Resistance to MTB infection in HIV infected individuals in Uganda and S. Africa

 描述(由申请人提供)： 项目概述大多数严重接触结核分枝杆菌的人都会被感染，并发展成潜伏的结核分枝杆菌感染。然而，在乌干达城市的一项结核病接触研究中，大约9%的密切成人接触者在两年的随访中没有发生下呼吸道合并症。在南非，尽管在世界上最大的结核杆菌感染压力下工作了20多年，一些矿工仍然没有发生肺结核病。这些没有LTBI证据的人，尽管有强烈的结核分枝杆菌暴露，似乎是LTBI抵抗者(RSTR)。结核分枝杆菌感染后的单核细胞表现出转录特征，将RSTR与LTBI的HHC区分开来。这些数据支持这样的假设，即宿主因素可以区分RSTR，并且它们具有在巨噬细胞中表达的保护性先天免疫反应。艾滋病毒对结核分枝杆菌的免疫反应失调，因此艾滋病毒感染者比艾滋病毒感染者更依赖先天免疫来控制结核分枝杆菌。因此，我们假设，在HIV+RSTR中更容易识别对LTBI的先天耐药机制。其目标是：1.在乌干达的结核病家庭接触者和南非的矿工中，识别、表征和招募抵抗潜在结核分枝杆菌感染的艾滋病毒+个人队列。方法：跟踪以前确定的HIV+RSTR，并在乌干达从结核病HHC中招募新的RSTR，南非招募新的HIV+RSTR队列。HIV+LTB、HIV-RSTR和HIV-LTB将作为对照。假设：HIV+和HIV-RSTR可以在暴露于不同结核分枝杆菌的人群中识别，但宿主抵抗机制将是共同的，因此能够为先天性免疫功能研究聚集强大的队列。目的2.确定与HIV+人群对结核分枝杆菌潜伏感染的抵抗力相关的巨噬细胞转录特征。方法：比较HIV+RSTR和LTB对巨噬细胞中结核分枝杆菌的早期转录反应。将这些与HIV-RSTR和HIV-LTBI的巨噬细胞的反应进行比较，并将研究扩展到肺泡巨噬细胞。假设：巨噬细胞中的生物途径调节对结核分枝杆菌感染的抵抗力，这些途径在HIV+患者中更容易识别。目的3.确定Mtb潜伏感染耐药的细胞学机制。方法：a.通过CRISPR/Cas9技术和小分子抑制剂敲除细胞，以研究AIM 2中确定的关键基因和途径如何允许巨噬细胞抵抗或清除结核杆菌；b.全血结核杆菌杀伤试验以区分HIV+RSTR和LTB I；c.流式细胞术检查先天非传统T细胞是否富含HIV+RSTR。假设：a.HIV+RSTR使用巨噬细胞特异性途径来控制结核分枝杆菌；b.WBA可以检测到RSTR和LTBI在控制结核分枝杆菌生长方面的差异；c.HIV+RSTR使用非传统的固有T细胞来控制结核分枝杆菌。在这个项目中，我们汇集了两个经验丰富的结核病临床研究地点(乌干达的穆拉戈医院和南非的奥鲁姆研究所)，以及乌干达Makerere大学、南非奥鲁姆研究所、华盛顿大学和凯斯西储大学的流行病学家、系统生物学家、遗传学家、免疫学家、微生物学家和临床医生。

 DESCRIPTION (provided by applicant): PROJECT SUMMARY Most people heavily exposed to Mycobacterium tuberculosis become infected and develop latent Mtb infection. However, in a TB contact study in urban Uganda, approximately 9% of close adult contacts did not develop LTBI during two years of follow-up. In S. Africa, some miners do not develop LTBI despite more than 20 years of employment under the highest Mtb infection pressure in the world. These persons without evidence of LTBI despite intense Mtb exposure appear to be LTBI resisters (RSTRs). Monocytes after Mtb infection revealed transcriptional signatures that distinguished RSTRs from HHC with LTBI. These data support the hypothesis that host factors can distinguish RSTRs and that they have protective innate immune responses expressed in macrophages. HIV dysregulates immune responses to Mtb, thus HIV infected persons depend more on innate immunity to control Mtb than HIV- persons. Thus we hypothesize that innate mechanisms of resistance to LTBI are more readily identified in HIV+ RSTRs. The aims are: 1. Identify, characterize and recruit cohorts of HIV+ individuals who resist latent Mtb infection among TB household contacts in Uganda and among miners in South Africa. Approach: Follow-up previously identified HIV+ RSTRs and recruit new RSTRs from among TB HHC in Uganda, and i S. Africa recruit a new cohort of HIV+ RSTRs. HIV+ LTBIs, and HIV- RSTRs and HIV- LTBIs will serve as controls. Hypothesis: HIV+ and HIV- RSTRs can be identified in populations with different Mtb exposures but host resistance mechanisms will be shared and thus enable assembly of robust cohorts for innate immune function studies. Aim 2. Determine macrophage transcriptional signatures associated with resistance to latent Mtb infection in HIV+ persons. Approach: Compare early transcriptional responses to Mtb in macrophages between HIV+ RSTRs and LTBIs. Compare these to the responses of macrophages from HIV- RSTRs and HIV- LTBIs, and extend studies to alveolar macrophages. Hypothesis: Biological pathways in macrophages regulate resistance to Mtb infection and these pathways are more readily identified in HIV+ persons. Aim 3. Determine cellular mechanisms of resistance to latent Mtb infection. Approach: a. Cellular knockouts through CRISPR/Cas9 technology and small molecule inhibitors to investigate how key genes and pathways identified in Aim 2 allow macrophages to resist or clear Mtb; b. Whole blood assays of Mtb killing to distinguish HIV+ RSTRs from LTBIs; c. Flow cytometry to examine whether innate unconventional T-cells are enriched in HIV+ RSTRs. Hypothesis: a. HIV+ RSTR use macrophage-specific pathways to control Mtb; b. A WBA can detect differences in control of Mtb growth between RSTRs and LTBIs; c. HIV+ RSTRs use unconventional innate T cells to control Mtb. For this project, we have assembled two experienced TB clinical research sites (Mulago Hospital in Uganda & the Aurum Institute in S. Africa) with epidemiologists, systems biologists, geneticists, immunologists, microbiologists, and clinicians at Makerere University in Uganda, Aurum Institute in S. Africa, University of Washington and Case Western Reserve University.