Supplementary Material for: Twist2 Reduced NLRP3-Induced Inflammation of Infantile Pneumonia via Regulation of Mitochondrial Permeability Transition by FOXO1

<b><i>Background:</i></b> Infantile pneumonia is an acute inflammatory lesion of the lung caused by mycoplasma pneumonia. Indeed, Twist2 signaling pathway controls inflammatory reaction, oxidative stress, and other biological reaction. However, the regulation of Twist2 on the inflammation in infantile pneumonia remains unclear. This study explained that the function and mechanism of Twist2 in infantile pneumonia. <b><i>Methods:</i></b> The subjects included the serum samples of 12 patients with infantile pneumonia and normal healthy volunteers from <i>Hunan Children’s Hospital</i>. Besides, mice were given with lipopolysaccharide (LPS) into the lung. Moreover, RAW264.7 macrophages were stimulated with LPS for 4 h and added to the culture medium. <b><i>Results:</i></b> In present study, in serum of patients with infantile pneumonia or lung tissue of mice model with infantile pneumonia, TWIST2 expression was lessened. Apart from that, TWIST2 protein could reduce the inflammatory reaction in mice model with infantile pneumonia, resulting in an inhibition in lung injury. Conversely, over-expression of TWIST2 also decreased inflammatory reaction in macrophages model via the regulation of FOXO1/NLRP3 pathway. Downregulation of TWIST2 promoted the inflammation in macrophages model by the regulation of FOXO1/NLRP3 pathway. <b><i>Conclusion:</i></b> According to the findings, present study have identified that the TWIST2 could reduce the inflammation of infantile pneumonia by NLRP3 inflammasome through the regulation of mitochondrial permeability transition and the induction of FOXO1 expression.

<b><i>背景：</i></b> 婴幼儿肺炎是由肺炎支原体引发的肺部急性炎症性病变。事实上，Twist2信号通路（Twist2 signaling pathway）可调控炎症反应、氧化应激等多种生物学过程。然而，Twist2对婴幼儿肺炎炎症反应的调控作用目前仍不明确。本研究旨在阐明Twist2在婴幼儿肺炎中的功能与作用机制。<b><i>方法：</i></b> 本研究纳入湖南省儿童医院（Hunan Children’s Hospital）的12例婴幼儿肺炎患者血清样本与健康志愿者血清样本作为研究对象。此外，通过向小鼠肺内注射脂多糖（lipopolysaccharide, LPS）构建肺炎模型；同时将RAW264.7巨噬细胞以LPS刺激4小时后，置于培养基中培养。<b><i>结果：</i></b> 本研究发现，在婴幼儿肺炎患者的血清以及婴幼儿肺炎小鼠模型的肺组织中，TWIST2的表达水平均显著降低。此外，TWIST2蛋白可减轻婴幼儿肺炎小鼠模型的炎症反应，进而抑制肺部损伤。反之，过表达TWIST2可通过调控FOXO1/NLRP3通路抑制巨噬细胞模型中的炎症反应；而TWIST2低表达则通过调控FOXO1/NLRP3通路加剧巨噬细胞模型的炎症反应。<b><i>结论：</i></b> 综上，本研究证实，TWIST2可通过调控线粒体通透性转换、诱导FOXO1表达，进而经由NLRP3炎症小体（NLRP3 inflammasome）减轻婴幼儿肺炎的炎症反应。