目前研究的热点。针对变应性疾病的传统药物治疗重要局限在TH12型免疫反应的效应阶段,包括激活的T细胞、肥大细胞和嗜酸细胞。在变应性免疫反应的启动阶段,以TSLP和或OX40L为治疗靶点的新概念的出现,为变应性疾病的治疗开辟了新的研究方向。由于SLIT具有借助粘膜途径对机体TH2型免疫反应进行调控的特点,而TSLP-OX40L信号通路介导的TH2型免疫反应也是以粘膜的应激反应为启动阶段,进而引起包括粘膜免疫和系统免疫在内的一系列的免疫级联反应,因此尘螨变应原SLIT与TSLP-OX40L介导的TH2型免疫反应之间是否存在相互影响及作用关系值得期待,这也是本研究拟探讨的主要问题,而目前尚无相关报道。
研究目的
探讨尘螨变应原SLIT治疗12个月对持续性中重度变应性鼻炎患者机体TSLP-OX40L信号通路介导的TH2免疫反应占优势的免疫状态的影响,包括鼻腔局部粘膜免疫反应的变化和机体系统免疫的变化。同时结合对患者临床症状的动态观察和评估,进一步分析尘螨变应原SLIT对机体TH2型免疫反应的影响和作用机制,为尘螨SLIT临床治疗和临床疗效评估及作用机制研究提供指导和帮助依据。
研究方法’
本研究以中国华南地区对尘螨过敏的持续性中重度变应性鼻炎患者为研究对象,以尘螨变应原舌下特异性免疫治疗为干预因素,以免疫治疗12个月为研究的时间截点,采取病例对照的研究方法,分别在免疫治疗开始前及免疫治疗12个月对机体TH2型免疫反应相关客观指标进行检测和分析,在此期间,同时对患者变应性鼻炎相关临床症状进行动态的观察和分析。首先,本研究项目总结了尘螨变应原SLIT治疗12个月内相关临床症状变化特点及规律；在此基础上,以TH2型免疫反应及变应原舌下特异性免疫治疗方面的最新的研究进展为依托,选取TSLP-OX40L信号通路为切入点,进一步分析患者鼻粘膜免疫和机体系统免疫相关免疫指标的变化情况。其中包括：酶联免疫吸附测定(Enzyme-linked immuno sorbent assay,ELISA)法检测鼻腔灌洗液中IL-4、IL-13、干扰素-γ(Interferon-gama, INF-γ)及TSLP的表达；实时荧光定量PCR(Real-time Quantitative PCR,RT-PCR)检测外周血中转录因子T-bet mRNA(transcription factor T-bet mRNA,T-bet mRNA)、转录因子GATA-3mRNA(transcription factor GATA-3mRNA,GATA-3mRNA).及OX40L mRNA的表达；流式细胞检测外周血单个核细胞(peripheral blood monouclear cells,PBMC)中OX40L的表达和PBMC来源的DCs(CD11c+CD86+)表面OX40L的表达；免疫组化检测部分患者鼻粘膜TSLP的表达。从上述各项客观指标的变化情况反应TSLP-OX40L介导的TH2型免疫反应是否发生改变,包括反应局部鼻粘膜免疫的指标和反应机体系统免疫的指标。最后将临床观测结果与实验室检查的客观结果进行分析汇总,解释临床症状变化的理论依据,探讨尘螨变应原SLIT治疗持续性中重度变应性鼻炎的作用机制。
研究结果
(一)临床部分(第一章)：
经过12个月的尘螨变应原SLIT,对46例持续性中重度变应性性鼻炎患者临床症状评分(包括鼻腔总体症状评分和鼻腔各个单项症状评分)和药物使用评分进行整体比较,结果都呈现明显下降,差异有统计学意义(P<0.001)。其中鼻腔总体症状评分由免疫治疗前9.065±1.162降至4.413±0.717(X2=82.667,P<0.001)；在鼻腔单项症状评分之中,鼻痒症状评分由免疫治疗前2.391±0.493降至1.022±0.257(X2=86.681,P<0.001)；鼻塞症状评分由免疫治疗前2.000±0.633降至1.196±0.401(X2=60.057,P<0.001)；打喷嚏症状评分由免疫治疗前2.457±0.504降至1.109±0.315(X2=74.146,P<0.001)；流鼻涕症状评分由免疫治疗前2.217±0.417降至1.087±0.354(X2=83.532,P<0.001)。药物使用评分由治疗前2.870±0.341降至1.109±0.315(X2=79.590,P<0.001)。结果显示尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者临床症状显著改善,患者对药物的依赖明显降低。治疗期间无严重的不良事件的发生,无症状加重的病例报告。
动态观察临床症状变化发现,鼻腔总体症状评分在尘螨变应原SLIT治疗12个月内总体呈现逐步的下降趋势。其中免疫治疗1个月,鼻腔总体症状的评分由免疫治疗前9.065±1.162降至7.132±1.554,差异有统计学意义(Z=-5.264,P<0.001)。在免疫治疗前6个月,患者鼻腔总体症状评分的降幅较大,下降趋势明显,呈陡降型变化曲线,由免疫治疗前9.065±1.162降至4.935±1.063,差异有统计学意义(Z=-5.962,P<0.001)。而从免疫治疗6个月至免疫治疗12个月期间,鼻腔总体症状评分降幅不明显,呈现平台型变化曲线,变化差异无统计学意义(Z=-2.449,P=0.014>0.0125)(此处需调整检验水准,以P<0.0125为差异有统计学意义)。至免疫治疗12个月,患者鼻腔总体评分大约为治疗前的50%左右。鼻腔单项症状评分的变化趋势与鼻腔总体症状评分的变化趋势基本一致。只有鼻塞症状评分和药物使用评分在免疫治疗6个月与免疫治疗12个月比较时显示有统计学差异(P<0.0125)(检验水准需调整,以P<0.0125为差异有统计学意义)。
此外在研究中还发现,部分患者(13.04%,6/46)在鼻腔总体症状评分逐步下降的过程中出现评分的一过性反弹,呈现波浪状的下降趋势,但其症状反弹时的鼻腔总体症状评分始终低于治疗前的评分,反弹发生的时间在免疫治疗后3-7个月内。
(二)实验部分(第二章、第三章)：
HE染色(第二章)：采用HE染色对鼻分泌物涂片中嗜酸性粒细胞计数在尘螨变应原SLIT开始前、免疫治疗6个月和免疫治疗12个月三个时间点进行整体比较,结果显示差异有统计学意义(Cochran's Q=23.330,P<0.001).其后分别将三个时间点鼻分泌物嗜酸性粒细胞计数分别进行两两比较发现(此处需调整检验水准,以P<0.0125为差异有统计学意义)：免疫治疗后鼻分泌物嗜酸性粒细胞计数较免疫治疗前显著下降,差异有统计学意义(免疫治疗6个月：Z=-3.162,P<0.0125；免疫治疗12个月：Z=-3.873,P<0.001)；免疫治疗6个月与免疫治疗12个月相比,差异无统计学意义(Z=-2.236,P>0.0125)。
ELISA检测(第二章、第三章)：鼻腔灌洗液中的细胞因子IL-4、IL-13、INF-y及TSLP在尘螨SLIT治疗12个月后都显著下降,差异有统计学意义(P<0.05)。其中IL-4由76.010±9.394(pg/ml)降至32.910±8.752(pg/ml)(t=27.010,P<0.05); IL-13由55.680±12.860(pg/ml)降至30.000±9.453(pg/ml)(t=14.050,P<0.05); INF-γ由30.600±6.755(pg/ml)降至21.710±5.457(pg/ml)(1=8.087,P<0.05); TSLP由36.390±6.610(pg/ml)降至21.780±4.231(pg/ml)(t=11.110,P<0.05)。此外,IL-4/INF-γ比值也显著下降,由治疗前2.584±0.581降至治疗后1.611±0.593,差异有统计学意义(t=8.987,P<0.05)。
血清TIgE和sIgE(第二章)：在尘螨SLIT治疗12个月前后的表达分别为：TIgE治疗前为423.900±29.990(kU/L),治疗后为420.200±39.110(kU/L);sIgE治疗前为54.920±5.217(kU/L),治疗后56.790±6.750(kU/L),差异无统计学意义(P>0.05)。
QRT-PCR检测(第三章)：外周血中,T-bet mRNA表达在SLIT治疗12个月后显著升高,由治疗前3.458±0.620,提高至治疗后5.389±1.228差异有统计学意义(t=12.230,P<0.05);GATA-3mRNA表达显著降低,治疗前为12.440±2.536,治疗后为7.246±1.953,差异有统计学意义(t=11.920,P<0.05);T-bet mRNA/GATA-3mRNA的比值显著升高,治疗前为0.288±0.071,治疗后为0.785±0.251差异有统计学意义(t=13.890, P<0.05);OX40LmRNA的表达在SLIT治疗12个月后显著下降,由治疗前1.401±0.361,下降至治疗后0.772±0.301,差异有统计学意义(t=12.300,P<0.05)；外周血OX40L mRNA表达水平与T-bet mRNA/GATA-3mRNA比率相关性分析显示,免疫治疗12个月前后,OX40L mRNA表达水平与T-bet mRNA/GATA-3mRNA的比率都呈负相关,差异有统计学意义(免疫治疗前：r=-0.440,P<0.05；免疫治疗12个月后：r=-0.565,P<0.05)；外周血OX40L mRNA表达水平与鼻腔灌洗液TSLP表达水平相关性分析显示,免疫治疗12个月前后,OX40L mRNA表达水平与TSLP表达水平呈正相关(免疫治疗前：r=0.491,P<0.05；免疫治疗12个月后：r=0.548,P<0.05),差异有统计学意义。
流式细胞检测(第三章)：外周血单个核细胞表面OX40L的表达在SLIT治疗12个月后显著下降,由免疫治疗前(2.712±0.511)%,下降至免疫治疗12个月后(1.630±0.471)%,差异有统计学意义(t=13.100,P<0.05)；外周血单个核细胞来源的DCs(CD11c+CD86+)表面OX40L的表达也显著下降,由免疫治疗前(5.839±2.841)%,下降至免疫治疗12个月后(2.098±0.870)%,差异有统计学意义(t=9.946,P<0.05)。
免疫组化检测(第三章)：本研究中有6例患者在免疫治疗前和免疫治疗12个月后接受了鼻粘膜HE染色和TSLP免疫组化染色。研究发现在SLIT治疗前鼻粘膜HE染色见：鼻粘膜上皮细胞之间的间隙宽,部分上皮细胞层变薄,上皮层下大量嗜酸性粒细胞浸润；TSLP染色见：上皮细胞TSLP表达阳性染色比例为83.33%,镜下见多数上皮细胞胞浆内大量粗大的棕黄色颗粒染色。SLIT治疗12个月后,鼻粘膜HE染色：上皮细胞间排列较前紧密,上皮层下嗜酸性粒细胞浸润减少,部分上皮层变薄未见明显改善；TSLP染色见：上皮细胞TSLP表达阳性33.33%,上皮细胞胞浆内棕黄色染色颗粒变小,颜色变浅。
研究结论
1.尘螨变应原SLIT治疗12个月,对于持续性中重度变应性鼻炎具有显著的临床疗效和安全性。其鼻腔总体症状约为免疫治疗前的50%左右。
2.尘螨变应原SLIT治疗12个月期间,患者临床症状的缓解呈现逐步改善的趋势。其中免疫治疗前6个月,临床症状改善幅度大,呈现陡降型临床症状变化曲线；免疫治疗后6个月,临床症状相对稳定,变化不大呈现平台型临床症状变化曲线。部分患者(13.04%)在免疫治疗的前6个月左右,临床症状会出现一定程度的反弹,总体呈现为波浪状下降的临床症状变化曲线。
3.尘螨变应原SLIT治疗早期(免疫治疗开始后1个月)即可产生显著的临床疗效,该结果早于本研究对临床疗效起效时间的预期。
4.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者鼻粘膜免疫的TH2型免疫反应占优势的免疫状态得到下调,表现为鼻粘膜局部TH2型免疫反应强度的下调,TH1/TH2免疫反应之间的平衡状态向TH1反向偏倚。
5.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者机体的系统免疫中TH2型免疫反应占优势的免疫状态实现下调,表现为调控TH2型免疫维持或免疫记忆功能的OX40L表达水平的下调,TH1/TH2免疫反应之间的平衡状态向TH1反向偏倚。
6.尘螨变应原SLIT治疗12个月,能实现对持续性中重度变应性鼻炎患者粘膜免疫和系统免疫中TH2型免疫反应占优势的免疫状态的下调。
7.尘螨变应原SLIT治疗12个月,持续性中重度变应性鼻炎患者临床症状显著改善,机体OX4OL和粘膜TSLP表达水平的下调,结合TH2型免疫反应在粘膜免疫和系统免疫的下调和TH1/TH2免疫平衡向TH1方向偏倚,提示着患者机体TSLP-OX40L信号通路介导的TH2型免疫反应的下调。
8.尘螨变应原SLIT的临床疗效能在免疫治疗早期的实现,来源于鼻腔粘膜免疫中TH2型免疫反应下调的结果；而免疫治疗后期,临床疗效的相对稳定源于系统免疫中维持TH2型免疫记忆的OX40L下调的结果。
9.对于持续性中重度变应性鼻炎患者,尘螨变应原SLIT治疗后鼻粘膜TSLP表达下调可以作为预测患者系统免疫中TH2型免疫反应下调的指标。
Background
WHO had pointed "allergic disease would be one of the biggest challenges for human health in21-century". Allergic rhinitis belongs to allergic disease. The pathophysiology of allergic rhinitis is occurred in nasal mucosa and originated from systemic immune abnormalities. After in contact with allergens, TH2type immune responses were started by IgE mediated eosinophil reaction and dominated by a variety of inflammatory cells and cytokines involved in. The common features of allergic disease were so-called TH2type immune response.
In our knowledge of TH2type immune responds, many kinds of cells take part in this sophisticated network of immune responds. There are included T cell, B cell, Eosinophils, Basophil, APC, innate helper cell and so on. Type2responses are characterized by the induction of CD4+T helper (TH)2cells, which secrete cytokines such as interleukin-4(IL-4), IL-5, IL-9, and IL-13. TH2cells promote B cell responses and immunoglobulin E (IgE) secretion through their production of IL-4. IgE immune complexes bind to high-affinity IgE receptors (FceRl) on basophils and mast cells, leading to their activation and secretion of several cytokines and inflammatory mediators such as histamine, heparin, and serotonin. These factors mediate a range of effector functions characteristic of type2inflammation, including recruitment of alternatively activated macrophages and granulocytes, smooth muscle contractility, and mucus hypersecretion. There are four stages in the courses of the formation of TH2type immune responses in allergic rhinitis. There are so-called immune recognition, sensitization, immune responses and immune maintenance. At the same time, mucosa immune and system immune are all involved in TH2type immune responses. A marked feature of type2responses is their diversity. There is diversity in terms of the array of stimuli that trigger type2responses, the mechanisms by which the innate immune system senses such stimuli, and the cellular and molecular pathways that orchestrate the response. Indeed, there even appears to be diversity in the cytokine profiles of responding cells in a TH2cell response. In summary, although many attentions have been pay in the field of TH2type immune responses, but the mechanisms that initiate and control type2responses remain enigmatic.
Allergen-specific immunotherapy has been used more than100years as a desensitizing therapy for allergic diseases and represents the potentially curative and specific method of treatment. Specific immunotherapy is the only treatment modality with capacity of change the natural course of allergic diseases. According to the guideline of ARIA, allergen specific immunotherapy is referred to consistent moderate-severe allergic rhinitis. Now days, specific immunotherapy are applied in clinic treatment for allergic rhinitis with two ways, subcutaneous immunotherapy and sublingual immunotherapy. Although many big samples randomized double-blind placebo-controlled trails have confirmed the clinic efficiency of SIT in patients with allergic rhinitis. But the mechanism of SIT is unclear. Many conclusions about SIT were originated from SCIT. SLIT was first applied in clinic treatment in1986. Compared with the consensus about SCIT, many disputations are still existed about SLIT. The mainly parts are clinic efficiency and mechanism. Allergen specific sublingual immunotherapy has recently received much attention around the world as a treatment for allergic rhinitis and is now widely used to replace the subcutaneous route. To date, many randomized double-blind placebo-controlled trials have confirmed the clinic efficiency of SLIT for patients with allergic rhinitis.
The knowledge of SLIT is mainly originated from Europe study team and the patients received SLIT are mainly with seasonal allergic rhinitis sensitive to grass or pollen allergen. Although clinic efficiency of SLIT have already been confirmed in seasonal allergic rhinitis. But for consistent moderate-severe allergic rhinitis, whether house dust mite sublingual immunotherapy would possess the same result or not were uncertain. On account of new progressions of SLIT and the finds of innate immune system, increasing interest in SLIT has immerged in recent years.
The recent identification of previously unrecognized innate cell populations, termed innate helper cell. Studies show that innate helper cell has ability to induce TH2type immune responses by directly secreting TH2type cytokine. Thymic stromal lymphopoietin (TSLP), a distant paralog of IL-7, is a type I cytokine that is part of the IL-2cytokine family. TSLP are produced primarily by epithelial cell in airway during airway's allergic inflammation. Recently study shows that TSLP can induce a robust expansion of human TH2memory cells while maintaining their central memory phenotype and TH2commitments. OX40L and its receptor OX40are members of the TNF and TNF receptor super families, respectively. OX40L is expressed by APCs, endothelial cells and T cells, whereas OX40is found mainly on activated T cells. The expression of OX40L on dendritic cells is mainly induced by thymic stromal lymphopoietin. Many factors of promoting mature of APC could improve the expression of OX40L. TSLP has ability to induce naive T cell difference into TH2cell by polarizing dendritic cells and this role is accomplice by inducing the expression of OX40L on the surface of dendritic cells. New results demonstrated the critical roles of OX40/OX40L interaction on the maintenance and re-activation of TH2memory responses during TSLP-mediated allergic responses. Targeting the TSLP-induced inflammatory TH2cells or allergen-specific TH2memory cells by blocking OX40/OX40L interaction may be one of the new therapeutic approaches for the prevention and treatment of human-allergic diseases. The important roles of TSLP-OX40L signal in TH2type immune responses have been reported in many allergic disease trials. The previous studies had demonstrated that TSLP-OX40L signal play a crucial role in pathogenesis of allergic disease. Until now, there is no research to concern the influence of house dust mite sublingual immunotherapy in TSLP-OX40L mediated TH2type Immanuel responds in patient with allergic rhinitis. Memory TH2cell is main cell mass that could maintain chronic inflammation stage in allergic responses and insure allergic responses reappearance when allergen was exposure again. The expression of TSLP can conduce to maintain the function of memory TH2cell. So TSLP-OX40L signal would become a potential molecular target in treatment of allergic diseases.
In conclusion, TSLP-OX40L signal has become a hot spot in treatment of allergic diseases. The traditional medicine treatment strategy is mainly focused on the effector phase of allergic responses. Its role is mainly on been activated cells, such as T cell, Eosinophils and Basophil. Since the ability of SLIT to regulate TH2-type immune response is characterized by mucosal route, and TH2-type immune response mediated by TSLP-OX40L signaling pathway is also in the mucosal start-up phase, thereby causing the body's immune cascade series. Whether there exist interaction between house dust mite allergen SLIT and TH2type immune response mediated by TSLP-OX40L signal or not, is worth the wait. This is the main purpose of this study to investigate. But until now, no related reports were published.
Objective
To investigate the impact of house dust mite allergen sublingual immunotherapy on TSLP-OX40L mediated TH2type immune responds in patient with moderate to severe allergic rhinitis, including changes in local mucosal immune of nasal mucosa and the system immune. Combined with changes in the patients clinic symptoms of observation and assessment, further analysis of the impact of dust mite allergen SLIT on TH2-type immune response in patients with moderated to severe allergic rhinitis and mechanism were carried out. We look forward to provide guidance and assistance for the clinical treatment of dust mite SLIT and the research of mechanism of SLIT.
Methods
In this study, according to the guideline of ARIA,46cases of allergic rhinitis patients in southern China were enrolled in our study. All of the patients had moderate to severe persistent allergic rhinitis with or without asthma. All patients received house dust mite allergen specific sublingual immunotherapy. The cut-off point of trials is12months immunotherapy. The case-control study is employed in trials. Each patient was treated for a12-month period. Mixture extracts of D.f. and D.p.(Zhejiang Wolwo Bio-Pharmaceutical Co., Ltd., China) were used in our study. Changes of TH2-type immune response and clinical symptoms in patients with allergic rhinitis in our trials were dynamic observed and analyses respectively at the times before the start of immune therapy and immunotherapy for12months. First, the characteristics and rules of changes related to clinical symptoms were summarized within12months'dust mite allergen SLIT; On the basis of the progress in TH2-type immune response and sublingual allergen-specific immunotherapy, TSLP-OX40L signaling pathway was selected as a starting point for further analysis of the changes both occurred in the nasal mucosa immune and system immune by impact of SLIT. There are included the expressions of IL-4, IL-13, INF-γ and TSLP in nasal lavage detected by ELISA; The expressions of T-bet mRNA, GATA-3mRNA and OX40L mRNA in blood detected by QRT-PCR; The expressions of OX40L in PBMC detected by flow cytometric analysis; The expression of TSLP detected by immunohistochemistry and the counts of EOS in nasal secretion smear detected by HE staining. From changes in the above objective indicators, we can deduce whether TH2-type immune response mediated by TSLP-OX40L signaling pathway would be changed or not, including the mucosa immune response and system immune response. Finally, the results of clinical observations and laboratory tests are analyzed.
Results
Outcomes of syndrome score
After12-month treatment, TNSS, medication score and all INSS (nasal rhinorrhea, itching, sneezing and nasal congestion) significantly decreased compared with baseline value by overall evaluation (P<0.001). TNSS significantly decreased after12-month SLIT compared with the baseline value from9.065±1.162to4.413±0.717(X2=82.667, P<0.001); Medication score decreased from2.870±0.341to1.109±0.315(X2=79.590, P<0.001). Among all INSS, itching score decreased from2.391±0.493to1.022±0.257(X2=86.681, P<0.001); nasal congestion score decreased from2.000±0.633to1.196±0.401(X2=60.057,P<0.001); sneezing score decreased from2.457±0.504to1.109±0.315(X2=74.146,P<0.001);nasal rhinorrhea score decreased from2.217±0.417to1.087±0.354(X2=83.532, P<0.001). All show significant difference. The results showed that a significant improvement of clinical symptoms for patients with moderate to severe persistent allergic rhinitis had achieved after12-month dust mite allergen SLIT treatment. The dependence on drugs for all patients was reduced significantly. No serious adverse events and no symptoms aggravated case were reported during12-month SLIT treatment, symptoms aggravated case report.
Additionally, by dynamic observation of clinic syndromes, the changes of TNSS showed a gradual decline trend during12-month dust mite allergen SLIT. Among this trend, TNSS decreased significantly after1-month dust mite allergen SLIT from9.065±1.162to7.132±1.554(Z=-5.264, P<0.001). During the first6-month dust mite allergen SLIT, TNSS declined obviously and showed a steep drop trend from9.065±1.162to4.935±1.063. The difference was statistically significant(Z=-5.962, P <0.001). But during the second6-month SLIT, the changes of TNSS were relatively stable and the trend of TNSS presented a platform-type curve. The difference was not statistically significant(Z=-2.449, P=0.014>0.0125)(The significance level in here needed to be adjusted to P<0.0125for the statistical significant difference). Until to the endpoint of12-month SLIT, TNSS had been declined50%compared with baseline. The changes of INSS during12-month dust mite allergen SLIT were basically as same as TNSS. Only the differences of nasal congestion score and medicine score compared at the time of6-month SLIT and12-month SLIT were statistical significant(P<0.0125)(significance level was adjusted). The study proved the safety and efficiency of dust mite allergen SLIT.
The results also showed that the trend of syndromes improvement was gradual and wavy during12-month dust mite allergen SLIT period. The wavy or the transient rebound of syndromes presented mainly mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline.
Outcomes of laboratory parameter
HE staining:The counts of EOS in nasal secretion smear were decreased significantly by overall evaluation(Cochran's Q=23.330, P<0.001) after12-month dust mite allergen SLIT. After that, pair-wise comparisons of the counts of EOS in nasal secretion smear were carried respectively at the three time points (baseline,6-month and12-month) to evaluate the differences. The results showed that the counts of EOS in nasal secretion smear at the times of6-month SLIT and12-month SLIT decreased significantly compared with baseline(6-month SLIT: Z=-3.162, P<0.0125;12-month SLIT:Z=-3.873, P<0.001). But there were no statistical difference of the counts of EOS in nasal secretion smear between6-month SLIT with12-month SLIT(Z=-2.236,P>0.0125).
ELISA:The levels of IL-4, IL-13, INF-y and TSLP in nasal lavage were decreased significantly after12-month dust mite allergen SLIT (P<0.05). The level of IL-4decreased from76.010±9.394(pg/ml) to32.910±8.752(pg/ml)(t=27.010, P <0.05); The level of IL-13decreased from55.68±12.86(pg/ml) to30.000±9.453(pg/ml)(t=14.050, P<0.05); The level of INF-y decreased from30.600±6.755(pg/ml) to21.710±5.457(pg/ml)(t=8.087, P<0.05); The level of TSLP decreased from36.390±6.610(pg/ml) to21.780±4.231(pg/ml)(t=11.110, P<0.05). The ratio of IL-4/INF-γ decreased from2.584±0.581tol.611±0.593. The difference was statistically significant (t=8.987, P<0.05).
The expressions of TIgE and sIgE in serum didn't showed significant changes compared with baseline (TIgE:423.900±29.990(kU/L) vs420.200±39.110(kU/L); slgE:54.920±5.217(kU/L)vs56.790±6.750(kU/L)(P>0.05).
QRT-PCR:The expressions of T-bet mRNA in blood significantly increased from3.458±0.6202to5.389±1.228fold after12-month SLIT compared with baseline (t=12.230, P<0.05). The expressions of GATA-3mRNA in blood significantly decreased from12.440±2.536fold to7.246±1.953fold after12-month SLIT compared with baseline (t=11.920, P<0.05). The ratio of T-bet mRNA/GATA-3mRNA increased significantly from0.288±0.071to0.785±0.251(t=13.890, P<0.05). The level of OX40L mRNA in blood decreased significantly after12-month SLIT from1.401±0.361fold to0.772±0.301fold compared with baseline (t=12.300, P<0.05). The negative associations of the levels of OX40L mRNA and the ratios of T-bet mRNA/GATA-3mRNA in blood were observed before and after12-month SLIT(before SLIT:r=-0.440, P<0.05;after12-month SLIT:r=-0.565, P<0.05). The difference was statistically significant. The positive associations of the levels of OX40L mRNA in blood and the levels of TSLP in nasal lavage fluid were observed before and after12-month SLIT(before SLIT:r=0.491, P<0.05; after12-month SLIT:r=0.548, P<0.05). The difference was statistically significant.
Flow cytometry:The expressions of OX40L on the surface of peripheral blood mononuclear cells after12-month SLIT were decreased significantly (t=13.100, P<0.05), from before treatment (2.712±0.511)%, down to12-month treatment (1.630±0.471)%. The expressions of OX40L on the surface of CDllc+CD86+cells in peripheral blood mononuclear cells also significantly decreased after12-month SLIT from the former (5.839±2.841)%, reduced to (2.098±0.870)%. The difference was statistically significant (t=9.946, P<0.05).
Immunohistochemical staining:There were6cases of nasal mucosa biopsies be obtained to study the HE staining and the expression of TSLP before and after12-month SLIT. The results showed that positive staining of TSLP in nasal mucosa was weakened after12-month SLIT compared with baseline from83.33%to33.33%. From the HE staining, the results showed that epithelial cells arranged closely and eosinophil infiltration under the epithelium decreased after12-month SLIT.
Conclusion
1. Dust mite allergen SLIT treatment for patients with moderate to severe persistent allergic rhinitis has significant clinical efficacy and safety. After12-month dust mite allergen SLIT, the TNSS was about to decrease50%compared with before immunotherapy.
2. During12-month dust mite allergen SLIT, the remission of clinical symptoms showed a gradual improvement trend. An obviously clinic improvement was found in the first6-month dust mite allergen SLIT. The changes of clinic symptoms showed a steep drop trend in the first six months. During the second six months, the changes of clinic symptoms were relatively stable and showed a platform-type curve. The transient rebounds of syndromes were presented in some patients mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline. Overall clinic symptoms showed a waving decline trend.
3. The significant clinical efficacy could be achieved in early stage of dust mite allergen SLIT (one month), earlier than the onset time being expected.
4. TH2type immune responses in nasal mucosa immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the intensity of TH2type immune responses was weakened and the balance of TH1/TH2immune responses was shifted to TH1.
5. TH2type immune responses in system immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the level of OX40L which was in charge of immune memory of TH2type immune responses was down-regulated and the balance of TH1/TH2immune responses was shifted to TH1.
6. The down-regulation of OX40L in system immune, TSLP in mucosa immune and TH2type immune responses in both of system and mucosa system implied the signaling pathway conducted by TSLP-OX40L in TH2type immune responses was down-regulated after12-month dust mite allergen SLIT.
7. TH2type immune responses both in mucosa immune and in system immune were down-regulated by12-month dust mite allergen SLIL in patients with moderate to severe persistent allergic rhinitis.
8. The achievement of clinic efficiency in early stages of dust mite allergen SLIT was derived from the down-regulation of TH2immune responses in nasal mucosa immune. While the maintaining the clinical efficacy was derivered from the down-regulation of OX40L in system immune.
9. The expressions of TSLP could be a predictor of the down-regulation of TH2type immune responses in system immune responses for patients with moderate to severe persistent allergic rhinitis after being received house dust mite SLIT.
引文
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