Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.

炎症性肠病 (IBD) 被认为是由宿主对遗传易感个体的共生肠道菌群的异常反应引起的。树突状细胞 (DC) 在对肠道共生体取样时在调节这种反应中发挥着关键作用。在健康个体中，DCs 在识别这些常驻细菌后积极促进耐受，而在 IBD 个体中，DCs 会引发炎症反应。为了在体外模拟疾病反应，人类单核细胞衍生的 DCs 用大肠杆菌成熟导致细胞产生高水平的促炎细胞因子 IL-12/IL-23p40 (p40) 和低水平的抗炎细胞因子 IL-10。开发并筛选了一种基于 siRNA 的筛选试验，以识别潜在的治疗靶点，将这种平衡转变为具有较低水平 p40 和较高水平 IL-10 的免疫抑制状态。使用非靶向对照和靶向IL12B和TLR4 的阳性对照 siRNA 对筛选试验进行了优化和质量控制成绩单。在初步筛选中，smartpool siRNA 被筛选为降低 p40 表达、诱导 IL-10 水平或增加 IL-10:p40 比率而不影响细胞活力。所有潜在的目标都被转移到不同 DC 供体的确认筛选中，其中每个目标筛选了四个单独的 siRNA。每个靶标至少有两个 siRNA 应具有效果才能被视为有效靶标。该筛选产生了十个基因的简明列表，目前正在研究它们在 DC 成熟中的作用。