ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides
ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides
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ATF4 缺乏通过减少谷氨酰胺的摄取和抗菌肽的表达促进小鼠肠道炎症
DOI：
10.1053/j.gastro.2018.11.033
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发表时间：
2019-03-01
期刊：
影响因子：
29.4
通讯作者：
Guo, Feifan
中科院分区：
文献类型：
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作者：
Hu, Xiaoming;Deng, Jiali;Guo, Feifan
背景与目的：激活转录因子4(ATF4)调控炎症反应、氨基酸代谢、自噬和内质网应激等相关基因。方法：对21例活动期克罗恩病(CD)患者、22例活动期溃疡性结肠炎(UC)患者和38例正常对照(对照组)、19例活动期克罗恩病(CD)患者和8例非活动期溃疡性结肠炎(UC)患者的回肠组织和19例活动期CD患者和8例非IBD患者的结肠组织进行胃镜检查。ATF4在肠上皮细胞中被破坏的小鼠(Atf4DIEC小鼠)和ATF4漂白的小鼠(对照组)被给予葡聚糖硫酸钠(DSS)诱导结肠炎。部分小鼠在饮水中注射重组防御素A1和补充L丙氨酰谷氨酰胺或谷氨酰胺。用实时定量聚合酶链式反应、免疫印迹和免疫组织化学方法分析人和小鼠的回肠和结肠组织。采用高效液相色谱-串联质谱法测定血清和肠上皮细胞(IEC)氨基酸含量。在IEC-18细胞中，ATF4的水平被小干扰RNA下调。结果：活动期CD和活动期UC患者炎症性肠黏膜中ATF4的表达水平明显低于非炎症区或正常对照组。结肠炎小鼠与非结肠炎小鼠相比，结肠炎小鼠结肠上皮细胞ATF4表达也降低。给予DSS后，Atf4DIEC小鼠出现自发性小肠结肠炎和结肠炎，其程度比对照组小鼠更严重。Atf4DIEC小鼠血清谷氨酰胺水平降低，回肠Paneth细胞抗菌肽水平降低，如Defa1、Defa4、Defa5、Camp和Lyz1。与对照组相比，Atf4DIEC小鼠的回肠微生物群发生了变化；这些变化被给予谷氨酰胺逆转。注射DEFA1可降低Atf4DIEC小鼠自发性肠炎和DSS诱导的结肠炎的严重程度。我们发现细胞中的谷氨酰胺转运蛋白溶质载体家族1成员5(SLC1A5)的表达受ATF4的直接调控。在IEC-18或原代IEC细胞中过表达SLC1A5可增加谷氨酰胺摄取和抗菌肽的表达。在IEC-18细胞中ATF4基因敲除增加了炎性细胞因子的表达，而SLC1A5在敲除细胞中过表达则减少了细胞因子的表达。CD和UC患者炎症肠黏膜中SLC1A5水平降低，且与ATF4水平呈正相关。结论：活动期CD和UC患者炎症肠黏膜中ATF4水平降低。在小鼠中，ATF4缺乏通过减少SLC1a5的转录来减少肠上皮细胞对谷氨酰胺的摄取和抗菌肽的表达。因此，ATF4可能成为治疗IBD的靶点。
BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis.METHODS: We obtained biopsy samples during endoscopy from inflamed and/ or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4DIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin a1 (DEFA1) and supplementation of L-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by highperformance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing.RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4DIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4DIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4DIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4DIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4.CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.