Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes.

T cells mediate skin immune surveillance by secreting specific cytokines and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between T cell cytokines and actomyosin machinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-γ-induced migration of HPKs by inhibiting the actomyosin machinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the IL-17A-mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-γ- and IL-17A-induced phosphorylation of myosin L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-γ or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratinocytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies.

Glycated collagen - a 3D matrix system to study pathological cell behavior.

A hyperglycemic condition like diabetes in patients renders them with an increased risk of developing breast cancer. Hyperglycemia and ageing increase the non-enzymatic glycosylation (glycation) of nearly all proteins in our body including collagen type I, which is an important extracellular matrix (ECM) component. This results in the formation of advanced glycated end products (AGEs), which can form covalent crosslinks in collagen fibers and change the overall architecture and stiffness of the matrix. In this study we have used MDA-MB-231 breast cancer cells to study the interaction of tumor cells with glycated collagen and have explored the role of matrix architecture and RAGE-mediated signaling in cellular behavior. We mimicked the non-enzymatic glycation of protein by treating collagen I with glucose or ribose and found that crosslinking due to AGEs induces collagen fiber bundling and an increase in pore size and stiffness of the matrix. We also observed that AGE formation triggers AGE-RAGE signaling playing a role in the morphology and migration of cells. Furthermore, our study suggests an interplay of the pore size of the collagen matrix and RAGE mediated signaling in 3D invasion of cells and our findings demonstrate that the effect of the AGE-RAGE interaction is more pronounced than that of an altered matrix architecture. This study has helped us develop a 3D system using glycated collagen to study the effects of pathological conditions such as diabetes on extracellular matrix proteins, which may have downstream effects on cell behavior and dysfunction.