背景:急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)是由于弥漫性肺部炎症和水肿引起的常见急性呼吸衰竭。对巨噬细胞活化的精细调节对于控制这种炎症过程和维持组织稳态是必不可少的。在过去的几十年里,巨噬细胞的代谢重编程已成为调节其生物学和功能的主要作用。这里,我们观察到肉碱棕榈酰转移酶1A(CPT1A)的表达降低,脂肪酸氧化的关键限速酶(FAO),在脂多糖(LPS)诱导的ALI小鼠模型的巨噬细胞中。我们假设CPT1A及其调节的FAO参与巨噬细胞极化的调节,白细胞介素-10(IL-10)可正向调节。
方法:经鼻吸入rIL-10和/或LPS后,野生型(WT),IL-10-/-,将Cre-CPT1Afl/fl和Cre+CPT1Afl/fl小鼠处死以收获支气管肺泡灌洗液,血清和肺部检查细胞浸润,细胞因子产生,肺损伤严重程度和IHC。从小鼠中提取骨髓来源的巨噬细胞(BMDM),并通过外源性rIL-10和/或LPS刺激。qRT-PCR,SeahorseXFe96和FAO代谢物相关试剂盒用于测试BMDM中的糖酵解和FAO水平。免疫印迹测定,共聚焦显微镜和荧光微孔板用于检测巨噬细胞极化以及线粒体结构和功能损伤。
结果:在体内实验中,我们发现缺乏CPT1A或IL-10的小鼠对LPS刺激产生加重的炎症反应.然而,添加rIL-10能有效缓解小鼠肺部炎症。IHC结果显示,在Cre+CPT1Afl/fl小鼠中,肺巨噬细胞中的IL-10表达显著降低。体外实验显示CreCPT1Afl/fl和IL-10-/-BMDMs变得更加“糖酵解”,但当受到外部攻击时,“粮农组织”就更少了。然而,向巨噬细胞中补充rIL-10显示出相反的作用。CPT1A和IL-10可以驱动BMDM从M1表型向M2表型分化,CPT1A-IL-10轴也参与维持线粒体稳态的过程。
结论:CPT1A调节LPS刺激下巨噬细胞的代谢重编程和极化。CPT1A的保护作用可能部分归因于IL-10/IL-10受体表达的诱导。
BACKGROUND: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common acute respiratory failure due to diffuse pulmonary inflammation and oedema. Elaborate regulation of macrophage activation is essential for managing this inflammatory process and maintaining tissue homeostasis. In the past decades, metabolic reprogramming of macrophages has emerged as a predominant role in modulating their biology and function. Here, we observed reduced expression of carnitine palmitoyltransferase 1A (CPT1A), a key rate-limiting enzyme of fatty acid oxidation (FAO), in macrophages of lipopolysaccharide (LPS)-induced ALI mouse model. We assume that CPT1A and its regulated FAO is involved in the regulation of macrophage polarization, which could be positive regulated by interleukin-10 (IL-10).
METHODS: After nasal inhalation rIL-10 and/or LPS, wild type (WT), IL-10-/-, Cre-CPT1Afl/fl and Cre+CPT1Afl/fl mice were sacrificed to harvest bronchoalveolar lavage fluid, blood serum and lungs to examine cell infiltration, cytokine production, lung injury severity and IHC. Bone marrow-derived macrophages (BMDMs) were extracted from mice and stimulated by exogenous rIL-10 and/or LPS. The qRT-PCR, Seahorse XFe96 and FAO metabolite related kits were used to test the glycolysis and FAO level in BMDMs. Immunoblotting assay, confocal microscopy and fluorescence microplate were used to test macrophage polarization as well as mitochondrial structure and function damage.
RESULTS: In in vivo experiments, we found that mice lacking CPT1A or IL-10 produced an aggravate inflammatory response to LPS stimulation. However, the addition of rIL-10 could alleviate the pulmonary inflammation in mice effectively. IHC results showed that IL-10 expression in lung macrophage decreased dramatically in Cre+CPT1Afl/fl mice. The in vitro experiments showed Cre+CPT1Afl/fl and IL-10-/- BMDMs became more \"glycolytic\", but less \"FAO\" when subjected to external attacks. However, the supplementation of rIL-10 into macrophages showed reverse effect. CPT1A and IL-10 can drive the polarization of BMDM from M1 phenotype to M2 phenotype, and CPT1A-IL-10 axis is also involved in the process of maintaining mitochondrial homeostasis.
CONCLUSIONS: CPT1A modulated metabolic reprogramming and polarisation of macrophage under LPS stimulation. The protective effects of CPT1A may be partly attributed to the induction of IL-10/IL-10 receptor expression.