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

描述（由申请人提供）：老年人特别容易受到空气中纳米颗粒物质（nPM）引起的心肺病理的影响。抗氧化酶在吸入nPM后被转录因子Nrf2诱导，而不是在老年小鼠中。抗氧化诱导能力的丧失与年龄相关的c-Myc和Bach1的升高有关，两者都抑制了nrf2对抗氧化酶转录的调节。在衰老过程中，炎症细胞因子的产生会随着nPM暴露而增加。我们假设nrf2抑制蛋白在衰老过程中对nPM的反应是抑制抗氧化酶诱导的原因。我们进一步假设，衰老过程中nrf2依赖性抗氧化反应的减少允许npm诱导的、nf - kb调节的炎症细胞因子的增加。来自四个年龄段个体的原代人支气管上皮（NHBE）和从外周血单核细胞（PBMC）分化的M1和M2巨噬细胞将用于模拟人类nPM暴露。环境nPM是不同颗粒形状、大小和组成的混合物。因此，我们将制造明确的、可重复的nPM模型，其物理化学特征来自nPM健康风险最高的地区之一的高分辨率显微镜和环境nPM光谱学。目的1是证明Nrf2调节的抗氧化酶（谷氨酸半胱氨酸连接酶、NAD(P)H：醌氧化还原酶1和血红素氧化酶1）在衰老过程中的诱导能力的丧失是由Nrf2抑制蛋白的升高引起的。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调节的抗氧化酶在nPM反应中的表达。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下降显著有助于nPM反应中炎症细胞因子的产生升高。我们将确定沉默或过表达Nrf2、c-Myc和/或Bach1对nPM对NHBE和M1细胞中促炎细胞因子NF-kB激活的影响。衰老和nPM暴露对M2细胞中TNF α诱导的抗炎IL-10表达的影响也将被检测。目的3是证明还原型谷胱甘肽二乙酯（GSH-E）或靶向Bach1的microRNAs （miRNA）可以逆转衰老过程中诱导抗氧化防御的衰减。GSH-E在细胞或bach1定向mirna中转化为谷胱甘肽，将用于不同年龄的NHBE和M1、M2巨噬细胞。基础和npm诱导的NF-kB活化和细胞因子的诱导将被确定。使用人体模型和具有代表性的可重复性nPM将为解决nPM诱导的炎症、抗氧化防御和衰老之间的联系提供一个新的机制框架。获得这一新知识对于实现确定新方法以减少易感个体（其中包括不断增加的美国老年人口）中npm诱导的氧化损伤和炎症的最终目标至关重要。

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.