IL-12~+重组卡介苗新生期接种对RSV感染后实验性哮喘小鼠影响及其机制研究
|
摘要
背景与目的支气管哮喘是儿童最常见的慢性疾病,其发病率呈上升趋势。目前哮喘的防治方法难于达到世界卫生组织(WHO)所提倡的一级预防要求。卡介苗(BCG)是包括中国在内的发展中国家在新生儿期广泛接种的减毒活疫苗。动物实验研究显示BCG可诱导Th1细胞反应和抑制哮喘发生,但未能在人类得到证实,其原因与机制有待进一步研究。我们前期的研究发现BCG新生期接种可减轻OVA致敏激发的实验性哮喘小鼠气道炎症和气道高反应性,对哮喘有保护作用,如果小鼠OVA致敏前感染引起婴幼儿下呼吸道感染最主要与重要的病原体-呼吸道合胞病毒(RSV)后,其保护作用消失,机制尚不清楚。
近年来运用分子生物学技术对分枝杆菌进行改造,制备出能表达IL-12的重组BCG疫苗(rBCG)。新生期接种分泌型rBCG能否在增强诱导Th1免疫反应作用的同时,增强机体抗RSV能力,从而达到预防过敏性疾病的作用值得研究。
Th17细胞是最近发现的一类新型Th细胞亚群,IL-17是其特征性细胞因子,无论在人类哮喘患者还是动物哮喘模型均发现IL-17表达增加,提示IL-17在哮喘发病中扮演重要角色。新生期BCG接种能显著降低OVA诱导的IL-17,RSV感染是否通过作用于IL-17逆转BCG抗哮喘作用值得进一步研究。
本研究旨在探讨IL-17在实验性哮喘模型发生及RSV逆转BCG抗哮喘中的作用,明确rBCG新生期接种能否对抗RSV的干扰,实现抗哮喘的作用。研究结果将有助于明确RSV逆转BCG抗哮喘作用的机制,为开发更有效的抗哮喘疫苗奠定理论基础。
第一部分IL-17在呼吸道合胞病毒感染逆转卡介苗抗哮喘作用中的机制研究
目的探讨IL-17在哮喘发生及RSV逆转BCG抗哮喘中的作用。
方法新生小鼠分五组:Control组,OVA组,BCG/OVA组,RSV/OVA组,BCG/RSV/OVA组。BCG/OVA组和BCG/RSV/OVA组小鼠新生期接种BCG;RSV/OVA组,BCG/RSV/OVA组小鼠在第3周时通过滴鼻感染RSV。除Control组外,其余四组均给予OVA致敏和激发。观察支气管肺泡灌洗液中细胞总数及分类,肺组织HE染色进行炎症病理评分,ELISA法检测支气管肺泡灌洗液中IFN-γ、IL-5、IL-10、IL-17、IL-12含量和血清OVA特异性IgE水平。
结果各哮喘组BALF中细胞总数,淋巴细胞、嗜酸性粒细胞、中性粒细胞比例均高于Control组(P<0.01或P<0.05),BCG/RSV/OVA组BALF中细胞总数及淋巴细胞比例均较BCG/OVA组显著增高(P<0.05 or P<0.01)。肺组织病理评分显示各哮喘组支气管细支气管周围炎症、血管周围炎症、肺泡炎症评分明显高于Control组(P<0.01),病毒组和OVA组肺泡炎症和血管周围炎症评分明显高于BCG/OVA组(P<0.01或P<0.05)。各哮喘组BALF中IFN-γ水平明显低于Control组(P<0.01),IL-5水平明显高于Control组(P<0.01或P<0.05),哮喘组之间无显著差异。除BCG/OVA,各哮喘组IL-17水平明显高于Control组(P<0.05),BCG/RSV/OVA组IL-17水平明显高于BCG/OVA组(P<0.05),各组IL-10、IL-12水平无统计学差异。各哮喘组血清OVA特异性IgE水平明显高于Control组(P<0.01),而哮喘组之间无显著差异。
结论IL-17在哮喘发病中起作用,BCG新生期接种能够降低实验性哮喘小鼠气道IL-17产生,减轻肺部炎症, RSV感染导致气道IL-17水平增高逆转新生期BCG接种抗哮喘作用。
第二部分IL-12+重组BCG新生期接种对RSV感染后实验性哮喘小鼠的影响及其机制研究
目的探讨rBCG新生期接种能否对抗RSV的干扰,实现对哮喘的保护作用。
方法新生期小鼠分五组:Control组,OVA组,rBCG/OVA组,RSV/OVA组,rBCG/RSV/OVA组。其中:rBCG/OVA组和rBCG/RSV/OVA组小鼠新生期接种rBCG;RSV/OVA组,rBCG/RSV/OVA组小鼠在第3周通过滴鼻感染RSV。除Control组外,其余四组均给予OVA致敏和激发。观察支气管肺泡灌洗液中细胞总数及分类,肺组织HE染色进行炎症病理评分,ELISA法检测支气管肺泡灌洗液中IFN-γ、IL-5、IL-10、IL-12含量和血清OVA特异性IgE水平。
结果各哮喘组BALF中细胞总数,淋巴细胞、嗜酸性粒细胞、中性粒细胞比例均高于Control组(P<0.01或P<0.05),rBCG/RSV/OVA组细胞总数及淋巴细胞比例较rBCG/OVA组显著增高(P<0.01)。肺组织病理评分显示各哮喘组支气管细支气管周围炎症、血管周围炎症、肺泡炎症评分明显高于Control组(P<0.01),rBCG/RSV/OVA组肺泡炎症和血管周围炎症评分明显高于rBCG/OVA组(P<0.01或P<0.05)。各哮喘组BALF中IFN-γ水平明显低于Control组(P<0.01),IL-5水平明显高于Control组(P<0.01或P<0.05),哮喘组之间无显著差异。rBCG/OVA组IL-12水平明显高于其它组(P<0.01),IFN-γ/IL-5比值明显高于其它哮喘组(P<0.01或P<0.05),rBCG/RSV/OVA组IFN-γ/IL-5比值明显低于rBCG/OVA组(P<0.05),各组IL-10水平无统计学差异。各哮喘组血清OVA特异性IgE水平明显高于Control组(P<0.01),而哮喘组之间无显著差异。
结论rBCG新生期接种可促进实验性哮喘小鼠肺部IL-12产生,但RSV感染后IL-12产生受到抑制,肺部炎症细胞浸润和组织病理改变明显,rBCG新生期接种不能对抗RSV对BCG保护哮喘的干扰。RSV逆转rBCG抗哮喘作用是否也和IL-17密切相关,还需深入研究,
Background and Objective Asthma is one of the most common chronic inflammation diseases of airway. So far, prevention and treatment of asthma have not met WHO’s demands of primary prevention. Balillus Calmette-Guerin(BCG) is an attenuated live vaccine which has been broadly vaccinated in developing countries including China. Animal experiments have showed BCG can induce Th1 immunity response and inhibit the development of asthma, but that has not been proved in human being. Our previous researches have showed that neonatal BCG could attenutate airway inflammation and AHR in OVA-induced murine model of asthma. However, RSV infection could reverse the anti-asthma effects of BCG vaccination.
These years mycobacterium has been recombinated via molecular biology techniques and it can express IL-12. It is worth to study to learn if recombinant BCG vaccination can induce Th1 immunity response and interrupt reversed anti-asthma effect of RSV infection.
Th17 cell is a new Th cells subset recently discovered, producing IL-17. Increased lever of IL-17 has been found both in human asthmatics and in animal models of asthma, which suggest IL-17 plays an important role in the pathogenesis of asthma. It is meaningful to know whether neonatal BCG vaccination can reduce the lever of IL-17 induced by OVA and whether RSV infection interrupts anti-asthma effect of BCG via IL-17.
Our purpose is to know the role of IL-17 in the pathogenesis of experimental asthma and in the reversed anti-asthma effect of RSV infection and whether neonatal recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection so that anti-asthma. This is very helpful to understand the mechanism of reversed anti-asthma effect of RSV infection and is the basis of developing new anti-asthma vaccination.
Part I. Impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccination
Objective To explore the role of IL-17 in the pathogenesis of experimental asthma and impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccination
Methods Neonatal BALB/c mice were divided into five groups: Control, OVA, BCG/OVA, RSV/OVA, BCG/RSV/OVA groups. Neonatal mice were vaccinated with BCG in BCG/OVA and BCG/RSV/OVA groups. Mice of RSV/OVA, BCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.
Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group(P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in BCG/RSV/OVA group were statistically increased compared with BCG/OVA group(P<0.01 or P<0.05). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). All groups infected RSV and OVA group had severer perivasculitis and alveolifis than BCG/OVA group(P<0.01 or P<0.05), while there was no significant difference among themselves. The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group (P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-17 was significantly higher in all asthmatic groups except BCG/OVA group compared with control groups (P<0.05). The level of IL-17 in BCG/RSV/OVA group was statistically increased compared with BCG/OVA group. The level of IL-10 and IL-12 in BALF among all groups was respectively no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01), while no significant difference between all OVA-induced groups.
Conclusions IL-17 played the role in the pathogenesis of asthma. Treatment of neonatal BALB/c mice with BCG can reduce the level of IL-17 in BALF and can attenuate lung inflammation, but this effect can be reversed by RSV infection. It may be led to following increasing IL-17 of airway on RSV infection, and the exact mechanism need to be further studied.
Part II. Combined effects of neonatal recombinant Bacillus Calmette-Guerin vaccination secreting IL-12 and RSV infection on OVA-induced murine model of asthma
Objective To explore whether neonatal IL-12 recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection or not.
Methods Neonatal BALB/c mice were divided into five groups: Control, OVA, rBCG/OVA, RSV/OVA, rBCG/RSV/OVA groups. Neonatal mice were vaccinated with rBCG only in rBCG/OVA and rBCG/RSV/OVA groups. Mice of RSV/OVA, rBCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.
Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group (P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in rBCG/RSV/OVA group were statistically increased compared with rBCG/OVA group ((P<0.01). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). rBCG/RSV/OVA group had severer perivasculitis and alveolifis than rBCG/OVA group (P<0.01 or P<0.05). The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group(P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-12 was significantly higher in rBCG/OVA group compared with other groups (P<0.01). The ratio of IFN-γ/IL-5 in in rBCG/OVA group was statistically increased compared with other asthmatic group (P<0.01 or P<0.05). The level of IL-10 in BALF among all groups was no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01),while no significant difference among all OVA-induced groups.
Conclusions Neonatal recombinant BCG vaccination secreting IL-12 can improve the lever of IL-12 in the lung of asthmatic mice. However, IL-12 can be inhibited and infiltration of inflammatory cells and pathological changes of lung are significant on RSV infection. Neonatal recombinant BCG vaccination secreting IL-12 can not interrupt reversed anti-asthma effect of RSV infection. It is still needed to study whether reversed anti-asthma effect of RSV infection is associated with IL-17 and this is helpful to develop more effective vaccination of anti-asthma.
近年来运用分子生物学技术对分枝杆菌进行改造,制备出能表达IL-12的重组BCG疫苗(rBCG)。新生期接种分泌型rBCG能否在增强诱导Th1免疫反应作用的同时,增强机体抗RSV能力,从而达到预防过敏性疾病的作用值得研究。
Th17细胞是最近发现的一类新型Th细胞亚群,IL-17是其特征性细胞因子,无论在人类哮喘患者还是动物哮喘模型均发现IL-17表达增加,提示IL-17在哮喘发病中扮演重要角色。新生期BCG接种能显著降低OVA诱导的IL-17,RSV感染是否通过作用于IL-17逆转BCG抗哮喘作用值得进一步研究。
本研究旨在探讨IL-17在实验性哮喘模型发生及RSV逆转BCG抗哮喘中的作用,明确rBCG新生期接种能否对抗RSV的干扰,实现抗哮喘的作用。研究结果将有助于明确RSV逆转BCG抗哮喘作用的机制,为开发更有效的抗哮喘疫苗奠定理论基础。
第一部分IL-17在呼吸道合胞病毒感染逆转卡介苗抗哮喘作用中的机制研究
目的探讨IL-17在哮喘发生及RSV逆转BCG抗哮喘中的作用。
方法新生小鼠分五组:Control组,OVA组,BCG/OVA组,RSV/OVA组,BCG/RSV/OVA组。BCG/OVA组和BCG/RSV/OVA组小鼠新生期接种BCG;RSV/OVA组,BCG/RSV/OVA组小鼠在第3周时通过滴鼻感染RSV。除Control组外,其余四组均给予OVA致敏和激发。观察支气管肺泡灌洗液中细胞总数及分类,肺组织HE染色进行炎症病理评分,ELISA法检测支气管肺泡灌洗液中IFN-γ、IL-5、IL-10、IL-17、IL-12含量和血清OVA特异性IgE水平。
结果各哮喘组BALF中细胞总数,淋巴细胞、嗜酸性粒细胞、中性粒细胞比例均高于Control组(P<0.01或P<0.05),BCG/RSV/OVA组BALF中细胞总数及淋巴细胞比例均较BCG/OVA组显著增高(P<0.05 or P<0.01)。肺组织病理评分显示各哮喘组支气管细支气管周围炎症、血管周围炎症、肺泡炎症评分明显高于Control组(P<0.01),病毒组和OVA组肺泡炎症和血管周围炎症评分明显高于BCG/OVA组(P<0.01或P<0.05)。各哮喘组BALF中IFN-γ水平明显低于Control组(P<0.01),IL-5水平明显高于Control组(P<0.01或P<0.05),哮喘组之间无显著差异。除BCG/OVA,各哮喘组IL-17水平明显高于Control组(P<0.05),BCG/RSV/OVA组IL-17水平明显高于BCG/OVA组(P<0.05),各组IL-10、IL-12水平无统计学差异。各哮喘组血清OVA特异性IgE水平明显高于Control组(P<0.01),而哮喘组之间无显著差异。
结论IL-17在哮喘发病中起作用,BCG新生期接种能够降低实验性哮喘小鼠气道IL-17产生,减轻肺部炎症, RSV感染导致气道IL-17水平增高逆转新生期BCG接种抗哮喘作用。
第二部分IL-12+重组BCG新生期接种对RSV感染后实验性哮喘小鼠的影响及其机制研究
目的探讨rBCG新生期接种能否对抗RSV的干扰,实现对哮喘的保护作用。
方法新生期小鼠分五组:Control组,OVA组,rBCG/OVA组,RSV/OVA组,rBCG/RSV/OVA组。其中:rBCG/OVA组和rBCG/RSV/OVA组小鼠新生期接种rBCG;RSV/OVA组,rBCG/RSV/OVA组小鼠在第3周通过滴鼻感染RSV。除Control组外,其余四组均给予OVA致敏和激发。观察支气管肺泡灌洗液中细胞总数及分类,肺组织HE染色进行炎症病理评分,ELISA法检测支气管肺泡灌洗液中IFN-γ、IL-5、IL-10、IL-12含量和血清OVA特异性IgE水平。
结果各哮喘组BALF中细胞总数,淋巴细胞、嗜酸性粒细胞、中性粒细胞比例均高于Control组(P<0.01或P<0.05),rBCG/RSV/OVA组细胞总数及淋巴细胞比例较rBCG/OVA组显著增高(P<0.01)。肺组织病理评分显示各哮喘组支气管细支气管周围炎症、血管周围炎症、肺泡炎症评分明显高于Control组(P<0.01),rBCG/RSV/OVA组肺泡炎症和血管周围炎症评分明显高于rBCG/OVA组(P<0.01或P<0.05)。各哮喘组BALF中IFN-γ水平明显低于Control组(P<0.01),IL-5水平明显高于Control组(P<0.01或P<0.05),哮喘组之间无显著差异。rBCG/OVA组IL-12水平明显高于其它组(P<0.01),IFN-γ/IL-5比值明显高于其它哮喘组(P<0.01或P<0.05),rBCG/RSV/OVA组IFN-γ/IL-5比值明显低于rBCG/OVA组(P<0.05),各组IL-10水平无统计学差异。各哮喘组血清OVA特异性IgE水平明显高于Control组(P<0.01),而哮喘组之间无显著差异。
结论rBCG新生期接种可促进实验性哮喘小鼠肺部IL-12产生,但RSV感染后IL-12产生受到抑制,肺部炎症细胞浸润和组织病理改变明显,rBCG新生期接种不能对抗RSV对BCG保护哮喘的干扰。RSV逆转rBCG抗哮喘作用是否也和IL-17密切相关,还需深入研究,
Background and Objective Asthma is one of the most common chronic inflammation diseases of airway. So far, prevention and treatment of asthma have not met WHO’s demands of primary prevention. Balillus Calmette-Guerin(BCG) is an attenuated live vaccine which has been broadly vaccinated in developing countries including China. Animal experiments have showed BCG can induce Th1 immunity response and inhibit the development of asthma, but that has not been proved in human being. Our previous researches have showed that neonatal BCG could attenutate airway inflammation and AHR in OVA-induced murine model of asthma. However, RSV infection could reverse the anti-asthma effects of BCG vaccination.
These years mycobacterium has been recombinated via molecular biology techniques and it can express IL-12. It is worth to study to learn if recombinant BCG vaccination can induce Th1 immunity response and interrupt reversed anti-asthma effect of RSV infection.
Th17 cell is a new Th cells subset recently discovered, producing IL-17. Increased lever of IL-17 has been found both in human asthmatics and in animal models of asthma, which suggest IL-17 plays an important role in the pathogenesis of asthma. It is meaningful to know whether neonatal BCG vaccination can reduce the lever of IL-17 induced by OVA and whether RSV infection interrupts anti-asthma effect of BCG via IL-17.
Our purpose is to know the role of IL-17 in the pathogenesis of experimental asthma and in the reversed anti-asthma effect of RSV infection and whether neonatal recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection so that anti-asthma. This is very helpful to understand the mechanism of reversed anti-asthma effect of RSV infection and is the basis of developing new anti-asthma vaccination.
Part I. Impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccination
Objective To explore the role of IL-17 in the pathogenesis of experimental asthma and impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccination
Methods Neonatal BALB/c mice were divided into five groups: Control, OVA, BCG/OVA, RSV/OVA, BCG/RSV/OVA groups. Neonatal mice were vaccinated with BCG in BCG/OVA and BCG/RSV/OVA groups. Mice of RSV/OVA, BCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.
Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group(P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in BCG/RSV/OVA group were statistically increased compared with BCG/OVA group(P<0.01 or P<0.05). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). All groups infected RSV and OVA group had severer perivasculitis and alveolifis than BCG/OVA group(P<0.01 or P<0.05), while there was no significant difference among themselves. The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group (P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-17 was significantly higher in all asthmatic groups except BCG/OVA group compared with control groups (P<0.05). The level of IL-17 in BCG/RSV/OVA group was statistically increased compared with BCG/OVA group. The level of IL-10 and IL-12 in BALF among all groups was respectively no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01), while no significant difference between all OVA-induced groups.
Conclusions IL-17 played the role in the pathogenesis of asthma. Treatment of neonatal BALB/c mice with BCG can reduce the level of IL-17 in BALF and can attenuate lung inflammation, but this effect can be reversed by RSV infection. It may be led to following increasing IL-17 of airway on RSV infection, and the exact mechanism need to be further studied.
Part II. Combined effects of neonatal recombinant Bacillus Calmette-Guerin vaccination secreting IL-12 and RSV infection on OVA-induced murine model of asthma
Objective To explore whether neonatal IL-12 recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection or not.
Methods Neonatal BALB/c mice were divided into five groups: Control, OVA, rBCG/OVA, RSV/OVA, rBCG/RSV/OVA groups. Neonatal mice were vaccinated with rBCG only in rBCG/OVA and rBCG/RSV/OVA groups. Mice of RSV/OVA, rBCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.
Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group (P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in rBCG/RSV/OVA group were statistically increased compared with rBCG/OVA group ((P<0.01). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). rBCG/RSV/OVA group had severer perivasculitis and alveolifis than rBCG/OVA group (P<0.01 or P<0.05). The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group(P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-12 was significantly higher in rBCG/OVA group compared with other groups (P<0.01). The ratio of IFN-γ/IL-5 in in rBCG/OVA group was statistically increased compared with other asthmatic group (P<0.01 or P<0.05). The level of IL-10 in BALF among all groups was no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01),while no significant difference among all OVA-induced groups.
Conclusions Neonatal recombinant BCG vaccination secreting IL-12 can improve the lever of IL-12 in the lung of asthmatic mice. However, IL-12 can be inhibited and infiltration of inflammatory cells and pathological changes of lung are significant on RSV infection. Neonatal recombinant BCG vaccination secreting IL-12 can not interrupt reversed anti-asthma effect of RSV infection. It is still needed to study whether reversed anti-asthma effect of RSV infection is associated with IL-17 and this is helpful to develop more effective vaccination of anti-asthma.
引文
[1]李睿,刘恩梅,杨锡强等.卡介苗新生期接种和呼吸道合胞病毒感染对小鼠实验性哮喘的联合作用[J].中华儿科杂志,2006,44(6):420-424.
[2]林立,李昌崇.呼吸道合胞病毒感染发病机制[J].中华儿科杂志,2006,44(9):673-675.
[3] Larra?aga CL, Ampuero SL, Luchsinger VF, et al. Impaired immune response in severe human lower tract respiratory infection by respiratory syncytial virus[J]. Pediatr Infect Dis J. 2009 Oct;28(10):867-73.
[4] Hung LY, Velichko S, Huang F, et al. Regulation of airway innate and adaptive immune responses: the IL-17 paradigm[J]. Crit Rev Immunol. 2008; 28(4):269-79.
[5] Peebles RS Jr, Sheller JR, Johnson JE, et al. Respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice[J]. J Med Virol. 1999 Feb;57(2):186-92.
[6]周娟,胡敏,王莉佳等.普通小鼠和裸鼠呼吸道合胞病毒感染模型比较[J].重庆医科大学学报,2006,31(5):631-635
[7] Shen HH, Zhang GS, Wang PL. Mycobacterium bovis-Bacillus Calmette-Guerin and asthma[J]. Chin Med J (Engl). 2005, Jun 5;118(11):942-7.
[8] Barlan IB, Bahceciler N, Akdis M, et al. Role of bacillus Calmette-Guérin as an immunomodulator for the prevention and treatment of allergy and asthma[J]. Curr Opin Allergy Clin Immunol. 2005 Dec;5(6):552-7.
[9] Ran D. Balicer, Itamar Grotto, Marc Mimouni et al. Is Childhood Vaccination Associated With Asthma? A Meta-analysis of Observational Studies[J]. Pediatrics. 2007, 120:1269-1277.
[10] Mejías A, Chávez-Bueno S, Jafri HS, et al. Respiratory syncytial virus infections: old challenges and new opportunities[J]. Pediatr Infect Dis J. 2005 Nov;24(11 Suppl):S189-96.
[11] Sigurs N. A cohort of children hospitalised with acute RSV bronchiolitis: impact on later respiratory disease[J]. Paediatr Respir Rev. 2002 Sep;3(3):177-83
[12]Sigurs N, Bjarnason R, Sigurbergsson F et al. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7[J]. Am J Respir Crit Care Med. 2000 May;161(5):1501-7
[13]Schauer U, Hoffjan S, Bittscheidt J, et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life[J]. Eur Respir J. 2002 Nov;20(5):1277-83
[14]Chen Dong. TH17 cells in development: an updated view of their molecular identity and genetic programming[J]. Nat Rev Immunol. 2008 May;8(5):337-48.
[15] Afshar R, Medoff BD, Luster AD. Allergic asthma: a tale of many T cells[J]. Clin Exp Allergy. 2008 Dec;38(12):1847-57.
[16] Chávez-Bueno S, Mejías A, Gómez AM, et al. Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: an experimental murine model[J]. Virol J. 2005 May 25;2:46.
[17]Jafri HS, Chavez-Bueno S, Mejias A, et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice[J]. J Infect Dis. 2004 May 15;189 (10):1856-65.
[18]Bettelli E, Korn T, Oukka M, et al. Induction and effector functions of T(H)17 cells[J]. Nature. 2008 Jun 19;453(7198):1051-7
[19]Chuanwang Song, Liqiong Luo, Zhang Lei, Li B, Liang Z, Liu G, et al. IL-17-Producing Alveolar Macrophages Mediate Allergic Lung Inflammation Related to Asthma[J]. J Immunol. 2008; 181;6117-6124.
[1]何云.T细胞水平干预防治哮喘的临床和实验研究[D].重庆:重庆医科大学附属儿童医院儿科研究所免疫室,2008
[2] Bermúdez-Humarán LG, Cortes-Perez NG, Ah-Leung S, et al. Current prophylactic and therapeutic uses of a recombinant Lactococcus lactis strain secreting biologically active interleukin-12[J]. J Mol Microbiol Biotechnol. 2008;14(1-3):80-9.
[3]Bryan SA, O'Connor BJ, Matti S, et al. Effects of recombinant human interleukin-12 on eosinophils, airway hyper-responsiveness, and the late asthmatic response[J]. Lancet, 2000,356:2149-53.
[4] Wu C, Yang G, Bermudez-Humaran LG, et al. Immunomodulatory effects of IL-12 secreted by Lactococcus lactis on Th1/Th2 balance in ovalbumin (OVA)-induced asthma model mice[J]. Int Immunopharmacol, 2006,6:610-5.
[5] Pinto RA, Arredondo SM, Bono MR, et al. T helper 1/T helper 2 cytokine imbalance in respiratory syncytial virus infection is associated with increased endogenous plasma cortisol[J]. Pediatrics. 2006 May;117(5):e878-86.
[6] Vieira PL, Kaliński P, Wierenga EA, et al. Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential[J]. J Immunol. 1998 Nov 15;161(10):5245-51.
[7] Jartti T, Paul-Anttila M, Lehtinen P, et al. Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study[J]. Respir Res. 2009 Sep 25;10:85.
[8] Dodd J, Riffault S, Kodituwakku JS, et al. Pulmonary V gamma 4+ gamma delta T cells have proinflammatory and antiviral effects in viral lung disease[J]. J Immunol. 2009 Jan 15;182(2):1174-81.
[9] Ostler T, Hussell T, Surh CD, et al. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus[J]. Eur J Immunol. 2001 Sep;31(9):2574-82.
[10] DiNapoli JM, Murphy BR, Collins PL, et al. Impairment of the CD8+ T cell response in lungs following infection with human respiratory syncytial virus is specific to the anatomical site rather than the virus, antigen, or route of infection[J]. Virol J. 2008 Sep 24;5:105.
[11] Durrant DM, Metzger DW. IL-12 can alleviate Th17-mediated allergic lung inflammation through induction of pulmonary IL-10 expression[J]. Mucosal Immunol. 2010 Mar 17. [Epub ahead of print]
[1] Welliver RC. Respiratory syncytial virus and other respiratory viruses. Pediatr Infect Dis J. 2003 Feb;22(2 Suppl):S6-12
[2] Giorgio Trinchieri. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003 Feb;3(2):133-46
[3] Legg JP, Hussain IR, Warner JA, et al. Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med. 2003 Sep 15;168(6):633-9
[4]李睿,刘恩梅,杨锡强等,新生期小鼠卡介苗接种和呼吸道合胞病毒感染对肺部炎症和免疫状态的影响.临床儿科杂志.2006;24(1):28-30
[5] Sigurs N. A cohort of children hospitalised with acute RSV bronchiolitis: impact on later respiratory disease. Paediatr Respir Rev. 2002 Sep;3(3):177-83
[6] Sigurs N, Bjarnason R, Sigurbergsson F et al. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7. Am J Respir Crit Care Med. 2000 May;161(5):1501-7
[7] Schauer U, Hoffjan S, Bittscheidt J, et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life. Eur Respir J. 2002 Nov;20(5):1277-83
[8] Stein RT, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet. 1999 Aug 14;354(9178):541-5
[9] Kaneko H, Matsui E, Asano T, et al. Suppression of IFN-gamma production in atopic group at the acute phase of RSV infection. Pediatr Allergy Immunol.2006: ;17:370-375
[10]MarionBarends, AnitaBoelen, LiadeRond, et al. Respiratory Syncytial Virus Enhances Respiratory Allergy in Mice Despite the Inhibitory Effect of Virus-Induced Interferon-γ. Journal of Medical Virology .2003;69:156-162
[11] Naseer T, Minshall EM, Leung DY, et al. Expression of IL-12 and IL-13 mRNA in asthma and their modulation in response to steroid therapy. Am J Respir Crit CareMed. 1997 Mar;155(3):845-51
[12] Chou CC, Huang MS, Hsieh KH, et al. Reduced IL-12 level correlates with decreased IFN-gamma secreting T cells but not natural killer cell activity in asthmatic children[J]. Ann Allergy Asthma Immunol. 1999 May;82(5):479-84
[13] Ricardo A. Pinto, Sonia M. Arredondo, Maria R. Bono, et al. T Helper 1/T Helper 2 Cytokine Imbalance in Respiratory Syncytial Virus Infection Is Associated With Increased Endogenous Plasma Cortisol. Pediatrics.2006;117;878-886
[14] Tang YW, Graham BS. Interleukin-12 treatment during immunization elicits a T helper cell type 1-like immune response in mice challenged with respiratory syncytial virus and improves vaccine immunogenicity. J Infect Dis. 1995 Sep;172(3):734-8
[15] Tracy Hussell, Usman Khan, Peter Openshaw. IL-12 Treatment Attenuates T Helper Cell Type 2 and B Cell Responses but Does Not Improve Vaccine-Enhanced Lung Illness. J Immunol. 1997 Jul 1;159(1):328-34
[16] Stephan Ehl, Ruth Bischoff, Tobias Ostler et al. The role of Toll-like receptor 4 versus interleukin-12 inimmunity to respiratory syncytial virus. Eur.J.Immunol. 2004.34:1146-1153
[17]鲍一笑,吕婕,董晓艳等. IL-12P40(-/-)小鼠感染呼吸道合胞病毒后肺部的免疫炎症反应.临床儿科杂志2006;24(6):507-511
[18] Marion Barends, Anita Boelen, Liade Rond, et al. Respiratory Syncytial Virus Enhances Respiratory Allergy in Mice Despite the Inhibitory Effect of Virus-Induced Interferon-γ. Journal of Medical Virology. 2003;69:156-162
[2]林立,李昌崇.呼吸道合胞病毒感染发病机制[J].中华儿科杂志,2006,44(9):673-675.
[3] Larra?aga CL, Ampuero SL, Luchsinger VF, et al. Impaired immune response in severe human lower tract respiratory infection by respiratory syncytial virus[J]. Pediatr Infect Dis J. 2009 Oct;28(10):867-73.
[4] Hung LY, Velichko S, Huang F, et al. Regulation of airway innate and adaptive immune responses: the IL-17 paradigm[J]. Crit Rev Immunol. 2008; 28(4):269-79.
[5] Peebles RS Jr, Sheller JR, Johnson JE, et al. Respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice[J]. J Med Virol. 1999 Feb;57(2):186-92.
[6]周娟,胡敏,王莉佳等.普通小鼠和裸鼠呼吸道合胞病毒感染模型比较[J].重庆医科大学学报,2006,31(5):631-635
[7] Shen HH, Zhang GS, Wang PL. Mycobacterium bovis-Bacillus Calmette-Guerin and asthma[J]. Chin Med J (Engl). 2005, Jun 5;118(11):942-7.
[8] Barlan IB, Bahceciler N, Akdis M, et al. Role of bacillus Calmette-Guérin as an immunomodulator for the prevention and treatment of allergy and asthma[J]. Curr Opin Allergy Clin Immunol. 2005 Dec;5(6):552-7.
[9] Ran D. Balicer, Itamar Grotto, Marc Mimouni et al. Is Childhood Vaccination Associated With Asthma? A Meta-analysis of Observational Studies[J]. Pediatrics. 2007, 120:1269-1277.
[10] Mejías A, Chávez-Bueno S, Jafri HS, et al. Respiratory syncytial virus infections: old challenges and new opportunities[J]. Pediatr Infect Dis J. 2005 Nov;24(11 Suppl):S189-96.
[11] Sigurs N. A cohort of children hospitalised with acute RSV bronchiolitis: impact on later respiratory disease[J]. Paediatr Respir Rev. 2002 Sep;3(3):177-83
[12]Sigurs N, Bjarnason R, Sigurbergsson F et al. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7[J]. Am J Respir Crit Care Med. 2000 May;161(5):1501-7
[13]Schauer U, Hoffjan S, Bittscheidt J, et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life[J]. Eur Respir J. 2002 Nov;20(5):1277-83
[14]Chen Dong. TH17 cells in development: an updated view of their molecular identity and genetic programming[J]. Nat Rev Immunol. 2008 May;8(5):337-48.
[15] Afshar R, Medoff BD, Luster AD. Allergic asthma: a tale of many T cells[J]. Clin Exp Allergy. 2008 Dec;38(12):1847-57.
[16] Chávez-Bueno S, Mejías A, Gómez AM, et al. Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: an experimental murine model[J]. Virol J. 2005 May 25;2:46.
[17]Jafri HS, Chavez-Bueno S, Mejias A, et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice[J]. J Infect Dis. 2004 May 15;189 (10):1856-65.
[18]Bettelli E, Korn T, Oukka M, et al. Induction and effector functions of T(H)17 cells[J]. Nature. 2008 Jun 19;453(7198):1051-7
[19]Chuanwang Song, Liqiong Luo, Zhang Lei, Li B, Liang Z, Liu G, et al. IL-17-Producing Alveolar Macrophages Mediate Allergic Lung Inflammation Related to Asthma[J]. J Immunol. 2008; 181;6117-6124.
[1]何云.T细胞水平干预防治哮喘的临床和实验研究[D].重庆:重庆医科大学附属儿童医院儿科研究所免疫室,2008
[2] Bermúdez-Humarán LG, Cortes-Perez NG, Ah-Leung S, et al. Current prophylactic and therapeutic uses of a recombinant Lactococcus lactis strain secreting biologically active interleukin-12[J]. J Mol Microbiol Biotechnol. 2008;14(1-3):80-9.
[3]Bryan SA, O'Connor BJ, Matti S, et al. Effects of recombinant human interleukin-12 on eosinophils, airway hyper-responsiveness, and the late asthmatic response[J]. Lancet, 2000,356:2149-53.
[4] Wu C, Yang G, Bermudez-Humaran LG, et al. Immunomodulatory effects of IL-12 secreted by Lactococcus lactis on Th1/Th2 balance in ovalbumin (OVA)-induced asthma model mice[J]. Int Immunopharmacol, 2006,6:610-5.
[5] Pinto RA, Arredondo SM, Bono MR, et al. T helper 1/T helper 2 cytokine imbalance in respiratory syncytial virus infection is associated with increased endogenous plasma cortisol[J]. Pediatrics. 2006 May;117(5):e878-86.
[6] Vieira PL, Kaliński P, Wierenga EA, et al. Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential[J]. J Immunol. 1998 Nov 15;161(10):5245-51.
[7] Jartti T, Paul-Anttila M, Lehtinen P, et al. Systemic T-helper and T-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study[J]. Respir Res. 2009 Sep 25;10:85.
[8] Dodd J, Riffault S, Kodituwakku JS, et al. Pulmonary V gamma 4+ gamma delta T cells have proinflammatory and antiviral effects in viral lung disease[J]. J Immunol. 2009 Jan 15;182(2):1174-81.
[9] Ostler T, Hussell T, Surh CD, et al. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus[J]. Eur J Immunol. 2001 Sep;31(9):2574-82.
[10] DiNapoli JM, Murphy BR, Collins PL, et al. Impairment of the CD8+ T cell response in lungs following infection with human respiratory syncytial virus is specific to the anatomical site rather than the virus, antigen, or route of infection[J]. Virol J. 2008 Sep 24;5:105.
[11] Durrant DM, Metzger DW. IL-12 can alleviate Th17-mediated allergic lung inflammation through induction of pulmonary IL-10 expression[J]. Mucosal Immunol. 2010 Mar 17. [Epub ahead of print]
[1] Welliver RC. Respiratory syncytial virus and other respiratory viruses. Pediatr Infect Dis J. 2003 Feb;22(2 Suppl):S6-12
[2] Giorgio Trinchieri. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003 Feb;3(2):133-46
[3] Legg JP, Hussain IR, Warner JA, et al. Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am J Respir Crit Care Med. 2003 Sep 15;168(6):633-9
[4]李睿,刘恩梅,杨锡强等,新生期小鼠卡介苗接种和呼吸道合胞病毒感染对肺部炎症和免疫状态的影响.临床儿科杂志.2006;24(1):28-30
[5] Sigurs N. A cohort of children hospitalised with acute RSV bronchiolitis: impact on later respiratory disease. Paediatr Respir Rev. 2002 Sep;3(3):177-83
[6] Sigurs N, Bjarnason R, Sigurbergsson F et al. Respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7. Am J Respir Crit Care Med. 2000 May;161(5):1501-7
[7] Schauer U, Hoffjan S, Bittscheidt J, et al. RSV bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life. Eur Respir J. 2002 Nov;20(5):1277-83
[8] Stein RT, Sherrill D, Morgan WJ, et al. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet. 1999 Aug 14;354(9178):541-5
[9] Kaneko H, Matsui E, Asano T, et al. Suppression of IFN-gamma production in atopic group at the acute phase of RSV infection. Pediatr Allergy Immunol.2006: ;17:370-375
[10]MarionBarends, AnitaBoelen, LiadeRond, et al. Respiratory Syncytial Virus Enhances Respiratory Allergy in Mice Despite the Inhibitory Effect of Virus-Induced Interferon-γ. Journal of Medical Virology .2003;69:156-162
[11] Naseer T, Minshall EM, Leung DY, et al. Expression of IL-12 and IL-13 mRNA in asthma and their modulation in response to steroid therapy. Am J Respir Crit CareMed. 1997 Mar;155(3):845-51
[12] Chou CC, Huang MS, Hsieh KH, et al. Reduced IL-12 level correlates with decreased IFN-gamma secreting T cells but not natural killer cell activity in asthmatic children[J]. Ann Allergy Asthma Immunol. 1999 May;82(5):479-84
[13] Ricardo A. Pinto, Sonia M. Arredondo, Maria R. Bono, et al. T Helper 1/T Helper 2 Cytokine Imbalance in Respiratory Syncytial Virus Infection Is Associated With Increased Endogenous Plasma Cortisol. Pediatrics.2006;117;878-886
[14] Tang YW, Graham BS. Interleukin-12 treatment during immunization elicits a T helper cell type 1-like immune response in mice challenged with respiratory syncytial virus and improves vaccine immunogenicity. J Infect Dis. 1995 Sep;172(3):734-8
[15] Tracy Hussell, Usman Khan, Peter Openshaw. IL-12 Treatment Attenuates T Helper Cell Type 2 and B Cell Responses but Does Not Improve Vaccine-Enhanced Lung Illness. J Immunol. 1997 Jul 1;159(1):328-34
[16] Stephan Ehl, Ruth Bischoff, Tobias Ostler et al. The role of Toll-like receptor 4 versus interleukin-12 inimmunity to respiratory syncytial virus. Eur.J.Immunol. 2004.34:1146-1153
[17]鲍一笑,吕婕,董晓艳等. IL-12P40(-/-)小鼠感染呼吸道合胞病毒后肺部的免疫炎症反应.临床儿科杂志2006;24(6):507-511
[18] Marion Barends, Anita Boelen, Liade Rond, et al. Respiratory Syncytial Virus Enhances Respiratory Allergy in Mice Despite the Inhibitory Effect of Virus-Induced Interferon-γ. Journal of Medical Virology. 2003;69:156-162