A novel proteolytic system of pulmonary inflammation

描述(由申请人提供)：在这项提案中，我们描述了一种新的通路，信号转导中性粒细胞(PMN)进入和损伤呼吸道，可能起到启动或辅助因子的作用，导致慢性炎症性肺疾病，如囊性纤维化(CF)。我们先前已经证明，脯氨酸-甘氨酸-脯氨酸(PGP)多肽可以引起PMN强劲地流入小鼠的呼吸道；这种细胞特异性是由于结构上与CXC趋化因子的受体结合区域有关，如IL-8。等电点研究表明，PGP是通过一个分步过程从胶原中释放出来的，最初涉及基质金属蛋白酶(MMP)-8和/或9，最后由Pro内肽酶(PE)催化。最近，PI的实验室发现，PE存在于中性粒细胞中，当刺激的PMN与胶原蛋白孵育时，可以引起显著的PGP生成。尽管PI的研究小组的工作已经证明在CF的呼吸道分泌物中发现了PGP，但PGP多肽在增强CF肺部疾病的炎症反应中的影响目前尚不清楚。这项拨款申请旨在解决这一问题，首先研究CXC配体(包括Pgp和N-(-Pgp))在体外研究中作为一种新的Pgp产生机制的影响，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。然后，这些观察将被带到一种独特的CF肺病小鼠模型-ENaC过度表达小鼠(特定目标2)，以确定这一炎症途径的成分对在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，即使在疾病稳定期，CXC趋化因子等炎性刺激也会增加。我们的初步证据表明，在住院患者CF恶化开始时，Pgp水平升高，但在住院结束时确实下降。然而，即使在临床上有所改善，与非疾病对照组相比，Pgp水平仍然较高，这表明这些呼吸道继续存在持续的炎症。在这项建议的最终目的中，我们检查了临床稳定的CF患者是否有痰和血清中Pgp和蛋白水解酶水平升高。如果是这样的话，它们的升高和持续性可能预测未来疾病恶化的发展，作为一种独特的CF肺部疾病的生物标志物。由这些目的产生的发现可能不仅对CF肺部疾病有深远的翻译意义，而且可能对其他中性粒细胞炎性疾病具有潜在的意义。 公共卫生相关性：我们提出了一系列互补的实验目标，以检查含有Pgp的多肽及其产生的蛋白水解酶在囊性纤维化(CF)中的影响。这些发现的意义可能广泛适用于其他中性粒细胞炎症和细胞外基质破坏明显的情况。

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.