Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Vic., Australia.
Department of Pediatric Respiratory Medicine, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Scand J Immunol. 2021 Jul;94(1):e13040. doi: 10.1111/sji.13040. Epub 2021 Apr 3.
Cystic Fibrosis (CF) is primarily a progressive lung disease, characterized by chronic pulmonary infections with opportunistic pathogens. Such infections typically commence early in life, producing an inflammatory response marked by IL-8 chemokine production and neutrophilic infiltration, major contributory factors in CF progression. Studying this inflammation, especially early in life, is critical for developing new strategies for preventing or slowing disruption to the structural integrity of the CF airways. However, evaluating the immune responses of bronchoalveolar lavage (BAL) cells from children with CF faces technical challenges, including contamination carried from the lung due to pre-existing infections and low cell number availability. Here, we describe a technique for preparing BAL cells from young children with CF and using those cells in a bacterial stimulation assay. Initial antibiotic treatment proved essential for preventing resident bacteria from overgrowing BAL cell cultures, or non-specifically activating the cells. ACTB, identified as an optimal reference gene, was validated for accurate analysis of gene expression in these cells. Pseudomonas aeruginosa and Staphylococcus aureus were used as bacterial stimulants to evaluate the immune response of BAL cells from young children with CF. Addition of gentamicin prevented bacterial overgrowth, although if added after 3 hours of culture an extremely variable response resulted, with the bacteria causing a suppressive effect in some cultures. Addition of gentamicin after 1 hour of culture completely prevented this suppressive effect. This technique was then able to reproducibly measure the IL-8 response to stimulation with S. aureus and P. aeruginosa, including co-stimulation with both bacteria.
囊性纤维化（CF）主要是一种进行性肺部疾病，其特征是慢性肺部感染机会性病原体。这种感染通常在生命早期开始，产生炎症反应，表现为白细胞介素-8（IL-8）趋化因子的产生和中性粒细胞浸润，这是 CF 进展的主要促成因素。研究这种炎症，特别是在生命早期，对于开发预防或减缓 CF 气道结构完整性破坏的新策略至关重要。然而，评估 CF 儿童支气管肺泡灌洗液（BAL）细胞的免疫反应面临技术挑战，包括由于先前存在的感染和细胞数量有限而从肺部携带的污染。在这里，我们描述了一种从 CF 儿童中制备 BAL 细胞的技术，并在细菌刺激测定中使用这些细胞。最初的抗生素治疗对于防止定植细菌过度生长或非特异性激活细胞是必不可少的。ACTB 被鉴定为最佳参考基因，用于准确分析这些细胞中的基因表达。铜绿假单胞菌和金黄色葡萄球菌被用作细菌刺激物，以评估 CF 儿童的 BAL 细胞的免疫反应。加入庆大霉素可防止细菌过度生长，尽管如果在培养 3 小时后添加，则会导致非常可变的反应，细菌在某些培养物中产生抑制作用。在培养 1 小时后加入庆大霉素可完全防止这种抑制作用。然后，该技术能够可靠地测量 S. aureus 和 P. aeruginosa 刺激的 IL-8 反应，包括与两种细菌的共刺激。
