Immunology Graduate Program, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
J Immunol. 2012 Nov 1;189(9):4528-36. doi: 10.4049/jimmunol.1201116. Epub 2012 Sep 24.
Hematopoietic stem cell transplant therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), after bone marrow transplantation (BMT) have impaired host defense against Gram-negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of PGE(2) by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE(2) from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized that epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5'-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared with control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) after treatment with 5-aza-2'-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 698-bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because TGF-β1 is elevated in lungs post-BMT, we tested whether TGF-β1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-β1-induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant-negative form of the TGF-βRII on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE(2) elevation post-BMT are due to hypomethylation of the COX-2 gene, which is at least partly regulated by TGF-β1.
造血干细胞移植治疗受到肺部感染的限制。骨髓移植（BMT）后，具有完全重建的造血隔室（包括肺泡巨噬细胞（AMs））的小鼠对革兰氏阴性铜绿假单胞菌的宿主防御受损。先天免疫受损与 AMs 中 PGE（2）产量增加有关。环氧化酶（COX）-2 是从花生四烯酸合成 PGE（2）的限速酶，BMT 后 AMs 中 COX-2 的表达升高。我们假设表观遗传机制可能负责 AMs 中 COX-2 的上调。通过亚硫酸氢盐测序，我们观察到与对照相比，BMT 小鼠 AMs 中 COX-2 基因的 5'-非翻译区和外显子 1 低甲基化。在用 5-氮杂-2'-脱氧胞苷（一种甲基转移酶抑制剂）处理后，原代 AMs 和 AM 细胞系（MHS）中的 COX-2 表达增加。SssI 甲基转移酶对包含外显子 1 起始的 COX-2 启动子的 698bp 区域进行甲基化，驱动荧光素酶报告基因沉默荧光素酶表达。由于 BMT 后肺部 TGF-β1 升高，我们测试了 TGF-β1 是否可以在高度甲基化的 COX-2 载体中促进 COX-2 的表达，并观察到 TGF-β1 诱导 COX-2 的适度表达，表明其具有启动子去甲基化的能力。最后，用在 CD11c 表达细胞（包括 AMs）上表达 TGF-βRII 显性负形式的骨髓进行 BMT 实验，结果显示宿主防御和 AM 功能得到改善。我们的研究结果表明，BMT 后先天免疫受损和 PGE（2）升高是由于 COX-2 基因的低甲基化，至少部分受 TGF-β1 调节。
