Commensal microbiome impact on CD4 T cell lymphopenia in HIV-1 infection

项目摘要/摘要 艾滋病毒感染的免疫无反应(INR)患者患共病和死亡的风险比 在抗逆转录病毒治疗(ART)后恢复其CD4T细胞计数(IR)的免疫应答者(IR)。INR CD4T细胞计数低(&lt；350c/ul)，全身炎症加剧，CD4T细胞周期增加 (Ki67)。我们之前报道了INR CD4T细胞通过转录和流动功能障碍的线粒体 细胞分析。在这里，我们报告了分选记忆CD3、CD4、CD45RO T细胞的代谢特征 来自健康对照组(HC)、IR和INR，并发现三羧酸循环(TCA)失调 电子显微镜成像(EMI)证实线粒体功能障碍的中间体。 高尔基体结构紊乱，线粒体数量减少，线粒体脊不活跃，有证据表明 在INR EMI切片中检测到有丝分裂。我们报告了一项新的观察结果，即记忆CD4T细胞和 来自几个队列的INR血浆样本被浓缩以检测肠道来源的细菌毒素p- 甲酚硫酸盐(PCS)和吲哚硫酸盐(IS)均与CD4T细胞计数呈负相关。孵化 来自HC的外周血单个核细胞(PBMC)与PCS或IS在体外阻断CD4T细胞的增殖， 诱导细胞凋亡，减少线粒体蛋白(COX-IV和MTFA)的表达。EMI 揭示了线粒体网络的扰动，与在INR中发现的类似 来自HC的记忆CD4T细胞与PCS或提示PCS和IS可能是导致PCS减少的原因 INR CD4T细胞线粒体适合性的研究用细菌16S rDNA分析发现大便中有丰富的 与代谢酪氨酸的蛋白水解菌属(双歧杆菌、瘤胃球菌、乳杆菌) 和苯丙氨酸氨基酸来产生PCS。我们认为肠道细菌菌群毒素可能损害CD4。 ART过程中T细胞的恢复，可能与INR所特有的CD4T细胞减少有关。我们 从机制上提出PCS/IS对线粒体网络有毒性，线粒体DNA是 释放在细胞质中，激活SING，触发线粒体死亡途径的半胱氨酸酶，IRF3， 干扰素敏感反应元件(ISRE)基因参与细胞凋亡和诱导下睑下垂 (AIM1)。我们将讨论血浆PCS/IS在大量INR队列中的丰富是否与Low相关 一组体外实验中的CD4T细胞计数、细胞凋亡/STING水平和线粒体功能障碍 (AIM2)。然后，我们将通过执行元基因组学、元蛋白质组学和代谢分析来探索， INR受试者肠道共生细菌是否处于菌群失调和菌种丰富的状态 PCS和IS生产过剩(AIM3)。

Project Summary/Abstract HIV-infected Immune non-responders (INR) persons are at greater risk of comorbidity and mortality than are immune responders (IR) who restore their CD4 T cells count (IR) after anti- retroviral therapy (ART). INR have low CD4 T cell counts (<350 c/ul), heightened systemic inflammation, and increased CD4 T cell cycling (KI67+). We previously reported dysfunctional mitochondria in INR CD4 T cells by transcriptomic and flow cytometric assays. In here we report the metabolic profiles of sorted memory CD3+CD4+CD45RO+T cells from healthy controls (HC), IR, and INR and found dysregulation of the tricarboxylic acid cycle (TCA) intermediates suggestive of mitochondrial dysfunction confirmed by Electron Microscopy Imaging (EMI). Disrupted Golgi structure, reduced mitochondrial numbers, inactive mitochondrial cristae, and evidence of mitophagy were detected in INR EMI sections. We report the novel observation that memory CD4 T cells and plasma samples of INR from several cohorts are enriched for levels of the gut-derived bacterial toxins p- cresol-sulfate (PCS) and indoxyl sulfate (IS) that both negatively correlate with CD4 T cell counts. Incubation of peripheral blood mononuclear cells (PBMCs) from HC with PCS or IS blocks CD4 T cell proliferation in vitro, induces apoptosis, and diminishes the expression of the mitochondrial proteins (COX-IV and mTFA). EMI reveals perturbations of mitochondria network similar to those found in INR following incubation of sorted memory CD4 T cells from HC with PCS or IS suggesting that PCS and IS may contribute to the diminished mitochondrial fitness of INR CD4 T cells. Using bacterial 16S rDNA assay INR stool were found enriched with proteolytic bacterial genera (Bifidobacterium, Ruminococcus, Lactobacillus) that metabolize tyrosine and phenylalanine amino acids to produce PCS. We propose that gut bacterial flora toxins may impair CD4 T cell recovery during ART and may contribute to CD4 T cell lymphopenia characteristic of INR. We propose, mechanistically, that PCS/IS induce toxicity to the mitochondrial network, mitochondrial DNA is then released in the cytosol and activates STING that triggers caspases of the mitochondrial death pathway, IRF3, and Interferon-Sensitive- Response Element (ISRE) genes implicated in apoptosis and pyroptosis induction (AIM1). We will address whether enrichment of plasma PCS/IS in a large cohort of INR is correlated to low CD4 T cell count, apoptosis/STING levels, and mitochondrial dysfunction in a set of ex-vivo experiments (AIM2). We will then explore, by performing metagenomics, metaproteomics, and metabolic analyses, whether commensal bacteria in the gut of INR subjects are in dysbiosis and enriched in bacterial strains overproducing PCS and IS (AIM3).