PPARβ/δ-dependent MSC metabolism determines their immunoregulatory properties

Mesenchymal stem cell (MSC)-based therapy is being increasingly considered a powerful opportunity for several disorders based on MSC immunoregulatory properties. Nonetheless, MSC are versatile and plastic cells that require an efficient control of their features and functions for their optimal use in clinic. Recently, we have shown that PPARβ/δ is pivotal for MSC immunoregulatory and therapeutic functions. However, the role of PPARβ/δ on MSC metabolic activity and the relevance of PPARβ/δ metabolic control on MSC immunosuppressive properties have never been addressed. Here, we demonstrate that PPARβ/δ deficiency forces MSC metabolic adaptation increasing their glycolytic activity required for their immunoregulatory functions on Th1 and Th17 cells. Additionally, we show that the inhibition of the mitochondrial production of ATP in MSC expressing PPARβ/δ, promotes their metabolic switch towards aerobic glycolysis to stably enhance their immunosuppressive capacities significantly. Altogether, these data demonstrate that PPARβ/δ governs the immunoregulatory potential of MSC by dictating their metabolic reprogramming and pave the way for enhancing MSC immunoregulatory properties and counteracting their versatility.

In the present study, we investigate the role of PPARβ/δ on MSC metabolic activity and the relevance of PPARβ/δ metabolic control on MSC immunoregulatory functions.

Results
PPARβ/δ knockdown enhances the capacity of MSC to inhibit the proliferation and the functions of Th1 and Th17 cells. As we previously described 2 , PPARβ/δ inhibition or knockdown in MSC enhances their capacity to inhibit T lymphocyte proliferation. To go a step further, we addressed the effect of PPARβ/δ on MSC immunoregulatory properties focusing on specific T cell subsets including Th1 and Th17 cells. To that end, naïve T-CD4 cells induced to differentiate into both Th1 or Th17 cells were co-cultured with either wild-type MSC (MSC PPARβ/δ +/+ ) or MSC deficient for PPARβ/δ (MSC PPARβ/δ −/− ). While PPARβ/δ knockout significantly increased the capacity of MSC to inhibit the proliferation of Th1 and Th17 (Fig. 1A,D) and to decrease the percentage of Th1 and Th17 cells (Fig. 1B,E), it also enhanced the capacity to generate regulatory T cells (Treg) (Fig. 1C,F). The immunosuppressive activity of MSC depends on the production of molecules including IL6 and PD-L1. The release of these mediators of MSC immunosuppressive functions is stimulated after 24 h of MSC treatment with TNFα and IFNγ 12 . Therefore, we evaluated the effect of pro-inflammatory   Regarding the ratio of ECAR to OCR, it was also significantly higher in MSC PPARβ/δ −/− than in MSC PPARβ/δ +/+ (Fig. 2F). These results revealed that MSC PPARβ/δ −/− display a higher glycolytic metabolism than MSC PPARβ/δ +/+ . Then, we evaluated the glucose consumption ( Fig. 2G) and lactate production (Fig. 2H) by the MSC and determine their GLUT1 expression profile (Fig. 2I,J). Our results showed that the highly immunosuppressive MSC PPARβ/δ −/− exhibit a significantly higher glucose consumption, lactate production and GLUT1 expression level than their wild-type counterpart suggesting that PPARβ/δ deficiency reprograms MSC metabolism while increases their immunoregulatory functions.
Oligomycin treatment induces a potent metabolic switch of MSC. Since PPARβ/δ is a master regulator of MSC immunosuppressive property and that the induction of MSC glycolytic metabolism enhanced this latter property, we investigated the effect of a pharmacological metabolic switch on poorly immunoregulatory MSC expressing high level of PPARβ/δ. To that end, we first treated MSC PPARβ/δ +/+ for 24 h with oligomycin, an inhibitor of ATP synthase that blocks oxidative phosphorylation, and compared their bioenergetics status to untreated MSC PPARβ/δ +/+ and MSC PPARβ/δ −/− . MSC PPARβ/δ +/+ treated with oligomycin displayed a significantly lower OCR compared to untreated cells (Fig. 3A). Moreover, the treatment of MSC PPARβ/δ +/+ with oligomycin significantly increased their glycolytic capacity at a similar level than MSC PPARβ/δ −/− (Fig. 3B). The analysis of the ECAR/OCR ratio of oligomycin treated MSC PPARβ/δ +/+ indicated a ~ 30-fold increase compared to untreated PPARβ/δ +/+ and a ~ tenfold increase compared to MSC PPARβ/δ −/− (Fig. 3C). Then, we evaluated the glucose consumption (Fig. 3D) and lactate production (Fig. 3E) and showed that oligomycin treatment significantly increased the capacities of MSC PPARβ/δ +/+ to consume glucose and produce lactate to the same extent as MSC deficient for PPARβ/δ. To define whether the modulation of MSC metabolism was also associated with PPARβ/δ expression level, we analyzed the expression profile of PPARβ/δ −/− on untreated and oligomycintreated MSC. PPARβ/δ +/+ -oligomycin MSC driven toward a glycolytic metabolism exhibited a reduced PPARβ/δ www.nature.com/scientificreports/ expression level compared to untreated MSC PPARβ/δ +/+ (Fig. 3F). These data confirm that the metabolic switch of MSC towards a glycolytic metabolism control PPARβ/δ expression level.
PPARß/δ-dependent MSC metabolic status partly controls their immunoregulatory potential. Going further, we decided to study whether the metabolic modulation of MSC PPARβ/δ +/+ could enhance their immunosuppressive functions. We performed a co-culture experiments with T-CD4 cells induced to differentiate into Th1 and Th17 cells with either MSC PPARβ/δ +/+ , oligomycin-treated MSC PPARβ/δ +/+ or MSC PPARβ/δ −/− . The enhancement of MSC PPARβ/δ +/+ glycolytic metabolism significantly increased their immunosuppressive activity on Th1 and Th17 cell proliferation and pro-inflammatory phenotypes (Fig. 4A,B,D,E) to the same level as MSC PPARβ/δ −/− without modifying their capacity to generate Treg cells (Fig. 4C,F). The treatment of MSC PPARβ/δ −/− with a glucose analogue, 2-deoxiglucose (2-DG), that induces OXPHOS metabolism, reduced the capacity of MSC to inhibit the proliferation of T-CD4 cells (Sup. Fig. 2A). Hence, our data demonstrate that the metabolic status of MSC significantly control their immunosuppressive activities partially www.nature.com/scientificreports/ through a PPARβ/δ-dependent manner. Furthermore, in order to determine whether glucose consumption was responsible for the enhanced immunosuppressive activity of both MSC deficient for PPARβ/δ and MSC pretreated with oligomycin, we supplemented the culture media with glucose every 24 h. Glucose addition in the media did not modify the proliferation profile suggesting that glucose deprivation is not involved in MSCmediated immunosuppression (Sup. Fig. 2B). Remarkably, the enhanced immunosuppressive effect of MSC pretreated with oligomycin was still observed using a transwell system to physically separate activated T-CD4 cells and MSC (Sup. Fig. 2C). Finally, we evaluated the expression profile of some of the mediators associated with the immunosuppressive functions of MSC such as PDL1, IL6, VCAM, ICAM, NO 2 and TGFβ1. For that purpose, PPARβ/δ +/+ and PPARβ/δ −/− MSC were pretreated or not with oligomycin and activated, when indicated, with TNFα and IFNγ. The treatment with oligomycin neither modified the capacity of MSC to express PDL1, VCAM and ICAM nor to produce IL6 (Fig. 4G-J). In contrast, MSC treated with oligomycin produced higher levels of NO 2 and TGFβ1 as compaerd to the non-treated MSC that reached the levels produced by PPARβ/δ −/− MSC (Fig. 4K,L). Thus, a ranking of MSC immunomodulatory levels according to their metabolic status can be proposed (Fig. 4M).

Discussion
In the present study, we demonstrated that the enhanced immunoregulatory properties of MSC PPARβ/δ −/− are associated with significantly higher glycolytic capacity, lactate production and higher glucose consumption than wild-type MSC. This result reveals that PPARβ/δ inhibition is a key switch of MSC immunoregulatory functions that acts by promoting MSC metabolic reprogramming towards glycolysis. Our findings identifies a novel mechanism underlying MSC immunoregulatory properties in which the high glucose consumption by the highly immunosuppressive MSC PPARβ/δ −/− might deprive T cell of glucose and thus impairing their phenotype and functions. Indeed, in lymphoid tissues T cells are primed prior to traffic to sites of inflammation where they will compete for resources with other cell types. Since glucose is pivotal for T cell proliferation and functions, its consumption by MSC and in particular by MSC PPARβ/δ −/− can metabolically restrict T cells, directly altering their function leading to immunosuppression, but this remains to be demonstrated.
While MSC expressing PPARβ/δ did not exhibit any preventive or therapeutic properties in an experimental model of arthritis, MSC deficient for PPARβ/δ exert potent beneficial effects. The inhibition of PPARβ/δ on MSC PPARβ/δ +/+ using a selective and irreversible pharmacological inhibitor generates therapeutic cells with both preventive and curative properties in experimental arthritis 2 . Similarly, in the present study we show that the pharmacologically-induced glycolytic switch of MSC PPARβ/δ +/+ significantly enhanced their immunoregulatory potential to an even greater extent than MSC PPARβ/δ −/− . Thus, these results suggest that the immunoregulatory potential of MSC involves other metabolic pathways than those related to PPARβ/δ. Indeed, recently it has been demonstrated that mitochondrial transfer from MSC to T cells represents a novel mechanism of immunosuppression involved in the inhibition of Th17 cell proliferation and function as well as in the generation of regulatory T cells to restrain inflammation 13,14 . Thus, the improved immunosuppressive activity of MSC PPARβ/δ −/− could be associated to their higher mitochondrial transfer capacity as compared to MSC PPARβ/δ +/+ . This hypothesis provides the basis for further investigations.
The enhancement of MSC immunoregulatory activity either using pro-inflammatory cytokines or inhibiting PPARβ/δ expression is associated with an increase glycolytic activity of MSC. This is in line with a recent study showing that IFNγ and hypoxia double priming of MSC enhances twice more their immunosuppressive properties than a single priming through a glycolytic switch of dual-primed MSC 15 . Therefore, this metabolic switch of MSC towards glycolysis leading to lactate production and the inhibition of T cells proliferation might be the key that permits, extends and guarantees MSC therapeutic effects regardless the window of injection.
These findings highlight the importance of such metabolic reprogramming for MSC immunoregulatory potential and pave the way for an enhanced MSC-based therapy for inflammatory and auto-immune disorders.

Material and methods
Bioethics. All methods were carried out in accordance with relevant guidelines and regulations for using animals. All the procedures presented in this work were approved by the Ethics Committee of Universidad de los Andes (Folio CEC Nº201630, Universidad de los Andes, Santiago, Chile).
Isolation and culture of MSC. Murine MSC were obtained from bone marrow of 129/Sv PPARβ/δdeficient mice (Ppard fl/fl sox2cre tg ) referred as PPARβ/δ −/− MSC and their wild-type littermates (Ppard fl/+ ) referred as PPARβ/δ +/+ MSC. Murine MSCs were characterized as previously described 16 . Murine MSC were cultured in Dulbecco's modified eagle medium (DMEM) high glucose (Corning, USA), and supplemented with 10% Fetal Bovine Serum, 1% Pen/Strep and 1% glutamine (Gibco, Thermo Fisher, USA). All the procedures presented in this work were approved by the Ethics Committee of Universidad de los Andes.
To assess immunosuppressive properties of murine MSCs, CD4 + T cells were cultured alone or in the presence of MSCs (control vs pretreated) at a cell ratio of 1 MSC per 10 lymphocytes in MLR media. After 72 h, proliferation and CD4 + T cell differentiation was quantified by flow cytometry.
Flow cytometry. Proliferation and differentiation of lymphocytes were quantified by flow cytometry. T cells were stimulated with phorbolmyristate acetate (PMA) (50 ng/ml; Merck, Germany) and ionomycin (1 mg/ ml; Merck, Germany), in the presence of brefeldin A (10 mg/ml; Sigma, Merck, Germany) for 4 h. Then surface staining was performed together with LIVE/DEAD Fixable near-IR stain (Invitrogen, Thermo Fisher, USA) in order to achieve the analysis only in live cells. Then, cells were fixed at 4 °C with the FoxP3 Cytofix/Cytoperm buffer (eBioscience, USA) and subsequently stained with intracellular fluorochrome-conjugated antibodies diluted in Perm/Wash buffer (eBioscience, USA) according to manufacturer's specifications.
Seahorse assay. Using the XF96 analyzer (Seahorse Biosciences, North Billerica, MA, USA), we measured the Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR), associated to oxidative phosphorylation and secretion of lactic acid as a metabolic product of glycolysis, respectively. Pre-stimulated murine MSC (20.000 cells/well) were plated on 96 well plates and analyzed according to manufacturer's recommended protocol. Three independent readings were performed after each sequential injection. Instrumental background was measured in separate control wells using the same conditions without biologic material.
Basal glycolytic rate was measured following the injection of glucose injection. Maximal glycolytic level was assessed following the injection of oligomycin and glycolytic capacity as the difference of oligomycin-induced ECAR and 2DG-induced ECAR. In XF media, OCR has been quantified under different conditions including basal conditions, in response to 1 μM oligomycin, 1 μM of FCCP (carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone) or 1 μM of antimycin A and rotenone (Sigma Aldrich).