Role of cockroach proteases in airway inflammation

描述（由申请方提供）：蟑螂（CR）暴露是发生哮喘的诱发因素。 虽然许多空气过敏原含有蛋白酶活性，但蛋白酶在调节炎症反应中的作用尚不清楚。我们已经表明，蟑螂提取物含有蛋白水解活性，这是负责协同增加肿瘤坏死因子（TNF）α介导的白细胞介素（IL）-8和IL-6的表达。 此外，我们有初步的数据表明，蟑螂蛋白酶激活气道上皮细胞上的蛋白酶激活受体（PAR）-2，以增强细胞外调节激酶（ERK）激活的信号级联反应。 为了了解活性蟑螂蛋白酶在体内外调节气道炎症的机制，我们提出以下具体目的：1。确定CR丝氨酸蛋白酶活性对PAR的作用及其对促炎反应的影响。我们将通过RT-PCR、流式细胞术和免疫组化检测TNF α刺激是否调节PAR-2的表达。 我们将用激活肽SLIGKV激活PAR-2，并研究其在细胞因子表达中的作用。我们将在Cos-1细胞中过表达PAR-2，并确定其在炎症中的作用。 我们将从粗CR提取物中纯化丝氨酸蛋白酶。2.建立介导CR诱导的IL-8调节的下游信号传导事件。我们有证据表明ERK在CR治疗后被激活。 我们将研究转录因子NF-IL 6和AP-1对CR诱导的协同作用的作用。 这将通过启动子缺失研究、DMA结合和反式激活研究来完成。我们将确定NF-IL 6或AP-1是否需要或足以调节IL-8。我们将确定ERK和NF-κ B通路之间是否发生串扰。最后，我们将确定CR提取物是否转录调节其他细胞因子/趋化因子。 3.建立蟑螂丝氨酸蛋白酶活性在调节气道炎症和反应性中的体内作用。我们将通过在治疗前体外抑制蛋白酶活性来确定蛋白酶在调节气道炎症中的作用。 我们将使用PAR-2基因敲除小鼠建立PAR-2在调节气道反应中的作用。 最后，我们将使用TNF α中和抗体和TNF α敲除小鼠研究内源性TNF α在气道中的作用。

DESCRIPTION (provided by applicant): Cockroach (CR) exposure is a predisposing factor for the development of asthma. While many aeroallergens contain protease activity, the role of proteases in modulating the inflammatory response is unknown. We have shown that cockroach extract contains proteolytic activity which is responsible for synergistically increasing tumor necrosis factor (TNF) alpha-mediated interleukin (IL)-8 and IL-6 expression. In addition, we have preliminary data suggesting that cockroach proteases activate the protease activated receptor (PAR)-2 on airway epithelium to elicits signaling cascade to extracellular regulated kinase (ERK) activation. To understand the mechanism by which active cockroach proteases modulate airway inflammation in vitro and in vivo, we propose the following Specific Aims: 1. Establish the role of CR serine protease activity on PARs and its effects on the pro-inflammatory response. We will examine whether TNF alpha stimulation regulates PAR-2 expression by RT-PCR, flow cytometry and immunohistochemistry. We will activate PAR-2 with the activating peptide SLIGKV and investigate its role in cytokine expression. We will overexpress PAR-2 in Cos-1 cells and determine its role in inflammation. We will purify the serine protease from the crude CR extract. 2. Establish the downstream signaling events mediating CR-induced regulation of IL-8. We have evidence that ERK is activated following CR treatment. We will examine the role of transcription factors NF-IL6 and AP-1 on CR-induced synergy. This will be done by promoter deletion studies, DMA binding and transactivation studies. We will determine if NF-IL6 or AP-1 is required or sufficient for the regulation of IL-8. We will determine if crosstalk occurs between ERK and NF-kappa B pathways. Finally we will determine if CR extract transcriptionally regulates other cytokines/chemokines. 3. Establish an in vivo role for cockroach serine protease activity in modulating airway inflammation and reactivity. We will determine the role of proteases in modulating airway inflammation by ex vivo inhibition of the protease activity prior to treatment. We will establish a role for PAR-2 in modulating airway responses using PAR-2 knockout mice. Finally, we will investigate the role of endogenous TNF alpha in the airways using a TNF alpha neutralizing antibody and TNF alpha knockout mice.