The Role of ARNO-Arf6 signaling in barrier stability and chronic rhinosinusitis

 描述(申请人提供)：慢性鼻-鼻窦炎(CRS)在美国是一个严重的健康问题。据推测，常见的病因学途径可能涉及鼻腔上皮屏障的一过性或慢性改变，可能允许使抗原暴露、细胞因子释放增加和炎症持续循环的药物通过。CRS有两种组织病理学亚型，一种没有鼻息肉，一种有鼻息肉(分别为CRSsNP和CRSwNP)。CRNsNP和CRSwNP背后的细胞和分子机制仍然知之甚少。我们的合作者Dean Li博士的实验室最近在《自然》杂志上发表的一项研究表明，炎症细胞因子白介素1β(IL-1ç)激活不依赖于NF-κB的腺苷二磷酸核糖化因子核苷酸交换因子(Arno)-腺苷二磷酸核糖化因子6(Arf6)途径，从而减少细胞间连接蛋白在血管中的细胞表面定位。我们的初步研究表明，通过靶向抑制Arno-Arf6通路，可以增强鼻腔上皮屏障的稳定性，减少CRS的指标。我们假设，正如先前在血管系统中证明的那样，Arf6是细胞因子诱导的鼻窦上皮连接蛋白细胞表面定位和屏障通透性失稳的中央调节因子。我们假设CRSsNP和CRSwNP患者的鼻窦上皮细胞屏障在基线水平以及通过增加Arf6活性的炎性细胞因子信号而不稳定，并且抑制Arno-Arf6信号将以一种不依赖于NF-κB的方式恢复这两个环境中的稳定性。最后，我们假设抑制Arno-Arf6信号将增加屏障的稳定性，并将减少CRS的指标。为了验证这些假设，我们首先将在目标1中检查鼻窦粘膜，并表征基线和细胞因子诱导的非慢性鼻窦炎、CRSsNP和CRSwNP患者SNEC连接不稳定的差异。我们还将确定抑制Arf6活性是否稳定鼻窦上皮屏障并降低细胞因子诱导的屏障通透性。我们还将确定在细胞因子处理的SNEC中，Arno-Arf6的抑制是否独立于NF-κB信号发挥作用。在目标2中，我们将利用转基因小鼠和CRS的卵蛋白小鼠模型来确定通过Arno-Arf6途径的基因缺失来稳定上皮或内皮屏障是否可以减少鼻窦炎。从根本上讲，这些研究试图了解屏障不稳定性和通透性在CRSsNP和CRSwNP中所起的病理作用，并评估Arno-Arf6通路作为治疗靶点。由于对抑制Arno-Arf6系统的屏障稳定作用的阐明在很大程度上仅限于稳定血管屏障，这一建议具有潜在的意义和适用性。 鼻窦炎与其他影响上皮屏障稳定性的情况有关。

 DESCRIPTION (provided by applicant): Chronic rhinosinusitis (CRS) is a significant health problem in the United States. It has been hypothesized that a common etiologic pathway may involve transient or chronic alterations to the sinonasal epithelial barrier perhaps allowing passage of agents that perpetuate a cycle of antigen exposure, increased cytokine release, and inflammation. There are two histopathologic subtypes of CRS, one without and one with nasal polyps (CRSsNP and CRSwNP respectively). The cellular and molecular mechanisms that underlie CRNsNP and CRSwNP remain poorly understood. A recent study published in Nature from the lab of our collaborator Dr. Dean Li demonstrated that the inflammatory cytokine interleukin-1 beta (IL-1ß) activates the ADP ribosylation factor nucleotide exchange factor (ARNO)-ADP-ribosylation factor 6 (Arf6) pathway independently of NF-κB to reduce inter-cellular junction protein cell surface localization in the vasculature. Our preliminary studies suggest that one can enhance sinonasal epithelial barrier stability and reduce indicies of CRS through targeted inhibition of the ARNO-Arf6 pathway. We hypothesize that as previously demonstrated in the vascular system, Arf6 is a central regulator of cytokine-induced destabilization of sinonasal epithelial junction protein cell surface localization and barrier permeability. We hypothesize that the sinonasal epithelial cell barrier in CRSsNP and CRSwNP patients is destabilized at baseline as well as through inflammatory cytokine signaling via increased Arf6 activity, and that inhibition of ARNO-Arf6 signaling will restore stability in both settings in an NF-κB-independent manner. Lastly, we hypothesize that inhibition of ARNO-Arf6 signaling will increase barrier stability and will reduce indicies of CRS. To test these hypotheses we will initially, in aim 1, examine sinus mucosa and characterize baseline and cytokine-induced differences in SNEC junction destabilization in patients without chronic sinusitis, CRSsNP and CRSwNP. We will also determine whether inhibition of Arf6 activity stabilizes the sinonasal epithelial barrier and reduces cytokine-induced barrier permeability. We will also determine whether inhibition of ARNO-Arf6 acts independently of NF-κB signaling in cytokine-treated SNECs. In aim 2, we will utilize genetically modified mice and the ovalbumin mouse model of CRS to determine whether stabilization of the epithelial or endothelial barrier through genetic deletion of the ARNO-Arf6 pathway reduces sinusitis. Fundamentally the studies proposed here seek to understand the pathologic role that barrier instability and permeability play in CRSsNP and CRSwNP and to evaluate the ARNO-Arf6 pathway as a therapeutic target. As elucidation of the barrier stabilizing effects of inhibition of the ARNO-Arf6 system has largely been limited to stabilizing the vascular barrier, this proposal has potential significance and applicability beyond sinusitis to other conditions affecting epithelial barrier stability.