The Role Of Cytokines In The Developing Immune System

细胞因子在免疫系统发育中的作用

• 批准号: 6681928
• 负责人: Dori R Germolec
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6681928
• 关键词: age difference; apoptosis; autoantibody; autoimmunity; cellular pathology; cytokine; dioxins; disease /disorder model; endotoxins; environmental exposure; environmental toxicology; gender difference; gene environment interaction; glomerulonephritis; hemotoxin; immune system; interferon gamma; laboratory rat; major histocompatibility complex; oxidative stress; pathologic process; prenatal stress; respiratory infections; sudden infant death syndrome; urinalysis

It has been suggested that SIDS may be a patho-physiological response elicited by combinations of microbial products and/or other environmental factors such as environmental tobacco smoke (ETS) at a time when the developing immune system is more vulnerable to the effects of inflammatory mediators. We have developed an animal model to mimic dual infection, using a non-lethal strain of Influenza A virus and a sub-lethal dose of endotoxin. We hypothesized that exposure to Influenza A virus prior to bacterial infection can mimic the pathology of SIDS and alter cytokine-mediated T-helper cell responses. More specifically, we postulate that at critical windows of immune system development exposure to Influenza A virus changes the regulation of the immune response to bacterial products by altering the kinetics of expression and increasing the production of IL-6, IL-10, TNFa and other inflammatory cytokines that normally promote a protective response. The immature immune system is either unable to tolerate these changes and/or unable to adequately coordinate the response resulting in death. We are using this model to examine the relationship between environmental risk factors for sudden unexplained death in infants including age, prone sleeping and exogenous factors such as ETS or mycotoxins. Rat adapted Influenza A virus (RAIV), developed from Influenza A/Port-Chalmers/1/73 (H3N2) virus was used for infectivity in these experiments. Using time points that correspond to critical points in immunological development and the peak age for SIDS in humans (2-6 months), preliminary studies were conducted to ascertain optimal timing between doses and endotoxin levels to model the mortality and pathology of SIDS. These studies established that inoculation with RAIV at 10 days of age followed by endotoxin 0.2 mg/kg 2 days post RAIV caused mortality with the lowest morbidity. These elements became the defining parameters of the model. Mortality only occurred when specific criteria such as timing between infectious insults and developmental age of the pup were met and age was a key risk factor related to mortality in the model. Adult rats were examined following similar challenges and no mortality was observed. To examine the mechanisms underlying this mortality, a series of experiments were conducted to examine pathology and evaluate immune parameters 2-8 hours following endotoxin administration. Immunophenotyping was performed on cells isolated from the spleen, blood and bronchoalveolar lavage fluid. Tissues were examined for histologic evidence of SIDS-like pathology and inflammatory mediator gene expression. Levels of inflammatory mediators in serum and lavage fluid were also measured. The data from these studies suggests that mortality is due to a rapid systemic shock event rather than lung specific damage. Gross pathologic findings such as lung petechiae and liquid blood around the heart on necropsy were consistent with those seen in infants dying of SIDS. Histopathologic lesions including sub-endocardial hemorrhage and mild cortical thymocyte necrosis were found with greater severity and frequency in dually challenged animals. Vascular congestion in the liver and spleen was consistent with endotoxin exposure and with the release of inflammatory cytokines and reactive nitrogen species (RNS) that may have led to circulatory collapse. Endotoxin induced an immediate release of IL-6 and IL-10 in virally infected animals, as compared to age-matched endotoxin only controls. Macrophage numbers in RAIV-inoculated animals were significantly elevated in the spleen at time of death. A statistically significant decrease in serum IFN-gamma levels was observed in animals that received endotoxin and RAIV. Cytokine mRNA expression was dysregulated in the lungs and livers of the RAIV infected animals. We have defined similarities of SIDS pathology in human infants and that seen in this model and examined basic immune factors associat ed with dual challenge. These studies suggest that the developing immune system can be primed to respond in an exaggerated way to a second immune challenge resulting in unexpected death. It is well documented that autoimmune disease susceptibility is largely dependent upon the presence of particular major histocompatibility complex (MHC) genotypes, however, development of disease symptoms also appears to require critical environmental factors, such as bacterial or viral infections or chemical exposure. MRL/lpr mice exhibit severe renal necrosis (glomerulonephritis) similar to that observed in human patients with systemic lupus erythematosus, with females normally presenting circulating autoantibodies against nucleoprotein particles and subsequent renal complement-antibody immune complexes at approximately 14-16 weeks of age vs. males at 20-22 weeks. Neonatal exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to alter thymocyte maturation and significantly impair immune function in adult animals. We hypothesized that prenatal exposure to TCDD may increase or exacerbate post-natal development of autoimmune disease in genetically-prone MRL/lpr mice. We have observed significant, dose-dependent increases in anti-ssDNA, urinary protein, and renal abnormalities in TCDD-exposed female MRL/lpr mice as compared to controls at six, ten, and 12 weeks of age, respectively. Similar results were noted in TCDD-exposed males at eight, ten and 12 weeks of age.

已经表明，SIDS可能是由微生物产物和/或其他环境因素（例如环境烟草烟雾（ETS））的组合引起的病理生理反应，此时发育中的免疫系统更容易受到炎症介质的影响。我们已经开发了一种动物模型来模拟双重感染，使用非致死性甲型流感病毒株和亚致死剂量的内毒素。我们假设，在细菌感染前暴露于甲型流感病毒可以模拟SIDS的病理学，并改变精氨酸介导的T辅助细胞反应。更具体地，我们假设在免疫系统发育的关键窗口，暴露于甲型流感病毒通过改变表达动力学和增加IL-6、IL-10、TNF α和通常促进保护性应答的其他炎性细胞因子的产生来改变对细菌产物的免疫应答的调节。不成熟的免疫系统要么无法耐受这些变化，要么无法充分协调导致死亡的反应。我们正在使用这个模型来研究婴儿不明原因猝死的环境风险因素之间的关系，包括年龄，俯卧和外源性因素，如ETS或真菌毒素。 在这些实验中，使用从甲型流感/Port-Chalmers/1/73（H3 N2）病毒开发的大鼠适应的甲型流感病毒（RAIV）用于感染性。使用对应于免疫发育中的临界点和人类SIDS的峰值年龄（2-6个月）的时间点，进行初步研究以确定剂量和内毒素水平之间的最佳时间，以模拟SIDS的死亡率和病理学。这些研究证实，在10日龄时接种RAIV，然后在RAIV后2天接种内毒素0.2 mg/kg，导致死亡率最低。这些元素成为模型的定义参数。仅当满足特定标准（如感染性损伤之间的时间和幼仔的发育年龄）时才会发生死亡，并且年龄是模型中与死亡率相关的关键风险因素。在类似的攻击后检查成年大鼠，未观察到死亡。 为了检查导致这种死亡的机制，进行了一系列实验以检查病理学并评估内毒素施用后2-8小时的免疫参数。对从脾、血液和支气管肺泡灌洗液中分离的细胞进行免疫表型分析。检查组织中SIDS样病理学和炎症介质基因表达的组织学证据。同时测定血清和灌洗液中炎症介质的水平。这些研究的数据表明，死亡率是由于快速的全身性休克事件，而不是肺特异性损伤。大体病理结果，如肺瘀点和心脏周围的液体血液尸检是一致的婴儿死于SIDS。组织学病变包括内膜下出血和轻度皮质胸腺细胞坏死，发现双重攻击动物的严重程度和频率更高。肝脏和脾脏中的血管充血与内毒素暴露以及可能导致循环衰竭的炎性细胞因子和活性氮物质（RNS）的释放一致。与年龄匹配的仅内毒素对照相比，内毒素诱导病毒感染动物中IL-6和IL-10的立即释放。巨噬细胞数量在RAIV接种的动物在脾脏死亡时显着升高。在接受内毒素和RAIV的动物中观察到血清IFN-γ水平的统计学显著降低。RAIV感染动物的肺和肝脏中细胞因子mRNA表达失调。我们已经确定了人类婴儿SIDS病理学的相似性，并在该模型中观察到，并检查了与双重攻击相关的基本免疫因子艾德。这些研究表明，发育中的免疫系统可能会对第二次免疫挑战产生过度反应，导致意外死亡。 有充分的证据表明，自身免疫性疾病的易感性在很大程度上取决于特定的主要组织相容性复合体（MHC）基因型的存在，然而，疾病症状的发展似乎也需要关键的环境因素，如细菌或病毒感染或化学暴露。MRL/lpr小鼠表现出与在患有系统性红斑狼疮的人类患者中观察到的相似的严重肾坏死（肾小球肾炎），雌性小鼠通常在约14-16周龄时呈现针对核蛋白颗粒的循环自身抗体，随后呈现肾补体-抗体免疫复合物，而雄性小鼠在20-22周龄时呈现。新生儿暴露于环境污染物2，3，7，8-四氯二苯并对二恶英（TCDD）已被证明会改变胸腺细胞的成熟，并显着损害成年动物的免疫功能。我们假设，产前暴露于TCDD可能会增加或加剧出生后的自身免疫性疾病的遗传倾向MRL/lpr小鼠的发展。我们已经观察到显着的，剂量依赖性增加抗ssDNA，尿蛋白，和肾脏异常TCDD暴露的女性MRL/lpr小鼠相比，在6，10，12周龄，分别。在8周、10周和12周大时接触TCDD的雄性中也观察到类似的结果。