A New Pathway for Neutrophil-induced Airway Inflammation

描述（由申请人提供）：在本更新申请中，我们描述了一种新的信号通路，该通路在母基金中描述了中性粒细胞（PMN）流入和气道损伤，可能作为慢性肺病（如阻塞性肺病（COPD）、囊性纤维化（CF）和慢性肺移植排斥反应）的启动因子或辅助因子发挥作用。具体而言，胶原蛋白的化学或酶促分解释放三肽、PGP和/或在体外对PMN具有趋化性的相关含PGP序列。我们证明，PGP进入气道的引入会导致中性粒细胞的大量涌入，但不是单核细胞。值得注意的是，PGP的PMN趋化活性可能是由于与ELR+ CXC趋化因子如IL-8的受体结合结构域的显著结构相关性，所述ELR+ CXC趋化因子含有该胶原序列或接近的类似物。气道长时间暴露于这种肽会导致肺泡扩大和右心室肥大，从而重现COPD的各个方面。我们已经开发了一种有效的拮抗剂，Arg-Thr-Arg，称为RTR，它结合PGP在溶液中，并阻止其在体外的趋化活性和在体内的病理生理作用。可能由于与PGP共享结构，RTR也阻断IL-8活性。在LPS诱导的COPD小鼠模型中，RTR完全抑制肺泡扩大和右心室肥大。胶原蛋白的降解和PGP的释放是一个逐步的过程，最初涉及基质金属蛋白酶（MMP）-8和/或9与脯氨酰内肽酶（PE）催化的最终反应。我们发现一种高度特异性的PE抑制剂，N-苄氧羰基（Z）脯氨酰脯氨醛（ZPP），可以阻断气道PGP的产生以及响应LPS的PMN内流。令人惊讶的是，ZPP对PMN流入的非常有效的钝化似乎不仅是由于PE抑制，而且是由于ZPP拮抗CXCR 1/2的结果，可能是由于ZPP和PGP之间的结构相似性。这表明了一种有趣的可能性，即可以开发一种既是蛋白酶抑制剂又是受体拮抗剂的单一化合物。如果成功的话，这将代表一种全新的嵌合型先导化合物，用于治疗一般的嗜酸性炎症，特别是肺部疾病。我们已经发现PGP存在于几乎所有CF、COPD或慢性肺移植排斥患者的支气管肺泡灌洗液（BALF）和/或痰中，但不存在于对照组或哮喘患者。此外，来自COPD和CF患者而非对照个体的痰含有从纯化的胶原蛋白离体产生PGP所必需的所有酶机制，并且这种PGP产生可以被PE抑制剂ZPP阻断。总的来说，这些发现使我们假设PGP和PE代表可能导致疾病的COPD的新生物标志物，并且PGP和PE代表在配体、受体和生成酶水平上有吸引力的治疗靶点。 COPD现在是美国第四大死亡原因，预计到2020年将成为第三大死亡原因。这一疾病造成许多人的痛苦以及巨大的经济负担。由于缺乏生物标志物来诊断和用作临床试验中的终点，以及缺乏对疾病过程本身的基本理解，因此对COPD的理解导致成功的治疗受到阻碍。如果成功，目前的提案将产生对疾病过程的基本见解和COPD的新的特异性生物标志物，并提出新的治疗靶点。

DESCRIPTION (provided by applicant): In this renewal application, we describe a new pathway signaling neutrophil (PMN) influx and damage to the airways that was delineated in the parent grant and that may play a role as an initiator or cofactor for chronic lung diseases such as obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic lung transplant rejection. Specifically, chemical or enzymatic breakdown of collagen releases a tripeptide, PGP, and/or a related PGP-containing sequence that is chemotactic for PMN in vitro. We demonstrate that introduction of PGP into the airways causes a robust influx of PMN, but not monocytes. Remarkably, the PMN chemotactic activity of PGP may be due to a marked structural relatedness to a receptor binding domain of ELR+ CXC chemokines such as IL-8 which contain this collagen sequence or a close analog. Prolonged airway exposure to this peptide causes alveolar enlargement and right ventricular hypertrophy and thus recapitulates aspects of COPD. We have developed a potent antagonist, Arg-Thr-Arg, termed RTR, which binds PGP in solution and blocks its in vitro chemotactic activity and in vivo pathophysiologic effects. Likely as a result of shared structure with PGP, RTR also blocks IL-8 activity. In a mouse model of LPS-induced COPD, RTR completely inhibited alveolar enlargement and right ventricular hypertrophy. The degradation of collagen and release of PGP is shown to be a stepwise process initially involving matrix metalloproteases (MMP)-8 and/or 9 with prolyl endopeptidase (PE) catalyzing the final reaction. We have found that a highly specific PE inhibitor, N-benzyloxycarbonyl (Z) prolyl prolinal (ZPP), can block airway PGP production as well as PMN influx in response to LPS. Surprisingly, the very potent blunting of PMN influx by ZPP appears to be due not only to PE inhibition but also as a result of ZPP antagonism of CXCR 1/2 likely as a result of structural similarity between ZPP and PGP. This suggests the intriguing possibility, to be explored in this proposal, that a single compound can be developed that is both a protease inhibitor and receptor antagonist. If successful, this would represent an entirely new chimeric type of lead compound for the treatment of neutrophilic inflammation, in general, and lung diseases in particular. We have found that PGP is present in bronchoalveolar lavage fluids (BALF) and/or sputum from virtually all CF, COPD or chronic lung transplant rejection patients but not controls or asthmatics. Furthermore, sputum from COPD and CF patients but not control individuals contains all the enzymatic machinery necessary for the ex vivo generation of PGP from purified collagen and such PGP production can be blocked by the PE inhibitor, ZPP. Collectively, these findings lead us to hypothesize that PGP and PE represent novel biomarkers for COPD that may contribute to disease and that PGP and PE represent attractive therapeutic targets at the level of the ligand, receptor, and generating enzyme. PUBLIC HEALTH RELEVANCE: COPD is now the fourth leading cause of death in the U.S. and is expected to be the third by 2020. This disease causes much human suffering as well as a large monetary burden. An understanding of COPD that leads to successful therapeutics has been hampered by a lack of biomarkers to diagnose and use as endpoints in clinical trials as well as a lack of a fundamental understanding of the disease process itself. If successful, the current proposal will yield fundamental insights into the disease process and a new and specific biomarker for COPD as well as suggesting new therapeutic targets.