Poster Z7 - Shear Stress Initiates Signal Two in NLRP3 Inflammasome Activation Via Piezo1

Introduction: The effect of shear stress is widely studied in literature where it affects cell morphology, cytokine secretion, and gene expression, however, little is known about how it plays a role in inflammatory responses of immune cells. Few studies have mechanistically investigated how physiologically relevant shear stress can activate the NLRP3 inflammasome in a microfluidic device. Calcium staining, qPCR, and inhibitor studies determined the mechanism through which shear stress induces NLRP3 inflammasome activation. In this study, we aim to gain a deeper understanding of how shear stress regulates the inflammatory response in innate immune cells.

Materials and

Methods: Macrophages were suspended in a collagen gel and seeded in a microfluidic device system where fluid shear stress was applied to cells. Confocal microscopy studies assessed the formation of ASC-CFP specks and intracellular calcium, hallmarks of inflammasome activation. For qPCR experiments, the collagen gel in the microfluidic devices was digested using Collagenase D in Basal DMEM then RNA was extracted, reverse transcribed, and the gene expression was quantified relative to β-actin mRNA. For the inhibitor studies performed, LPS-primed iBMDMs were treated with Dooku1 before shear-stress-activating where imaging and qPCR studies were performed.

Results, Conclusions, and Discussions: Results and

Discussion: To determine the effect of shear-stress exposure on Signal 2 of NLRP3 inflammasome activation, iBMDMs were primed with lipopolysaccharide (LPS) followed by treatment with physiologically relevant magnitudes of shear stress. We discovered that ASC-CFP speck formation was positively correlated to shear stress magnitude in wild-type iBMDMs, with no significant changes in NLRP3 and caspase-1 knock-out iBMDMs compared to controls, indicating NLRP3 and caspase-1 are both necessary for shear-stress-induced NLRP3 inflammasome activation (Fig. 1A). The mechanism of shear-stress-induced NLRP3 inflammasome activation was investigated through quantifying calcium staining (Fig. 1B) and qPCR which determined an increase in the relative expression of Piezo1 and known players for NLRP3 inflammasome activation. Additionally, we performed an inhibitor study using Dooku1, a well-known inhibitor of mechanosensitive ion channel Piezo1, where 50 μM treatment before shear stress exposure showed significant reductions in speck formation, Piezo1 expression, and NLRP3 expression (Fig. 1C).

Conclusion: In conclusion, we determined the mechanism through which shear stress can selectively induce NLRP3 inflammasome activation. We supported these observations with imaging, qPCR analysis, and a study with an inhibitor of Piezo1. This study determined that physiologically relevant shear stress exhibits a positive correlation on intracellular signaling mechanisms and the activation of the inflammatory response in innate immune cells.

Acknowledgements (Optional):