Fall in circulating mononuclear cell mitochondrial DNA content in human sepsis

Abstract

Purpose: Loss of mitochondrial DNA (mtDNA) has been described in whole blood samples from a small number of patients with sepsis, but the underlying mechanism and clinical implications of this observation are not clear. We have investigated the cellular basis of the mtDNA depletion in sepsis, and determined clinical correlates with mtDNA depletion.

Methods: Whole blood samples were obtained from 147 consecutive patients with severe sepsis admitted to a General Critical Care Unit in a University Hospital and 83 healthy controls. In a separate study of 13 patients with severe sepsis, blood was obtained for immediate cell sorting by flow cytometry. MtDNA content was determined in whole blood DNA by PCR methods, and subsequently in the 13 samples where white cell subtypes were separated.

Results: The mtDNA content of peripheral blood in human subjects was lower in patients with sepsis than controls (P < 0.0001). By studying leukocyte subsets in a subgroup of 13 patients, we showed that this was largely due to an increase in the proportion of circulating neutrophils, which contained approximately 3-fold less mtDNA than mononuclear leukocytes. However, isolated monocytes (P = 0.041) and lymphocytes (P = 0.021) from septic patients showed clear evidence of mtDNA depletion, which correlated with the APACHE II score (P = 0.015).

Conclusions: In severe sepsis much of the apparent whole blood mtDNA depletion is due to a change in the differential leukocyte count. However mtDNA depletion in mononuclear cells occurs in patients with sepsis and correlates with disease severity.

Fig. 1

Relative mtDNA/nDNA ratio in whole blood DNA from 147 sepsis patients and 83 controls. Horizontal lines represent mean value

Fig. 2

Relative mtDNA/nDNA ratio in whole blood DNA from sepsis patient survivors (n = 77) and non-survivors (n = 70) 180 days after ICU admission. Horizontal lines represent mean value

Fig. 3

Absolute amount of mtDNA measured in fluorescence activated (FACS) sorted cells taken from whole blood. Monocytes (triangles), lymphocytes (circles), and granulocytes (diamonds) were FACS sorted from whole blood taken from three control subjects. MtDNA content was determined by real time PCR in 10, 50, 100 and 1,000 cells of each subset. Each symbol represents the mean value of one control sample taken from a triplicate real time measurement. Each sample was measured in triplicate. Lines represent linear regression (r² = 0.99, P < 0.001 in each case)

Fig. 4

Absolute amount of mtDNA measured in fluorescence activated (FACS) sorted monocytes (triangles), lymphocytes (circles), and granulocytes (diamonds) from 14 control subjects (filled symbols) and 13 sepsis patients (open symbols). Horizontal lines represent mean values

Fig. 5

Correlation of APACHE II score of severity of illness in 13 sepsis patients and absolute mtDNA copy number from FACS sorted mononuclear sub-sets. Line represents linear regression (r² = 0.222, P = 0.015). Open squares represent lymphocytes and filled squares represent monocytes