Prolonged Loss of Oxidative Phosphorylation and Mitochondrial Mass Characterize CD66b+ Leukocytes from Patients with Sepsis
Introduction: Sepsis leads to expansion of myeloid-derived suppressor cells (MDSC) and their subtypes. These normally transitory MDSC suppress T cell activation and alter T cell cytokine production while simultaneously promulgating systemic low-grade inflammation. Immune metabolism has been demonstrated to shape cell responses and regulate immune suppression or enhance effector activity. While the metabolism of MDSC has been extensively studied in cancer, the metabolic phenotype of this heterogeneous population in sepsis remains unclear. Our goal was to assess metabolic flux in MDSC during and after sepsis and stratify these patients by characteristics and outcome to determine differences that may guide treatment decisions. Methods: Peripheral blood mononuclear cells (PBMC) from healthy subjects and sepsis patients at four days, 2-3 weeks, and 6 months underwent CD66b+ or CD3+ isolation, followed by assessment of metabolic flux, flow cytometry, mRNA sequencing, and epigenetic analysis. Results: Mitochondrial basal oxygen consumption rates (OCR) and maximal oxygen consumption rates (SRC) were decreased in MDSC from septic patients at four days after infection and persisted for up to six months after sepsis onset. Glycolysis was not impacted by sepsis. In contrast, oxidative metabolism in CD3+ T cells was similar between sepsis patients and healthy subjects. Reduced MDSC oxidative metabolism was linked to adverse clinical outcomes. The decline in oxygen consumption from MDSC in septic patients was also linked to significant reductions in MDSC mitochondrial content. Transcriptomic analysis of CD66b+ cells isolated from PBMC healthy participants and patients with sepsis at four days, 2-3 weeks, and 6 months revealed 19 differentially expressed genes and two long non-coding RNAs as potentially responsible for this decline in mitochondrial mass. Specifically, NR4A3, NR4A2, and TAMLIN/NR4A1 (all critical for mitochondrial biogenesis) expression were persistently decreased with reduced chromatin accessibility indicative of gene silencing. Discussion: After sepsis, PBMC CD66b+ cells present with a unique metabolic signature that is not shared with CD3+ T-cell subsets in these patients or other disease cohorts. These changes in mitochondrial function result from a reduced content of these organelles. We have identified gene silencing, reduced gene expression of key transcription factors that regulate mitochondrial biogenesis, as well as increased long non-coding RNA as potential drivers of this unique metabolic phenotype. These results highlight a need to target metabolism in sepsis to promote immune homeostasis and recovery. Overall design: A total of 383 samples were collected from septic patients: 9 samples at day four, 6 at 2-3 weeks, and 6 samples at six months, as well as individual samples from 12 healthy participants. CD66b+ cells were isolated as described above and total RNA isolated using RNeasy Mini kit (Qiagen 74104) following manufacturers' recommendations.  RNA was quantified using Qubit RNA Broad Range Assay Kit (Ref# Q10210; Invitrogen, MA) and RNA integrity was assessed using RNA ScreenTape kit (Ref# 5067-5576) on TapeStation 4200 (Agilent). Illumina Stranded mRNA Prep and Ligation kit (Ref# 20040534; Illumina, CA) were used to prepare sample libraries following the manufacturer's recommendations. Very briefly, 300 ng/uL high quality RNA was captured and purified, followed by reverse transcription to convert the captured mRNA into first and second strands of cDNA. The resulting double stranded cDNA molecules were then enzymatically fragmented in preparation for unique dual indexing using IDT for Illumina RNA UD Set Ligation Indexes (Ref# 20091655; Illumina, CA). The ligated samples were subsequently enriched through PCR amplification on Bio-Rad Thermal cycler (T100; Bio-Rad, CA) and purified using AMPure XP magnetic beads (Ref# A63881; Beckman Coulter, CA). The average fragment length of the library insert was measured to be ~300–400 bp using the dsDNA 1000 kit (Ref# 5067-5582) on Agilent TapeStation. Optimized libraries were pooled to a final concentration of 150pM. The libraries were sequenced on NovaSeq X Plus Platform (Illumina, CA) at UF's Interdisciplinary Center for Biotechnology Research (ICBR).