Trained immunity refers to the capacity of the innate immune system to develop a non-specific, memory-like response following prior exposure to microbial stimuli. This phenomenon, which involves epigenetic and metabolic reprogramming, is characterized by an enhanced response of myeloid cells, which exhibit increased production of proinflammatory cytokines upon secondary stimulation.

In this study, we induce trained immunity by incubating mouse bone marrow-derived macrophages (BMDMs) for 7 days with β-glucan or the Bacillus Calmette-Guérin (BCG) vaccine and then analysed the activation of different inflammasomes by measuring pyroptosis by lactate dehydrogenase (LDH) release and interleukin (IL)-1β and IL-18 release. Specifically, NLRP3 inflammasome was activated using lipopolysaccharide (LPS) and the K⁺-ionophore nigericin, the pyrin inflammasome with LPS and Clostridium difficile toxin B (TcdB), AIM2 with LPS and double-stranded DNA, and NLRC4 inflammasome with LPS and the fusion protein system LFN-PA, composed of the N-terminal domain of anthrax lethal factor (LFN) together with protective antigen (PA).

We first confirmed the effectiveness of trained immunity by observing a significant increase in the production of IL-6 and TNFα in macrophages exposed to β-glucan or BCG prior to LPS stimulation compared to the production in controls. Specifically, BCG induced stronger training in BMDMs than β-glucan in terms of IL-6 release. Then, trained macrophages were stimulated to activate the NLRP3, pyrin, AIM2, or NLRC4 inflammasomes, resulting in increased inflammasome-dependent IL-1β release compared with untrained controls. These results showed that trained immunity induced a higher increase in IL-1β release than in IL-6 or TNFα release. Although BMDMs trained with BCG or β-glucan produced higher amounts of IL-1β for all the inflammasomes tested, there were no significant differences in terms of IL-1β release across them when expressed as fold change. The enhanced activation of inflammasomes by trained immunity was further confirmed by increased levels of IL-18 release in trained macrophages and increased levels of LDH release as a hallmark of pyroptosis. Nevertheless, the results show that IL-1β release is a more robust indicator of inflammasome activation in trained immunity than IL-18.

In conclusion, these results demonstrate that trained immunity induced by BCG or β-glucan enhances the activity of multiple inflammasomes in macrophages, including NLRP3, pyrin, AIM2 and NLRC4, with a strong release of IL-1β.

Acknowledgements

This work was supported by Ministerio de Ciencia, Innovación y Universidades proyectos PID2023-147531OB-I00 and PRE2023-000343.