Control of Macrophage Fusion By Sensing the Physical Properties of Fibrin(ogen) Matrices

Macrophage fusion is a salient and unwanted characteristic of implanted biomaterials. We have recently shown that macrophage fusion requires fibrinogen, as fibrinogen-deficient mice do not support this process. We also found that this is not fibrinogen itself but rather its clotting product, the fibrin polymer deposited on the surface of biomaterials, which drives macrophage fusion. This is evidenced by the data showing that macrophage fusion on biomaterials implanted into FibAEK mice expressing a mutated form of fibrinogen incapable of thrombin-mediated polymerization was strongly reduced. This phenotype was similar to fibrinogen-deficient mice, even though FibAEK is structurally similar to wild-type fibrinogen and normally adsorbs on the surface. It is well established that the physical properties of substrates control cellular functions through integrin-mediated cell adhesion and signaling. Fibrinogen adsorption on various surfaces at its physiological concentrations is known to reduce integrin-mediated adhesion and signaling in leukocytes and platelets by forming the nanoscale extensible multilayer matrix. To test the possibility that the fibrinogen multilayer cannot support macrophage fusion due to its inability to mediate firm adhesion and, consequently, transduce a competent mechanotransduction response in fusing macrophages, we examined the matrices prepared from wild-type fibrinogen and FibAEK. Adhesion, spreading, and podosome formation in macrophages were strongly reduced on both fibrinogens adsorbed on the biomaterial surface. We also used immunostaining to examine the localization of YAP, a sensor, and a molecular reporter of the ECM rigidity. On rigid substrates, YAP undergoes dephosphorylation and relocalization from the cytoplasm to the nucleus, and this regulation requires the tension of the actomyosin cytoskeleton developed in spread cells. We found that YAP localized exclusively in the cytoplasm of poorly spread macrophages attached to the fibrinogen multilayer matrix. In contrast, YAP was redistributed to the perinuclear region in macrophages that firmly adhered and spread on the fibrinogen monolayer, a model rigid substrate prepared by adsorption of low-density fibrinogen. IL-4-induced macrophage fusion exhibited a similar pattern with a lack of fusion in cells seeded on the fibrinogen multilayer and robust fusion in cells adherent to the monolayer. Furthermore, YAP remained in the cytoplasm in the non-fused mononucleated cells attached to the multilayer matrix while fully localized to the nucleus in multinuclear macrophages undergoing fusion on the monolayer. These data indicate that fibrinogen adsorption as a nonadhesive multilayer matrix on the surface of biomaterials discourages macrophage fusion, which has implications for controlling the foreign body reaction by modulating the mechanical properties of biomaterials.