1Universitätsklinikum Essen Institut für Pharmakologie Essen, Deutschland
Background: Pro-inflammatory signalling through the NLRP3 inflammasome and its major effector interleukin-1β (IL-1β) is causally linked to the pathophysiology of atrial fibrillation (AF). The presence of increased pericardial fat and fibro-fatty infiltration into the atria have been associated with an enhanced susceptibility to AF. There is evidence suggesting that an overactive NLRP3 inflammasome in adipocytes contributes to metabolic remodelling, but a paracrine interaction with cardiac cells has not been demonstrated. Here, we established an indirect cell co-culture model to study the role of the NLRP3 inflammasome in the pro-inflammatory cross-talk between adipocytes and atrial cardiomyocytes.
Methods: Pericardial fat was obtained from right atrial appendages of patients undergoing cardiac surgery who either developed post-operative AF (poAF) or remained in sinus rhythm (Ctl). To assess inflammatory cross-talk between adipocytes and cardiomyocytes, an indirect co-culture model was designed. Fully differentiated 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS, 100 ng/ml, 2 h) and the potassium ionophore nigericin (5 µg/ml, final 1 h) to transcriptionally prime and trigger the NLRP3 inflammasome. Conditioned supernatant of LPS-stimulated and non-stimulated adipocytes (diluted 1:3.5 in Claycomb medium) was added to mouse atrial cardiomyocytes (HL-1 cells) pretreated ± anakinra
(IL-1 receptor antagonist, 100 ng/ml, 1 h) for 12 h. Functional expression of the NLRP3 inflammasome components was assessed by qPCR, Western blot, IL-1β ELISA and custom-designed and validated caspase-1 and caspase-11 activity assays.
Results: Abundance of NLRP3, caspase-1, caspase-11, and IL-1β is higher in pericardial fat from poAF-prone patients vs. Ctl. Conditioned supernatant from LPS-stimulated adipocytes contained higher amounts of IL-1β compared to medium from non-stimulated adipocytes and more strongly upregulated NLRP3 mRNA in recipient HL-1 cells, reflecting inflammasome priming. Transcript levels and activity of both caspase-1 and caspase-11 also increased in HL-1 cells exposed to supernatant from LPS-stimulated versus non-stimulated adipocytes, as did the intracellular levels of mature IL-1β. This constellation indicates enhanced triggering by both the caspase-1 (canonical) and caspase-11 (non-canonical) NLRP3-inflammasome pathways. The presence of the IL-1 receptor antagonist anakinra blunted these stimulatory effects of adipocyte-conditioned supernatant on recipient HL-1 cells.
Conclusion: Atrial fat from patients susceptible to poAF overexpresses components of the NLRP3 inflammasome. We show that adipocytes are a culprit source of inflammasome-derived IL-1β that transmits the pro-inflammatory signal to atrial cardiomyocytes via the IL-1 receptor, leading to priming and triggering of the canonical and non-canonical inflammasome pathways. Our data positions the IL-1β pathway as a potential drug target in future AF therapy.