The effect of LCN2 treatment on anti-inflammatory bone marrow derived macrophages

Allogeneic hematopoietic stem cell transplantation remains the most efficacious treatment for many hematological malignancies. However, its therapeutic potential is affected by the most prominent side effect graft versus host disease. Despite advances in the treatment of graft versus host disease in recent years, morbidity and mortality remains high, which requires the development of new treatment approaches. We therefore implemented mouse models to assess potential treatment options for graft versus host disease. In in vivo experiments, we had observed a protective effect of LCN2 on graft versus host disease of the gastrointestinal tract. We also observed higher numbers of anti-inflammatory macrophages in the intestinal tissues of these animals. Therefore, we aimed to determine potentially regulated genes in these cells by using an in vitro approach of LCN2-treated macrophages. Bone marrow from healthy C57BL/6 mice was collected and seeded in cell culture dishes with complete media (10 % low endotoxin FCS, 1 % PenStrep, 20 ng/ml mCSF). Cells were cultured for seven days with or without supplementation of 150 ng/ml rmLCN2. On day 7, cells were stimulated with 20 ng/ml rmIL-4 towards an anti-inflammatory phenotype. A third group was treated with LCN2 at this time point. After 24 hours of stimulation, cells were harvested for further processing.

异基因造血干细胞移植（Allogeneic hematopoietic stem cell transplantation）仍是诸多血液系统恶性肿瘤最有效的治疗手段。然而其治疗潜力受限于最突出的不良反应——移植物抗宿主病（graft versus host disease）。尽管近年来移植物抗宿主病的治疗已取得进展，但其发病率与死亡率仍居高不下，亟需开发全新的治疗策略。为此，本研究构建小鼠模型以评估移植物抗宿主病的潜在治疗方案。在体内实验中，我们观察到LCN2对胃肠道移植物抗宿主病具有保护作用，同时还发现这些动物的肠道组织中抗炎巨噬细胞数量显著升高。基于此，本研究拟通过体外实验（即用LCN2处理巨噬细胞）明确此类细胞中受调控的潜在基因。收集健康C57BL/6小鼠的骨髓，接种于含完全培养基（10%低内毒素胎牛血清（FCS）、1%青霉素链霉素混合液（PenStrep）、20 ng/ml小鼠集落刺激因子（mCSF））的细胞培养皿中；将细胞分为两组，分别添加或不添加150 ng/ml重组小鼠LCN2（rmLCN2），连续培养7天；于第7天，用20 ng/ml重组小鼠白细胞介素4（rmIL-4）刺激细胞使其向抗炎表型分化，另有第三组细胞于此时点加入LCN2进行处理；刺激24小时后，收集细胞用于后续实验处理。