miR-29b and autophagy regulate alveolar macrophage function post-BMT

 描述(申请人提供)：造血干细胞移植(HSCT)用于治疗各种遗传缺陷和恶性肿瘤，但其有效性受到肺部感染的限制。感染并发症可能发生在异体和自体移植环境中，尽管进行了造血重建，但感染的易感性仍然很高。为了更好地了解HSCT的先天免疫缺陷，我们建立了一种同基因骨髓移植后细菌感染的小鼠模型。我们之前已经证明，即使在造血系统重建后，这些小鼠也更容易感染铜绿假单胞菌。我们发现环氧合酶-2(COX-2)的上调和PGE2的过度产生是这些小鼠先天免疫功能受损的主要因素。我们发现肺泡巨噬细胞(AM)和中性粒细胞(PMN)在吞噬、杀菌和细胞因子分泌等先天免疫功能方面存在缺陷。此外，骨髓移植后肺泡巨噬细胞上清道夫受体的分布发生了改变，具有胶原性结构的巨噬细胞受体(MARCO)丢失，MARCO是识别南美斑潜蝇的关键受体。我们确定PGE2通过这些AM上升高的E-前列腺素2(EP2)受体来抑制它们的功能。重要的是，我们的小鼠研究表明，对BMT后COX-2的药物或遗传抑制可以恢复肺的先天免疫和AM对铜绿假单胞菌的功能。这些结果令人兴奋，因为它们表明抑制PGE2信号可以作为一种治疗方法来提高移植后的宿主防御能力。然而，在全球范围内阻止所有前列腺素合成的治疗策略存在系统性问题。因此，这项建议的一个方面将是测试一种新开发的EP2拮抗剂(PF-044148948)，这是我们从辉瑞获得的。我们相信这将是一种更特异和有效的治疗方法来阻断抑制的PGE2信号。该应用程序还试图提供对以下未回答问题的见解。1)为什么BMT后COX-2的升高导致PGE2的过量产生？2)这些先天免疫缺陷是否是异基因(Allo)BMT的特征？3)这些缺陷是否使BMT后的小鼠更容易受到革兰氏阳性感染(如肺炎链球菌)和革兰氏阴性感染？4)我们能否确定自噬功能受损是否是BMT后杀伤功能受损的一种机制？5)我们在小鼠模型中注意到的缺陷是否也存在于人类HSCT患者中？我们的总体假设是：骨髓移植预适应诱导肺上皮细胞分泌转化生长因子(TGF)。这增加了miR-29b的表达，阻断了DNA甲基转移酶(DNMT)的合成，导致COX-2的低甲基化，导致AM中PGE2的过度表达。此外，PGE2-EP2诱导的自噬改变削弱了BMT后宿主对铜绿假单胞菌和肺炎链球菌的防御，我们推测BMT后的宿主防御可以通过COX抑制剂、EP2拮抗剂或通过诱导自噬来改善。这些假设将在以下具体目标中进行探讨。目的1)确定同种和异基因骨髓移植小鼠是否对铜绿假单胞菌和肺炎链球菌的感染更敏感，以及易感性是否与PGE2信号通过EP2在小鼠和人中有关。目的2：确定骨髓移植后转化生长因子诱导的miR-29b是否导致环氧合酶-2去甲基化增加PGE2的产生。目的3：确定自噬在同种和异基因骨髓移植小鼠中是否受损，以确定这是否与PGE2-EP2信号有关，以及自噬对骨髓移植后宿主防御的重要性。

 DESCRIPTION (provided by applicant): Hematopoietic stem cell transplantation (HSCT) is used to treat a variety of genetic defects and malignancies, but its usefulness is limited by pulmonary infections. Infectious complications can occur both in allogeneic and autologous transplant settings and susceptibility to infection remains elevated despite hematopoietic reconstitution. To better understand innate immune deficiencies that characterize HSCT, we developed a murine model of bacterial infection post-syngeneic (syn) bone marrow transplant (BMT). We have previously shown that these mice are more susceptible to infection with Pseudomonas aeruginosa even after the hematopoietic system is reconstituted. We identified the upregulation of cyclooxygenase-2 (COX-2) and the overproduction of PGE2 as major contributing factors to the impaired innate immune function in these mice. We identified that alveolar macrophages (AMs) and neutrophils (PMNs) had defects in innate immune functions such as phagocytosis, bacterial killing and cytokine secretion. In addition, the profile of scavenger receptors on AMs were altered post-BMT, with loss of macrophage receptor with collagenous structure (MARCO), a critical receptor for recognition of P. aeurignosa. We determined that PGE2 signaled via elevated E prostanoid 2 (EP2) receptors on these AMs to inhibit their functions. Importantly, our murine studies have shown that pharmacologic or genetic inhibition of COX-2 post-BMT restores lung innate immunity and AM function against P. aeruginosa. These results are exciting because they suggest inhibition of PGE2 signaling can be a therapeutic to improve host defense post-transplant. However, there are systemic problems with a therapeutic strategy that globally blocks all prostaglandin synthesis. Thus, one aspect of this proposal will be to test a newly developed EP2 antagonist (PF-044148948) which we have obtained from Pfizer. We believe this will be a much more specific and effective therapeutic to block the inhibitory PGE2 signaling. The application also seeks to provide insight into the following unanswered questions. 1) Why is COX-2 elevated post-BMT leading to overproduction of PGE2? 2) Do these same innate immune defects characterize allogeneic (allo) BMT? 3) Do these defects post-BMT make mice more susceptible to Gram positive infections (like Streptococcus pneumoniae) as well as Gram negative ones? 4) Can we determine whether impairment of autophagy is one mechanism for impaired killing post-BMT? 5) Are the defects we have noted in our murine model also present in human HSCT patients? Our overall hypothesis is: BMT conditioning induces transforming growth factor (TGF) ß secretion from lung epithelial cells. This augments miR-29b expression to block synthesis of DNA methyltransferases (DNMTs) causing hypomethylation of COX-2 leading to PGE2 overexpression in AMs. Furthermore, PGE2-EP2 induced alterations in autophagy impair host defense against P. aeruginosa and S. pneumoniae post-BMT and we speculate that host defense post-BMT can be improved via treatment with a COX inhibitor, an EP2 antagonist or via induction of autophagy. These hypotheses will be explored in the following specific aims. Aim 1) To determine if syn BMT and allo BMT mice are more susceptible to P. aeruginosa and S. pneumoniae infection and if susceptibility is related to PGE2 signaling via EP2 in mice and man Aim 2: To determine whether TGFß-induced miR-29b causes COX-2 hypomethylation to increase PGE2 production post-BMT Aim 3: Aim 3: To determine if autophagy is impaired in syn and allo BMT AMs, to determine whether this is related to PGE2- EP2 signaling and the importance of autophagy to host defense post-BMT