IL-17在中性粒细胞性哮喘中的作用及其调控气道炎症表型的机制研究

发布时间：2018-01-07 05:18

本文关键词：IL-17在中性粒细胞性哮喘中的作用及其调控气道炎症表型的机制研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

更多相关文章： 中性粒细胞性哮喘 IL-17 CD4+Th17细胞

【摘要】：研究背景与目的支气管哮喘(简称哮喘)本质上属于气道慢性炎症疾病,遗传因素和外界环境因素均可影响其发病。全球约有三亿哮喘患者,中国就占十分之一,而且中国是哮喘致死率最高的国家之一。支气管哮喘还具有明显异质性,不同病人之间及同一病人不同时期其哮喘临床表现、病情严重程度、治疗反应等可能不同。这种“不同”其实是个体内因(遗传因素)和外因(环境因素)共同作用的结局。以往认为嗜酸性粒细胞(Eos)在气道内聚集和浸润是哮喘的主要炎症特征或表型,但近期研究发现,哮喘病人气道内并非全部以嗜酸性粒细胞浸润为主,大量的中性粒细胞(Neu)出现在哮喘急性发作期和一些致死性哮喘发作的病人气道内。目前认为中性粒细胞浸润气道是重症哮喘气道炎症的重要表型之一,而且与糖皮质激素治疗反应性差相关。虽然只占哮喘病例的5%至10%不等,但重症哮喘病人对现有的治疗药物如吸入性糖皮质激素联合长效β2肾上腺素能受体激动剂(ICS+LABA)、白三烯受体拮抗剂(LTRA)、茶碱、长效胆碱能受体拮抗剂(LAMA)等反应差,因此构成了哮喘防治的巨大医疗支出负担。研究中性粒细胞性哮喘表型及其病理生理机制对于哮喘的预防和治疗,尤其是对于重症哮喘、激素抵抗性哮喘的防治有着非常重要的意义。过去认为过敏性哮喘发病的免疫学机制为“Th1/Th2失衡”,气道内Th2反应增强,Th2细胞因子IL-4、IL-5和IL-13等增高,形成嗜酸性粒细胞气道炎症。在此理论基础上,研究者研发了针对Th2因子的靶向治疗药物,如抗IL-5和IL-13的单克隆抗体及人重组IL-4受体等,试图逆转Th1/Th2失衡,但一些临床试验结果并不令人满意。另一方面,使用Th1细胞因子治疗哮喘也基本无效。因此,“Th1/Th2失衡”学说不能完全阐明嗜酸性粒细胞性哮喘的发病机制,更不能解释中性粒细胞性哮喘的病理生理改变,后者可能存在不同免疫学机制。在随后的临床研究中观察到,IL-17、IL-1β、IL-6、IL-8等炎症因子在中性粒细胞性哮喘病人的诱导痰上清、支气管肺泡灌洗液(BALF)及支气管肺组织标本中表达增高,且IL-17可能与中性粒细胞性哮喘病人气道高反应性(AHR)正相关,但确切的机制仍不明。CD4+辅助性T淋巴细胞(Th)是几乎参与机体所有免疫反应的重要免疫细胞之一。IL-17及其分泌细胞CD4+Th17细胞参与了机体对病原菌如细菌、真菌、支原体、衣原体等的清除,并可引起中性粒细胞和/或单核细胞向炎症部位聚集。因此我们推测,在中性粒细胞性哮喘中,CD4+Th17细胞及其分泌的IL-17是引起气道中性粒细胞性炎症的重要机制,对此机制的深入研究可能给临床上重症哮喘的防治提供新的思路。目的本课题拟使用卵清蛋白(ovalbumin,OVA)联合脂多糖(lipopolysaccharide,LPS)对C57BL/6J小鼠进行致敏以建立哮喘中性粒细胞性炎症模型,并与单纯OVA致敏的嗜酸性粒细胞性炎症模型相比较:气道炎症特点、AHR、BALF中炎症因子表达及脾脏、淋巴结CD4+Th细胞分化情况。最后利用IL-17基因敲除小鼠探讨Th17/IL-17在哮喘中性粒细胞性炎症形成中的作用及机制。方法1.C57BL/6J小鼠30只随机分为3组:对照组(NS组),嗜酸性粒细胞性哮喘组(OVA组)和中性粒细胞性哮喘组(OVA+LPS组),每组10只。于实验第1天、7天致敏小鼠,麻醉小鼠后腹腔注射OVA(50μg)与等容积氢氧化铝混合液100μl,经鼻腔缓缓滴入0.2%OVA 50μl(OVA组)或加LPS 1μg(OVA+LPS组);NS组小鼠给予等体积的生理盐水致敏。实验第14天开始,用1%OVA液进行雾化激发小鼠(OVA组和OVA+LPS组),每次1小时,每天1次,连续5天,对照组用生理盐水代替OVA激发。2.完成激发后的小鼠行气道高反应性(AHR)测定及支气管肺泡灌洗(BAL),对BALF细胞进行计数并分类,BALF上清用ELISA方法检测IL-1β、IL-4、IL-5、IL-8、IL-17表达。3.摘取小鼠完整左肺行病理学检查,观察支气管及肺组织炎症(HE染色)和气道上皮细胞杯状化生程度(PAS染色)。4.无菌取脾及肺引流淋巴结(LN)制备单细胞悬液,用流式细胞技术分别检测其CD4+Th细胞分化情况。5.参照OVA+LPS组小鼠建模方法用IL-17基因敲除小鼠复制中性粒细胞性哮喘模型,检测气道中炎症细胞总数及分类、炎症因子表达水平,测定气道高反应性(AHR),并观察肺组织病理炎症损害及气道粘液分泌状态。6.流式细胞技术检测IL-17基因敲除小鼠脾脏及肺引流巴结CD4+Th细胞分化情况。结果1.由OVA联合LPS致敏建立的中性粒细胞性哮喘小鼠BALF中炎症细胞以中性粒细胞为主,与单纯OVA致敏建立的嗜酸性粒细胞性哮喘小鼠相比较,具有更高的气道反应性和更严重的肺组织炎症,但其气道上皮细胞杯状化生降低;2.中性粒细胞性哮喘小鼠BALF中IL-1β(P0.05)、IL-8(P0.001)和IL-17(P0.05)因子水平显著高于嗜酸性粒细胞性哮喘小鼠,而Th2细胞因子IL-4(P0.01)和IL-5(P0.001)水平显著低于嗜酸性粒细胞性哮喘小鼠;3.中性粒细胞性哮喘小鼠脾脏及肺引流淋巴结(LN)中CD4+Th细胞分化与嗜酸性粒细胞性哮喘小鼠相比较,CD4+IL-17+Th17细胞分化增多(脾:P0.05;LN:P0.05),CD4+IL-4+Th2细胞分化减少(脾:P0.001;LN:P0.001),而CD4+IFNγ+Th1细胞分化无明显变化(脾:P0.05;LN:P0.05);4.与野生型中性粒细胞性哮喘小鼠相比较,IL-17基因敲除小鼠气道反应性降低、BALF中细胞总数(P0.01)及中性粒细胞(P0.001)明显减少,肺组织炎症减轻,但BALF中嗜酸性粒细胞数(P0.01)增多,气道上皮杯状化生增加;5.IL-17基因敲除小鼠BALF中Th2细胞因子IL-4(P0.05)和IL-5(P0.05)表达高于野生型中性粒细胞性哮喘小鼠,IL-1β(P0.05)、IL-8(P0.001)和IL-17(P0.001)明显低于野生型中性粒细胞性哮喘小鼠;6.与野生型中性粒细胞性哮喘组小鼠相比较,IL-17基因敲除鼠脾脏及肺引流淋巴结中CD4+IL-17+Th17细胞分化明显降低(P0.001),CD4+IL-4+Th2细胞分化升高(脾:P0.01;LN:P0.001);脾CD4+IFNγ+Th1细胞分化减少(P0.05),而淋巴结CD4+IFNγ+Th1细胞分化无变化(P0.05)。结论及意义1.用OVA联合LPS复制的中性粒细胞性哮喘小鼠模型,表现为典型的中性粒细胞性炎症,与仅用OVA致敏的嗜酸性粒细胞性哮喘小鼠相比较,有更高的气道反应性和更重的肺组织炎症损害。2.CD4+IL-17+Th17细胞分化增加及其分泌的IL-17是引起哮喘中性粒细胞性炎症表型的重要机制;3.CD4+IL-17+Th17细胞的增高及IL-17的高表达抑制了CD4+IL-4+Th2细胞分化,并进一步抑制了哮喘的Th2反应,即抑制了气道内嗜酸性粒细胞浸润、粘液高分泌及Th2细胞因子过表达。本文结果的意义在于,在动物模型中证实了,反复细菌感染接触内毒素可以使哮喘气道炎症表型从嗜酸性粒细胞性炎症转变为中性粒细胞性炎症,其机制依赖于CD4+Th17细胞的分化及其分泌的IL-17。此机制的发现为“卫生假说”补充了免疫学机制的证据。Th17细胞途径是哮喘中性粒细胞性炎症发生发展的关键环节,是治疗中性粒细胞性哮喘的潜在靶点。
[Abstract]:Background and objective bronchial asthma (asthma) belongs to chronic inflammatory airway diseases, affecting its incidence may be genetic factors and environmental factors. There are about three hundred million asthma patients worldwide, China accounted for 1/10, and Chinese is one of the country the highest rate of fatal asthma. Asthma has obvious heterogeneity, different clinical manifestations between patients and the same patient in different periods of their asthma severity, treatment response may be different. This "different" is the individual internal factors (genetic factors) and external factors (environmental factor) interaction outcome. The past that eosinophils (Eos) in airway inflammation is the main accumulation and infiltration characteristics the phenotype of asthma, but recent studies have found that not all patients with asthma airway eosinophil infiltration of a large number of neutrophils (Neu) in acute asthma. For the period and some fatal asthma patients. The airway neutrophil infiltration in the airway is one of the important phenotypes of severe asthma airway inflammation, and treatment with glucocorticoid responsiveness. Although the difference accounted for only 5% to 10% of asthma cases differ, but in patients with severe asthma to drugs such as inhaled existing corticosteroids and long-acting beta 2 adrenergic receptor agonist (ICS+LABA), leukotriene receptor antagonist (LTRA), theophylline, long-acting muscarinic antagonist (LAMA) and poor response, therefore constitutes a huge burden of medical expenses for prevention and treatment of asthma. Study of neutrophilic asthma phenotype and its pathophysiological mechanism for prevention and for the treatment of asthma, especially for severe asthma, has a very important significance for prevention and treatment of steroid resistant asthma. In the past that the immunological mechanism of the incidence of allergic asthma is Th1/Th2 Hengshui, enhanced Th2 reaction in airway, Th2 cytokines IL-4, IL-5 and IL-13 increased, the formation of eosinophilic airway inflammation. On the basis of this theory, the researchers developed for Th2 factor targeted therapy, such as anti IL-5 and anti IL-13 monoclonal antibody and recombinant human IL-4 receptor, trying to reverse the balance of Th1/Th2, but some clinical trial results are not satisfactory. On the other hand, the use of Th1 cell factor in the treatment of asthma is invalid. Therefore, "Th1/Th2 imbalance" theory can not fully explain the pathogenesis of eosinophilic asthma, but can not explain the pathophysiology of neutrophilic asthma changes, which may have different immunological to observe the mechanism. And in subsequent clinical studies in IL-17, IL-1, IL-6, beta, IL-8 and other inflammatory cytokines in induced sputum supernatant of neutrophils in patients with asthma, bronchoalveolar lavage fluid (BALF) and bronchial lung Increased expression of tissue samples, and IL-17 may be related to neutrophil cells of patients with asthma and airway hyperresponsiveness (AHR) positive correlation, but the exact mechanism is still unknown.CD4+ T helper lymphocytes (Th) is involved in almost all the immune response is one of the important immune cells and the secretion of.IL-17 cells and CD4+Th17 cells in the body of the pathogen bacteria such as bacteria, fungus, mycoplasma, chlamydia and clear, can cause neutrophil and / or aggregation of monocytes to sites of inflammation. Therefore we speculate that in neutrophilic asthma, CD4+Th17 cells and the secretion of IL-17 is an important mechanism of airway neutrophilic inflammation caused by deep research on this mechanism may provide new ideas for clinical prevention and treatment of severe asthma. The purpose of this project intends to use ovalbumin (ovalbumin, OVA) and lipopolysaccharide (lipopolysaccharide, LPS) of C57BL/6J in mice For sensitization to establish asthma neutral model granulocytic inflammation and eosinophilic inflammation model sensitivity compared with OVA: AHR, features of airway inflammation, the expression of inflammatory cytokines in BALF and spleen, lymph node differentiation of CD4+Th cells. The IL-17 gene knockout mice to investigate the effect and mechanism of Th17/IL-17 formation in neutrophilic inflammation in asthma. Methods 30 1.C57BL/6J mice were randomly divided into 3 groups: control group (group NS), eosinophilic asthma group (OVA group) and neutrophilic asthma group (OVA+LPS group), 10 rats in each group. On the first day, 7 day of sensitization mice, mice were anesthetized after intraperitoneal injection of OVA (50 g) and the volume of the mixture of aluminum hydroxide 100 L, slowly through the nose drops 0.2%OVA 50 L (OVA group) or LPS 1 g (OVA+LPS group); NS group were given an equal volume of saline sensitization. The fourteenth day of the experiment, for the fog with 1%OVA solution Of challenged mice (OVA group and OVA+LPS group), 1 hours each time, 1 times a day, for 5 consecutive days, the control group with saline instead of OVA.2. mice were stimulated airway hyperresponsiveness after excitation (AHR) determination and bronchoalveolar lavage (BAL), count the number of BALF cells and BALF. The supernatant of IL-1 beta, ELISA was detected by IL-4, IL-5, IL-8, IL-17 expression of.3. mice at the left lung pathology, lung tissue and bronchial inflammation were observed (HE staining) and airway epithelial goblet cell metaplasia degree (PAS staining).4. of spleen and lung draining lymph node (LN) for preparing single the cell suspension by flow cytometry was used to detect the differentiation of CD4+Th cells of.5. mice according to OVA+LPS modeling method in IL-17 gene knockout mice model replication of neutrophilic asthma, detection of total number of inflammatory cells in the airways and the classification, the level of expression of inflammatory cytokines, determination of airway hyperresponsiveness (of AHR), and to observe the pathological damage of lung tissue inflammation and airway mucus secretion.6. flow cytometry IL-17 knockout mice spleen and lung draining lymph node CD4+Th cell differentiation. Results 1. by OVA combined with LPS induced inflammatory cells to establish the sensitivity of neutrophilic asthma mice BALF in neutrophils, and the simple OVA established by sensitization of eosinophils of asthmatic mice compared with airway hyperresponsiveness and more severe inflammation of lung tissue, but the goblet cell metaplasia of lower airway epithelium; 2. of neutrophilic asthma mice BALF IL-1 beta (P0.05), IL-8 (P0.001) and IL-17 factor (P0.05) level was significantly higher than that of mouse eosinophils eosinophil asthma, and Th2 cell factor IL-4 (P0.01) and IL-5 (P0.001) was significantly lower than that of mouse eosinophils eosinophil asthma; 3. neutrophils in spleen and lung cells of asthmatic mice lymph drainage Node (LN) in the differentiation of CD4+Th cells and eosinophils of asthmatic mice, CD4+IL-17+Th17 cells increased (spleen: P0.05; LN:P0.05), CD4+IL-4+Th2 cell differentiation decreased (spleen: P0.001; LN:P0.001), and CD4+IFN gamma differentiation of +Th1 cells had no obvious change (spleen: P0.05; LN:P0.05; 4.) and wild type neutrophilic asthma mice compared to IL-17 knockout mice airway reactivity decreased, the total cell number in BALF (P0.01) and neutrophil (P0.001) significantly reduced the lung tissue inflammation, but BALF in the number of eosinophils (P0.01) increased airway goblet metaplasia increased; 5.IL-17 gene knockout in addition to Th2 cytokines in BALF mice IL-4 (P0.05) and IL-5 (P0.05) were higher than that of the wild type mouse granulocyte neutral asthma, IL-1 beta (P0.05), IL-8 (P0.001) and IL-17 (P0.001) was significantly lower than wild-type neutrophils in asthmatic mice and wild-type neutrophils; 6. Cells of asthmatic mice compared to IL-17 knockout mice spleen and lung drainage in the differentiation of CD4+IL-17+Th17 cells in lymph nodes was significantly lower (P0.001), CD4+IL-4+Th2 cell differentiation increased (spleen: P0.01; LN:P0.001); spleen CD4+IFN gamma +Th1 cell differentiation (P0.05), and the reduction of lymph node CD4+IFN gamma +Th1 cell differentiation (P0.05 no change). Conclusion and significance of 1. OVA combined with LPS replication in a mouse model of neutrophilic asthma, is characterized by neutrophilic inflammation, compared with sensitized with only OVA eosinophils of asthmatic mice, IL-17.2.CD4+IL-17+Th17 cells increased and the secretion of airway responsiveness and more the inflammation of lung tissue damage is an important mechanism of asthma neutrophil inflammatory phenotype by overexpression of 3.CD4+IL-17+Th17 and IL-17 increased; the cells inhibit the differentiation of CD4+IL-4+Th2 cells, and further inhibit asthma Th2 reaction of asthma, which inhibits airway eosinophilia, mucus hypersecretion and overexpression of Th2 cytokines. This is the significance of the results was confirmed in the animal model, can make the airway inflammation of asthma phenotype transition from eosinophils inflammation to neutrophilic inflammation contact endotoxin repeated bacterial infection the mechanism depends on the differentiation, CD4+Th17 cells and IL-17. secretion mechanism the findings add to the "hygiene hypothesis" evidence.Th17 cell pathway immunological mechanism is a key link in asthma neutrophilic inflammation development, is a potential target for the treatment of neutrophilic asthma.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R562.25

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