Lactate: an alternative pathway for the immunosuppressive properties of mesenchymal stem/stromal cells.

BACKGROUND: The metabolic reprogramming of mesenchymal stem/stromal cells (MSC) favoring glycolysis has recently emerged as a new approach to improve their immunotherapeutic abilities. This strategy is associated with greater lactate release, and interestingly, recent studies have proposed lactate as a functional suppressive molecule, changing the old paradigm of lactate as a waste product. Therefore, we evaluated the role of lactate as an alternative mediator of MSC immunosuppressive properties and its contribution to the enhanced immunoregulatory activity of glycolytic MSCs. MATERIALS AND METHODS: Murine CD4+ T cells from C57BL/6 male mice were differentiated into proinflammatory Th1 or Th17 cells and cultured with either L-lactate, MSCs pretreated or not with the glycolytic inductor, oligomycin, and MSCs pretreated or not with a chemical inhibitor of lactate dehydrogenase A (LDHA), galloflavin or LDH siRNA to prevent lactate production. Additionally, we validated our results using human umbilical cord-derived MSCs (UC-MSCs) in a murine model of delayed type 1 hypersensitivity (DTH). RESULTS: Our results showed that 50 mM of exogenous L-lactate inhibited the proliferation rate and phenotype of CD4+ T cell-derived Th1 or Th17 by 40% and 60%, respectively. Moreover, the suppressive activity of both glycolytic and basal MSCs was impaired when LDH activity was reduced. Likewise, in the DTH inflammation model, lactate production was required for MSC anti-inflammatory activity. This lactate dependent-immunosuppressive mechanism was confirmed in UC-MSCs through the inhibition of LDH, which significantly decreased their capacity to control proliferation of activated CD4+ and CD8+ human T cells by 30%. CONCLUSION: These findings identify a new MSC immunosuppressive pathway that is independent of the classical suppressive mechanism and demonstrated that the enhanced suppressive and therapeutic abilities of glycolytic MSCs depend at least in part on lactate production.

The ATP synthase inhibition induces an AMPK-dependent glycolytic switch of mesenchymal stem cells that enhances their immunotherapeutic potential.

Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro. Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo, MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy.

Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions.

Hypoxia-inducible factor 1 α (HIF1α), a regulator of metabolic change, is required for the survival and differentiation potential of mesenchymal stem/stromal cells (MSC). Its role in MSC immunoregulatory activity, however, has not been completely elucidated. In the present study, we evaluate the role of HIF1α on MSC immunosuppressive potential. We show that HIF1α silencing in MSC decreases their inhibitory potential on Th1 and Th17 cell generation and limits their capacity to generate regulatory T cells. This reduced immunosuppressive potential of MSC is associated with a metabolic switch from glycolysis to OXPHOS and a reduced capacity to express or produce some immunosuppressive mediators including Intercellular Adhesion Molecule (ICAM), IL-6, and nitric oxide (NO). Moreover, using the Delayed-Type Hypersensitivity murine model (DTH), we confirm, in vivo, the critical role of HIF1α on MSC immunosuppressive effect. Indeed, we show that HIF1α silencing impairs MSC capacity to reduce inflammation and inhibit the generation of pro-inflammatory T cells. This study reveals the pivotal role of HIF1α on MSC immunosuppressive activity through the regulation of their metabolic status and identifies HIF1α as a novel mediator of MSC immunotherapeutic potential.

Author Correction: Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

When a Little Bit More Makes the Difference: Expression Levels of GKRP Determines the Subcellular Localization of GK in Tanycytes.

Glucose homeostasis is performed by specialized cells types that detect and respond to changes in systemic glucose concentration. Hepatocytes, ß-cells and hypothalamic tanycytes are part of the glucosensor cell types, which express several proteins involved in the glucose sensing mechanism such as GLUT2, Glucokinase (GK) and Glucokinase regulatory protein (GKRP). GK catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6P), and its activity and subcellular localization are regulated by GKRP. In liver, when glucose concentration is low, GKRP binds to GK holding it in the nucleus, while the rise in glucose concentration induces a rapid export of GK from the nucleus to the cytoplasm. In contrast, hypothalamic tanycytes display inverse compartmentalization dynamic in response to glucose: a rise in the glucose concentration drives nuclear compartmentalization of GK. The underlying mechanism responsible for differential GK subcellular localization in tanycytes has not been described yet. However, it has been suggested that relative expression between GK and GKRP might play a role. To study the effects of GKRP expression levels in the subcellular localization of GK, we used insulinoma 832/13 cells and hypothalamic tanycytes to overexpress the tanycytic sequences of Gckr. By immunocytochemistry and Western blot analysis, we observed that overexpression of GKRP, independently of the cellular context, turns GK localization to a liver-like fashion, as GK is mainly localized in the nucleus in response to low glucose. Evaluating the expression levels of GKRP in relation to GK through RT-qPCR, suggest that excess of GKRP might influence the pattern of GK subcellular localization. In this sense, we propose that the low expression of GKRP (in relation to GK) observed in tanycytes is responsible, at least in part, for the compartmentalization pattern observed in this cell type. Since GKRP behaves as a GK inhibitor, the regulation of GKRP expression levels or activity in tanycytes could be used as a therapeutic target to regulate the glucosensing activity of these cells and consequently to regulate feeding behavior.

Adenovirus-mediated suppression of hypothalamic glucokinase affects feeding behavior.

Glucokinase (GK), the hexokinase involved in glucosensing in pancreatic ß-cells, is also expressed in arcuate nucleus (AN) neurons and hypothalamic tanycytes, the cells that surround the basal third ventricle (3V). Several lines of evidence suggest that tanycytes may be involved in the regulation of energy homeostasis. Tanycytes have extended cell processes that contact the feeding-regulating neurons in the AN, particularly, agouti-related protein (AgRP), neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC) neurons. In this study, we developed an adenovirus expressing GK shRNA to inhibit GK expression in vivo. When injected into the 3V of rats, this adenovirus preferentially transduced tanycytes. qRT-PCR and Western blot assays confirmed GK mRNA and protein levels were lower in GK knockdown animals compared to the controls. In response to an intracerebroventricular glucose injection, the mRNA levels of anorexigenic POMC and CART and orexigenic AgRP and NPY neuropeptides were altered in GK knockdown animals. Similarly, food intake, meal duration, frequency of eating events and the cumulative eating time were increased, whereas the intervals between meals were decreased in GK knockdown rats, suggesting a decrease in satiety. Thus, GK expression in the ventricular cells appears to play an important role in feeding behavior.

Hypothalamic Neurogenesis as an Adaptive Metabolic Mechanism.

In the adult brain, well-characterized neurogenic niches are located in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the hippocampus. In both regions, neural precursor cells (NPCs) share markers of embryonic radial glia and astroglial cells, and in vitro clonal expansion of these cells leads to neurosphere formation. It has also been more recently demonstrated that neurogenesis occurs in the adult hypothalamus, a brain structure that integrates peripheral signals to control energy balance and dietary intake. The NPCs of this region, termed tanycytes, are ependymal-glial cells, which comprise the walls of the infundibular recess of the third ventricle and contact the median eminence. Thus, tanycytes are in a privileged position to detect hormonal, nutritional and mitogenic signals. Recent studies reveal that in response to nutritional signals, tanycytes are capable of differentiating into orexigenic or anorexigenic neurons, suggesting that these cells are crucial for control of feeding behavior. In this review, we discuss evidence, which suggests that hypothalamic neurogenesis may act as an additional adaptive mechanism in order to respond to changes in diet.

Inhibition of hypothalamic MCT1 expression increases food intake and alters orexigenic and anorexigenic neuropeptide expression.

Hypothalamic glucosensing, which involves the detection of glucose concentration changes by brain cells and subsequent release of orexigenic or anorexigenic neuropeptides, is a crucial process that regulates feeding behavior. Arcuate nucleus (AN) neurons are classically thought to be responsible for hypothalamic glucosensing through a direct sensing mechanism; however, recent data has shown a metabolic interaction between tanycytes and AN neurons through lactate that may also be contributing to this process. Monocarboxylate transporter 1 (MCT1) is the main isoform expressed by tanycytes, which could facilitate lactate release to hypothalamic AN neurons. We hypothesize that MCT1 inhibition could alter the metabolic coupling between tanycytes and AN neurons, altering feeding behavior. To test this, we inhibited MCT1 expression using adenovirus-mediated transfection of a shRNA into the third ventricle, transducing ependymal wall cells and tanycytes. Neuropeptide expression and feeding behavior were measured in MCT1-inhibited animals after intracerebroventricular glucose administration following a fasting period. Results showed a loss in glucose regulation of orexigenic neuropeptides and an abnormal expression of anorexigenic neuropeptides in response to fasting. This was accompanied by an increase in food intake and in body weight gain. Taken together, these results indicate that MCT1 expression in tanycytes plays a role in feeding behavior regulation.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology.

Stem cells are considered a valuable cellular resource for tissue replacement therapies in most brain disorders. Stem cells have the ability to self-replicate and differentiate into numerous cell types, including neurons, oligodendrocytes and astrocytes. As a result, stem cells have been considered the "holy grail" of modern medical neuroscience. Despite their tremendous therapeutic potential, little is known about the mechanisms that regulate their differentiation. In this review, we analyze stem cells in embryonic and adult brains, and illustrate the differentiation pathways that give origin to most brain cells. We also evaluate the emergent role of the well known anti-oxidant, vitamin C, in stem cell differentiation. We believe that a complete understanding of all molecular players, including vitamin C, in stem cell differentiation will positively impact on the use of stem cell transplantation for neurodegenerative diseases.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology

Stem cells are considered a valuable cellular resource for tissue replacement therapies in most brain disorders. Stem cells have the ability to self-replicate and differentiate into numerous cell types, including neurons, oligodendrocytes and astrocytes. As a result, stem cells have been considered the "holy grail" of modern medical neuroscience. Despite their tremendous therapeutic potential, little is known about the mechanisms that regulate their differentiation. In this review, we analyze stem cells in embryonic and adult brains, and illustrate the differentiation pathways that give origin to most brain cells. We also evaluate the emergent role of the well known anti-oxidant, vitamin C, in stem cell differentiation. We believe that a complete understanding of all molecular players, including vitamin C, in stem cell differentiation will positively impact on the use of stem cell transplantation for neurodegenerative diseases.