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

Abstract

Aims: 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.

Methods: 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.

Results: 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.

Conclusions: 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.