Substrate stiffness modulates bone marrow-derived macrophage polarization through NF-κB signaling pathway

Abstract

The stiffness of the extracellular matrix (ECM) plays an important role in regulating the cellular programming. However, the mechanical characteristics of ECM affecting cell differentiation are still under investigated. Herein, we aimed to study the effect of ECM substrate stiffness on macrophage polarization. We prepared polyacrylamide hydrogels with different substrate stiffness, respectively. After the hydrogels were confirmed to have a good biocompatibility, the bone marrow-derived macrophages (BMMs) from mice were incubated on the hydrogels. With simulated by the low substrate stiffness, BMMs displayed an enhanced expression of CD86 on the cell surface and production of reactive oxygen species (ROS) in cells, and secreted more IL-1β and TNF-α in the supernatant. On the contrary, stressed by the medium stiffness, BMMs expressed more CD206, produced less ROS, and secreted more IL-4 and TGF-β. In vivo study by delivered the hydrogels subcutaneously in mice, more CD68⁺CD86⁺ cells around the hydrogels with the low substrate stiffness were observed while more CD68⁺CD206⁺ cells near by the middle stiffness hydrogels. In addition, the expressions of NIK, phosphorylated p65 (pi-p65) and phosphorylated IκB (pi-IκB) were significantly increased after stimulation with low stiffness in BMMs. Taken together, these findings demonstrated that substrate stiffness could affect macrophages polarization. Low substrate stiffness promoted BMMs to shift to classically activated macrophages (M1) and the middle one to alternatively activated macrophages (M2), through modulating ROS-initiated NF-κB pathway. Therefore, we anticipated ECM-based substrate stiffness with immune modulation would be under consideration in the clinical applications if necessary.

Keywords: Macrophage polarization; NF-κB signaling pathway; Substrate stiffness.

Graphical abstract

Fig. 1

Characterizations of the polyacrylamide hydrogels. (a) The images of the polyacrylamide hydrogels with different formula; (b) Compression test to measure the elastic modulus of polyacrylamide hydrogels with different formula, namely low (L), medium (M) and high (H) group according to its elastic modulus; (c) The Young's modulus of polyacrylamide hydrogels with different formula (n = 3); (d) The proliferation of Raw264.7 cells cultured in the leachate of polyacrylamide hydrogels (n = 5); (e) The proliferation of Raw264.7 cells seeded on the polyacrylamide hydrogels (n = 5); (f) The images of Raw264.7 cells cultured in the leachate for 5d stained by Live/dead cell staining kit. C: TCP, L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness.

Fig. 2

The morphology and actin cytoskeleton of BMMs seeded on the polyacrylamide hydrogels with different stiffness. (a) The morphology of BMMs cultured for 3d and 5d, respectively, observed by SEM; (b) The F-actin cytoskeleton of BMMs cultured for 5d were observed by the fluorescent microscope with phalloidin-FITC (in green) and DAPI (in blue) staining. L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness.

Fig. 3

Substrate stiffness monitored CD86 and CD206 expression of BMMs. BMMs were cultured on the polyacrylamide hydrogels with different stiffness. (a) and (b) the representative analysis of the expression of CD68⁺CD86⁺ and CD68⁺CD206⁺ BMMs by FCA for 3d and 5d, respectively; (c) and (d) the quantitative analysis of CD68⁺CD86⁺ and CD68⁺CD206⁺ BMMs for 3d and 5d, respectively; (e) (f) The expression of CD86 and CD206 of BMMs for 3d and 5d by immunofluorescence staining; (g) (h) Quantifying CD86⁺ and CD206⁺ immunofluorescence density by Image J for 3d and 5d (Rawak Software, Germany). L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness, n = 3, *p < 0.05.

Fig. 4

Substrate stiffness regulated genes and cytokines expressions of BMMs. BMMs were cultured on the polyacrylamide hydrogels with different stiffness for 3 and 5d, respectively. (a) Gene expressions levels were normalized to β-actin of BMMs by qRT-PCR; (b) Secretion of cytokines in the supernatant of BMMs by ELISA. L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness, n = 3, *p < 0.05.

Fig. 5

Substrate stiffness controlled ROS production of BMMs. BMMs incubated on the polyacrylamide hydrogels with different substrate stiffness for 3d and 5d, respectively. (a) Images by the fluorescence microscope; (b) FCA analysis of ROS production; L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness, Solid red line: negative control, dash blue line: ROS, n = 3, *p < 0.05.

Fig. 6

Substrate stiffness monitored macrophage polarization in vivo. The polyacrylamide hydrogels were subcutaneously implanted into mice for 14d. (a) Representative slices with H&E staining; (b) and (c) Representative images with immunofluorescence staining of CD68⁺CD86⁺ and CD68⁺CD206⁺ subcutaneously; (d) and (e) Quantification analysis of CD68⁺CD86⁺ and CD68⁺CD206⁺ immunofluorescence density by Bioquant osteo; S: sham, L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness, n = 3, *p < 0.05.

Fig. 7

Substrate stiffness affected NF-κB signaling pathway in BMMs. BMMs were cultured on the polyacrylamide hydrogels for 24h. (a) The representative expression of NIK, p65, pi-p65, IκB and pi-IκB in nuclear extracts of BMMs; (b) Quantitative analysis of the target proteins. C: TCP, L: low substrate stiffness, M: middle substrate stiffness, H: high substrate stiffness, n = 5, *p < 0.05.