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Succinate beyond metabolism in blood
investigations have shown that this is not always the case and that the impact of GPR91 depends on the cellular con- text. For example, GPR91 may indirectly protect mice from low-grade systemic inflammation under high-fat-diet con- ditions, since GPR91-deficient mice show progressive hyperglycemia and reduced insulin secretion.33 This was related to a function of GPR91 in white adipose tissue, a tissue where GPR91 is highly expressed.33
Monocytes that egress from the bone marrow (BM) seem to lack expression of GPR91, yet the ability to respond to activation and differentiation signals is influ- enced by extracellular succinate.12 Unlike immature mono- cytes, both naive dendritic cells and macrophages express high levels of GPR91, and its activation promotes inflam- mation.12 In vitro, succinate failed to influence TNF-α and IL-1b expression by dendritic cells. However, higher expression of these cytokines is achieved by addition of succinate in combination with TLR-3 and TLR-7 ligands compared to use of TLR agonists alone.12 Macrophages respond to extracellular inflammatory signals like LPS in similar ways (Figure 2A), with GPR91-mediated signal transduction yielding a strong pro-inflammatory pheno- type that results in increased production of IL-1b.2,34 Furthermore, LPS-stimulated macrophages release succi- nate into the cell culture medium.34 This effect seems to be independent of GPR91, as extracellular succinate is found more abundantly in cultures of GPR91 deficient macrophages than in corresponding wild-type (WT) cells.34 The underlying mechanism for this succinate transport still needs to be revealed. In this regard, future studies examin- ing the activity and expression of the SLC13 transporters in blood cells will be of great interest.
In addition, GPR91 seems to be required for dendritic cell activation and licensing in vivo. It leads dendritic cell migra- tion to lymph nodes, as this function is impaired in GPR91- deficient mice. However, after maturation, GPR91 expres- sion rapidly declines, making dendritic cells unresponsive to subsequent succinate stimulation after 24 hours of stim- ulation with LPS or the cytokine cocktail of TNF-α and IL- 1b.12 Conversely, activated macrophages become more responsive to succinate stimulation.12
In both human cell lines and monocyte-derived dendritic cells, succinate activates the extracellular signal-regulated kinases Erk1 and Erk2,12,30 which are downstream signaling components of TLR pathways. In macrophages, HIF-1α is one key mediator of their response to succinate.3,25 Accumulation of succinate in the cytosol results in compet- itive inhibition of PHD enzymes and subsequent stabiliza- tion of HIF-1α, even in the presence of normal oxygen lev- els (Figure 2A).3,6 In turn, HIF-1α activation contributes to IL-1b expression.3,25 Later, succinate oxidation in mitochon- dria proved central to macrophage pro-inflammatory phe- notype, given that competitive inhibition of SDH abro- gates IL-1b and HIF-1α expression.25 In accordance with the IL-1b findings previously described, HIF-1α stability is similar in macrophages derived from both GPR91-deficient and WT mice in presence of extracellular succinate alone, whereas GPR91 promotes pseudohypoxia upon LPS stim- ulation (Figure 2A).34 Recently, this mechanism has been related to pathogenesis in an experimental model of anti- gen-induced arthritis, where GPR91-deficient mice display reduced macrophage activation and IL-1b production.34
Surprisingly, the inflammatory impact of GPR91 defi- ciency varies in different myeloid cell subsets. Animal models of allergic contact dermatitis revealed increased
reactions in the absence of GPR91, and this was related to hyperactive mast cells in GPR91-deficient mice by means of increased TNF-α expression.35 This effect seems to be dependent on defective mast cell differentiation from BM precursors, as augmented mast cell responses could be recapitulated in WT mast cells differentiated in vitro in the absence of succinate.35 Unfortunately, no further efforts were made to explore the mechanisms involved.
Intriguingly, GPR91 may induce anti-inflammatory effects through non-cell-autonomous effects (Figure 2B). In an in vivo model of autoimmune encephalomyelitis, succi- nate produced by type I mononuclear phagocytes accumu- lates in the cerebrospinal fluid of the chronically inflamed central nervous system.20 Transplanted neural stem cells sense this extracellular succinate through GPR91. In vitro, GPR91 activation leads neural stem cells to secretion of prostaglandin E2, and upregulation of members of the SLC13 family of transporters (i.e. SLC13A3 and SLC13A5) that uptake and scavenge extracellular succinate. In vivo, succinate scavenging seems to be the main mediator of the anti-inflammatory effects of transplanted neural stem cells. As a result, the beneficial effects of this strategy include reduction of succinate levels in the cerebrospinal fluid, shifting from type 1 inflammatory mononuclear phago- cytes to anti-inflammatory cells, reduced post mortem tissue pathology, and improvement of behavioral defects.20 However, GPR91 is essential for these effects in vivo, as GPR91-deficient neural stem cells show reduced ability to protect from chronic neuroinflammation upon transplanta- tion.20 It remains to be seen how broadly applicable this succinate-mediated anti-inflammatory mechanism will be to additional regulatory cells and inflammatory disease systems. However, it is interesting to hypothesize that suc- cinate may play dual roles in inflammation both as an early driver, through cell-autonomous mechanisms, and as a late terminator by non-cell autonomous processes. Future stud- ies should clarify the validity of this exciting hypothesis.
In addition, given the contribution of GPR91 to tipping the inflammatory balance, studies are required to better understand how its modulation may provide a useful clin- ical tool for the control of inflammatory responses and maintenance of immune homeostasis. In this regard, recent development of GPR91-specific agonists, up to 20-fold more powerful compared to succinate,36 and antagonists,37 opens new avenues to explore the promising therapeutic value of GPR91 control with any accuracy.
New insights into hematopoiesis: what role does succinate play?
Early investigations into hematopoiesis have provided hints of a role for succinate signaling in blood stem cell function, i.e. blood formation or hematopoiesis. GPR91 is expressed in monocyte-derived cell subsets, as previously discussed, while it is absent in immature monocytes, and T cells and B cells,38 indicative of selective roles for GPR91 on different blood cell subsets. In contrast, others have found expression of GPR91 transcript in purified popula- tions of blood CD14+ monocytes and platelets, whereas its absence was confirmed in CD4+ T cells, CD8+ T cells, CD16+ granulocytes and CD19+ B cells.39 Unexpectedly, western blot analysis of protein expression showed pres- ence of GPR91 in all blood cells except granulocytes.39 Interestingly, human BM CD34+ progenitor cells have been
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