(M-494) Shear Stress Selectively Regulates NLRP3 Inflammasome Activation Through Signal 1

Introduction:: The effect of shear stress on cells is widely studied in literature where it effects cell morphology, cytokine secretion, and gene expression, however, little is known about how shear stress plays a role in inflammatory responses of immune cells. No studies have mechanistically investigated how physiologically relevant shear stress magnitudes in a microfluidic device system can prime NLRP3 inflammasome activation. We also utilized qPCR for observing changes in the gene expression of NLRP3-associated proteins and a small-molecule inhibitor to determine the mechanism through which shear stress induces priming for 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 shear stress was applied to cells by flowing DMEM. After shear stress exposure, cells were imaged with a confocal microscope to assess the formation of ASC-CFP specks (Fig. 1A), a well-known hallmark 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, iBMDMs seeded in a microfluidic device were pretreated with Bay 11-7082 before shear-stress-priming. After subsequent nigericin treatment, the cells were imaged with a confocal microscope to determine the fraction of ASC-CFP speck formation.


Results, Conclusions, and Discussions:: Results and Discussion: To determine the effect of shear-stress exposure on NLRP3 inflammasome activation, iBMDMs were primed with physiologically relevant magnitudes of shear stress followed by nigericin treatment. We discovered that ASC-CFP speck formation was positively correlated to shear stress magnitude in wild-type cells, with no significant changes in knock-out cells (Fig. 1B). This observation indicated that NLRP3 and caspase-1 are both necessary in the mechanism of shear-stress-primed NLRP3 inflammasome activation. The mechanism of shear-stress-primed NLRP3 inflammasome activation was investigated through performing qPCR which determined an increase in the relative expression of known players for NLRP3 inflammasome activation. Additionally, we performed an inhibitor study using Bay 11-7082, a well-known inhibitor of the NLRP3 inflammasome, where 12.5 μM treatments showed significant reductions in the formation of ASC-CFP specks.

Conclusion: In conclusion, we determined the mechanism through which shear stress can selectively prime for NLRP3 inflammasome activation. We supported these observations with imaging and qPCR analysis as well as a study with an inhibitor of the NLRP3 inflammasome activation pathway. 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.


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