Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Formation of ASC-specks in mature adipocytes and stromal vascular fraction in different depots of murine adipose tissue

J. Wissemann1, A. Heidenreich1, L. Karnbrock2, C. Bode1, D. Dürschmied3, P. Stachon1, J. Merz1

1Klinik für Kardiologie und Angiologie I, Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH, Freiburg im Breisgau; 2Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH, Freiburg im Breisgau; 3I. Medizinische Klinik, Universitätsklinikum Mannheim, Mannheim;

Background The metabolic syndrome is characterized by a chronic inflammation of the adipose tissue, which increases the risk for major cardiovascular events. Studies suggest that this is caused by hypertrophic adipocytes releasing danger signals like ATP upon their death. ATP further leads to the activation of the NLRP3-inflammasome by activating certain purinergic receptors. The formation of “apoptosis-associated speck like protein containing a caspase activation and recruitment domain” (ASC)-specks is a crucial mediator between activation of the NLRP3-inflammasome and release of Interleukin-1β, which then acts as an important proinflammatory cytokine. Purpose We aim to answer the question, which cells residing in visceral, subcutaneous and brown adipose tissues are capable of forming ASC-specks and thereby initiate, promote or maintain inflammation. Methods Adipocytes and stromal vascular fraction (SVF) of the epididymal white adipose tissue (eWAT), perirenal WAT and brown adipose tissue where isolated from ASC-Citrine inflammasome reporter mice. The SVF was cultured for 14d, while adipocytes where stimulated on the day following isolation. The NLRP3-inflammasome was primed using LPS and assembled through incubation with ATP or Nigericin. Formation of ASC-specks was assessed using fluorescence microscopy and flow cytometry. Results ASC-speck formation upon stimulation (6h 100ng/ml LPS, 1h Nigericin 10µM) of the eWAT derived SVF was significantly higher after 14 days of culture compared to freshly isolated SVF (n=3, speck-frequency after 0d=0.013 Mean ± 0.008 SEM, after 14d= 6.983 ± 1.104, p=0,003). Incubation with 3µM of the specific NLRP3 inhibitor MCC950 blocked speck-production, proving activation of the NLRP3-inflammasome to be the underlying mechanism. Regression analysis showed a strong correlation between speck-frequency and capase-1-activity (r²=0,9266) as well as IL-1β release (r²=0,8029). Fluorescence microscopy showed formation of ASC-specks in less than 1% of white adipocytes upon stimulation while no specks where found in brown adipocytes. In contrast to our findings in the SVF, viability staining showed that this is not associated with pyroptosis. Flow cytometry confirmed the presence of specks, but the current sample size does not allow for evaluation of significance between different conditions. Discussion We showed that both the stromal vascular fraction and adipocytes are capable of forming ASC-specks, but the depots differ in the extent to which they do so. However, mature SVF has the strongest capability to produce active ASC-Specks. As the metabolic syndrome becomes even more prevalent, a better understanding of the molecular mechanisms is key to finding new therapeutic tools. Therefore, the contribution of adipocytes and stromal vascular fraction to inflammasome activity needs to be investigated further.

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