Intra-cellular lactate concentration in T lymphocytes from septic shock patients - a pilot study

Abstract

Background: Sepsis-associated hyperlactatemia is a widely used biomarker, associated with initial severity and poor outcomes. This increased circulating lactate concentration has been proposed to result in part from a mismatch between oxygen delivery and demand in organs. However, other mechanisms may participate. In particular, a metabolic reprogramming similar to the Warburg effect initially described in cancer cells could lead to increased lactate production by immune cells such as T lymphocytes after sepsis. The objective of this study was to set up a protocol for lactate measurement in T lymphocytes, and to evaluate whether lactate production by T lymphocytes was increased in septic shock patients.

Methods: We first optimized protocols for lactate and pyruvate measurements in T lymphocytes purified from healthy volunteers' blood, either stimulated with phytohaemagglutinine (PHA) or left untreated. We then conducted a pilot study to confirm the feasibility of this protocol in samples from septic shock patients.

Results: PHA stimulation induced aerobic glycolysis in human lymphocytes ex vivo, with increased lactate and pyruvate productions. To correctly measure this phenomenon, minimal cell number was 250,000 and optimal culture duration was 40 h. We also observed a significant correlation between lactate concentration in T lymphocytes and in their culture supernatants. We were able to measure lactate concentration in T lymphocytes from septic shock patients. Our preliminary results showed that intra-lymphocyte lactate concentration was not different between patients and healthy volunteers.

Conclusion: This protocol should now be tested in a larger cohort of patients. The association between immune cell metabolic reprogramming as measured by lactate concentration in T cells and functionality represents an exciting field for research.

Keywords: Bioenergetics; Lactate; Lymphocyte; Sepsis; Technique optimization; Warburg effect.

Fig. 1

Optimal cell stimulation duration determination. T lymphocytes were purified from four healthy donors’ blood. Cells were cultured in complete RPMI medium at a concentration of one million cells/mL and were stimulated with 4 μg/mL PHA (black circles) or left unstimulated (NS, open circles). After various duration of incubation, ranging from overnight (OVN) to approximately 62 h (OVN + 48 h), one million T cells were aliquoted and intra-lymphocyte lactate and pyruvate concentrations were measured. Results are expressed as the quantity of lactate (Fig. 1a) or pyruvate (Fig. 1b) per well of the lecture plate, which contains 50 μL of the obtained cell lysate. Each point represents the mean of the duplicates for each donor, horizontal bars represent the median value

Fig. 2

Effect of PHA-stimulation on intra-T cell metabolites concentration. T lymphocytes were purified from eight healthy donors’ blood. Cells were cultured in complete RPMI medium at a concentration of one million cells/mL and were stimulated with 4 μg/mL PHA (grey box) or left unstimulated (NS, white box). After a 40-h incubation, one million T cells were aliquoted and intra-lymphocyte lactate and pyruvate concentrations measured. Results are expressed as the concentration of lactate (Fig. 2a) or pyruvate (Fig. 2b) per well of the lecture plate, which contains 50 μL of the obtained cell lysate, or as the lactate/pyruvate ratio (Fig. 2c), which is known to reflect anaerobic metabolism when exceeding 10 (horizontal dotted line). Each point represents the mean of the duplicates for each donor. Data are also presented as Tukey boxplots. Bottom and top of the box represent the first and third quartiles, respectively. The horizontal bar in the box represents the median value. Lower and higher extremities of the whiskers respectively represent the lowest datum still within 1.5 inter-quartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile. Difference between NS and PHA groups was assessed with Wilcoxon test

Fig. 3

Correlation between intracellular and supernatant lactate concentration in healthy volunteer’s T lymphocytes. T lymphocytes were purified from six healthy donors’ blood. Cells were cultured in complete RPMI medium at a concentration of one million cells/mL, and stimulated with 4 μg/mL PHA (black circles) or left unstimulated (NS, open circles). After the previously determined 40-h incubation period, supernatant and one million T cells were aliquoted and lactate concentration was measured in supernatants and in T cells. Results are expressed as the quantity of lactate per well of the lecture plate, which contains 50 μL of the obtained cell lysate or supernatant. Each point represents the mean of the duplicates for each donor. Dotted line represents the linear regression model and grey zone represents the 95% confidence interval of the regression. Correlation between intra-lymphocyte and supernatant lactate concentration was explored with Pearson correlation

Fig. 4

Minimal cell number for lactate measurement in healthy volunteers’ T lymphocytes. T lymphocytes were purified from four healthy donors’ blood. Cells were cultured in complete RPMI medium at a concentration of one million cells/mL and were stimulated with 4 μg/mL PHA (black circles) or left unstimulated (NS, open circles). After the previously determined 40-h incubation period, different number of T cells (ranging from 100,000 to 1,000,000) were aliquoted and intra-lymphocyte lactate concentrations measured. Results are expressed as the quantity of lactate per well of the lecture plate, which contains 50 μL of the obtained cell lysate. Each point represents the mean of the duplicates for each donor and horizontal bars represent the median value

Fig. 5

Intra-cellular lactate concentration in T lymphocytes in healthy volunteers versus septic shock patients. T lymphocytes were purified from 10 healthy donors’ and 7 septic shock patients’ blood. After the purification process, 250,000 T cells were aliquoted, lysed, and deproteinised. One septic patient was too lymphopenic to harvest 250,000 T cells, leading to one missing data. Results are expressed as the quantity of lactate per well of the lecture plate, which contains 50 μL of the obtained cell lysate. Each point represents the mean of the duplicates for each donor. Data are also presented as Tukey boxplots (white for HV, grey for SS). Bottom and top of the box represent the first and third quartiles, respectively. The horizontal bar in the box represents the median value. Lower and higher extremities of the whiskers respectively represent the lowest datum still within 1.5 inter-quartile range (IQR) of the lower quartile, and the highest datum still within 1.5 IQR of the upper quartile