879 IL-9 Mediated Human Primary Keratinocytes Invasion is Dependent on MLC Controlled Contractility and Independent of MMP Activity

T-helper 9 cells are recently discovered IL-9 secreting CD4+T helper cells and their presence is described in healthy and skin inflammatory diseases. However, it remains poorly examined how IL-9 impacts the cellular responses in the skin during homeostasis and disease pathogenesis. In this study, we examined the roles of IL-9 in regulating the human primary keratinocytes (HPKs) biophysical properties such cellular morphology, invasion and migration potential, which are critical for maintaining skin homeostasis in healthy and diseased individuals. IL-9 promoted the random HPKs motility in 2D space as demonstrated by wound healing and 2-D motility assays. In contrast, IL-9 down-regulated the HPKs invasion when cells were embedded in 3D collagen matrix. Surprisingly, IL-9 mediated inhibition of cell invasion was independent of matrix-metalloproteinase (MMPs) as neither the RNA levels nor MMP activity changed upon IL-9 stimulation of HPKs. Further, we investigated the effect of IL-9 on HPKs stiffness and contractility by atomic force microscopy and de-adhesion assay respectively. Interestingly, IL-9 reduced the HPKs stiffness and also increased the cell circularity which corroborated with reduction in its contractility. Finally, pharmacological inhibition of MLC kinases demonstrated that IL-9 mediated loss of contractility and invasion potential are dependent on Rho associated kinase (ROCK) and independent of MMP mediated pathways. In conclusion, we show a novel mechanism by which IL-9 controls the HPKs migration and invasion potential.