A125: Immunomodulatory Factors Produced by Mesenchymal Stem Cells After in vitro Priming with Danger Signals

Abstract

BACKGROUND/PURPOSE: We have previously shown that pre-exposure to IL-1β augments the immunosuppressive activity of MSC using the mixed lymphocyte reaction as a readout assay. In this project, we studied the profiles of inflammatory cytokines secreted by MSCs at the protein level to identify biomarkers in in vitroprimed MSCs. The objective of this study was to determine the MSC response to "danger signals."METHODS: MSCs were isolated from a healthy donor and cultured as per a protocol published earlier by the Skeletal Research Center. After three passages, 200,000 cells per 35 mm culture dish were incubated in 2 ml DMEM with 10% fetal bovine serum at 37°C for 5 days until 90% confluent. The MSCs were then treated with LPS (10 μg/ml), IL-1β (25 ng/ml), TNF-α (50 ng/ml), or poly (I:C) (30 ng/ml) overnight (18 hours). Control and treatment groups were done in triplicate. The culture media were then collected and analyzed for secreted mediators using the RayBiotech® Human Cytokine Array (2000), as per the manufacturer's protocol. Chemiluminescense was detected using Kodak BioMax MR film, and was analyzed using the ImageJ program (rsbweb.nih.gov). Normalization and fold-difference computations were then performed in MS Excel. Pathway analysis for biomarker detection was performed using the Ingenuity (ingenuity.com) program by the CWRU proteomics center.RESULTS: There were clear distinctions in the patterns of inflammatory mediators produced by MSC upon exposure to LPS versus poly (I:C). LPS stimulated the production of mediators that affect mostly neutrophil chemotaxis and activation, including IL-8 (1.8 fold), CXCL7 (2 fold), CXCL5 (2.4 fold), CXCL1 (1.6 fold). However, poly (I:C) induced mediators with activities on T cells and NK cells including IL-15 (18 fold), IL-4 (23 fold), TGF-β3 (9.7 fold), CCL-18 (4.6 fold). While poly (I:C) stimulated IL-1β production (2.6 fold), LPS did not (0.7 fold), and neither agent increased TNF-α significantly (1.3 and 2.1 fold). Interestingly, there was marked production of CCL1 (12 fold) upon exposure of MSC to IL-1β but not to LPS (0.6 fold), TNF-α (1.3) or poly (I:C) (2.1 fold).CONCLUSION: Priming MSC by TLRs ligands triggered different cytokine and chemokine expression profiles. These profile patterns may signify framework of the upcoming immune responses in the tissue. Accordingly, the candidate biomarkers for ongoing MSC immunomodulatory activities should include those for neutrophils versus those for T/NK cells. Furthermore, our results strongly suggest involvement of IL-1β in MSCmediated activation of T-regulatory cells by induction of the CCR-8 ligand, CCL-1. CCR-8 is a known biomarker identifying CD4(+) memory T-cells enriched for FoxP3(+) T-regulatory cells and is involved in delivery of immunosuppression.