Preclinical study for therapy with siRNA targeting cell-to-cell communications in obstructive lung diseases

在COPD和哮喘中，多种炎症细胞和组织结构细胞之间的信号在疾病的发展中起着关键作用。这些信号部分是通过与共刺激分子的相互作用来调节的。B7家族分子CD86表达于树突状细胞(DC)表面，为与T细胞的相互作用提供共刺激信号。采用基于小干扰RNA(SiRNA)的方法，研究CD86基因敲除对哮喘小鼠模型的治疗作用。我们制备了骨髓来源的树突状细胞(BMDCs)。从DO11大鼠的脾组织中分离出CD4+细胞。10只小鼠采用磁珠分选法，与抗原提呈细胞、重组IL-4、抗IL-12单抗共培养，作为OVA特异性Th2细胞。将CD86siRNA或对照siRNA导入BMDCs，用OVA多肽和脂多糖冲击，然后与Th2细胞共培养24小时。收集培养上清液进行细胞因子酶联免疫吸附试验。CD86 siRNA可显著降低BMDCs培养上清液中IL-4、IL-5和IL-13的水平。接下来，我们测试了CD86 siRNA对OVA诱导的小鼠哮喘模型的治疗作用。CD86siRNA或对照siRNA在OVA激发期间经气管内给药。在显微镜研究中，荧光探针siRNA被成功地沉积在呼吸道粘膜下层。给予CD86 siRNA可显著降低嗜酸性粒细胞数量和支气管肺泡液中IL-5和IL-13水平，降低吸入性乙酰胆碱引起的气道高反应性，以及血清中OVA特异性IgE水平。这些发现提示，靶向呼吸道树突状细胞的siRNA治疗可能对开发治疗阻塞性肺疾病的新的核酸疗法有价值。

In COPD and asthma, the signalings among a variety of inflammatory cells and tissue-structural cells play a pivotal role in the development of diseases. These signalings are partly mediated via interaction with costimulatory molecules. The B7-family molecule CD86, expressed on the surface of dendritic cells(DCs), delivers costimulatory signals for the interaction with T cells. Using small interfering RNA(siRNA)-based approach, we investigated the therapeutic effect of CD86 knockdown on a mouse model of asthma. We prepared bone marrow-derived dendritic cells(BMDCs). CD4+ cells were isolated from the spleens of DO11. 10 mice by magnetic beads sorting method(MACS), cocultured with antigen-presenting cells, recombinant IL-4, and anti-IL-12 mAb, and then used as OVA-specific Th2 cells. BMDCs were transfected with CD86 siRNA or control siRNA, pulsed with OVA peptides and LPS, and then cocultured with Th2 cells for 24hr. The culture supernatant was collected for cytokine ELISA. The levels of IL-4, IL-5, and IL-13 in culture supernatant were significantly reduced by CD86 siRNA treatment of BMDCs. Next, we tested the therapeutic effects of CD86 siRNA for OVA-induced mouse model of asthma. CD86 siRNA or control siRNA was administrated via the intratracheal route during the OVA challenge. In microscopic study, The fluorescent-probed siRNA was successfully deposited beneath airway submucosa. The administration of CD86 siRNA significantly decreased eosinophil number and levels of IL-5 and IL-13 in the bronchoalveolar fluid, airway hyperreactivity to inhaled acetylcholine, and a level of OVA-specific IgE in the serum. These findings suggest that siRNA treatment targeting airway DCs might be of value in the development of new nucleic acid therapies for obstructive lung diseases.