Dysfunctional Myelopoiesis and Myeloid-Derived Suppressor Cells in Sepsis Pathobiology

摘要 脓毒症仍然是影响我们成年人口的最灾难性的挑战之一。我们认为， 创伤、脓毒症和危重病是骨髓疾病，代表骨髓的“病理性激活”。 骨髓细胞群骨髓源性抑制细胞（MDSC）的优先骨髓生成和扩增是 这是危重病的标志，无论是由于严重创伤还是败血症。我们花了十年时间 以大量的血液和骨髓免疫反应以及造血细胞为特征， 骨髓中的反应。全血转录组学和荧光激活细胞分选策略可以 仅捕获大量富集的细胞群体。推进这些研究需要更多的 单个干细胞、骨髓和淋巴细胞群的功能和表型的颗粒评估 随着时间的推移，在脓毒症患者中具有不同的结局。在过去的十年里，我们一直依赖于 使用基于微阵列的方法，最初由Glue Grant使用GeneChip™开发的转录组学 技术.最近，我们已经转移到下一代测序（NGS）以获得外显子水平的细胞DNA。 转录组学和表观遗传学，以及血浆组蛋白结合的游离DNA序列。我们现在还 使用新的第二代多组学技术实施单细胞RNA-seq技术： 通过测序的转录组和表位的细胞索引（CITE-seq）。在程序中，RM 1 GM 139690， 在脓毒症病理生物学中，我们打算使用 血液白细胞上的NGS和从300例败血症患者获得的血液单核细胞（PBMC）级分上的CITE-seq 以及在多个时间点具有不同临床结果的创伤患者。从历史上看，我们依赖于 外部实体（布罗德研究所）或UF共享资源（跨部门生物医学研究中心）， NGS。然而，由于测序需求的需求大幅增加， 以及败血症和危重病研究中心（SCIRC）的其他NIGMS资助项目， 使用商业实体或学院范围内的共享单元不再是辩护。我们不仅付更高的价钱 包括利润和管理费用在内的成本，但高质量、快速的分析回报率随着我们的增长而下降 在数量上。最重要的是，我们不存在NIH要求的确保严格性和可重复性的能力， 利用现有的外部实体。在这里，我们建议购买Illumina NextSeq 1000™测序仪， SCIRC内部的共享资源，以提供NGS（包括CITE-seq）来支持RM 1计划和其他 NIGMS资助的创伤和败血症项目。我们设想每年分析200多个样本， 提出的目标。医学院（COM）保证的额外机构资源将支付 服务合同（每年约2.5万美元），用于资助终身，并支持专门的生物科学家 （每年约6万美元）来操作仪器（见所附信件）。通过国家地理信息系统和 的UF COM，可以实现高严谨性和再现性的方案目标。

ABSTRACT Sepsis remains one of the most catastrophic challenges affecting our adult population. We have argued that trauma, sepsis and critical illness are diseases of the bone marrow, and represent ‘pathologic activation’ of myeloid populations. Preferential myelopoiesis and expansion of myeloid-derived suppressor cells (MDSC) are a hallmark of critical illness, whether due to severe trauma or sepsis. We have spent the past decade characterizing in bulk both the blood and bone marrow immunological response, as well as the hematopoietic response in bone marrow. Whole blood transcriptomics and fluorescently-activated cell sorting strategies can only capture large cohorts of enriched cell populations. Moving forward with these studies requires a more granular assessment of the function and phenotype of individual stem cell, myeloid and lymphoid cell populations over time, in patients with differential outcomes to sepsis. For the past decade, we have relied on bulk transcriptomics using a microarray-based approach originally developed by the Glue Grant using GeneChip™ technology. More recently, we have moved to next generation sequencing (NGS) to obtain both exon-level cell transcriptomics and epigenetics, as well as plasma histone-bound free-DNA sequences. We are also now implementing single-cell RNA-seq technologies using a novel second generation multi-omics technology: Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq). In the program, RM1 GM139690, Dysfunctional Myelopoiesis and Myeloid-Derived Suppressor Cells in Sepsis Pathobiology, we intend to use NGS on blood leukocyte and CITE-seq on blood mononuclear cell (PBMC) fractions obtained from 300 sepsis and trauma patients with different clinical outcomes at multiple time points. Historically, we have relied on either outside entities (Broad Institute) or UF-shared resources (Interdepartmental Center for Biomedical Research) for NGS. However, as the need for sequencing requirements has increased substantially secondary to this program as well as other funded NIGMS-funded programs in the Sepsis and Critical Illness Research Center (SCIRC), use of a commercial entity or college-wide shared unit is no longer defensible. Not only are we paying higher costs that include profit and overhead, but high quality, rapid return on analyses has declined with our increases in volume. Most importantly, our ability to assure rigor and reproducibility as required by NIH is nonexistent with use of the current outside entities. Here we propose the purchase of an Illumina NextSeq 1000™ sequencer as a shared-resource within SCIRC to provide NGS (including CITE-seq) to support the RM1 program and other NIGMS-funded programs in trauma and sepsis. We envision analyzing over 200 samples annually based on the proposed goals. Additional institutional resources guaranteed from the College of Medicine (COM) will pay the service contract (~$25k annually) for the life of the grant award(s) and support a dedicated biological scientist (~$60k annually) to operate the instrument (see attached letters). Through combined support from NIGMS and the UF COM, the programmatic goals can be achieved with high rigor and reproducibility.