(69d) Mitochondrial Metabolism in the Regulation of Human Mesenchymal Stem Cell Immunomodulatory Property

Human mesenchymal stem cells (hMSCs) isolated from various adult tissues are primary candidates in cell therapy and tissue regeneration. The recovery and regenerative effect of hMSCs is largely attributed to their release of immunosuppressive factors and growth factors that coordinately modulate the progression of inflammation, and enhance the propagation and differentiation of endogenous progenitor cells. Similar to macrophage polarization into the proinflammatory M1 or anti-inflammatory M2 phenotype, hMSCs are sensors and switchers of inflammation via their ability to adopt a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype in order to either promote or inhibit an immune response, depending on the severity of the inflammation, and the types of inflammatory cytokines present [1]. While much effort has been put on the interplay of hMSCs and immune cells via paracrine signaling, the regulatory mechanisms that mediate the phenotypic switch from MSC1 to MSC2 remain to be fully explored. Recent studies suggest that extensive metabolic reconfiguration of hMSCs occurs and plays central role in hMSCs fate decision and adaptation to environmental stimuli, such as during hypoxia, ischemic stress, and chemokine or cytokines stimulus. Identification of the metabolic profile and key metabolic components that control the immunophenotype of hMSCs and their response to inflammatory environment could potentially be used to enhance therapeutic applications of hMSCs. The major metabolic consequences of environmental stress involve the sequential inhibition of mitochondrial respiration and activation of ROS mediated redox signaling. Indeed, inflammatory cytokines IFN-γ and TNF-α have been reported to insert synergetic effect on the production of nitric oxide. Whether these metabolic events and in particular mitochondrial ROS are connected to IFN-γ-induced immunosuppressive cytokines production and act in concert to help establish or enhance the MSC2 phenotype is unknown. Our preliminary data demonstrated the important role of mitochondrial metabolism in hMSC â??stemnessâ?? decision that mitochondrial electron transport chain (ETC) and adaptive metabolic changes are not only correlated but also instrumental for MSCs pluripotent gene expression. The objective of present study is to explore the role of mitochondrial metabolism in hMSCs immunomodulatory property with aim to develop approachable strategy to enhanced hMSCs therapeutic potential.

Materials and Methods

To obtain MSC2 phenotype, hMSCs were incubated with IFN-γ (40 ng/ml) containing complete culture medium for 24 to 48 hours. The key immune suppressive factor released by hMSCs, indoleamine 2,3-dioxygenase (IDO), was measured for mRNA level and protein level. Kynurenine level in culture conditioned medium was quantified to assess the activity of IDO, which catalyzes the conversion of tryptophan to kynurenine. A metabolic profile for glucose metabolism was generated for unstimulated MSCs and MSC2 using ¹³C-labeling based GCMS. Several metabolic modulators were used to probe the impact of specific metabolic pathway in the regulation of hMSCs IDO activity.

Results and Conclusion

We observed spontaneous induction of mRNA for IDO and increased kynurenine level in MSCs conditioned medium after IFN-γ (40ng/ml) treatment. In addition, we observed metabolic changes of hMSCs associated with the raise of MSC2 that both glucose consumption and lactate release were increased. MSC2 were associated with changes in mitochondrial respiration that ETC complex II succinate dehydrogenase complex, subunit D (SDHD) was lower expressed in MSC2, with occurrence of mild decrease in mitochondrial membrane potential and accumulation in intracellular succinate level, indicating reduced ETC activity. As a result of interruption of mitochondrial respiration, both mitochondrial reactive oxygen species (ROS) and total cellular ROS level were increased in MSC2. Importantly, enhancement of mitochondrial function by PDK inhibitor DCA or reducing ROS level by mitochondrial targeted antioxidant Mito-Q both potently reduced IDO activity in hMSCs treated with IFN-γ. In contrast, restriction of cell respiration by hypoxia or ETC complex I and III inhibitor antimycin A and rotenone shifted MSCs into a MSC2-like phenotype in that it strongly potentiated IFN-γ mediated IDO activation. Together, the results revealed the mechanistic connection between metabolic regulation and MSC2 phenotype, and demonstrate the regulation of metabolism as a strategy in potentiating MSCs properties for cell therapy.

References:

[1] Wang Y, Chen XD, Cao W et al. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol. 2014;15:1009-1016.