A novel proteolytic system of pulmonary inflammation

描述（由申请人提供）：在本提案中，我们描述了一种介导中性粒细胞（PMN）内流和气道损伤的新途径，该途径可能作为慢性炎症性肺部疾病（如囊性纤维化（CF））的启动剂或辅助因子。我们之前已经证明脯氨酸-甘氨酸-脯氨酸（PGP）肽可以导致PMN大量流入小鼠气道；这种细胞特异性是由于CXC趋化因子（如IL-8）的受体结合域在结构上的相关性。PI表明，PGP从胶原蛋白中释放是一个循序渐进的过程，最初涉及基质金属蛋白酶(MMP)-8和/或9，脯氨酸内肽酶（PE）催化最终反应。最近，PI的实验室已经证明，PE存在于中性粒细胞和受刺激的PMNs中，当在胶原蛋白上孵育时，可以引起显著的PGP生成。尽管PI小组的工作已经证明在CF气道分泌物中发现了PGP，但PGP肽在CF肺部疾病中增强炎症反应的影响目前尚不清楚。该资助申请旨在通过首先研究CXC配体（包括PGP和N-(-PGP)）作为体外研究中PGP产生的新机制的影响，并进一步研究用于中性粒细胞释放蛋白酶的特定细胞内途径来解决这一问题。然后，这些观察结果将被带到一种独特的CF肺部疾病小鼠模型，(-ENaC过表达小鼠（Specific Aim 2），以确定该炎症途径的成分对该小鼠模型中观察到的表型和炎症反应的影响。确定这些驱动蛋白酶调节/释放的基本途径对慢性炎症性疾病（如CF）患者具有重要意义，其中炎症刺激（如CXC趋化因子）即使在疾病稳定期间也会增加丰度。我们的初步证据表明，在住院CF恶化开始时，PGP水平升高，但在住院结束时确实下降。然而，即使有这种临床改善，与非疾病对照相比，PGP水平仍然升高，这表明这些气道中仍存在持续的炎症。在本提案的最终目的中，我们检查临床稳定的CF患者是否有痰和血清中PGP和蛋白酶水平升高。如果是这样，它们的升高和持续可能预测未来疾病恶化的发展，作为CF肺病的独特生物标志物。从这些目标产生的发现可能具有深远的转化意义，不仅对CF肺病，而且可能对其他中性粒细胞炎症性疾病。

DESCRIPTION (provided by applicant): In this proposal, we describe a new pathway signaling neutrophil (PMN) influx and damage to the airways that may play a role as an initiator or cofactor for chronic inflammatory lung diseases such as cystic fibrosis (CF). We have previously demonstrated that proline-glycine-proline (PGP) peptides can cause a robust PMN influx into the airways of mice; this cellular specificity is due to structural relatedness to a receptor binding domain of CXC chemokines such as IL-8. The PI has shown that PGP is released from collagen by a stepwise process initially involving matrix metalloproteases (MMP)-8 and/or 9 with prolyl endopeptidase (PE) catalyzing the final reaction. Recently, the PI's lab has shown that PE is found in neutrophils and stimulated PMNs, when incubated on collagen, can cause notable PGP generation. Although work from the PI's group has demonstrated that PGP is found in CF airway secretions, the impact of PGP peptides in augmenting inflammatory responses in CF lung disease is not currently known. This grant application aims to address this by first investigating the impact of CXC ligands (including PGP and N-(-PGP) as a novel mechanism for the generation of PGP in ex vivo studies and further examining the specific intracellular pathways utilized for protease release from neutrophils. Then, these observations will be taken to a unique mouse model of CF lung disease, the (-ENaC overexpressor mouse (Specific Aim 2), to determine the impact of components of this inflammatory pathway on the phenotype and inflammatory response observed in this murine model. Determination of these fundamental pathways driving protease regulation/release have important implications to patients with chronic inflammatory disease, such as CF, where inflammatory stimuli such as CXC chemokines, are in increased abundance even during periods of disease stability. Our preliminary evidence suggests that at the beginning of an inpatient CF exacerbation, PGP levels are elevated but do decline by the end of hospitalization. However, even with this clinical improvement, PGP levels are still elevated compared to non-disease controls, suggesting that there continues to be ongoing inflammation in these airways. In the final aim of this proposal, we examine if clinically stable CF individuals have elevated levels of PGP and proteases in both sputum and serum. If so, it is possible that their elevation and persistence may predict the development of future disease exacerbations, serving as a unique biomarker for CF lung disease. The findings generated from these aims may have profound translational implications not only to CF lung disease but potentially to other neutrophilic inflammatory conditions. PUBLIC HEALTH RELEVANCE: We have proposed a series of complementary experimental aims to examine the impact of PGP-containing peptides and their generating proteases in cystic fibrosis (CF). The implications of these findings may be broadly applicable to other conditions with prominent neutrophilic inflammation and extracellular matrix breakdown.