Abstract 687 Extracellular ASC specks are released after marathon running and induce proinflammatory effects in endothelial cells in vitro

Background:
The NLRP3 inflammasome has emerged as an important regulator of inflammatory and acute stress conditions. In the vascular wall, NLRP3 inflammasome can be activated by pathogens such as cholesterol crystals, hypoxia, and hypercholesterinaemia. The effects of other stressors, such as acute high-intensity exercise, on the inflammasome are not known. This study aims to characterize the acute effects of exercise on the release of extracellular apoptosis-associated speck-like protein (ASC) particles and the effects of ASC specks on endothelial cells.

Methods:
We established flow cytometry to quantitate extracellular ASC specks in human serum and measured ASC specks in 46 marathon runners 24-72 hours before, immediately after, and again 24-58 hours after the run. For mechanistic characterization, ASC specks were isolated from a fluorescent THP1-ASC-eGFP cell line to treat primary human coronary artery endothelial cells. These cells were then examined by microscopy for their ability to internalize extracellular inflammasome particles and the subsequent response.

Results:
Athletes showed a significant increase in serum concentration of circulating ASC specks immediately after the marathon (+46 % compared to baseline, p<0.05) and a decrease during follow-up (11 % reduction compared to immediately after the run, p<0.01). Exploratory analysis of quartiles of baseline ASC speck concentrations at time t0 revealed that the exercise-induced regulation correlates with the baseline levels. Athletes with low baseline ASC speck levels showed a marked upregulation at t1 that persisted until t2. In contrast, individuals with baseline ASC specks in the highest quartile showed no additional upregulation after the marathon. The ASC specks showed a significant positive correlation with stress hormones such as cortisone (R=0.22) and cortisol (R=0.19) as well as with aldosterone (R=0.24) and progesterone (R=0.24). In contrast to the stress hormones, there was no correlation of the concentration of extracellular ASC specks with circulating microparticles. These included endothelial, platelet, and leukocyte microparticles. The lack of correlation of ASC specks and the different types of microparticles is an indicator for the specificity of the circulating ASC particles as released by inflammasome activation rather than more general cell damage. In addition, there was a significant negative correlation with the number of circulating lymphocytes that are known to decrease after severe endurance exercise (R=0.27).

Confocal microscopy revealed that human endothelial cells can internalize extracellular ASC specks. After internalization, human endothelial cells showed an inflammatory response with higher expression of the cell adhesion molecule ICAM1 (2.7-fold, p<0.05) and increased adhesion of monocytes (1.5-fold, p<0.05).

Conclusion:
The three main findings of the study are the increase of circulating extracellular ASC specks in human serum immediately after a marathon run, their uptake by endothelial cells in vitro, and the induction of endothelial cell inflammation. These findings identify extracellular inflammasome specks as novel systemic mediators of cell-cell communication after acute exercise. The proinflammatory effects on endothelial cells in vitro may contribute to the increased cardiovascular risk mediated by high loads of exercise.

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5
Extracellular ASC specks are released after marathon running and induce proinflammatory effects in endothelial cells in vitro
S. Gaul¹, A. Kogel¹, S. Fikenzer¹, L. Uhlmann¹, L. Opitz¹, J. Kneuer¹, K. G. Häusler², J. Herm³, M. Endres⁴, J. Kratzsch⁵, V. F. Schwarz⁶, C. Werner⁶, H. Kalwa⁷, U. Laufs¹
¹Klinik und Poliklinik für Kardiologie, Universitätsklinikum Leipzig, Leipzig; ²Neurologische Klinik und Poliklinik, Universitätsklinikum Würzburg, Würzburg; ³Department of Neurology and Center for Stroke Research Berlin, Charité - Universitätsmedizin Berlin, Berlin; ⁴Abt. Neurologie, Charité - Universitätsmedizin Berlin, Berlin; ⁵Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Universitätsklinikum Leipzig, Leipzig; ⁶Innere Medizin III - Kardiologie, Angiologie und internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Homburg/Saar; ⁷Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig;
Background: The NLRP3 inflammasome has emerged as an important regulator of inflammatory and acute stress conditions. In the vascular wall, NLRP3 inflammasome can be activated by pathogens such as cholesterol crystals, hypoxia, and hypercholesterinaemia. The effects of other stressors, such as acute high-intensity exercise, on the inflammasome are not known. This study aims to characterize the acute effects of exercise on the release of extracellular apoptosis-associated speck-like protein (ASC) particles and the effects of ASC specks on endothelial cells. Methods: We established flow cytometry to quantitate extracellular ASC specks in human serum and measured ASC specks in 46 marathon runners 24-72 hours before, immediately after, and again 24-58 hours after the run. For mechanistic characterization, ASC specks were isolated from a fluorescent THP1-ASC-eGFP cell line to treat primary human coronary artery endothelial cells. These cells were then examined by microscopy for their ability to internalize extracellular inflammasome particles and the subsequent response. Results: Athletes showed a significant increase in serum concentration of circulating ASC specks immediately after the marathon (+46 % compared to baseline, p<0.05) and a decrease during follow-up (11 % reduction compared to immediately after the run, p<0.01). Exploratory analysis of quartiles of baseline ASC speck concentrations at time t0 revealed that the exercise-induced regulation correlates with the baseline levels. Athletes with low baseline ASC speck levels showed a marked upregulation at t1 that persisted until t2. In contrast, individuals with baseline ASC specks in the highest quartile showed no additional upregulation after the marathon. The ASC specks showed a significant positive correlation with stress hormones such as cortisone (R=0.22) and cortisol (R=0.19) as well as with aldosterone (R=0.24) and progesterone (R=0.24). In contrast to the stress hormones, there was no correlation of the concentration of extracellular ASC specks with circulating microparticles. These included endothelial, platelet, and leukocyte microparticles. The lack of correlation of ASC specks and the different types of microparticles is an indicator for the specificity of the circulating ASC particles as released by inflammasome activation rather than more general cell damage. In addition, there was a significant negative correlation with the number of circulating lymphocytes that are known to decrease after severe endurance exercise (R=0.27). Confocal microscopy revealed that human endothelial cells can internalize extracellular ASC specks. After internalization, human endothelial cells showed an inflammatory response with higher expression of the cell adhesion molecule ICAM1 (2.7-fold, p<0.05) and increased adhesion of monocytes (1.5-fold, p<0.05). Conclusion: The three main findings of the study are the increase of circulating extracellular ASC specks in human serum immediately after a marathon run, their uptake by endothelial cells in vitro, and the induction of endothelial cell inflammation. These findings identify extracellular inflammasome specks as novel systemic mediators of cell-cell communication after acute exercise. The proinflammatory effects on endothelial cells in vitro may contribute to the increased cardiovascular risk mediated by high loads of exercise.
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