Chronic inflammation is associated with the development of human hepatocellular carcinoma (HCC), an essentially incurable cancer. Anti-inflammatory nutraceuticals have emerged as promising candidates against HCC, yet the mechanisms through which they influence the cell signaling machinery to impose phenotypic changes remain unresolved. Herein we implemented a systems biology approach in HCC cells, based on the integration of cytokine release and phospoproteomic data from high-throughput xMAP Luminex assays to elucidate the action mode of prominent nutraceuticals in terms of topology alterations of HCC-specific signaling networks. An optimization algorithm based on SigNetTrainer, an Integer Linear Programming formulation, was applied to construct networks linking signal transduction to cytokine secretion by combining prior knowledge of protein connectivity with proteomic data. Our analysis identified the most probable target phosphoproteins of interrogated compounds and predicted translational control as a new mechanism underlying their anticytokine action. Induced alterations corroborated with inhibition of HCC-driven angiogenesis and metastasis.

Purpose: To investigate the interactions between allogeneic articular chondroprogenitors and immune cells in vitro by comparing them to allogeneic MSC Methods: Primary articular chondroprogenitors were isolated from the knees of Dark Agouti rats. These cells were used in co-culture systems with allogeneic lymphocytes to determine the immune response elicited by and immunosuppressive ability of the chondroprogenitors. Specific inhibitors of signalling molecules were used to determine the mechanism of chondroprogenitor immunosuppression. Flow cytometric assays were used to assay the allogeneic T cell response to chondroprogenitors. Chondroprogenitors ability to alter the polarisation of allogeneic macrophages was also investigated by flow cytometry, qPCR and zymography. Results: We found that allogeneic chondroprogenitors elicit allogeneic T cell proliferation similar to Mesenchymal Stem Cells (MSC), indicating that these cells are weakly immunogenic. The chondroprogenitors were not targeted any more than MSC by allo-specific cytotoxic T cells and these cytotoxic T cells expressed low levels of the cytotoxic molecule Granzyme-B. The immunogenicity of the chondroprogenitors was not increased by exposure to an in vitro inflammatory environment. Interestingly we found that allogeneic chondroprogenitors were capable of suppressing allogeneic T cell proliferation. This inhibition was not dependent on cell contact, as is the case with MSC. We found that chondroprogenitors suppress T cell proliferation by IL1b induced nitric oxide production. Chondroprogenitors, like MSC, also produce significant amount of the immunosuppressive molecule PGE2. Macrophages play an important role in OA, therefore we analysed the effects of chondroprogenitors on macrophage polarisation. We found that chondroprogenitors exert effects on the expression of cytokines IL10 and IL12 in macrophages similar to allogeneic MSC. Additionally similar MMP secretion was observed in chondroprogenitor/macrophage co-culture as MSC/macrophage co-culture. Conclusions: These data indicate that allogeneic articular chondroprogenitors are weakly immunogenic, are capable of suppressing allogeneic T cells and altering the polarisation of allogeneic macrophages. This raises the potential for use of allogeneic chondroprogenitors to treat cartilaginous defects in vivo. Such a treatment would greatly reduce the costs of the procedure and indeed, due to the fact that allogeneic chondroprogenitors produce immunosuppressive molecules in an inflammatory environment, may be preferable to autologous treatments as the allogeneic cells may ameliorate the inflammatory component of diseases such as OA, while also repairing the damaged tissue.