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Piezo1 expression in neutrophils regulates shear-induced NETosis.

ABSTRACT: Neutrophil infiltration and subsequent extracellular trap formation (NETosis) is a contributing factor in sterile inflammation. Furthermore, neutrophil extracellular traps (NETs) are prothrombotic, as they provide a scaffold for platelets and red blood cells to attach to. In circulation, neutrophils are constantly exposed to hemodynamic forces such as shear stress, which in turn regulates many of their biological functions such as crawling and NETosis. However, the mechanisms that mediate mechanotransduction in neutrophils are not fully understood. In this study, we demonstrate that shear stress induces NETosis, dependent on the shear stress level, and increases the sensitivity of neutrophils to NETosis-inducing agents such as adenosine triphosphate and lipopolysaccharides. Furthermore, shear stress increases intracellular calcium levels in neutrophils and this process is mediated by the mechanosensitive ion channel Piezo1. Activation of Piezo1 in response to shear stress mediates calpain activity and cytoskeleton remodeling, which consequently induces NETosis. Thus, activation of Piezo1 in response to shear stress leads to a stepwise sequence of cellular events that mediates NETosis and thereby places neutrophils at the centre of localized inflammation and prothrombotic effects.

SUBMITTER: Baratchi S

PROVIDER: S-EPMC11341855 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Piezo1 expression in neutrophils regulates shear-induced NETosis.

Baratchi Sara S Danish Habiba H Chheang Chanly C Zhou Ying Y Huang Angela A Lai Austin A Khanmohammadi Manijeh M Quinn Kylie M KM Khoshmanesh Khashayar K Peter Karlheinz K

Nature communications 20240822 1

Neutrophil infiltration and subsequent extracellular trap formation (NETosis) is a contributing factor in sterile inflammation. Furthermore, neutrophil extracellular traps (NETs) are prothrombotic, as they provide a scaffold for platelets and red blood cells to attach to. In circulation, neutrophils are constantly exposed to hemodynamic forces such as shear stress, which in turn regulates many of their biological functions such as crawling and NETosis. However, the mechanisms that mediate mechan ...[more]

PMID: 39174529

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Project description:Modulation of endothelial traction is critical for the responses of endothelial cells to fluid shear stress (FSS), which has profound implications for vascular health and atherosclerosis. Previously, we demonstrated that under high FSS, endothelial cells rapidly increase traction forces, followed by relaxation, with traction aligning in the flow direction. In contrast, low shear preconditioning induces a modest short-term increase in traction (<30 min), followed by a secondary long-term (>14 hr) rise, with traction/cells aligning perpendicular to the flow. The upstream mechanosensors driving these responses, however, remain unknown. Here, we sought the roles of Piezo1 and TRPV4 ion channels in shear-induced traction modulation. We report that HUVECs with Piezo1 silencing reduced the initial traction rise in half under high FSS compared to those by WT cells, while not affecting the traction modulation in response to low FSS or traction/cell alignment to the flow direction. Conversely, cells with siTRPV4 fully abrogated the initial traction rise, as well as alignment of traction and cells, in response to both high and low FSS conditions. Dual inhibition of Piezo1 and TRPV4 further impaired both initial and long-term traction under high FSS. Interestingly, dual-inhibited cells displayed larger initial traction responses to low FSS compared to control cells, suggesting the involvement of alternative calcium-independent pathways that become dominant when both ion channels are nonfunctional. Additionally, either ion channel inhibition led to secondary long-term traction increase even under high FSS condition. These findings suggest that while both Piezo1 and TRPV4 channels contribute to shear mechanotransduction, TRPV4 plays more dominant role than Piezo1 in mediating the initial traction rise and sustaining long-term relaxation under high or low shear stress, highlighting their critical and distinct contributions to endothelial mechanotransduction and remodeling.

Project description:Neutrophils represent primary mobile phagocytes recruited to the site of infection, and their functions are essential to enhance animals' health performance. Neutrophils have an essential role in innate immunity and are able to kill the pathogens via the synthesis of neutrophil extracellular traps (NETs). The objective of the present work was the study of the in vitro NETosis of peripheral neutrophils isolated from dairy cows supplemented with olive pomace. Dairy cows (n = 16) balanced for parity (3.67 ± 1.5 for CON, 3.67 ± 1.9 for OP), milk yield (24.3 ± 4.5 kg d-1for CON and 24.9 ± 1.7 kg d-1 for OP), the number of days in milk (109 ± 83.5 for CON and 196 ± 51 for OP), and body weight (647 ± 44.3 kg for CON and 675 ± 70.7 kg for OP) were divided into two experimental groups fed with a control diet (CON) and supplemented with 6% of olive pomace (OP). Peripheral blood neutrophils were isolated and stimulated in vitro with phorbol-myristate-acetate (PMA) as a marker for activation and reactivity of the neutrophils. After isolation, both the viability and CD11b expression were analyzed by flow cytometry. Both NETosis by neutrophil elastase-DNA complex system and myeloperoxidase (MPO) activity were evaluated by ELISA. The specific antibodies against MPO and citrullination of Histone-H1 were used for investigating NETosis by immunofluorescence microscopy. The neutrophil elastase-DNA complexes produced during NETosis and MPO activity of neutrophil extracts were affected by OP supplementation. Furthermore, results from immunofluorescence analysis of NETosis depicted a similar result found by ELISA showing a higher expression of MPO and citrullination of Histone-H1 in OP than the CON neutrophils. In addition, all data showed that the OP diet resulted in a better response of neutrophils to PMA stimulation than the CON diet, which did not support the neutrophils' responses to PMA stimulation. Our results demonstrated that OP supplementation can enhance the neutrophil function in dairy cows leading to udder defense and inflammation response especially when an immunosuppression state can occur.

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Piezo1 expression in neutrophils regulates shear-induced NETosis.

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Piezo1 expression in neutrophils regulates shear-induced NETosis.

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