Extracellular inflammasome particles promote visceral adipose tissue inflammation
Authors: Wissemann J , Heidenreich A , Suchanek D , Zimmermann H , Hertle L , Engelmann J , Wolf D , Stachon P , Westermann D , Merz J 
Published: November 2023

Abstract

Background
The metabolic syndrome is characterized by a chronic low-grade inflammation that increases the risk for cardiovascular events. With its capability to activate IL-1β, the NLRP3-inflammasome is believed to affect the inflammatory process. When the inflammasome is primed and activated, cytosolic ASC aggregates into the ASC-speck. This inflammasome particle can persist in the extracellular space after the cell it originated from underwent pyroptosis.

Purpose
We aimed to assess the role of extracellular inflammasome particles as a potential target to reduce chronic inflammation in the metabolic syndrome.

Methods
Male C57BL6/J mice received either a chow or high-fat-diet (HFD) for 7, 14 or 21 weeks. Flow cytometry and fluorescence microscopy were used to detect inflammasome particles. Adipocytes and stromal vascular fraction (SVF) were isolated from subcutaneous (scWAT) and visceral white adipose tissue (vWAT) using collagenase.

Results
Mice subjected to HFD show increased NLRP3-inflammasome priming in the SVF of visceral but not subcutaneous WAT (vWAT: 4-fold p&lt;0.001, scWAT: 1-fold). The frequency of ASC-specks induced by the NLRP3-activating agent nigericin correlates with immune cell infiltration (macrophages: r=0.83) and disruption of metabolic homeostasis (bodyweight: r=0.8). Co-staining of nigericin-induced ASC-specks with CD45 and CD11b suggests their derivation from myeloid cells of the SVF. ASC-speck formation can be fully inhibited with the NLRP3-specific inhibitor MCC950. Adipocytes likely do not form ASC-specks as they, despite upregulating the required genes when primed with lipopolysaccharide (e.g. IL-1β: 8-fold), fail to activate the inflammasome in-vitro.
In chow-fed mice, cells carrying cytosolic ASC are evenly dispersed in the WAT and can be co-stained with the macrophage marker F4/80. When a metabolic syndrome is acquired, ASC-positive cells form crown-like-structures around dying adipocytes in which ASC-speck formation takes place. In the vWAT of HFD-fed mice, we found ASC-specks that were externalized and also taken up by adipocytes.
Co-culture of isolated ASC-specks with adipocytes induced ER-stress and resulted in upregulation of IL-6 (42-fold), CCL2 and TNFα after 24h. In addition, previously undifferentiated bone marrow derived macrophages internalized these particles, which lead to a pro-inflammatory shift.
Finally, also human endothelial cells responded to isolated ASC-specks with upregulation of the leukocyte adhesion molecules VCAM1 and ICAM1.

Conclusion
Our study suggests that extracellular inflammasome particles accumulate in metabolically altered visceral adipose tissue and contribute to a pro-inflammatory environment. The imbalance between pro- and anti-inflammatory immune response is an important contributor to the development of cardiovascular disease. Extracellular inflammasome particles could be an interesting target for novel therapeutic approaches to reduce chronic inflammation.