Inhibition of necroptosis during inflamm-aging and pneumonia

坏死性下垂是一种新发现的细胞死亡途径。受受体相互作用蛋白激酶(RIP)1调节 和RIP3，坏死性下垂涉及效应分子MLKL故意破坏细胞膜。 坏死性上睑下垂是一种炎症性疾病，是由于充当警报器的细胞质成分的释放所致。坏死性下垂是 被认为放大和促进导致慢性病的炎症性循环。坏死性下垂有什么作用 在衰老过程中发挥作用是未知的。例如，它是否会导致炎症老化？ 肺炎链球菌和其他呼吸道病原体产生的致孔毒素已被证明 引发肺细胞坏死性下垂，这是所观察到的大部分损伤的原因。老年人来了 特别容易感染肺炎，下呼吸道感染是第四大死因 在那些65岁的≥中。因此，抑制坏死性下垂是保护脆弱人群的潜在途径。 个人，如老年人，避免细菌性肺炎期间发生的肺损伤。 我们假设，随着年龄的增长，坏死性下垂导致的细胞死亡增加，从而导致炎症-- 衰老。此外，阻止坏死性下垂可以保护老年人免受肺炎期间的肺损伤。在这里，我们将 利用我们在衰老、坏死性下垂和细菌性肺炎方面的专业知识来验证这些假设并改进 我们对坏死性下垂及其在衰老过程中的影响的理解。我们会： 目的1.确定坏死性下垂在炎症衰老和年龄相关性功能减退中的作用。我们有 缺乏RIP3和MLKL的小鼠不能发生坏死性下垂。我们将比较促炎细胞因子 以及WT、杂合子和KO小鼠在6、14和24个月龄时的血清中的警蛋白谱。我们会 同时检查使用两种不同的坏死下垂抑制剂治疗7天的老年WT小鼠。对于所有的老鼠，我们都会 检测NFkB和MAPK在胸腺、肺、心、脾、肝、肾、脑和 免疫细胞(单核细胞、B细胞、T细胞)用流式细胞仪检测。我们还将 纵向测量活动、步态和肌肉力量，以了解阻止坏死性下垂是否会影响健康状况。 目的2.确定阻断坏死性下垂是否能保护老年动物免受肺炎的侵袭。牙槽骨 6、14和24月龄小鼠的巨噬细胞和骨髓来源的巨噬细胞将被检测其 暴露于致孔毒素肺溶素后发生坏死性下垂的倾向。不同年龄的人 WT、杂合子和MLKL KO小鼠，以及使用坏死下垂抑制剂治疗的WT小鼠将被 气管内感染肺炎溶血素或感染肺炎链球菌。肺损伤和疾病 严重程度将分别通过测量炎症细胞因子、病理和细菌负荷来评估。 这项建议是对PAR-14-191：T1翻译研究的回应：针对 年龄相关性疾病的预防和治疗。我们将确定坏死性下垂对 炎症-老化，并了解是否阻止坏死性下垂预防炎症-老化相关的衰退和肺炎。

Necroptosis is a newly discovered cell death pathway. Regulated by receptor-interacting protein kinase (RIP)1 and RIP3, necroptosis involves the purposeful disruption of the cell membrane by the effecter molecule MLKL. Necroptosis is inflammatory due to the release of cytoplasmic components that act as alarmins. Necroptosis is thought to amplify and promote inflammatory circles that contribute to chronic disease. What role necroptosis plays during aging is unknown. For example, does it contribute to inflamm-aging? Pore-forming toxins produced by Streptococcus pneumoniae and other airway pathogens have been shown to trigger lung cell necroptosis and this is responsible for much of the injury that is observed. The elderly are in particular vulnerable to pneumonia, with lower respiratory tract infections being the 4th leading cause of death in those ≥65 years of age. Thus, inhibition of necroptosis is potential way to protect vulnerable individuals, such as the elderly, from the lung damage that occurs during bacterial pneumonia. We hypothesize that cell death by necroptosis increases with advanced age and this contributes to inflamm- aging. Also, that blocking necroptosis can protect the elderly from lung injury during pneumonia. Herein, we will leverage our expertise on aging, necroptosis, and bacterial pneumonia to test these hypotheses and improve our understanding of necroptosis and its impact during aging. We will: AIM 1. Determine the role of necroptosis on inflamm-aging and age-related decline in function. We have mice deficient in RIP3 and MLKL that cannot undergo necroptosis. We will compare pro-inflammatory cytokine and alarmin profiles in serum from WT, heterozygote, and KO mice at 6, 14, and 24 months of age. We will also examine aged WT mice treated for 7 days with two different necroptosis inhibitors. For all mice, we will examine the activation status of NFkB and MAPK in the thymus, lungs, heart, spleen, liver, kidney, brain, and visceral adipose by western and in immune cells (monocytes, B cells, T cells) by flow cytometry. We will also longitudinally measure activity, gait, and muscle strength to learn if blocking necroptosis impacts health status. AIM 2. Determine if blocking necroptosis protects aged animals against pneumonia. Alveolar macrophages and bone marrow derived macrophages from 6, 14 and 24 month old mice will be tested for their propensity to undergo necroptosis following exposure to the pore-forming toxin pneumolysin. Different aged WT, heterozygote, and MLKL KO mice, along with WT mice treated with necroptosis inhibitors will be intratracheally challenged with pneumolysin or infected with S. pneumoniae. Lung damage and disease severity will be assessed by measuring inflammatory cytokines, pathology, and bacterial burden, respectively. This proposal is in response to PAR-14-191:T1 Translational Research: Novel Interventions for Prevention and Treatment of Age-Related Conditions. We will determine the contribution of necroptosis to inflamm-aging and learn if blocking necroptosis protects against inflamm-aging related decline and pneumonia.