Innate Immune Control of TB and HIV

据估计，结核病占艾滋病毒感染者总死亡率的三分之一到一半。 自艾滋病开始流行以来，患者就一直是艾滋病患者。目前治疗结核病/艾滋病毒合并感染的方法仍然存在 复杂的药物方案、药物间的相互作用和毒性以及耐药性。因此，小说 加强治疗的治疗方法对于结核病控制和控制的进展至关重要 结核病/艾滋病毒混合感染。为了确定感染，结核分枝杆菌(MTB)和艾滋病毒必须躲避宿主 先天免疫机制，包括检测病原体衍生的核酸的机制。我们最近 研究表明，I型干扰素和rna感应通路中的分子是至关重要的。 限制结核杆菌在人类细胞中生长的宿主因素。此外，我们已经证明，FDA- 批准的药物Nitazoxanide(NTZ)显著增强I型干扰素(干扰素)和RNA传感通路， 诱导细胞应激基因Gadd34，抑制细胞内结核分枝杆菌的生长。我们的初步数据显示， NTZ显著抑制原代人类细胞中HIV、MTB和HIV/TB混合感染的细胞内生长 (PBMC、单核细胞衍生巨噬细胞(MDM)和单核细胞)，在我们建立的一个模型中， 用HIV和MTB感染株建立人肺上皮细胞系。此外，使用CRISPR- 编辑或shRNA修饰的缺乏IFITM3或I型干扰素受体(IFNAR)和成像的细胞 我们已经优化了流式细胞术来评估结核杆菌和HIV的生长，我们已经表明IFITM3和 干扰素信号对结核分枝杆菌和艾滋病毒生长的先天控制至关重要。根据这些初步数据，我们 将在这里测试一个重要的假设，即一组重叠的先天性免疫分子相交 RNA感应、I型干扰素和细胞应激信号通路处于控制结核病和 艾滋病毒的生长，并负责非专利药品的能力，以抑制这两种病原体。在目标1中，我们将使用CRISPR 靶向检测关键RNA传感器和干扰素信号分子在宿主控制HIV和TB/HIV中的作用 我们的结核病/艾滋病毒模型和初级MDM中的感染。同时，我们将建立一种新的人类肺泡 应用单细胞RNA-SEQ研究结核、HIV和TB/HIV混合感染的AT2细胞有机物 询问感染细胞和旁观者细胞的转录本，以检验结核分枝杆菌感染细胞 和NTZ显著影响结核分枝杆菌未感染/HIV感染细胞的生长和幼稚病毒的感染性 旁观者细胞。在目标3中，我们将结合无偏见的rna-seq、CRISPR筛查和ntz- 诱导主要信号通路的磷酸化来验证我们的假设，即NTZ通过 一组分子，其功能在干扰素和细胞应激途径中汇聚。加在一起，这些 实验将识别抑制结核病和艾滋病毒的基因和分子，建立新的结核病有机模型 和艾滋病毒感染，确定宿主导向治疗的新靶点，并潜在地提供关键知识 将NTZ作为抑制结核病和艾滋病毒的补充疗法。

Tuberculosis (TB) is estimated to be responsible for a third to a half of all mortality in HIV-infected patients since the beginning of the AIDS epidemic. Current treatments of TB/HIV co-infection remain complicated by complex drug regimens, drug-drug interactions and toxicity, and drug resistance. Thus, novel treatment approaches to enhance therapy are of critical importance for advances in the control of TB and TB/HIV co-infection. To establish infection, Mycobacterium tuberculosis (MTb) and HIV must evade host innate immune mechanisms including those that detect pathogen-derived nucleic acids. Our recent discoveries have shown that molecules in the type I interferon (IFN) and RNA sensing pathways are critical host factors that restrict MTb growth in human cells. Furthermore, we have shown that that the FDA- approved drug nitazoxanide (NTZ) significantly enhances type I interferon (IFN) and RNA sensing pathways, induces the cell stress gene GADD34, and inhibits intracellular MTb growth. Our preliminary data show that NTZ significantly inhibits intracellular growth of HIV, MTb, and HIV/TB co-infection in primary human cells (PBMC, monocyte-derived-macrophages (MDM), and monocytes) and in a model we have established in a human pulmonary epithelial cell line using infectious isolates of HIV and MTb. Furthermore, using CRISPR- edited or shRNA modified cells that are deficient in IFITM3 or the type I IFN receptor (IFNAR) and imaging flow cytometry we have optimized for evaluation of MTb and HIV growth, we have shown that IFITM3 and IFN signaling are critical to innate control of both MTb and HIV growth. Based on these preliminary data, we will test here the overarching hypothesis that an overlapping set of innate immune molecules that intersect the RNA sensing, type I IFN, and cell stress signaling pathways are at the intersection of control of TB and HIV growth and are responsible for NTZ’s ability to inhibit both pathogens. In Aim 1, we will use CRISPR targeting to test the role of key RNA sensor and IFN signaling molecules in host control of HIV and TB/HIV infections in our TB/HIV models and primary MDM. In parallel, we will establish a novel human lung alveolar epithelial (AT2) cell organoid for study of TB, HIV, and TB/HIV co-infection using single-cell-RNA-seq to interrogate transcriptomes of infected cells and bystander cells to test the hypothesis that MTb-infected cells and NTZ significantly influence the growth of MTb-uninfected/HIV infected cells and the infectability of naïve bystander cells. In Aim 3, we will combine unbiased RNA-seq, CRISPR screening, and profiling of NTZ- induced phosphorylation of major signaling pathways to test our hypothesis that NTZ inhibits TB and HIV via a set of molecules whose function converges in the IFN and cell stress pathways. Together, these experiments will identify genes and molecules that inhibit TB and HIV, establish a new organoid model of TB and HIV infection, identify novel targets for host-directed therapy, and potentially provide critical knowledge for repurposing of NTZ as a complementary therapy to inhibit TB and HIV.