Dysfunctional Myelopoiesis and Myeloid-Derived Suppressor Cells in Sepsis Pathobiology

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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.