Mechanical Resistance of Fibrotic Extracellular Matrix Directs Myeloid Inflammation

Objectives: Inflamed dental pulp recruits monocytes and becomes fibrotic as it undergoes necrosis. The role of extracellular matrix (ECM) in mechanical regulation of monocytes remains unknown. Here, we study the bone marrow (BM) niche as a model, similar to dental pulp, which is normally a viscous solid; stress dissipates rapidly over-time while static deformation is applied. We hypothesized that viscous/elastic mechanical properties of ECM impact the fate of monocytes, and thus could contribute to inflammation.
Methods: Human BM, obtained from fracture hematomas, was mechanically characterized. An artificial ECM of a chemically-modified polysaccharide and type-I collagen was fabricated with viscous or elastic properties of stiffness similar to human BM and dental pulp. Human BM monocytes were isolated and encapsulated in viscous or elastic matrix. After three days, monocytes were retrieved and characterized by cytokines with Luminex and ELISA, gene expression with bulk RNA-sequencing and a nanoString myeloid panel, and surface markers with flow cytometry. Multi-photon confocal imaging monitored F-actin cytoskeleton of monocytes in artificial ECM tested with a panel of cytokines and inhibitors related to mechanotransduction. Gene expression of human BM biopsies with fibrosis was determined by a nanoString inflammation panel. Statistical significance was determined by appropriate tests, p<0.05. Monocyte experiments were repeated with at least 3 donors.
Results: At constant stiffness, solid-like ECM elasticity upregulated dendritic cell differentiation and pro-inflammatory markers, whereas monocytes in faster-relaxing viscous ECM expressed immature markers. Elasticity upregulated the cortical F-actin cytoskeleton, which was dependent on PI3K-gamma signaling. Inhibition of PI3K-gamma blocked the effects of elasticity on differentiation, as well as secretion of IL8, CCL2, IL6, and CCL4. Gene expression of IL8 and CCL2 was also upregulated in biopsies of advanced BM fibrosis.
Conclusions: Together, these data demonstrate how elasticity of ECM directs monocyte cell fate and polarization, and revealed PI3K-gamma as a potential therapeutic target in fibrosis and inflammation.