Selenium supplementation induces changes in body composition, increases CD4+ T cells frequency and is associated with the expression of genes responsible of naïve and memory CD8+ T cells function on treated HIV-infected individuals.

Background & aim: Micronutrient deficiencies, particularly those of zinc and selenium, are common in persons living with human immunodeficiency virus (PLWHIV), and have been associated with the development of non-AIDS related comorbidities, impaired immune system function, HIV disease progression and mortality. The implementation of intervention strategies on clinically stable long-term-treated PLWHIV could bring potential benefits on impeding or delaying the onset of non-AIDS associated comorbidities, improving their health status and overall quality of life. The aim of the present study is to analyze the effect of zinc and selenium supplementation on body composition, bone mineral density (BMD), lipids profile, glucose and immune system activation and function on PLWHIV on antiretroviral therapy (ART) without metabolic diseases. Methods: This pilot trial was composed of 60 PLWHIV on ART who were randomly assigned to either zinc, selenium, zinc + selenium supplementation or to a control group. Daily supplementation was prescribed during 6 months as follows: the zinc group received 30 mg of zinc gluconate, the selenium group received 200 mcg of selenium yeast and the zinc + selenium group received both micronutrients at the same doses and presentations. Individuals in the control group were followed during the same time without any nutritional supplementation. Body composition (weight, body mass index [BMI], fat mass and muscle mass), BMD, blood pressure, blood biochemical parameters (cholesterol, glucose and triglycerides), serum zinc and selenium concentrations, CD4+ T cell count and CD4+ and CD8+ T cells immune activation were assessed before and after supplementation. One individual of each supplementation group and one of the control group were analyzed for single cell transcriptomics before and after supplementation. Results: BMI (p=0.03), fat mass in kg (p=0.03) and percentage (p=0.02), as well as trunk fat (p=0.01), were significantly decreased after selenium supplementation. No changes were observed for body composition, BMD, biochemical determinations or blood pressure on the other intervention groups after supplementation (p>0.05 in all cases). The CD4+ T cells frequency and count significantly increased after selenium and zinc supplementation (p=0.03, p=0.05 respectively). CD4+ and CD8+ T-cell immune activation did not change after supplementation (p>0.05 in both cases). On the single cell transcriptome analysis, zinc and selenium supplementation significantly change de expression of genes associated with the function of naive and memory CD8+ T cells (adjusted p<0.05 in all cases). Conclusions: Selenium supplementation have beneficial effects on the body composition, while zinc and selenium supplementation increased CD4+ T cell replenishment of PLWHIV on long-term ART without metabolic diseases. Additionally, zinc and selenium supplementation modified the expression of genes associated with the function of naive and memory CD8+ T cells. Zinc and selenium supplementation are a simple, low-cost, and safe nutritional treatment that represents a complementary intervention to improve the health status and increase the quality of life of clinically stable PLWHIV without metabolic diseases. Registered: Under ClinicalTrails.gov identifier NCT03421314 Overall design: Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-anticoagulated peripheral blood by density gradient centrifugation. Vials with 25 million PBMCs were cryopreserved until used. Blood samples taken at baseline and after six months of treatment were used for targeted scRNA-seq library preparation; the samples came from a control patient (with no supplementation) and three patients that had received zinc, selenium, or zinc plus selenium supplementation, respectively.

研究背景与目的：人类免疫缺陷病毒感染者（PLWHIV）普遍存在微量营养素缺乏症，尤以锌与硒缺乏最为常见，该类缺乏与非艾滋病相关合并症发生、免疫系统功能受损、HIV疾病进展及病死率升高密切相关。对病情稳定的长期接受抗反转录病毒治疗（ART）的人类免疫缺陷病毒感染者实施干预策略，或可阻碍或延缓非艾滋病相关合并症的发作，改善其健康状况与整体生活质量。本研究旨在分析锌与硒补充干预，对无代谢性疾病、接受抗反转录病毒治疗的人类免疫缺陷病毒感染者的身体成分、骨密度（BMD）、血脂谱、血糖及免疫系统活化与功能的影响。

方法：本先导临床试验共纳入60名接受抗反转录病毒治疗的人类免疫缺陷病毒感染者，按随机分组原则分为锌补充组、硒补充组、锌硒联合补充组及对照组。干预周期为6个月，每日补充方案如下：锌补充组给予30mg葡萄糖酸锌，硒补充组给予200μg酵母硒，锌硒联合补充组给予相同剂量与剂型的两种微量营养素；对照组受试者在同期未接受任何营养补充，仅进行随访。分别于补充干预前后检测评估以下指标：身体成分（体质量、体质量指数[BMI]、脂肪量与肌肉量）、骨密度、血压、血液生化指标（胆固醇、血糖与甘油三酯）、血清锌与硒浓度、CD4+T细胞计数，以及CD4+与CD8+T细胞免疫活化状态。此外，每个补充组及对照组各选取1名受试者，在干预前后进行单细胞转录组学分析。

结果：硒补充干预后，受试者体质量指数（p=0.03）、脂肪质量（kg，p=0.03）及脂肪占比（p=0.02）均显著降低，躯干脂肪量亦显著降低（p=0.01）；其余干预组在干预后，身体成分、骨密度、生化指标及血压均未出现显著变化（所有指标p均>0.05）。锌与硒补充干预后，CD4+T细胞的频率与计数均显著升高（分别为p=0.03与p=0.05）；CD4+与CD8+T细胞的免疫活化状态在干预后未发生显著变化（两项指标p均>0.05）。单细胞转录组分析结果显示，锌与硒补充干预可显著改变初始型及记忆型CD8+T细胞功能相关基因的表达（所有校正后p均<0.05）。

结论：硒补充干预可改善身体成分指标；而锌与硒联合补充可提升无代谢性疾病的长期抗反转录病毒治疗人类免疫缺陷病毒感染者的CD4+T细胞补充水平。此外，锌与硒联合补充可调控初始型及记忆型CD8+T细胞功能相关基因的表达。锌与硒补充干预是一种简便、低成本且安全的营养治疗手段，可作为补充干预措施，用于改善无代谢性疾病、病情稳定的人类免疫缺陷病毒感染者的健康状况与生活质量。

注册信息：本研究在ClinicalTrials.gov注册，编号为NCT03421314。

整体实验设计：采用密度梯度离心法从乙二胺四乙酸（EDTA）抗凝外周血中分离外周血单个核细胞（PBMCs），将2500万外周血单个核细胞分装于冻存管中，置于液氮冻存待用。于干预基线及治疗6个月后采集的血液样本用于靶向单细胞RNA测序（scRNA-seq）文库构建；本次测序样本分别来自1名未接受补充干预的对照受试者，以及分别接受锌补充、硒补充、锌硒联合补充的3名受试者。