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

Area of Science:

• Biomaterials Science
• Immunology
• Cell Biology

Background:

• The extracellular matrix (ECM) is crucial for cellular programming.
• ECM mechanical properties, specifically stiffness, are under-investigated regarding their impact on cell differentiation.
• Understanding how ECM stiffness affects macrophage polarization is essential for regenerative medicine and immunology.

Purpose of the Study:

• To investigate the effect of extracellular matrix (ECM) substrate stiffness on macrophage polarization.
• To elucidate the underlying molecular mechanisms by which ECM stiffness modulates macrophage phenotypes.

Main Methods:

• Preparation of polyacrylamide hydrogels with varying substrate stiffness.
• Incubation of mouse bone marrow-derived macrophages (BMMs) on hydrogels with different stiffness.
• Assessment of macrophage polarization markers (CD86, CD206) and cytokine secretion (IL-1β, TNF-α, IL-4, TGF-β).
• In vivo subcutaneous implantation of hydrogels in mice to evaluate macrophage infiltration and phenotype.

Main Results:

• Low substrate stiffness promoted BMMs towards an M1-like phenotype, characterized by increased CD86 expression, higher reactive oxygen species (ROS) production, and elevated secretion of IL-1β and TNF-α.
• Medium substrate stiffness induced an M2-like phenotype, with increased CD206 expression, reduced ROS production, and enhanced secretion of IL-4 and TGF-β.
• In vivo studies confirmed that low stiffness hydrogels attracted CD68+CD86+ cells, while medium stiffness hydrogels attracted CD68+CD206+ cells.
• Low stiffness stimulation upregulated NIK, phosphorylated p65 (p-p65), and phosphorylated IκB (p-IκB) in BMMs, indicating activation of the NF-κB pathway.

Conclusions:

• Substrate stiffness significantly affects macrophage polarization.
• Low substrate stiffness promotes M1 polarization, while medium stiffness promotes M2 polarization, mediated by the ROS-initiated NF-κB pathway.
• ECM-based substrate stiffness offers a potential strategy for immune modulation in clinical applications.