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

描述（由申请方提供）：老年人特别容易受到空气中纳米颗粒物（nPM）诱导的心肺病理学的影响。抗氧化酶诱导的转录因子Nrf 2 nPM吸入后，在年轻的，但不是老年小鼠。抗氧化剂诱导的损失与年龄相关的c-Myc和Bach 1的升高相关，这两者都抑制了Nrf 2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增加，基础和响应于nPM暴露。我们推测，Nrf 2抑制蛋白在老化过程中响应nPM的增加是负责抑制抗氧化酶诱导。我们进一步假设，Nrf 2依赖性抗氧化反应在衰老过程中的减少，允许更大的nPM诱导的，NF-κ B调节的炎症细胞因子的增加。原代人支气管上皮细胞（NHBE）以及从4个年龄范围内个体的外周血单核细胞（PBMC）分化而来的M1和M2巨噬细胞将用于模拟人nPM暴露。环境nPM是不同颗粒形状、尺寸和成分的混合物。因此，我们将制造定义的，可重复的模型nPM，其物理化学特征来自高分辨率显微镜和光谱学的环境nPM，来自一个具有最高nPM健康风险的地区。目的1是证明衰老中Nrf 2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和血红素加氧酶-1）的诱导作用的丧失是由Nrf 2抑制蛋白的升高引起的。Bach 1和/或c-Myc将在NHBE或M1或M2巨噬细胞中沉默或过表达，并确定响应于nPM的Nrf 2调节的抗氧化酶的表达。目的2是证明在衰老中诱导Nrf 2调节的抗氧化酶的能力降低显著有助于响应于nPM的炎性细胞因子产生升高。我们将确定沉默或过表达Nrf 2，c-Myc和/或Bach 1对NHBE和M1细胞中促炎细胞因子响应于nPM的NF-κ B活化的影响，这些细胞来自不同年龄的供体。还将检查老化和nPM暴露对M2细胞中TNF α诱导的抗炎性IL-10表达的影响。目的3是证明还原型谷胱甘肽二乙酯（GSH-E）或靶向Bach 1的microRNA（miRNA）可以逆转衰老中诱导型抗氧化防御的衰减。在细胞中转化为谷胱甘肽的GSH-E或Bach 1定向的miRNA将用于不同年龄的NHBE和M1和M2巨噬细胞。将测定基础和nPM诱导的NF-κ B活化和细胞因子诱导。 使用人类模型和代表性的，可重复的nPM将提供一个新的机制框架，以解决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确定减少易感个体中nPM诱导的氧化损伤和炎症的新方法的最终目标至关重要，其中包括不断增加的美国老年人群。

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.