Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages

Abstract

Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow–derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness–regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p–NF–κB p65, MyD88, and p–IκBα expression as well as p–NF–κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages.

Deletion of calponin 2 in macrophages alters cytoskeleton-based functions and attenuates the development of atherosclerosis

“…Macrophages cultured on stiffer substrate exhibited increased spreading area and enhanced adhesion, accompanied with elevated classical activation than that of macrophages cultured on softer substrate (Blakney et al, 2012). Macrophage grown on soft substrates produced less proinflammatory cytokines with decreased TLR4 activity than that of the macrophages grown on rigid substrates (Previtera and Sengupta, 2015). The modulation of macrophage function by substrate rigidity is dependent on actin polymerization and RhoGTPase activation (Patel et al, 2012).…”

Biophysical regulation of macrophages in health and disease

“…Much of our knowledge in this area has stemmed from studies performed in the context of biomaterials engineering, since implanted materials clearly alter the biophysical environment, and are known to interact with abundant macrophages . Several groups have found that stiffer materials enhance inflammatory activation, and grooved or fibrous materials promote alternative activation . In some cases, these findings have been correlated with in vivo studies, where materials are implanted within animals and macrophages in the surrounding tissue are characterized .…”

“…A and B). With respect to phenotype polarization, several groups have found that culture of macrophages (THP‐1, RAW 264.7, or BMDM) on stiffer materials potentiates inflammatory activation, although there are some reported discrepancies . Nonetheless, subcutaneous implantation of polyethylene glycol hydrogels with different stiffnesses led to greater macrophage recruitment and fibrous capsule thickness in response to stiffer compared to softer materials, suggesting an important ramification of material stiffness on the immune‐mediated foreign body response in vivo .…”

“…In the context of cancer, a recent study showed that matrix stiffness potentiated the ability for tumor‐associated macrophages to regulate the invasive potential of tumor cells in co‐culture model of lung adenocarcinoma The presence of macrophages and increased matrix stiffness together enhanced the epithelial to mesenchymal transition of human adenocarcinoma cells and increased tumor cell proliferation and invasiveness. Given that matrix stiffness has also been shown to enhance the activation of macrophages themselves, tumor stiffness may contribute to the protective activity that is commonly associated with tumor‐associated macrophages . One possibility is that increased stiffness along with the presence of protective cytokines enhances macrophage alternative activation, although this hypothesis has yet to be tested.…”

“…(A, C, E, G) Schematics showing the interaction of macrophages with surfaces of different stiffness ( A ) or topology ( C ) and external mechanical forces including stretch ( E ) and interstitial flow ( G ). ( B ) Image of IgG‐coated latex beads (cyan) uptaken by RAW264.7 macrophages on stiff compared to soft surfaces (left), adapted from Patel et al., and TNFα secretion by primary murine macrophages on surfaces different stiffness (right), adapted from Previtera and Sengupta . ( D ) Immunofluorescence image of Arg1 (M2 marker, red) and nuclei (blue) of macrophages cultured on flat and 1D wrinkled surfaces (left), and quantification of Arg1 expression as measured by Western blot (right), adapted from Wang et al .…”