Stiffness regulates macrophage fusion via Piezo1 and metabolic responses

Introduction: : Foreign body response (FBR) is a prevalent but poorly understood pathological phenomenon in the field of biomedical device implantation. During FBR, the presence of an implant often triggers a chronic inflammatory state and formation of a fibrotic tissue capsule, leading to functional impairment and possible damage to tissues. Macrophages are the main orchestrators of the FBR. They fuse and differentiate into multinucleated giant cells (MGCs)- highly oxidative, inflammatory and destructive cells that also modulate fibroblast activation state. Implanted devices are often stiffer than the surrounding tissue; however, the mechanotransduction pathways that lead to MGCs formation remain largely unknown. Previously, we demonstrated that in macrophages Piezo1 is a stiffness mechanosensor that modulates Ca2+ signaling, cytoskeleton remodeling and inflammatory responses, but a role in macrophage fusion has not yet been reported. In parallel, emerging evidence suggests that macrophage fusion is a highly metabolically demanding process, relying in oxidative phosphorylation. Given the intricate relationship between metabolic processes and immune responses, we aim to elucidate the role of stiffness and Piezo1 in modulating macrophage fusion and metabolic responses.

Materials and

Methods: : Bone marrows were flushed from femurs and tibias of C57BL/6J, myeloid-specific Piezo1 knockout (Piezo1∆LysM), or Piezo1 gain-of-function mice (Piezo1GOFLysM) to obtain bone-marrow derived macrophages (BMDMs). Fusion was visualized by staining BMDMs with Red CMTPX or Green CMFDA CellTracker and cultured on fibronectin-coated polyacrylamide hydrogels of different stiffness and examining overlapping red and green fluorescence. After 3 days, GM-CSF and IL-4 (10 ng/mL) were added for 4 days to induce fusion. Cells were stained with Hoechst 33342, fixed, mounted and imaged using a confocal microscope. Fusion index was determined as the number of MGCs nuclei (>3 nuclei) divided by the total number of nuclei. For fluorescence lifetime imaging microscopy (FLIM), BMDMs from C57BL/6J mice were seeded on fibronectin-coated polyacrylamide hydrogels. After 3 days, GM-CSF and IL-4 (10 ng/mL) were added for 4 days. NADH fluorescence lifetime signals were acquired from live cells at different time points using a two-photon microscope coupled to a FLIM box.

Results, Conclusions, and Discussions:: To investigate the role of Piezo1 in macrophage fusion, we cultured either Piezo1GOFLysM or Piezo1∆LysM BMDMs in different stiffness substrates. As expected, increased stiffness elicited higher levels of fusion, but we observed no differences in fusion with Piezo1∆LysM macrophages (Figure 1a). However, we observed higher fusion in Piezo1GOFLysM macrophages cultured in soft substrates (Figure 1b). We explored the changes in metabolism during macrophage fusion by measuring NADH fluorescence lifetime – which measures free NADH, an indicator of glycolysis - after stimulation with fusion-inducing cytokines. We found that between day 0 and 2, free NADH is reduced, suggesting that macrophages shift their metabolism towards oxidative phosphorylation. Cells cultured on stiff gels showed significantly lower levels of free NADH on day 2 compared to cells cultured on soft gels (Figure 1c). Together, these data suggest that 1) Piezo1 activation is sufficient for macrophage fusion and 2) macrophage fusion in stiff environments is associated with a metabolic shift towards oxidative phosphorylation.

We showed that macrophage fusion is a stiffness-dependent process, with stiffer substrates leading to enhanced MGCs formation. Our data reveal that stiffness-dependent fusion does not require Piezo1, but that Piezo1 activation is sufficient to induce fusion even in soft environments. Moreover, our data showed that macrophage fusion is mainly driven by a metabolic shift towards oxidative phosphorylation, painting a new picture on the mechanisms underlying this process. Current work is focused on exploring the direct role of Piezo1 in the metabolism of fusing macrophages. The elucidation of mechanotransduction pathways underlying MGCs formation could set the ground for the development of strategies that dampen FBR.

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