Significance: Rho-associated kinase 2 (ROCK2) is implicated in the regulation of proinflammatory cytokines, such as IL-17 and IL-21, and the development of autoimmunity in mice. However, the role of ROCK2 signaling pathway in regulation of immune responses in humans is still an enigma. Here we show that targeted ROCK2 inhibition down-regulates proinflammatory responses via concurrent regulation of STAT3/STAT5 phosphorylation and shifting Th17/Treg balance in human T cells with a minimal effect on the rest of the immune response. This work provides previously unidentified insights into the molecular mechanism of ROCK2-mediated modulation of the immune response in man and has profound implications for development of a selective ROCK2 inhibitor as a new therapeutic target for autoimmunity treatment.

Nanofibrous scaffolds provide high surface area for cell attachment, and resemble the structure of the collagen fibers which naturally occur in the basement membrane and extracellular matrix. A label free and non-destructive method of assessing the interaction of cell tissue and scaffolds aids in the ability to discern the effective quality and magnitude of any scaffold modifications. Impedance cell spectroscopy is a biosensing method that employs a functional approach to assessing the cell monolayer. The electrical impedance barrier function of a cell monolayer represents the level of restriction to diffusion of charged species between all adjacent cells across an entire contiguous cellular monolayer. The impedance signals from many individual paracellular pathways contribute to the bulk measurement of the whole monolayer barrier function. However, the scaffold substrate must be entirely porous in order to be used with electrochemical cell impedance spectroscopy (ECIS) and cells must be closely situated to the electrodes. For purposes of evaluating cell-scaffold constructs for tissue engineering, non-invasive evaluation of cell properties while seeded on scaffolds is critical. A Transwell-type assay makes a measurement across a semi-permeable membrane, using electrodes placed on opposing sides of the membrane immersed in fluid. It was found that by suspending a nanofiber scaffold across a Transwell aperture, it is possible to integrate a fully functional nanofiber tissue scaffold with the ECIS Transwell apparatus. Salivary epithelial cells were grown on the nanofiber scaffolds and tight junction formation was evaluated using ECIS measurements in parallel with immunostaining and confocal imaging. The trans-epithelial resistance increased coordinate with cell coverage, culminating with a cell monolayer, at which point the tight junction proteins assemble and strengthen, reaching the peak signal. These studies demonstrate that ECIS can be used to evaluate tight junction formation in cells grown on nanofiber scaffolds and on effects of scaffold conditions on cells, thus providing useful biological feedback to inform superior scaffold designs.

Salivary glands are crucial organs that are responsible for secreting saliva which is important for the breakdown of carbohydrates, providing lubrication of the mouth, and maintenance of oral health. Patients diagnosed with Sjögren's Syndrome or receiving treatment for head and neck cancer, can permanently lose salivary gland function, which is referred to as salivary hypofunction. As a result, these patients develop Xerostomia, or the feeling of dry mouth, and suffer from an overall decreased quality of life. To combat the negative effects of salivary hypofunction, stem cell therapy is an attractive option, as implantation of specific cell populations can rescue salivary gland function. Cells expressing the tyrosine kinase, Kit, have previously been shown to restore function in irradiated murine salivary glands. For clinical transplantation, expansion of stem/progenitor cells is a limiting factor. Expansion of salivary progenitor cells has previously been accomplished through floating 3D cultures known as salispheres. However, further characterizing the cellular composition of salispheres could improve their regenerative capabilities, as these salispheres could be grown under conditions that enrich for cells expressing progenitor markers that replenish the acinar lineage. As the Rho-associated Kinase (ROCK) inhibitor, Y27632, has been used to promote survival of stem/progenitor cells grown ex vivo, we investigated whether application of Y27632 treatment in salisphere formation in different cell media contexts could be used to modulate the composition of adult epithelial progenitor cells in salispheres. In a simple serum-containing medium, addition of Y27632 increased the number of proliferative basal progenitor cells expressing Keratin 5. Salispheres cultured for three days in specialized Salisphere (or Sali) medium in the presence of Y27632, showed an increased number of proliferating Kit+ cells and Mist1+ acinar cells, demonstrating that inhibition of ROCK signaling can modulate the expansion and survival of different progenitor cell populations, which is modified by other signals.

Salisphere-derived adult epithelial cells enriched for progenitor cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) ¹ inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 in different cell media contexts could be used to improve expansion of adult epithelial progenitor cells or to affect their differentiation potential. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit ⁺ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. When tested in a 3D organoid assay, Y27632 enhanced the contribution of adult salispheres to salivary organoids expressing the secretory proacinar marker Aquaporin 5 (AQP5) in response to FGF2 dependent mesenchymal signals. Optimization of epithelial-mesenchymal interactions organoids can be used to improve application of adult salivary progenitor cells in regenerative medicine strategies. Highlights: Y27632 promotes Kit ⁺ salisphere cell proliferation in salisphere media. Y27632 promotes K5 expression in salispheres cultured in serum containing media. Y27632 treated Kit ⁺ salispheres form salivary organoids expressing AQP5.