DNA methylation demonstrates bronchoalveolar cell senescence in people living with HIV: An observational cohort study

Background: Age-related comorbidities remain a burden in People living with HIV (PLWH). DNA methylation may be a link between HIV, aging, and the increased risk of lung comorbidities. Here, we investigate whether bronchoalveolar lavage (BAL) cells of PLWH demonstrate epigenome wide disruptions and advanced epigenetic aging. Methods: BAL cell DNA methylation profiles from 25 PLWH and 16 uninfected controls were tested for differential methylation of transposable elements Alu and LINE-1, a marker of aging. We used a weighted gene correlation network analysis to identify co-methylation networks associated with HIV and age. We tested the effect of HIV on DNA methylation using a robust linear model. An enrichment analysis was conducted to identify differentially methylated pathways (false discovery rate <0.10). Results: The BAL cells of PLWH were marked by global hypomethylation in both Alu and LINE-1 elements. Six co-methylated CpG networks were identified that were significantly associated with age; of these, the red module was significantly differentially methylated in PLWH and enriched pathways associated with HIV, Ras signaling, T cell receptors, and bacterial invasion of epithelial cells. We identified 6,428 differentially methylated sites associated with HIV. Conclusions: We have shown here for the first time that alterations in the DNA methylation of BAL cells (mainly macrophages) in the lung HIV show in a pattern of advanced aging. In conjunction with our findings in blood cells and airway cells, this study strongly support that HIV may contribute to increase the risk of lung comorbidities through the epigenetic regulation of aging. 41 samples profiles for Bronchoalveolar lavage DNA methyaltion

**背景**：人类免疫缺陷病毒（HIV）感染者（People living with HIV, PLWH）仍深受年龄相关共病的困扰。DNA甲基化可能是连接HIV感染、衰老与肺部共病风险升高的纽带。本研究旨在探讨HIV感染者的支气管肺泡灌洗（bronchoalveolar lavage, BAL）细胞是否存在全表观基因组紊乱及表观遗传衰老加速现象。

**方法**：本研究对25名PLWH与16名未感染对照者的BAL细胞DNA甲基化谱进行分析，检测转座元件Alu与LINE-1（衰老标志物）的差异甲基化水平。采用加权基因共表达网络分析筛选与HIV感染及年龄相关的共甲基化网络；通过稳健线性模型检验HIV感染对DNA甲基化的影响；开展富集分析以鉴定差异甲基化通路（错误发现率<0.10）。

**结果**：PLWH的BAL细胞在Alu与LINE-1转座元件中均呈现全基因组低甲基化特征。共鉴定出6个与年龄显著相关的共甲基化CpG网络；其中红色模块在PLWH中呈现显著差异甲基化，且富集了与HIV感染、Ras信号通路、T细胞受体及细菌侵袭上皮细胞相关的通路。本研究共鉴定出6428个与HIV感染相关的差异甲基化位点。

**结论**：本研究首次证实，肺部HIV感染者的BAL细胞（主要为巨噬细胞）的DNA甲基化改变呈现表观遗传衰老加速的模式。结合本团队在血细胞与气道细胞中的研究发现，本研究有力支持HIV感染可通过表观遗传调控衰老过程，进而升高肺部共病风险这一结论。本研究共纳入41份支气管肺泡灌洗DNA甲基化样本谱。