Hyperglycemic Conditions Enhance the Mechanosensitivity of Proinflammatory RAW264.7 Macrophages

Macrophages are a primary contributor to the orchestration and severity of the foreign body response. As phagocytes and antigen-presenting cells, macrophages engage foreign objects, producing chemokines, degrading enzymes, and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). 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 by macrophages to the encapsulated islet allograft may cause rejection. Recent studies have shown that substrate stiffness affects macrophage activity, which can inform EIT capsule design. However, due to the dysregulation of glucose maintenance in diabetic patients, varying from normoglycemic to hypoglycemic or hyperglycemic conditions, it is imperative to determine if glucose dysregulation affects macrophage mechanosensitivity to EIT biomaterials. This study explores the relationship between glucose metabolism and mechanosensitivity and the ultimate impact on proinflammatory macrophage function in static hyperglycemic and normoglycemic conditions. Using a 2-dimensional (2D) polyacrylamide model of 3-order magnitude in stiffness, 2, 15, and 274 kPa Young's moduli, the effect of glycemic condition on the mechanosensitive characteristics of unstimulated and proinflammatory RAW264.7 macrophage function in vitro using lipopolysaccharide (LPS) was examined. Hyperglycemic conditions were found to impact macrophage response to substrate stiffness significantly. Notably, TNF-α secretion was significantly reduced as substrate stiffness increased in LPS-stimulated hyperglycemic conditions, whereas normoglycemic macrophages held similar secretion across all stiffnesses. Stiffness-influenced differences in cytokine secretion were also induced in IL-6 secretion by hyperglycemic conditions. Hyperglycemic conditions promoted a biphasic trend in IL-6 cytokine secretion and gene expression by proinflammatory macrophages with significantly decreased production when cultured on 15 kPa compared to production on 2 and 274 kPa. Although hyperglycemic conditions drastically increased IL-10 secretion, stiffness-influenced differences were not shown when compared to the same glycemic condition. Furthermore, under LPS stimulation, lactate secretion had an inverse relationship to TNF-α secretion. However, no significant stiffness-influenced difference was demonstrated in glucose transporter 1 (GLUT1) expression, glucose uptake, or GAPDH. These findings suggest that hyperglycemic conditions enhance the mechanosensitivity of proinflammatory macrophages and should be explored further. Impact statement The work presented increases our understanding of the effect of glycemic condition on macrophage mechanosensitivity related to substrate stiffness. This has ramifications on the design of material-based therapies, such as encapsulated islet transplantation, for type 1 diabetic patients who experience glycemic dysregulation.