Human models of the particulate-induced inflammatory/antioxidant axis in aging

描述(申请人提供)：老年人特别容易受到空气纳米颗粒(NPM)引起的心肺病变的影响。小鼠吸入NPM后，抗氧化酶由转录因子Nrf2诱导，但不是老年小鼠。抗氧化诱导性的丧失与c-Myc和Bach1的年龄相关，这两种基因都抑制了Nrf2对抗氧化酶转录的调节。在衰老过程中，炎性细胞因子的产生在基础上和对NPM暴露的反应中都会增加。我们假设，在衰老过程中，NRF2抑制蛋白对NPM的反应增加，这是抑制抗氧化酶诱导的原因。我们进一步假设，衰老过程中依赖于Nrf2的抗氧化反应的减少允许NPM诱导的、NF-kB调节的炎性细胞因子的更大增加。从四个年龄段的个体的外周血单核细胞(PBMC)分化出的原代人支气管上皮(NHBE)和M1和M2巨噬细胞将被用来模拟人类NPM暴露。环境NPM是不同颗粒形状、大小和组成的混合物。因此，我们将制造定义的、可重复的模型NPM，具有来自高分辨率显微镜和环境NPM光谱的物理化学特征，该环境NPM来自具有最高NPM健康风险的地区之一。目的1证明衰老过程中Nrf2调节的抗氧化酶(谷氨酸半胱氨酸连接酶，NAD(P)H：苯醌氧化还原酶1和血红素加氧酶-1)的诱导能力的丧失是由于Nrf2抑制蛋白的增加所致。Bach1和/或c-Myc将在NHBE或M1或M2巨噬细胞中沉默或过表达，并确定Nrf2调节的抗氧化酶对NPM的反应。目的2是证明在衰老过程中诱导Nrf2调节的抗氧化酶的能力降低显著地导致NPM反应中炎性细胞因子的产生增加。我们将确定沉默或过度表达Nrf2、c-Myc和/或Bach1在不同年龄的供者的NHBE和M1细胞中对NPM反应中促炎细胞因子NF-kB激活的影响。衰老和NPM暴露对肿瘤坏死因子α诱导M2细胞抗炎IL-10表达的影响也将被检测。目的3是证明针对Bach1的还原型谷胱甘肽二乙酯(GSH-E)或microRNAs(MiRNA)可以逆转衰老过程中诱导的抗氧化防御的减弱。GSH-E在细胞内被转化为谷胱甘肽或Bach1定向的miRNAs，将用于NHBE和不同年龄的M1和M2巨噬细胞。基础和NPM诱导的核因子-kB的激活和细胞因子的诱导将被确定。使用人类模型和代表性的、可复制的NPM将提供一个新的机制框架来解决NPM诱导的炎症、抗氧化防御和衰老之间的联系。获得这一新知识对于实现最终目标至关重要，即找到新的方法来减少NPM在易感人群中引起的氧化损伤和炎症，这些易感人群中的美国老年人口不断增加。

DESCRIPTION (provided by applicant): Elderly individuals are particularly susceptible to cardiopulmonary pathology induced by airborne nanoparticulate matter (nPM). Antioxidant enzymes are induced by the transcription factor Nrf2 upon nPM inhalation in young but not older mice. The loss of antioxidant inducibility correlates with age-related elevation of c-Myc and Bach1, both of which inhibit Nrf2-regulation of antioxidant enzyme transcription. In aging, inflammatory cytokine production increases both basally and in response to nPM exposure. We hypothesize that the increase in Nrf2-inhibitory proteins in response to nPM during aging is responsible for suppressing antioxidant enzyme inducibility. We further hypothesize that the decrease in Nrf2-dependent antioxidant response in aging permits a greater nPM-induced, NF-kB-regulated increase in inflammatory cytokines. Primary human bronchial epithelium (NHBE) and M1 and M2 macrophages differentiated from peripheral blood monocytes (PBMC) from individuals in four age ranges will be used to model human nPM exposure. Ambient nPM is a mixture of different particle shapes, sizes, and compositions. Thus, we will manufacture defined, reproducible model nPM with physico-chemical characteristics derived from high-resolution microscopy and spectroscopy of ambient nPM from a region with one of the highest nPM health risks. Aim 1 is to demonstrate that the loss in Nrf2-regulated inducibility of antioxidant enzymes (glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase 1, and heme oxygenase-1) in aging is caused by elevation of Nrf2- inhibitory proteins. Bach1 and/or c-Myc will be silenced or overexpressed in NHBE or M1 or M2 macrophages, and expression of Nrf2-regulated antioxidant enzymes in response to nPM determined. Aim 2 is to demonstrate that the decreased ability to induce Nrf2-regulated antioxidant enzymes in aging significantly contributes to elevated inflammatory cytokine production in response to nPM. We will determine the effects of silencing or overexpressing Nrf2, c-Myc and/or Bach1 on NF-kB activation of pro-inflammatory cytokines in response to nPM in NHBE and M1 cells from donors of different ages. The effects of aging and nPM exposure on TNF alpha-induced anti-inflammatory IL-10 expression in M2 cells will also be examined. Aim 3 is to demonstrate that reduced glutathione diethyl ester (GSH-E) or microRNAs (miRNA) that target Bach1 can reverse the attenuation of inducible antioxidant defense in aging. GSH-E, which is converted to glutathione in cells or Bach1-directed miRNAs will be used in NHBE and M1 and M2 macrophages of different ages. Basal and nPM-induced NF-kB activation and induction of cytokines will be determined. The use of human models and representative, reproducible nPM will provide a new mechanistic framework to resolve links among nPM-induced inflammation, antioxidant defense and aging. Acquiring this new knowledge is critical to achieving the ultimate goal of identifying novel means to reduce nPM-induced oxidative injury and inflammation in susceptible individuals, among who are the increasing US elderly population.