Q8 - Dynamic interplay of mechanosensitive ion channels in macrophage fusion

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Co-Author(s)

Daniela Enriquez-Ochoa, Master in Science

Graduate Student Researcher
University of California Irvine
Irvine, California, United States

Primary Investigator(s)

Wendy F. Liu, PhD

Professor
University of California, Irvine
Irvine, California, United States

Co-Author(s)

HN

Houston Nguyen

Student
University of California, Irvine, United States

Presenting Author(s)

Brandon Dao

Student
University of California, Irvine
Santa Ana, California, United States

Introduction: : Foreign body response (FBR) is a chronic inflammatory phenomenon that poses a major challenge to the success of biomedical device implantation. During FBR, macrophages accumulate at the tissue-implant interface and fuse into multinucleated foreign body giant cells (FBGCs) (1). These cells secrete enzymes, reactive oxygen species (ROS) and pro-inflammatory signals, contributing to fibrotic encapsulation of the implant and interfering with its functionality over time (2). Despite that it is widely accepted that macrophages play a key role in FBR and can recognize mechanical cues, such as stiffness, the mechanotransduction pathways involved in FBGCs formation remain poorly understood. Piezo1, a stiffness mechanosensor, modulates calcium signaling, cytoskeleton remodeling and inflammatory responses; critical processes in macrophage fusion. Previous research from our group has revealed that FBGCs formation is a stiffness-dependent process that occurs even in the absence of Piezo1. However, its overactivation is sufficient to induce fusion in soft environments. In parallel, recent studies have emerged showing that mechanosensitive ion channels can regulate each other in a dynamic and interdependent manner. For example, TREK1 channels can respond to changes in Piezo1 activity, and Piezo1 can influence the activity of other channels, such as TRPV4 (1) (3). Thus, we hypothesize that macrophage fusion persists even in the absence of Piezo1 due to compensatory mechanisms, where other ion channels activity increases to counterbalance the loss of Piezo1-mediated function. Our aim is to gain a deeper understanding of the dynamic interactions between Piezo1 and other mechanosensitive channels and how they contribute to macrophage fusion.

Materials and

Methods: : Bone marrows from femurs and tibias were harvested from myeloid-specific Piezo1 knockout (Piezo1∆LysM) or myeloid-specific Salsa6f Piezo1 knockout (Salsa6fPiezo1∆LysM) mice as well as their respective controls to obtain bone marrow-derived macrophages (BMDMs). BMDMs were cultured on fibronectin-coated polyacrylamide hydrogels of varying stiffness for 3 days. To assess gene expression, Piezo1∆LysM cells and their controls were stimulated with macrophage fusion-inducing cytokines, GM-CSF and IL-4 (10 ng/mL). After 2 hours, they were lysed and their RNA was extracted. cDNA was synthesized and qPCR was performed using the AzuraView GreenFast qPCR blue mix. To analyze calcium influx, Salsa6fPiezo1∆LysM cells and their controls were stimulated with GM-CSF and IL-4 (10 ng/mL) for 10 minutes and then imaged using a confocal microscope. Cells were maintained at 37°C and 5% CO2, and excited using sequential line scan at 488 and 561 nm (4). Ratiometric analysis was performed using ImageJ by evaluating the pixel intensities obtained over time for both the Ca2+ independent tdTomato and the green Ca2+ sensitive GCaMP6f probes.

Results, Conclusions, and Discussions:: To investigate changes in expression of other mechanosensitive channels in the absence of Piezo1 , we cultured Piezo1∆LysM and control BMDMs in different stiffness substrates and stimulated them with fusion-inducing cytokines. Our results showed that in Piezo1∆LysM BMDMs, the expression of the evaluated mechanosensitive channels increases significantly in comparison to the control (Figure 1a). As expected, cells cultured in stiffer substrates showed a higher expression of these channels. To explore the dynamic activity of these ion channels, we cultured Salsa6fPiezo1∆LysM and control BMDMs in soft and stiff substrates, stimulated them with fusion-inducing cytokines and evaluated their calcium influx. We quantified the single-cell GCaMP6f/tdTomato (G/R) ratios to measure cytosolic changes in calcium levels and found that in absence of Piezo1, calcium influx is significantly higher than in controls at both stiffness (Figure 1b). Surprisingly, both control and Piezo1∆LysM cultured in soft substrates show no significant calcium influx difference compared to stiff substrates. These results further confirm the observed increase in ion channel expression in Piezo1∆LysM BMDMs. Altogether, these results suggest that macrophages exhibit a dynamic and adaptive mechanosensory behavior during FBGC formation. Specifically, when Piezo1 is absent, other ion channels appear to compensate for its loss by increasing both their expression and activity. Interestingly, this compensation doesn’t restore baseline calcium influx, it enhances calcium influx beyond levels observed in the controls, indicating a potentially enhanced response that supports macrophage fusion in absence of Piezo1.

Our findings reveal that macrophages exhibit a dynamic and adaptive response during macrophage fusion, characterized by the interplay of different ion channels. Although Piezo1 potentially is not required for FBGCs formation, its absence triggers a compensatory increase in the expression and activity of other ion channels, resulting in an increase in calcium influx. To better understand the correlation between ion channel activation and macrophage fusion, current work is focused on quantifying calcium influx in Piezo1 gain-of-function BMDMs. Ultimately, a deeper understanding of mechanotransduction pathways underlying FBGCs formation could enable the development of new strategies to dampen FBR, improving biocompatibility and longevity of implanted biomedical devices.

Acknowledgements and/or References (Optional): : 1) Sandip M. Swain 1, et al. “PIEZO1 Acts Upstream of TRPV4 to Induce Pathological Changes in Endothelial Cells Due to Shear Stress.” Journal of Biological Chemistry, Elsevier, 4 Jan. 2021, www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1349461/full.

2) Piatnitskaia, Svetlana, et al. “Modelling of Macrophage Responses to Biomaterials in Vitro: State-of-the-Art and the Need for the Improvement.” Frontiers, Frontiers, 21 Feb. 2024, www.sciencedirect.com/science/article/pii/S0021925820001660?via%3Dihub.

3) Lewis, Amanda H. et al. "Piezo1 ion channels are capable of conformational signaling" Neuron, Volume 112, Issue 18, 3161 - 3175.e5, 25 Sept. 2024, www.cell.com/neuron/abstract/S0896-6273(24)00459-8

4) Jairaman, Amit, and Michael D Cahalan. “Calcium Imaging in T Lymphocytes: A Protocol for Use with Genetically Encoded or Chemical ca2+ Indicators.” Bio-Protocol, U.S. National Library of Medicine, 5 Oct. 2021, pmc.ncbi.nlm.nih.gov/articles/PMC8517641/#s6.