TGF-β1 requires IL-13 to sustain collagen accumulation and increasing tissue strength and stiffness

Fibrosis is a multifactorial process characterized by the excessive accumulation of extracellular matrix (ECM), increased tissue stiffness, and decreased elasticity. This study examined how individual cytokines and a cytokine combination alter collagen production and biomechanics in a 3D in vitro model of the human ECM. Cultured human fibroblasts were seeded into a circular agarose trough molded in 24 well plates. The fibroblasts aggregated and formed a 3D ring-shaped tissue that synthesized de novo a collagen-rich human ECM complete with collagen fibrils. Unlike existing models, no macromolecular crowders were added, nor artificial scaffolds or exogenous ECM proteins. Rings were treated with TGF-β1, IL-13 or the combination of TGF-β1 and IL-13 for up to 3 weeks. Morphology, histology, collagen, DNA, fibril formation, gene expression and tensile properties of the rings were measured. As the rings compacted, cellularity and total DNA decreased, whereas total collagen accumulated. TGF-β1 stimulated collagen accumulation and increased ring biomechanics at day 7, but these increases stalled and declined by day 21. When treated with IL-13, a cytokine exclusive to the immune system, there were no significant differences from control. However, when TGF-β1 was combined with IL-13, collagen levels and ring biomechanics increased over the entire three weeks to levels higher than TGF-β1 alone. Gene expression was differentially regulated by cytokine treatment over the entire three weeks suggesting that increased collagen accumulation was not due to upregulation of collagen gene expression. These results suggest that TGF-β1 requires a second signal, such as IL-13, to sustain the long-term pathological increases in collagen accumulation and biomechanics that can compromise the function of fibrotic tissues.