<i>In Vitro</i> Experimental Model of Trained Innate Immunity in Human Primary Monocytes

TLDR

Innate immune memory, or trained immunity, is a recently recognized property of innate immune cells. The study aimed to define optimal in‑vitro conditions for monocyte training using β‑glucan, BCG, and ox‑LDL. Monocytes were exposed to β‑glucan, BCG, or ox‑LDL for an initial training period, then washed, rested, and restimulated with bacterial stimuli, with training and resting intervals varied to optimize long‑term trained immunity, measured by cytokine production, ROS, morphology, and glycolysis. Training with β‑glucan, BCG, or ox‑LDL enhanced cytokine responses and induced a Warburg‑type glycolytic shift, most pronounced with 24‑h training and 6‑day rest; BCG and ox‑LDL also raised ROS production, whereas β‑glucan lowered it.

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 (ox-LDL). 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 antiinflammatory 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.