Innate Immune Control of TB and HIV

结核病和艾滋病毒的先天免疫控制

• 批准号: 10426882
• 负责人: ANNE GOLDFELD
• 金额: $ 64.61万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-16 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426882
• 关键词: Acquired Immunodeficiency Syndrome; Antiparasitic Agents; Antiviral Agents; Antiviral Response; Biological Models; Boston; CD4 Positive T Lymphocytes; CRISPR screen; CRISPR/Cas technology; Cell Line; Cell model; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complementary therapies; Complex; Critical Pathways; Data; Detection; Disease; Disease Progression; Double-Stranded RNA; Drug Interactions; Drug resistance; Drug toxicity; Effectiveness; Epidemic; Epithelial Cells; Evaluation; FDA approved; Flow Cytometry; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Growth; HIV; HIV Infections; HIV drug resistance; HIV/TB; Human; IFNAR1 gene; Image; Immune; Immunologics; Infection; Injury; Innate Immune Response; Integration Host Factors; Interferon Receptor; Interferon Type I; Interferons; Knowledge; Laboratories; Lead; Lung; Modeling; Mycobacterium tuberculosis; Nucleic Acids; Organism; Organoids; PRKR gene; Pathway interactions; Patients; Pediatric Hospitals; Peripheral Blood Mononuclear Cell; Phagosomes; Pharmaceutical Preparations; Phosphorylation; Play; Production; Protein Family; Proteins; RNA; RNA amplification; Regimen; Research Personnel; Resistance; Risk; Role; Signal Pathway; Signal Transduction; Signaling Molecule; T-Cell Depletion; Testing; Therapeutic; Tretinoin; Tuberculosis; Type II Epithelial Receptor Cell; Viral; alveolar epithelium; antimicrobial drug; base; co-infection; experimental study; innate immune mechanisms; macrophage; melanoma; member; monocyte; mortality; nitazoxanide; novel; novel strategies; pathogen; physiologic model; reactivation from latency; response; sensor; single-cell RNA sequencing; small hairpin RNA; transcriptome; transcriptome sequencing; tuberculosis treatment

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.

据估计，结核病（TB）占艾滋病毒感染者死亡率的三分之一至一半。 从艾滋病流行开始。目前对结核病/艾滋病毒合并感染的治疗仍然是 复杂的药物方案、药物间相互作用和毒性以及耐药性使其复杂化。因此，小说 加强治疗的治疗方法对于结核病控制的进展至关重要， 结核病/艾滋病毒合并感染。要建立感染，结核分枝杆菌（MTb）和艾滋病毒必须逃避宿主 先天免疫机制，包括检测病原体衍生核酸的机制。我们最近 一些发现表明，I型干扰素（IFN）和RNA传感途径中的分子是至关重要的 限制MTb在人类细胞中生长的宿主因素。此外，我们已经表明，FDA- 批准的药物硝唑尼特（NTZ）显著增强I型干扰素（IFN）和RNA传感途径， 诱导细胞应激基因GADD 34，抑制细胞内MTb生长。初步数据显示， NTZ显著抑制HIV、MTb和HIV/TB共感染原代人细胞的细胞内生长 (PBMC单核细胞衍生的巨噬细胞（MDM）和单核细胞），并在我们建立的模型中， 使用HIV和MTb感染性分离株的人肺上皮细胞系。此外，使用CRISPR- 编辑的或shRNA修饰的IFITM 3或I型IFN受体（IFNAR）缺陷的细胞和成像 我们已经优化了流式细胞术用于评估MTb和HIV生长，我们已经表明IFITM 3和 IFN信号传导对MTb和HIV生长的先天控制至关重要。根据这些初步数据，我们 我将在这里测试一个总体假设，即一组重叠的先天免疫分子， RNA传感、I型IFN和细胞应激信号传导途径处于结核病控制和 HIV的生长和负责NTZ的能力，以抑制这两种病原体。在目标1中，我们将使用CRISPR 靶向测试关键RNA传感器和IFN信号传导分子在宿主控制HIV和TB/HIV中的作用 我们的结核病/艾滋病模型和原发性MDM中的感染。与此同时，我们将建立一种新的人类肺泡 上皮（AT 2）细胞类器官，用于使用单细胞RNA-seq研究TB、HIV和TB/HIV共感染， 询问受感染细胞和旁观者细胞的转录组以检验MTb感染细胞 和NTZ显著影响MTb-未感染/HIV感染细胞的生长和幼稚细胞的感染性， 旁观者细胞在目标3中，我们将结合联合收割机无偏的RNA-seq、CRISPR筛选和NTZ-1的分析。 诱导的主要信号通路的磷酸化，以验证我们的假设，即NTZ抑制结核病和艾滋病毒通过 一组分子，其功能在IFN和细胞应激途径中会聚。所有这些 实验将确定抑制结核病和艾滋病病毒的基因和分子，建立一个新的结核病类器官模型， 和HIV感染，确定宿主导向治疗的新靶点，并可能提供关键知识 重新利用NTZ作为抑制TB和HIV的补充疗法。