RNA sequencing of Chlamydia trachomatis-stimulated HIV-1-infected CD4 T cells

Sexually transmitted infections (STIs) are commonly reported among HIV-1 infected patients. The increasing prevalence of the most common STI, Chlamydia trachomatis (CT), among HIV-1 infected people suggests a role in HIV-1 infectivity. However, the mechanisms modulating the enhancement of HIV-1 infectivity during HIV-1/STIs coinfection remain elusive. The stimulation of CD4 T cells during CT infection may modulate the expression of specific genes, which in turn enhance the susceptibility and infectivity of CT-specific CD4 T cells to HIV-1 infection. After three days of CT stimulation of PBMCs followed by 3 days of HIV-1 infection, we observed a significant increase in HIV-1 p24 levels among clinically diagnosed C. trachomatis-infected patients as compared to cells from healthy donors. Similarly, ex vivo CT antigen-stimulated PBMCs from healthy donors showed enhanced susceptibility to HIV-1 as compared to unstimulated PBMCs. CT-specific CD4 T cells also harbour more HIV-1 copy numbers as compared to healthy unstimulated CD4 T cells. RNA-seq data revealed the upregulation of CCR chemokine receptors and cytokines in CD4 T cells from CT-stimulated CD4 T cells infected with HIV-1. Overall design: PBMCs from healthy blood donors were extracted and stimulated with Chlamydia trachomatis (CT) for three days followed by infection with 50ng/ml of HIV-1 p24 for three days. CD4+ T cells were isolated using the EasySep Human CD4+ T cells isolation kit (Stemcell Technologies, Canada) according to the manufacturer's instruction followed by RNA extraction. Total RNAs from CT-HIV-1, HIV-1 only, and negative controls were used for RNA-seq at the Beijing Genome Institute (China).

性传播感染（Sexually transmitted infections, STIs）在HIV-1感染者中较为常见。在HIV-1感染者群体中，最常见的性传播病原体沙眼衣原体（Chlamydia trachomatis, CT）的患病率持续上升，提示其可能参与调控HIV-1的感染性。然而，HIV-1与性传播感染共感染期间，介导HIV-1感染性增强的具体分子机制仍尚不明确。沙眼衣原体感染过程中对CD4 T细胞（CD4 T cells）的刺激，可调控特定基因的表达，进而增强沙眼衣原体特异性CD4 T细胞对HIV-1感染的易感性与感染性。
在对健康供体细胞进行为期3天的沙眼衣原体刺激后，再予以HIV-1感染3天，我们观察到：临床确诊为沙眼衣原体感染者的样本中，HIV-1 p24水平较健康供体来源的细胞显著升高。类似地，与未受刺激的外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）相比，健康供体来源的经体外沙眼衣原体抗原刺激的外周血单个核细胞，对HIV-1的易感性显著增强。相较于健康未受刺激的CD4 T细胞，沙眼衣原体特异性CD4 T细胞内的HIV-1拷贝数也更高。RNA测序（RNA-sequencing, RNA-seq）数据显示，经沙眼衣原体刺激后再感染HIV-1的CD4 T细胞中，CC趋化因子受体与细胞因子的表达显著上调。
实验整体设计：提取健康献血者的外周血单个核细胞，用沙眼衣原体刺激3天，随后以50ng/ml的HIV-1 p24感染3天。按照制造商说明书，使用EasySep人CD4+ T细胞分离试剂盒（加拿大Stemcell Technologies公司）分离CD4+ T细胞，随后进行RNA提取。将CT-HIV-1共感染组、单纯HIV-1感染组以及阴性对照组的总RNA送至中国北京基因组研究所进行RNA测序。