Table 5_Interaction with IGF1 overrides ANXA2-mediated anti-inflammatory functions of IGFBP5 in vivo.docx

BackgroundIGFBP5 is a differentially expressed gene (DEG) between M1 and M2 macrophages. This study explained why it causes opposite effects in different circumstances.
MethodsGene expression profiles of various cell subsets were compared by mining a public database. THP-1 cells were treated by siRNAs, recombinant IGFBP5, lipopolysaccharide (LPS), picropodophyllin, IGF1 or the combinations. Clinical implication of IGFBP5 changes was investigated using rheumatoid arthritis (RA) and acute lung injury (ALI) models. IGFBP5-bound and differential proteins were identified by Liquid Chromatography Mass Spectrometry method.
ResultsIGFBP5 situated in the center of a network constructed by the DEGs of M0 and M1/2 macrophages. Its expression negatively correlated to inflammation in vitro. When IGFBP5 was silenced, monocytes released more IL-1β and IL-6. NF-κB downstream proteins were overexpressed. IGFBP5 interacted with ANXA2 directly. In ANXA2-silenced cells, it showed no anti-inflammatory effect. Monocytes of adjuvant-induced arthritis rats and RA patients expressed less IGFBP5 than normal controls, but its blood levels increased significantly. Adipocytes secreted large amounts of IGFBP5. This secretion was reinforced by the above sera. IGFBP5 decreased in ALI mice’s blood, while its supplement exacerbated inflammation. By binding to IGF1, IGFBP5 prevented its interaction with IGF1R. An IGF1R inhibitor picropodophyllin antagonized functions of IGF1/IGF1R too, but didn’t reinforce the effects of IGFBP5.
ConclusionIGFBP5 eases inflammation by interacting with ANXA2, an activator of NF-κB; as an antagonist of IGF1/IGF1R, IGFBP5 may disrupt immune homeostasis in vivo, due to impairment of the latter’s anti-inflammatory functions; excessive IGFBP from adipocytes would be a pathogenic factor in certain diseases.

背景：胰岛素样生长因子结合蛋白5（IGFBP5）是M1型与M2型巨噬细胞间的差异表达基因（differentially expressed gene, DEG）。本研究阐释了其在不同情境下产生相反效应的原因。
方法：通过挖掘公共数据库，比对多种细胞亚群的基因表达谱。将THP-1细胞分别用小干扰RNA（siRNA）、重组胰岛素样生长因子结合蛋白5（recombinant IGFBP5）、脂多糖（lipopolysaccharide, LPS）、匹克洛多菲林（picropodophyllin）、胰岛素样生长因子1（IGF1）或上述试剂的组合进行处理。采用类风湿关节炎（rheumatoid arthritis, RA）与急性肺损伤（acute lung injury, ALI）模型，探究IGFBP5表达变化的临床意义。通过液相色谱-质谱联用法（Liquid Chromatography Mass Spectrometry）鉴定与IGFBP5结合的蛋白及差异表达蛋白。
结果：IGFBP5位于M0、M1/2型巨噬细胞差异表达基因构建的调控网络的核心位置。其表达水平与体外炎症反应呈负相关。当IGFBP5被沉默后，单核细胞会释放更多的白细胞介素1β（IL-1β）与白细胞介素6（IL-6），核因子κB（NF-κB）下游蛋白的表达量显著上调。IGFBP5可与膜联蛋白A2（ANXA2）直接结合。在ANXA2被沉默的细胞中，IGFBP5未表现出抗炎效应。佐剂诱导性关节炎大鼠及类风湿关节炎（RA）患者的单核细胞中IGFBP5表达量低于正常对照，但外周血中IGFBP5水平显著升高。脂肪细胞可分泌大量IGFBP5，上述患者血清可增强这一分泌过程。急性肺损伤（ALI）模型小鼠外周血中IGFBP5水平降低，而外源性补充IGFBP5会加重炎症反应。IGFBP5可通过结合胰岛素样生长因子1（IGF1），阻断其与胰岛素样生长因子1受体（IGF1R）的相互作用。IGF1R抑制剂匹克洛多菲林同样可拮抗IGF1/IGF1R的功能，但并未增强IGFBP5的作用效果。
结论：IGFBP5可通过结合核因子κB（NF-κB）的激活剂膜联蛋白A2（ANXA2）发挥抗炎作用；作为IGF1/IGF1R的拮抗剂，IGFBP5可能通过削弱后者的抗炎功能，破坏体内免疫稳态；脂肪细胞分泌过量的IGFBP5可能成为部分疾病的致病因素。