(C-94) Substrate Stiffness-Related Mechanosensitivity of Unstimulated Macrophage Focal Adhesion and Phagocytic Functionality Enhanced by Hyperglycemic Conditions

Introduction:: Encapsulated islet transplantation (EIT) is a return of function therapy in which donor insulin-secreting cells are encased in a biomaterial and implanted into a diabetic patient to regulate blood glucose levels. However, the foreign body response (FBR) by macrophages causes rejection of the EIT, preventing commercial use. Macrophage adherence/activation is crucial to determining the foreign body response's extent. Macrophages adhere to surface-adsorbed proteins by integrins, which link with structural focal adhesion proteins, such as talin and vinculin, allowing the cells to sense the mechanical properties of the substrate.^1,2 This mechanosensing leads to mechanotransduction through the focal adhesion kinase (FAK) pathway, promoting various morphologic and functional changes in macrophages, including activation and phagocytosis. During FBR to EIT, due to the microcapsule size (600 – 1000 µm), macrophages undergo frustrated phagocytosis in attempts to remove the transplant, leading to chronic inflammation.³ Substrate stiffness has been demonstrated to alter F-actin construction leading to changes in morphology, phenotype, and phagocytic capabilities in macrophages.^4–6 However, the role of glucose dysregulation experienced by people with diabetes has not been revealed in the mechanosensitivity of focal adhesion and phagocytosis, which would be advantageous in diabetic tissue-engineered therapy design to increase clinical viability. In this study, we aim to elucidate this role by exploring the interplay between glycemic condition and substrate stiffness on the morphology, phenotype, and phagocytic functionality of macrophages.


Materials and Methods:: Polyacrylamide gels (PA) (2, 15, and 274 kPa) were synthesized using varying ratios of acrylamide and bis on glass coverslips. PA gels were activated with 0.5 mg/mL sulfo-SANPAH and coated with 0.1 mg/mL human fibronectin for cell adherence. For morphology, macrophages (1e5 RAW264.7) were seeded onto each gel and incubated for 1 hour and overnight (18 hours) at 37°C. Cells were cultured under either hyperglycemic (450 mg/dL) or normoglycemic (100 mg/dL) conditions. Cells were fixed and stained for phalloidin (TRITC) and DAPI and imaged under fluorescent microscopy. Cell size and circularity were quantified using ImageJ. For phenotype, macrophages (1e5 RAW264.7) were seeded on gel and incubated overnight at 37°C. Cells were fixed and stained for CD80, CD86, and CD206. To assess frustrated phagocytosis, sulfo-SANPAH-activated PA gels were coated with 0.1 mg/mL human IgG to initiate phagocytic functionality. Macrophages (1e5 RAW264.7) were seeded onto each gel and incubated for 1 hour at 37°C. Cells were fixed, stained for F-actin (TRITC) and cell nucleus (DAPI), and imaged under fluorescent microscopy. Macrophages with lamella extending radially symmetric were counted as positive for frustrated phagocytosis. The percentage of cells experiencing frustrated phagocytosis was compared by stiffness and glycemic condition. Glucose uptake was inhibited using 0.4mM 2-deoxy-d-glucose to confirm findings. n = 5 biological replicates and n = 5 technical replicates were conducted for all samples. Data were analyzed using ANOVA and Tukey tests.

Results, Conclusions, and Discussions:: Stiffness-influenced morphology changes of macrophages were independent of glycemic condition. At 1 hour, cell area and circularity were comparable across all stiffnesses and glycemic conditions. As cells incubated for 18 hours, macrophage morphology was significantly altered based on substrate stiffness. Macrophage size significantly increased as substrate stiffness increased from 2 – 274 kPa while circularity decreased. This trend held independent of glycemic condition. Macrophage phenotype also remained comparable independent of glucose and stiffness when comparing the proinflammatory CD86 marker and anti-inflammatory CD206 marker. However, when comparing the costimulatory CD80, expression decreased with increased stiffness under normoglycemic conditions, while expression was comparable across stiffness under hyperglycemic conditions. This will be further studied using flow cytometry. Furthermore, we found that glycemic condition impacted macrophage response to substrate stiffness, as seen in cells experiencing frustrated phagocytosis, Figure 1. The percentage of macrophages undergoing frustrated phagocytosis significantly increased proportionally with increased stiffness in both glycemic conditions. However, as glycemic conditions increased from normoglycemic to hyperglycemic, stiffness-derived mechanosensitivity enhanced drastically from a 6 – 12% range to a 6 – 22% range. Inhibiting glucose uptake using 2-deoxy-d-glucose resulted in insignificant differences across glycemic condition and stiffness, confirming our findings that glucose drives enhanced stiffness-derived mechanosensitivity in phagocytic functionality.

Our work demonstrated that glycemic condition plays a role in stiffness-derived macrophage mechanosensitivity, as seen in the phagocytic functionality of the macrophages. While morphology and phenotype changes remained comparable independent of glycemic conditions, these results elucidate the role of glucose metabolism in macrophage functionality and mechanotransduction and should be further studied. Future studies will further investigate FAK signaling and phagocytosis of stiffness beads to understand the mechanism of this relationship and determine the ideal stiffness for EIT design.


Acknowledgements (Optional): :

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