Table3_Aconitate decarboxylase 1 mediates the acute airway inflammatory response to environmental exposures.docx

BackgroundEnvironmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung disease. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Aconitate decarboxylase 1 (Acod1) was one of the most upregulated genes following LPS (vs. saline) exposure of murine whole lungs with transcriptomic profiling of sorted lung monocyte/macrophage subpopulations also highlighting its significance. Given monocyte/macrophage activation can be tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator ACOD1 in environmental exposure-induced lung inflammation.
MethodsWild-type (WT) mice were intratracheally (i.t.) instilled with 10 μg of LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 h post-exposure. Next, WT and Acod1−/− mice were instilled with LPS, 25% organic dust extract (ODE), or saline, whereupon serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Invasive lung function testing was performed 3 h post-LPS with WT and Acod1−/− mice.
ResultsAcod1−/− mice treated with LPS demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4+ T cells, decreased BALF and lung levels of TNF-α, and decreased BALF CXCL1 compared to WT animals. In comparison, Acod1−/− mice treated with ODE demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B-cell infiltrates with decreased BALF levels of TNF-α and IL-6 and decreased lung CXCL1 vs. WT animals. Mediators of tissue remodeling (TIMP1, MMP-8, MMP-9) were also decreased in the LPS-exposed Acod1−/− mice, with MMP-9 also reduced in ODE-exposed Acod1−/− mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyperresponsiveness in Acod1−/− animals.
ConclusionAcod1 is robustly upregulated in the lungs following LPS exposure and encodes a key immunometabolic regulator. ACOD1 mediates the proinflammatory response to acute inhaled environmental LPS and organic dust exposure-induced lung inflammation.

背景：
环境中的脂多糖（lipopolysaccharide, LPS）以及富含微生物组分的有机粉尘可引发严重肺部疾病。此类环境暴露可诱导肺部不同单核细胞/巨噬细胞亚群的招募与活化，这些亚群参与疾病的发病机制。顺乌头酸脱羧酶1（aconitate decarboxylase 1, Acod1）是脂多糖（与生理盐水对照）暴露于小鼠全肺后上调最显著的基因之一；对分选得到的肺部单核细胞/巨噬细胞亚群进行转录组分析，也证实了其重要性。鉴于单核细胞/巨噬细胞的活化与代谢密切相关，本研究旨在探究免疫代谢调控因子ACOD1在环境暴露诱导的肺部炎症中的作用。

方法：
将野生型（wild-type, WT）小鼠经气管内（intratracheally, i.t.）滴注10 μg脂多糖或生理盐水。对全肺进行批量RNA测序，或通过分选分离单核细胞/巨噬细胞亚群。暴露后48小时，采用NanoString先天免疫多重芯片对分选得到的亚群进行转录组学表征。随后，分别向野生型小鼠及Acod1基因敲除（Acod1−/−）小鼠滴注脂多糖、25%有机粉尘提取物（organic dust extract, ODE）或生理盐水，之后采集血清、支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）及肺组织。采用质谱法定量检测三羧酸循环（tricarboxylic acid, TCA）相关的支气管肺泡灌洗液代谢物。通过酶联免疫吸附试验（ELISA）定量检测细胞因子/趋化因子及组织重塑介质。采用流式细胞术对肺部免疫细胞进行表征。在脂多糖暴露后3小时，对野生型及Acod1基因敲除小鼠进行侵入性肺功能检测。

结果：
与野生型小鼠相比，经脂多糖处理的Acod1基因敲除小鼠的支气管肺泡灌洗液中衣康酸水平降低、三羧酸循环发生重编程，支气管肺泡灌洗液中性粒细胞减少，肺部CD4+ T细胞增多，支气管肺泡灌洗液及肺组织中肿瘤坏死因子-α（TNF-α）水平降低，支气管肺泡灌洗液中CXCL1水平下降。与之相比，经有机粉尘提取物处理的Acod1基因敲除小鼠，其血清中五聚蛋白2（pentraxin-2, PTX2）水平降低，支气管肺泡灌洗液中衣康酸水平降低，肺部总细胞、中性粒细胞、单核细胞及B细胞浸润减少，支气管肺泡灌洗液中TNF-α及白细胞介素-6（IL-6）水平降低，肺组织中CXCL1水平较野生型小鼠下降。在经脂多糖暴露的Acod1基因敲除小鼠中，组织重塑介质（金属蛋白酶组织抑制因子1（TIMP1）、基质金属蛋白酶8（MMP-8）、基质金属蛋白酶9（MMP-9））的水平也有所降低；而在经有机粉尘提取物暴露的Acod1基因敲除小鼠中，MMP-9的水平同样降低。肺功能检测结果显示，Acod1基因敲除小鼠对脂多糖诱导的气道高反应性的应答被削弱。

结论：
脂多糖暴露后，肺部的Acod1表达显著上调，其编码的蛋白是关键的免疫代谢调控因子。ACOD1可介导急性吸入环境脂多糖及有机粉尘暴露所引发的肺部炎症的促炎应答。