The immunoregulatory role of Alveolar Macrophages in Chronic Beryllium Disease

项目摘要/摘要： 本研究的目的是确定CD55和JAK/STAT信号转导通路异常在脑出血中的作用。 肺泡巨噬细胞(AM)对慢性铍病(CBD)进展的影响。CBD是一个重要的 器官特异性免疫介导性疾病，以肉芽肿性肺部炎症、纤维化和 死亡，死于终末期呼吸功能不全。因此，CBD仍然是一个重要的公共卫生问题。 这种疾病的前驱疾病(铍敏化；BES)以大约6-8%的比率进展到CBD 每年。尽管BE通过含有HLAGlu69的β-DP分子与致病性疾病结合和递呈 CD4+T细胞为DP2与CBD和BES的遗传连锁提供了解释，这不能解释 BES向CBD的进展。此外，在没有BES或BES证据的BE工人中，高达40%发现了DP2 CBD。因此，在BES向CBD发展的过程中必须涉及其他机制。我们的假设是 CBD AM下调负免疫调节基因CD55及其途径和过表达 正调节激活JAK/STAT通路，增强BE和BE的免疫应答 从BES到CBD的进展。在目标1中，我们将定义AM上的CD55是否在 BES患者AM对BE的免疫应答。具体来说，使用CD55的过度表达 使用腺病毒过表达系统和激动型抗体的基因或使用CD55基因的减少 腺病毒敲除系统和中和抗体的CD55，我们将确定改变的影响 CD55对相应功能变化的肿瘤坏死因子-α的产生、表型(CD16、CD40、CD80、CD86、HLA... DR)、JAK/STAT激活和BeLPT比较CBD、BES和对照。最后，使用rna-seq，我们将 与BES相比，调查CBD中CD55下调相关的调控网络； 目的2，我们将确定JAK/STAT通路是否是AM免疫反应的正调节因子 与BES相比，CBD受试者AM中AMS的表达增加。具体来说，来自CBD的AM将是 与BES和对照组相比，评估JAK/STAT的过表达 使用腺病毒过表达系统的途径基因或减少JAK/STAT途径基因 使用腺病毒敲除系统和药理JAK1-3抑制剂对BE免疫应答的影响。我们 将评估肿瘤坏死因子-α的产生、表型(CD16、CD40、CD80、CD86、人类白细胞抗原DR)和BeLPT的变化。 最后，使用rna-seq，我们将定义与过度激活有关的调控网络。 JAK/STAT通路。在目标3中，我们将研究CD55和JAK/STAT通路是否相关 与BES相比，使用纵向研究并验证这些结论 在更大的纵向人群中的发现，以确定这些基因和途径是否揭示了潜在的新奇 CBD诊断和预后的生物标志物。在该项目结束时，我们将确定关键的监管 暴露性AM中与疾病相关的免疫失调的基因和途径 研究进展，揭示临床预后和诊断的潜在生物标志物。从这个过程中得到的结果 研究将加深我们对CBD发展所涉及的因素以及目标的了解 治疗，并将作为其他暴露相关免疫反应和环境诱导的模型 慢性病。

Project Summary/Abstract: The goal of this study is to determine the impact of CD55 and JAK/STAT pathway dysregulation in alveolar macrophages (AMs) on chronic beryllium disease (CBD) progression. CBD is an important organ-specific immune-mediated disease, characterized by granulomatous lung inflammation, fibrosis, and death, due to end-stage respiratory insufficiency. Thus, CBD remains an important public health concern. The precursor to this disease (beryllium sensitization; BeS) progresses to CBD at a rate of approximately 6-8% per year. Although the binding and presentation of Be via βGlu69-containing HLA-DP molecules to pathogenic CD4+ T cells provides an explanation for the genetic linkage of DP2 to CBD and BeS, this does not explain the progression of BeS to CBD. Furthermore, DP2 is found in up to 40% of Be-workers without evidence of BeS or CBD. Thus, additional mechanisms must be involved in progression of BeS to CBD. Our hypothesis is that CBD AMs downregulate the negative immunoregulatory gene CD55 and its pathway and overexpress the positive regulatory activating JAK/STAT pathway, which augments the immune response to Be and progression from BeS to CBD. In Aim 1, we will define if CD55 on AMs is a negative regulator in the immune response to Be in AMs from BeS patients. Specifically, using either the overexpression of the CD55 genes using a adenovirus overexpression system and agonistic antibody or the reduction of CD55 genes using adenovirus knockdown system and neutralizing antibody of CD55, we will determine the impact of alteration of CD55 on consequential functional changes in TNF-α production, phenotype (CD16, CD40, CD80, CD86, HLA- DR), JAK/STAT activation and BeLPT comparing CBD, BeS and controls. Finally, using RNA-seq, we will investigate the regulatory networks associated with the downregulation of CD55 in CBD compared to BeS; In Aim 2, we will determine if the JAK/STAT pathway is a positive regulator of the immune response to Be in AMs and is increased in AMs from subjects with CBD compared to BeS. Specifically, AMs from CBD will be compared to those from BeS and controls subjects, assessing either the overexpression of the JAK/STAT pathway genes using a adenovirus overexpression system or the reduction of the JAK/STAT pathway genes using adenovirus knockdown system and pharmacological JAK1- 3 inhibitors on the Be immune response. We will evaluate the changes in TNF-α production, phenotype (CD16, CD40, CD80, CD86, HLA-DR), and BeLPT. Finally, using RNA-seq we will define the regulatory networks associated with the overactivation of the JAK/STAT pathway. In Aim 3, we will investigate whether the CD55 and JAK /STAT pathway are associated with CBD and progression from BeS to CBD, compared to BeS, using a longitudinal study and validate these findings in a larger longitudinal population to determine if these genes and pathways reveal potential novel biomarkers of diagnosis and prognosis for CBD. At the end of this project we will determine the key regulatory genes and pathways of exposure-mediated immune dysregulation in AMs that are associated with disease progression and reveal potential biomarkers for clinical prognosis and diagnosis. The results obtained from this study will improve our understanding of factors involved in the development of CBD, as well as targets for therapy, and will serve as a model of other exposure-related immune responses and environmentally-induced chronic diseases.