Keratin biomaterials augment anti-inflammatory macrophage phenotype in vitro

Tissue regeneration following injury is mediated by macrophage recruitment and differentiation in response to environmental signals. In general, macrophages adopt either a classically M1 (M[IFN-γ, LPS]) or alternatively activated M2 (M[IL-4, IL-13] or M[IL-10]) phenotype. Recent studies have highlighted the importance of alternatively activated macrophages in tissue remodeling and repair as well as the contribution of an imbalance of classically and alternatively activated macrophages to tissue degeneration and disease progression. Keratin biomaterials have recently demonstrated their ability to promote alternatively activated macrophage polarization in an in vitro model using a monocytic cell line. In the present study, the ability of extracted human hair keratins to influence alternative activation of human primary monocytes in vitro is assessed by evaluating changes in surface receptor expression, inflammatory cytokine secretion, and phagocytic activity. The impact of keratin molecular weight fractionation on these outcomes was also investigated. High and low molecular weight fractions of the oxidized form of extractable human hair keratins - referred to as keratose (KOS^H and KOS^P, respectively) - were characterized by size exclusion chromatography, mass spectrometry, and Western blot. Primary macrophages underwent traditional differentiation to the M[IFN-γ, LPS], M[IL-4, IL-13], or M[IL-10]) phenotypes or were plated on different molecular weight keratin coatings (KOS^H and KOS^P). Macrophages plated on keratin and analyzed via flow cytometry yielded the largest CD163⁺ cell populations and CD163 mean fluorescence intensities. Cells in the KOS^P group were significantly more phagocytic than all other cell types at the 1.5 and 3 h time points and exhibited behavior and a cytokine production profile most similar to the M[IL-10] treated group. These findings may have important implications for understanding and evaluating the ability of keratin biomaterials to influence inflammation and tissue regeneration in disease and injury models.

Biomaterials made from human hair keratins have previously been shown to elicit anti-inflammatory responses from naïve macrophages and polarize them toward an M2 phenotype. In this work we show for the first time that primary human cells respond similarly, that it is the M2c phenotype that predominates, that a sub-fraction of hydrolyzed keratin peptides are most likely responsible for the response, and that immobilization of the keratin peptides to a surface is required. Keratin biomaterials have been used to regenerate several tissues such as skin, muscle, bone, nerve, and cornea, in vitro and in animal studies. Our current findings will help guide the development of keratin-based biomaterials that seek to direct responses toward regenerative outcomes by attenuating inflammation.