Peripheral Blood Mononuclear Cells Demonstrate Mitochondrial Damage Clearance During Sepsis

Abstract

Objectives: Metabolic derangements in sepsis stem from mitochondrial injury and contribute significantly to organ failure and mortality; however, little is known about mitochondrial recovery in human sepsis. We sought to test markers of mitochondrial injury and recovery (mitochondrial biogenesis) noninvasively in peripheral blood mononuclear cells from patients with sepsis and correlate serial measurements with clinical outcomes.

Design: Prospective case-control study.

Setting: Academic Medical Center and Veterans Affairs Hospital.

Patients: Uninfected control patients (n = 20) and septic ICU patients (n = 37).

Interventions: Blood samples were collected once from control patients and serially with clinical data on days 1, 3, and 5 from septic patients. Gene products for HMOX1, NRF1, PPARGC1A, and TFAM, and mitochondrial DNA ND1 and D-loop were measured by quantitative reverse transcriptase-polymerase chain reaction. Proinflammatory cytokines were measured in plasma and neutrophil lysates.

Measurements and main results: Median (interquartile range) Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores were 21 (8) and 10 (4), respectively, and 90-day mortality was 19%. Transcript levels of all four genes in peripheral blood mononuclear cells were significantly reduced in septic patients on day 1 (p < 0.05), whereas mitochondrial DNA copy number fell and plasma D-loop increased (both p < 0.05), indicative of mitochondrial damage. D-loop content was directly proportional to tumor necrosis factor-α and high-mobility group protein B1 cytokine expression. By day 5, we observed transcriptional activation of mitochondrial biogenesis and restoration of mitochondrial DNA copy number (p < 0.05). Patients with early activation of mitochondrial biogenesis were ICU-free by 1 week.

Conclusions: Our findings support data that sepsis-induced mitochondrial damage is reversed by activation of mitochondrial biogenesis and that gene transcripts measured noninvasively in peripheral blood mononuclear cells can serve as novel biomarkers of sepsis recovery.

Figure 1.

Gene expression studies (qRT-PCR) in PBMCs in control subjects and septic subjects (paired samples only) on Days (D) 1, 3, and 5. Grey open circles represent individual control subjects. Black horizontal bars show median values. Line graphs represent individual septic subjects for which samples were collected at each time point (e.g. paired samples). Statistical analysis is Kruskal-Wallis ANOVA with Dunn’s post-hoc testing. *P<0.05 denotes statistically significant comparisons relative to D1, and ^# P<0.0.05 denotes statistically significant comparisons relative to control.

Figure 2.

Mitochondrial (mt) DNA copy number measured by qRT-PCR of MT-ND1 gene in PBMCs in control and septic subjects. (A) mtDNA copy number in all collected control and sepsis samples (grey open circles). (B) mtDNA copy number in each control subject (grey open circles) and each septic subject (black line graphs) for which samples were collected at each time point (e.g. paired samples). Black horizontal bars show median values. Statistical analysis is Kruskal-Wallis ANOVA with Dunn’s post-hoc testing. *P<0.05 denotes statistically significant comparisons relative to D1, and ^#P<0.0.05 denotes statistically significant comparisons relative to control. (C) Circulating mitochondrial DNA (mtDNA) D-loop measured by PCR in plasma of control (n=8) and septic subjects (n=12). All samples were collected on study day 1. Black bars show median and interquartile range. Statistical analysis is Mann-Whitney U test.

Figure 3.

Intensive care unit (ICU) freedom by 1 week as a function of PBMC gene expression on Days 1, 3, and 5. Closed grey circles depict individual subjects with sepsis that were discharged from the ICU within 1 week. Open black circles depict individual subjects with sepsis that had prolonged ICU stays (≥ 1 week) or early death (within 1 week of presentation). Left Y-axis shows gene expression (qRT-PCR) for HMOX1, NRF1, PPARGC1A, and TFAM. Right T-axis shows qRT-PCR of mtDNA copy number. Black horizontal bars depict median values. Statistical analysis is Mann-Whitney U test. *P<0.05 denotes statistically significant comparisons. Trends (P<0.1) are depicted on the graphs.

Figure 4.

Partial least-squares discriminant analysis. (A) 2D score plot shows the profiles of control (black circles) and septic (grey circles) subjects are distinct and separate. The grey zone in the score plot depicts outliers with 95% confidence. (B) The corresponding 2D loading plot depicts associations between clusters and the measured variables. The two principal components explain 85% of the variation of the model.