Role of mechanosensitive Piezo1 receptor expressed on macrophage in Periodontitis

Abstract Objectives The mechanosensitive Ca2+ ion channels, Piezo1, converts the mechanical stimulus into cell signals in a variety of immune cells. However, the pathophysiological property of Piezo1 in the oral polymicrobial infectious disease of periodontitis is largely unknown. This study investigated the role of Piezo1 in the inflammatory and phagocytic reactions by RAW264.7 macrophages in response to the exposures to periodontal pathogen, P.gingivalis. Experimental methods Short hairpin RNA (shRNA) vector for Piezo1 or control scrambled sequence was transfected to RAW264.7 cells. Then, RAW264.7 cells were incubated for 24 hours in the presence or absence of whole P.gingivalis (MOI; 10) or outer membrane vesicles (OMVs, 1 μg/ml) isolated from P.gingivalis, w or w/o Yoda1 (synthetic Piezo1 activator), or shear stress. qPCR was used to monitor the mRNA expressions of Piezo1, TNF-α, TLR-2, and TLR-4, whereas TNF-α protein was detected by ELISA. RAW264.7’s phagocytic ability was evaluated by cytoplasm-internalization of pHrodo-labeled whole P.gingivalis or OMVs using a flow cytometer. Results OMVs induced more TNF-α production and phagocytosis by RAW264.7 cells than whole P.gingivalis, both of which were further upregulated by Yoda1 as well as shear stress (p<0.05, ANOVA). Shear stress combined with OMV-stimulation upregulated the gene expression of TLR-4, while downregulating TLR-2 gene in RAW264.7 cells. The shRNA-targeting Piezo1 significantly suppressed the TNF-α production and internalization of OMVs by RAW264.7 cells compared control scramble shRNA (p<0.05, ANOVA). Conclusion The present study indicated that mechanical stress may dysregulate the macrophage-mediated innate immune responses in periodontitis. NIH NIDCR grants, DE-027851, DE-028715 and DE-029709