In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes

Abstract

Innate immune memory, or trained immunity, has recently been described to be an important property of cells of the innate immune system. Due to the increased interest in this important new field of immunological investigation, we sought to determine the optimal conditions for an in vitro experimental protocol of monocyte training using three of the most commonly used training stimuli from the literature: β-glucan, the bacillus Calmette-Guérin (BCG) vaccine, and oxidized low-density lipoprotein (oxLDL). We investigated and optimized a protocol of monocyte trained immunity induced by an initial training period with β-glucan, BCG, or oxLDL, followed by washing and resting of the cells and, thereafter, restimulation with secondary bacterial stimuli. The training and resting time intervals were varied to identify the optimal setting for the long-term induction of trained immunity. Trained immunity was assessed in terms of the secondary cytokine response, the production of reactive oxygen species, cell morphology, and induction of glycolysis. Monocytes primed with β-glucan, BCG, and oxLDL showed increased pro- and anti-inflammatory cytokine responses upon restimulation with nonrelated stimuli. Also, all three stimuli induced a switch to glycolysis (the Warburg effect). These effects were most pronounced when the training interval was 24 h and the resting time interval was 6 days. Training with BCG and oxLDL also led to the increased production of reactive oxygen species, whereas training with β-glucan led to the decreased production of reactive oxygen species. We describe the optimal conditions for an in vitro experimental model with human primary monocytes for study of the induction of trained innate immunity by microbial and metabolic stimuli.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Schematic overview of trained immunity methodology. Monocytes were trained for 2 h (2 hr-T), 4 h (4 hr-T), or 24 h (24 hr-T). After the training stimulus was washed away, the cells were rested for 24 h (24 h-R), 3 days (3d-R), or 6 days (6d-R), after which the cells were restimulated with RPMI, LPS, or Pam3Cys for 24 h.

FIG 2

Increased proinflammatory cytokine production is dependent on both the training interval and the resting time. (A) IL-6 production after restimulation. Cells were trained for 2 h, 4 h, or 24 h with β-glucan, BCG, or oxLDL and rested for 24 h, 3 days (3d), or 6 days (6d). (B) TNF-α production after restimulation. Cells were trained for 2 h, 4 h, or 24 h with β-glucan, BCG, or oxLDL and rested for 24 h, 3 days, or 6 days. Shown are the fold changes compared to the results for the RPMI control (n = 6; ^, P = 0.06 compared to the RPMI control; *, P < 0.05 compared to the RPMI control; **, P < 0.01 compared to the RPMI control).

FIG 3

Trained immunity effects on cell morphology and numbers. (A) Morphology of cells after 24 h of training and 6 days of rest when the cells were trained with RPMI (negative control), β-glucan, BCG, or oxLDL. Pictures were taken before restimulation at day 6. Magnification, ×20. (B) Size of cells after 24 h of training and 6 days of rest before restimulation at day 6 (n = 3, *, P < 0.05; compared to the RPMI control; ***, P < 0.001 compared to the RPMI control). (C) Relative cell counts before restimulation at day 6 (n = 6; *, P < 0.05, compared to the RPMI control; ns, not significant). (D) IL-6 and TNF-α production after restimulation for corrected cell counts (n = 6; *, P < 0.05 compared to the RPMI control; **, P < 0.01 compared to the RPMI control).

FIG 4

Anti-inflammatory cytokine production is increased in trained monocytes. (A) IL-10 production after restimulation. Cells were trained for 24 h with β-glucan, BCG, or oxLDL and rested for 6 days. The level of production of the anti-inflammatory cytokine IL-10 increased upon training. (B) IL-1Ra production. Cells were trained for 24 h with β-glucan, BCG, or oxLDL and rested for 6 days. The level of production of the anti-inflammatory cytokine IL-1Ra increased upon training not only after restimulation but also at the baseline (n = 6; ^, P = 0.06 compared to the RPMI control; *, P < 0.05 compared to the RPMI control; **, P < 0.01 compared to the RPMI control; ns, not significant). P3C, Pam3Cys.

FIG 5

ROS production is a component of trained immunity for some stimuli. The level of ROS production was significantly increased for monocytes trained with BCG and oxLDL; training with β-glucan decreased the level of ROS production (n = 6; *, P < 0.05 compared to the RPMI control; ***, P < 0.001 compared to the RPMI control; ns, not significant). Cells were trained for 24 h using β-glucan, BCG, or oxLDL, and normalized cell numbers were restimulated using zymosan. Luminol was added, and luminescence was measured for 1 h. RLU/s, relative light units per second.

FIG 6

Trained immunity induction of glycolysis. Cells were trained for 2 h (2hr-T), 4 h (4hr-T), or 24 h (24hr-T) with RPMI (negative control), β-glucan, BCG, or oxLDL and rested for 24 h (24hr-R), 3 days (3d-R), or 6 days (6d-R). The level of lactate production in the supernatants was measured before restimulation (n = 6; *, P < 0.05 compared to the RPMI control; **, P < 0.01 compared to the RPMI control; ns, not significant).