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PSRC 60th Annual Meeting
Program and Abstracts

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Microenvironmental Cues Enhance Indoleamine 2,3-dioxygenase Expression To Facilitate Mesenchymal Stem Cell-Mediated Immunosuppression
Rohini L. Kadle, BS, Marc A. Soares, MD, Nakul Rao, MD, April Duckworth, MD, Chin Park, MD, Joshua David, BS, Christopher Lopez, BS, Anna Zhou, BA, Nicholas Brownstone, BA, Rita Abigail Sartor, BS, Camille Kim, BS, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
NYU, New York, NY, USA.

PURPOSE:
Indoleamine 2,3-dioxygenase (IDO) has a powerful role in mesenchymal stem cell (MSC)-mediated immunosuppression, as previously demonstrated by our lab. However the environmental conditions that regulate IDO expression and thereby increase MSC immunosuppression remain unknown. Here we investigate approaches to enhance the role of IDO in MSC function. We hypothesize that MSC exposure to an inflammatory environment will increase IDO expression.
METHODS:
MSCs derived from Lewis rat bone marrow were expanded in either normoxia or hypoxia (5% O2 or 0.5% O2). During expansion, MSCs were exposed to IFNγ and/or TNFα for either 24 hours or 1 week. IDO expression was assessed using quantitative real-time PCR. Composite tissue allografts from Brown Norway rats were seeded ex-vivo with Lewis rat MSCs and transferred to Lewis rats and local inflammatory markers were measured using quantitative real-time PCR.
RESULTS:
MSCs grown in 5% O2 demonstrated a 4-fold increase in IDO expression versus normoxia (p<0.01). However at 0.5% O2, expression of IDO was virtually undetectable. In both normoxia and 5% O2 hypoxia, the addition of IFNγ increased IDO expression as compared to control (>20-fold at 24 hours, p<0.0001, and >127-fold higher at 1 week, p<0.001). Exposure to a combination of IFNγ/TNFα also showed a significant increase in IDO expression (>60-fold at 24 hours, p<0.05, and >1400-fold at 1 week, p<0.001). The combination of IFNγ/TNFα led to a significantly greater IDO expression than IFNγ alone in normoxia (p<0.001), however, no difference was present in hypoxia. MSCs exposed to longer treatment with IFNγ show a change in spindle morphology with decreased cytoplasmic processes, implicating a loss of “stemness” with prolonged exposure to inflammatory conditions. In composite tissue allografts seeded with MSCs, IDO expression was markedly increased and correlated with a significant decrease in the early inflammatory markers ICAM1, MCP-1 and CXCL9 (p<0.01).
CONCLUSION:
We demonstrate that hypoxic conditions similar to the native bone marrow environment of MSCs (5% O2) increase IDO expression. Oxygen tension is important to consider as IDO expression is negligible at very low oxygen levels. The exposure of MSCs to inflammatory molecules dramatically increases the expression of IDO. The increase is seen with just 24 hours of exposure, and is substantially enhanced with longer exposure. However, we also see a negative impact on MSC morphology with longer exposure to cytokines. In allografts, increased IDO expression by MSCs is associated with a decrease in early inflammatory markers. These results provide potential approaches to enhance IDO expression in order to facilitate MSC-driven immunosuppression.


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