How to reprogram microglia toward beneficial functions

Abstract Microglia, brain cells of nonneural origin, orchestrate the inflammatory response to diverse insults, including hypoxia/ischemia or maternal/fetal infection in the perinatal brain. Experimental studies have demonstrated the capacity of microglia to recognize pathogens or damaged cells activating a cytotoxic response that can exacerbate brain damage. However, microglia display an enormous plasticity in their responses to injury and may also promote resolution stages of inflammation and tissue regeneration. Despite the critical role of microglia in brain pathologies, the cellular mechanisms that govern the diverse phenotypes of microglia are just beginning to be defined. Here we review emerging strategies to drive microglia toward beneficial functions, selectively reporting the studies which provide insights into molecular mechanisms underlying the phenotypic switch. A variety of approaches have been proposed which rely on microglia treatment with pharmacological agents, cytokines, lipid messengers, or microRNAs, as well on nutritional approaches or therapies with immunomodulatory cells. Analysis of the molecular mechanisms relevant for microglia reprogramming toward pro‐regenerative functions points to a central role of energy metabolism in shaping microglial functions. Manipulation of metabolic pathways may thus provide new therapeutic opportunities to prevent the deleterious effects of inflammatory microglia and to control excessive inflammation in brain disorders.

Contributions of microglia to physiological brain function are underlined by functional deficits in neuronal connectivity and neurologic disorders in mice harboring dysfunctional microglia (Prinz & Priller, 2014), mutant for complement or CX 3 CR1 chemokine receptors (Paolicelli et al., 2011;Schafer et al., 2012).
Consolidated evidence indicates that the pro-inflammatory cytokine interleukin (IL)-1β, the toll-like-receptor (TLR)-4 agonist lipopolysaccharide (LPS), the TLR-3 agonist Poly I : C, and tumor necrosis factor-α (TNFα) are potent inducers of detrimental microglial phenotype in vitro. Functionally, pro-inflammatory microglia are characterized by microbicidal, antigen-presenting and immunepotentiating abilities, through synthesis of nitric oxide (NO), chemokines, and inflammatory cytokines (Amici, Dong, & Guerau-de-Arellano, 2017). Conversely, the anti-inflammatory cytokines IL-4 and IL-13, immune complexes, IL-1R ligands, IL-10, TGF-β, and glucocorticoids are typical agents which drive microglia toward pro-regenerative, immunomodulatory or deactivated phenotypes. Pro-regenerative microglia secrete anti-inflammatory mediators and express Arg1 instead of inducible NO synthase (iNOS), switching arginine metabolism from production of NO to ornithine and polyamines for collagen and extracellular matrix synthesis. However, detrimental and proregenerative microglia polarization in vitro is highly artificial and does not reflect the real in vivo situation where microglia activation follows a wide spectrum of phenotypes, which may be driven by diverse stimuli, as described for macrophages (Xue et al., 2014).
Using transcriptomic and proteomic approaches Gautier et al., 2012;Hickman et al., 2013;Krasemann et al., 2017) the molecular signature of microglia in vivo has begun to be defined under physiological and pathological conditions, including amyotrophic lateral sclerosis (ALS) (Chiu et al., 2013), multiple sclerosis (MS), Alzheimer's disease (AD) (Krasemann et al., 2017) or perinatal brain injury (Krishnan et al., 2017). However, our understanding of the molecular mechanisms that govern transition of microglia from detrimental to proregenerative phenotypes both in vitro and in vivo remains limited. Much more is known about cellular pathways controlling polarization in macrophages, the peripheral microglia counterparts, where it is increasingly clear that metabolic activities co-define their effector state. Specifically, in peripheral macrophages, mitochondrial oxidation is a determinant of pro-regenerative polarization. Blocking oxidative metabolism abrogates anti-inflammatory macrophage function without affecting the proinflammatory state (Ghesquiere, Wong, Kuchnio, & Carmeliet, 2014). For a detailed description of both classical and emerging pathways, including metabolic pathways, relevant for macrophage phenotypes we refer the readers to recent excellent reviews (Amici et al., 2017;Diskin & Palsson-McDermott, 2018).
Despite the genetic profile of microglia differs profoundly from that of macrophages (Yamasaki et al., 2014), recent evidence indicates that energy production in mitochondria may be a determinant of proregenerative/resolving differentiation also in microglia. In fact, reduced mitochondrial oxygen consumption, reduced electron transport, and mitochondrial ATP synthesis caused by deficiency in Clock (Clk)1, a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (Hekimi, 2013;Lapointe & Hekimi, 2008), has been linked to the acquisition of pro-inflammatory phenotype by microglia. Importantly, the inflammatory state of Clk1 deficient microglia relies on aerobic glycolysis, as it is abolished when glycolytic metabolism is inhibited by knocking down hexokinase 2 (HK2), a key rate-limiting enzyme for phosphorylating the glucose (J. J. Gu et al., 2018). However, whether metabolic pathways may take central stage in inducing a repair-supportive phenotype is not yet established.
In this review, we summarize emerging strategies able to redirect microglia from detrimental to beneficial functions. As we learn more about molecular mechanisms controlling microglia effector functions, a central role of cell metabolism emerges, opening novel approaches to target microglia therapeutically.

| RECEPTOR AND CHANNELS PROMOTING ANTI-INFLAMMATORY MICROGLIA FUNCTION
Multiple brain's signaling substances converge on microglia to actively maintain or alter their functional state through a variety of neurotransmitter receptors and channels (Domercq, Vazquez-Villoldo, & Matute, 2013). Here we focus on receptors and channels which are involved in microglia reprogramming toward anti-inflammatory phenotype with a known mechanism of action.

| Histamine and other neurotransmitter receptors
Histamine has a well-established role as neuron-to-glia alarm signal in the brain and microglia constitutively express all four histamine receptors (H1R, H2R, H3R, and H4R) (W. Hu & Chen, 2017). Apolloni et al. recently demonstrated that histamine counteracts proinflammatory microglia phenotype in the SOD1-G93A mouse model of ALS. They showed that histamine not only reduces expression of both NADPH oxidase 2 (NOX2), a super-oxide generating enzyme which forms reactive oxygen species (ROS), and of NF-κB, the classical transcription factor activated in pro-inflammatory microglia, but also increases the anti-inflammatory genes (IL-10, ARG1, P2Y12, CD163, and CD206) acting through H1R and H4R. Importantly, histamine exerts beneficial action only in inflammatory SOD1-G93A microglia, which are characterized by decreased H1R levels, while elicits a pro-inflammatory effect in nontransgenic cells. In support to these findings a recent work showed that histamine deficiency reduces the ramified/surveilling morphology of microglia and the production of anti-inflammatory factors in the striatum of knockout mice (Frick, Rapanelli, Abbasi, Ohtsu, & Pittenger, 2016). With respect to the signal transduction mechanisms involved downstream of H1R and H4R activation in ALS microglia, Apolloni, and colleagues demonstrated a persistent ERK1/2 phosphorylation, which accounts for downregulation of p-NF-κB ( Figure 1).

| Neuropeptide/growth factor receptors
Neuropeptides and neurotrophins acting through specific receptors are able to modulate microglial response by inhibiting the release of inflammatory mediators while favoring development of an alternative activation program (Carniglia et al., 2017;Rizzi et al., 2018). Specifically, the melanocortins α-MSH and NDP-MSH, both exerting their actions through G protein-coupled receptors (GPCRs), were found to induce expression of the metabolic enzyme Arg1 in microglia in the retina (Kawanaka & Taylor, 2011) and in rat primary culture (Carniglia, Durand, Caruso, & Lasaga, 2013). Arg1 induction was accompanied by enhanced expression of the nuclear receptor peroxisome proliferatoractivated receptor gamma (PPAR-γ) (Szanto et al., 2010), and by enhanced IL-10 release (Figure 1), known markers of pro-regenerative microglia (see below).
The classical neurotrophin NGF, through activation of Trk-A receptors was recently shown to steer microglia toward a neuroprotective and anti-inflammatory phenotype (Rizzi et al., 2018) (Figure 1), being particularly effective in reverting the pro-inflammatory state of FIGURE 1 Schematic representation of receptors and signaling pathways mainly contributing to anti-inflammatory polarization of microglia. Stimulation of histamine receptors H1R and H4R induces persistent ERK1/2 phosphorylation, that leads to pNF-κB downregulation and reduces the expression of NADPH oxidase 2 (NOX2), dampening pro-inflammatory responses. Similarly, the endocannabinoid type 2 receptor (CB2) induces sustained ERK1/2 phosphorylation causing pNF-κB downregulation. GABA receptors are also involved in reducing microglia reactivity through the activation of NOX4. The nicotinic α7 receptor decreases pNF-κB and its p65 subunit and inhibits p38-MAPK. Blocking the voltage independent KCa3.1 channels and the voltage-gated Kv1.3 also results in inhibition of p38-MAPK pathway and of iNOS and COX expression. IL-10, upon binding of its receptor, suppresses pro-inflammatory cytokine production through induction of suppressor of cytokine signalling (SOCS)1 and (SOCS)3 proteins. These proteins, in turn, inhibit the cytokine-activated Janus kinase (JAK)/ STAT-1 signaling pro-inflammatory pathway. The transcription factor signal transducer and activator of transcription (STAT) 3 is instead induced by the activation of sphingosine-1-phosphate (S1P) receptors through its ligand FTY720, resulting in attenuation of microglia-mediated neuroinflammation. NGF, through activation of Trk-A receptor, reduces cytokine/chemokine secretion, microglia motility, phagocytosis, and degradative pathways. The activation of the tyrosine kinase AXL receptor by pro-resolving lipid mediators suppresses the pro-inflammatory microglial phenotype by dampening type I interferon (IFN) signaling. The melanocortins α-MSH and NDP-MSH, through G protein-coupled receptors, enhance PPAR-γ, and Arg1 expression and promote IL-10 release. Upon stimulation with IL-4, IL-4 receptor (IL-4R) induces activation of STAT-6, with consequent increase in Arg1, mannose receptor CD206 and PPAR-γ expression and with induction (and release) of anti-inflammatory cytokines like IL-4, IL-10, IL-13. For specific references see text [Color figure can be viewed at wileyonlinelibrary.com] microglia induced by β-amyloid. Importantly, the receptor signaling activated by NGF in primary culture and ex vivo not only regulates a number of microglia activities, such as cytokine/chemokine secretion, motility, phagocytosis, and degradative pathways but also controls microglia-neuron interaction, protecting neurons against Aβ-induced spine alterations and synaptic dysfunction.

| Receptors of endogenous immune signals
IL-4 and IL-13 are endogenous immune signals which polarize microglia toward neuron protective, pro-regenerative phenotype. Despite microglia responsiveness to these cytokines have been studied mostly in culture, microglia have been shown to respond to IL-4 administration with the expression of genes typical of alternative activation also in vivo (Pepe, Calderazzi, De Maglie, Villa, & Vegeto, 2014). The phenotypic features of IL-4-treated microglia are controlled by a network of molecular pathways that impact gene expression and cellular metabolism ( Figure 1). Among them, the transcription factor STAT 6 accumulates in response to IL-4 and, along with its downstream effector PPAR-γ, has a central role in the regulation of transcription of anti-inflammatory and pro-resolving genes ( Figure 1) (Lan, Han, Li, Yang, & Wang, 2017). The specific role of PPAR-γ in driving positive metabolic and immune microglial functions will be discussed in a subsequent section. Additionally, IL-4 induces expression of CD36, a receptor of lipoproteins, whose uptake and metabolism may support fatty-acid oxidation, contributing to metabolic microglia reprogramming ( Figure 2); (S. C. Huang et al., 2014).
IL-10 drives microglia toward an alternative activated phenotype, which mainly participates in phagocytosis and removal of tissue debris (Chhor et al., 2013). IL-10 suppresses pro-inflammatory cytokine production from microglia via induction of suppressor of cytokine signaling (SOCS)1 and (SOCS)3 proteins. SOCS1,3 act in a negative feedback loop, inhibiting the cytokine-activated Janus kinase (JAK)/ STAT-1 signaling pathway that are activated upon binding of proinflammatory cytokines to their specific receptors ( Figure 1).
Both IL-4/IL-13 and IL-10 represent the challenging opportunities to convert microglia toward protective functions.

| Triggering receptor expressed on myeloid cells-2
Initial studies showed that protective microglial phenotype is induced by signals derived from apoptotic cells that activate the triggering receptor expressed on myeloid cells-2 (TREM-2). However, subsequent evidence (Krasemann et al., 2017) revealed that TREM-2 activation by apolipoprotein E (APOE), the major risk factor for AD, identified as a TREM2 ligand (Atagi et al., 2015;Bailey, DeVaux, & Farzan, 2015), drives a neurodegenerative phenotype in microglia, characterized by suppression of major transcription factors of homeostatic microglia (PU.1, MEF2a, SMAD3, and TGF-β signaling) and overlapping to some extent the classical pro-inflammatory microglia phenotype, featured by high expression of the pro-inflammatory miRNA miR-155 (Butovsky et al., 2012). Thus, targeting of the TREM2-APOE pathway might serve as a way to restore homeostatic microglia.
On the contrary, other studies revealed that TREM-2 deficiency, by limiting microglia activation and phagocytosis, facilitates Aβ plaque buildup, and injury of adjacent neurons in mouse model of AD (Jay, von Saucken, & Landreth, 2017;Ulrich et al., 2014; Y. Wang et al., 2016;Yuan et al., 2016) and results in impaired elimination of supernumerary synapses during brain development (Filipello et al., 2018). Interestingly the results coming from studies on TREM2-deficient microglia showed reduced mitochondrial mass and increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) (Ulland et al., 2017), a key regulator of energy metabolism that is activated in response of low glucose and inhibits a shift in the cellular metabolism from oxidative phosphorylation to glycolysis (Hardie, 2011a(Hardie, , 2011b. Importantly, these metabolic changes were reverted by supporting ATP synthesis, suggesting that microglia dysfunctions caused by TREM-2 deficiency depend on energetic compromise (Ghosh, Castillo, Frias, & Swanson, 2017).

| Potassium channels
The expression of Ca2 + -dependent K + channel (KCa3.1) and K + channels K v 1.3 varies during microglia activation and can be a determinant for microglia polarization (Nguyen et al., 2017). KCa3.1 channels are voltage independent and only require a small increase in intracellular calcium to open. By maintaining a negative membrane potential through K + efflux, the channels provide the driving force for prolong calcium influx which typically occurs during microglia respiratory burst (oxygen consumption by mitochondria) (Khanna, Roy, Zhu, & Schlichter, 2001), migration (Schilling, Stock, Schwab, & Eder, 2004), proliferation (Maezawa & Jin, 2010), and NO production (Maezawa & Jin, 2010), features of classical pro-inflammatory microglia. Also the voltage-gated potassium channel K v 1.3 through K + efflux helps to maintain a negative membrane potential (Maezawa et al., 2018)

| MOLECULAR MECHANISMS LINKING RECEPTOR/CHANNEL ACTIVATION TO THE BIOENERGETIC STATE OF MICROGLIA
Pharmacological analysis of microglia receptor/channel signaling cascades points to p-NF-κB, p38 -MAPK, iNOS, COX-2, and NOX2 as key inflammatory intracellular pathways downregulated by agents which dampen microglia activation under pathological stimuli. Intriguingly, emerging evidence indicates that at least p-NF-κB, iNOS and NOX2 activity are influenced by the bioenergetics state of microglia, as clearly described in a recent review (Ghosh et al., 2017) (Figure 3).
In fact, NADPH generated via glucose metabolism through the penthose phosphate pathway (PPP) is requisite cofactor for the pro- While SFA mainly act as pro-inflammatory stimuli (Z. Wang et al., 2012;Gao et al., 2014), N-3 PUFAs and their metabolites have emerged as anti-inflammatory modulators of microglial functions (Laye, Nadjar, Joffre, & Bazinet, 2018). Independent of the inflammatory challenge applied (e.g., hypoxia, interferon-y, amyloid-β), cultured microglia consistently show decreased production of pro-inflammatory factors (De Smedt-Peyrusse et al., 2008), decreased COX2 and iNOS activity (Pettit, Varsanyi, Tadros, & Vassiliou, 2013;Zendedel et al., 2015) while exibit typical features of anti-inflammatory microglia, like CD206 surface expression and autophagy when treated with n-3 PUFAs (Chhor et al., 2013;Inoue et al., 2017). Many in vivo studies confirm that N-3 PUFA supplementation reduces detrimental microglia function (summarized in (Laye et al., 2018). Although microglia express a wide range of lipidsensitive receptors and lipid metabolism-related genes (Mauerer, Walczak, & Langmann, 2009;Mecha et al., 2015), the molecular link between bioactive fatty acids and their effects in microglia has not been completely clarified yet. Emerging evidence suggests that PUFAs can drive a protective phenotype through the activation of CB2, which is among microglial receptors reprogramming microglia toward beneficial function (Guida et al., 2017;Mecha et al., 2015;Nunez et al., 2004)  playing crucial roles in both the brain (Cao, Li, & Chan, 2016;Schratt, 2009) and the immune system (Xiao & Rajewsky, 2009). Due to their ability to simultaneously modulate the fate of different genes, these molecules are particularly well suited to act as key regulators during neural and immune cell differentiation and activation.
The impact of miRNAs on microglial phenotype has been recently revealed by generating mice lacking microglial Dicer, an enzyme necessary for miRNA maturation. Dicer ablation resulted in hyperresponsiveness of adult microglia to stimuli (peripheral endotoxin), indicating that miRNAs overall limit microglia responses to challenge (Varol et al., 2017) Table 1).
MiR-124 was one of the first miRNA to be linked to proregenerative microglial phenotype (Y. Sun, Luo, Guo, Su, & Liu, 2015).  Metformin promotes oxidative phosphorylation through AMPK activation. Aldose reductase (AR) converts glucose into sorbitol using NADPH as a cofactor. Sorbitol, in turn, activates the phospholipase C/protein kinase C signaling pathway, resulting in downstream activation of pro-inflammatory NF-κB. By inhibiting AR, the small-molecule FMHM, Sorbinil, Zopolrestat, and Fidarestat, limit inflammatory microglia response. Furthermore, Fidarestat induces CREB phosphorylation to increase Arg1. SIRT1 activators, such as Resveratrol, inhibit the pro-inflammatory NF-kB pathway and activate fatty acid metabolism. The scheme also depicts how NF-kB, iNOS, and NOX activities are influenced by the bioenergetics state of microglia. NADPH generated via glucose metabolism through PPP is a cofactor for the production of NO, by iNOS, and of superoxide, by NOX. NADH generated through glycolysis promotes dimerization of C-terminal binding protein (CtBP), preventing the corepressor activity of the monomeric form on the p300 acetylase, a NK-kB activator, with consequent activation of NF-kB transcriptional activity [Color figure can be viewed at wileyonlinelibrary.com] of CCAAT/enhancer-binding protein (C/EBP)-α (P. Zhang et al., 2004), one of the major transcription factor that drives pro-inflammatory microglia polarization (A. Yu et al., 2017). Moreover, miR-124 suppresses p38-MAPK, that also has central role in coordinating inflammatory microglia responses (Lawson et al., 2013). Of relevance for metabolic microglia reprogramming, miR-124 (Qiu et al., 2015) directly controls the bioenergetic state of the cells. Specifically, it reduces lactate production and the PPP, that supports NO and superoxide production, by targeting mRNAs encoding phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and ribose-5-phosphate isomerase-A (RPIA) (Qiu et al., 2015); (Table 1).

MiR200b reduces iNOS expression and NO production and limits
the migratory potential of reactive microglia. Suppression of the glycolytic enzyme lactate dehydrogenase A (LDHA) and aerobic glycolysis likely underlies the miRNA action (S. Hu et al., 2016); (Table 1).
miR-181a targets the inflammatory genes IL-1α , TNF  and the transcriptional factor C/EBPα but also suppresses Kruppel-like factor 6 (KLF6) (Bi et al., 2016), a PPAR-γ inhibitor  Table 1). let 7b belongs to the let 7 family of miRNAs, that dampens production and release of inflammatory molecules from microglia (Table 1). p21-activated kinase 1 (PAK) and C/EBP have been disclosed as downstream inflammatory targets of let 7 miRNAs . However, let 7 miRNAs also serve as important mediators in energetic metabolism (Chu et al., 2015). They reduce glucose uptake and lactate production and prompt the use of fatty acid as energy source. However, further work is required to define how let 7b impacts microglia metabolism.
Finally, miR-125-a, known to be involved in macrophage differential activation through downregulation of TNF-α-induced protein 3 (TNFAIP3) (Graff, Dickson, Clay, McCaffrey, & Wilson, 2012) and of the inflammatory transcription factor Kruppel-like factor 13 (KLF13) , has been recently found to be upregulated in reactive microglia and in extracellular vesicles (EVs) thereof (Prada et al., 2018). Few glycolytic enzymes, including enolase-1 (ENO-1), HK2 (F. Jin et al., 2017;C. M. Sun, Wu, Zhang, Shi, & Chen, 2017) and phosphofructokinase (PFK1) are miR-125-a validated targets (Table 1) properties, their self-renewal, their multipotency and their minimally invasive accessibility that make them an attractive tool for regenerative medicine. A large part of MSC positive effects across models of brain injury is due to their secretion products which create a favorable regenerative microenvironment. MSC secretome includes soluble factors, released in a naked form, and molecules (proteins, lipids and genetic material) released in association with EVs. EVs, including exosomes and microvesicles, are fundamental mediators of intercellular communication. They shuttle bioactive molecules from one cell to another, causing the exchange of information and reprogramming of the recipient cells (Turola, Furlan, Bianco, Matteoli, & Verderio, 2012).
Previous findings from our group and others have shown that through their secretome MSCs re-direct microglia from detrimental toward pro-regenerative functions (Giunti et al., 2012;Zanier et al., 2014). By investigating post-traumatic changes of microglia in mice that had received MSCs through intracerebroventricular administration, we observed an increased number of Ym1(+) protective microglia, associated with early and persistent recovery of neurological functions. MSCs, indirectly co-cultured with microglia in vitro, directly counteract the pro-inflammatory response of cells activated with inflammatory cytokines and induce persistent pro-regenerative traits (Giunti et al., 2012;Zanier et al., 2014). More recent studies demonstrated that MSCs-derived EVs may be sufficient to significantly dampen microglia response and improve functional recovery upon intravenous administration in traumatic brain or spinal cord injuries (L. X. Zhang, Yin, Zhang, & Deng, 2015;J. W. Kim, Ha, Molon, & Kim, 2013;Ruppert et al., 2018) as well as in a mouse model of AD. Despite these findings offer a hopeful opportunity to advance novel cell-free therapeutic strategies that might prevail over the risks associated with the use of cells, a still unresolved issue is the molecular mechanism whereby the infused EVs reduce inflammation and rescue cognitive impairments in rodent models of neuroinflammatory diseases. Indeed, EVs contain many active components and it is difficult to identify those with significant effects on microglia and other brain cells (D. K. Kim et al., 2016).
An alternative source of stem cells able to redirect brain inflammatory myeloid cells (microglia and monocyte-derived cells) are neural precursor cells (NPCs) (De Feo et al., 2017). Martino and coworkers recently showed that intrathecal transplantation of NPCs in mice with EAE restrains microglia activation and impairs the accumulation of inflammatory monocyte-derived cells, favoring their switch toward an anti-inflammatory phenotype. Transcriptome analysis, combined to the use of TGF-β2 knockout neural progenitors, identified a secretory protein, TGF-β2, as the primary factor responsible for NPC immunomodulation and disease amelioration. TGF-β2 is a well-known inducer of Arg1, the key metabolic enzyme which distinguishes alternatively activated myeloid cells (Gordon, 2003). Consistent with the presence of TGF-β responsive elements in the flanking region of the metabolic gene (Peranzoni et al., 2007), Martino and colleagues found by transcriptome analysis an early upregulation of Arg1 in myeloid cells exposed in vitro to NPCs, followed by subsequent induction of antiinflammatory molecules (e.g., Hspa1a, IL-1 decoy receptor that blocks IL-1 [Il1r2] and the NF-κB inhibitor Ascc1). Moreover, a profound rearrangement of metabolic and synthetic processes was observed in vivo in myeloid cells FACS-sorted from EAE mice intrathecally injected with NPCs. In particular, oxidative phosphorylation was the most prominent pathway altered under NPC treatment.
Very recently a significant progress has been made toward understanding the molecular mechanism that underpins the capacity of NPCs to drive pro-inflammatory microglia and infiltrating macrophages toward beneficial functions in EAE (Peruzzotti-Jametti et al., 2018). Pluchino and colleagues clearly showed that NPCs counteract the metabolic changes of pro-inflammatory cells and reprogram them toward an oxidative phosphorylation anti-inflammatory phenotype.
Indeed NPCs restore basal oxygen consumption rate and extracellular acidification rate in pro-inflammatory macrophages. Furthermore, by performing an untargeted mass spectrometry analysis of the extracellular and intracellular metabolite content of macrophages, they identified the metabolite succinate as the main target of the proregenerative NPC action. Intracellular succinate is known to act as a key pro-inflammatory signal in phagocytes, by enhancing IL-1β generation (Littlewood-Evans et al., 2016;Tannahill et al., 2013) and favouring mitochondrial production of ROS over ATP synthesis (E. L. Mills et al., 2016). Extracellular succinate also exhibits pro-inflammatory activity though interaction with its specific receptor SUCNR1 and it is emerging as a biomarker of metabolic distress and inflammatory activity (Littlewood-Evans et al., 2016;E. Mills & O'Neill, 2014 ( Figure 3). DCA has been shown to shifts glucose metabolism toward the TCA cycle/oxidative phosphorylation in T cells in mice affected by EAE (Gerriets et al., 2015) and to promote a pro-regenerative microglia/macrophages phenotype in vitro and in a peripheral inflammation model (Kato et al., 2007). However, whether DCA shifts microglial phenotype by promoting oxidative phosphorylation has not been proven yet.
However, this intriguing hypothesis still remains to be validated.
Metformin is a widely used drug to treat diabetes and a wellknown activator of AMPK, already mentioned to be activated in response of low glucose and to promote oxidative phosphorylation (Hardie, 2011a(Hardie, , 2011b; (Figure 3) Metformin has been recently shown to promote pro-regenerative microglial phenotype and significantly ameliorate neurobehavioral function after stroke (Q. Jin et al., 2014) and traumatic brain injury (Tao et al., 2018), suggesting that it may be used to enhance pro-regenerative microglial function by promoting oxidative metabolism.
PPAR-γ activation is an alternative way to push oxidative metabolism in microglia, as already mentioned. PPARs-γ can be activated with thiazolidinediones (e.g., pioglitazone or rosiglitazone) a class of antidiabetic drugs proven to be effective in reducing the extent of neuroinflammation in different models of brain diseases and to attenuate neurodegeneration in patients with mild-to-moderate dementia (Heneka, Fink, & Doblhammer, 2015).
Protective microglial function may be also promoted by pharmacological inhibitors of aldose reductase (AR), a metabolic enzyme implicated in inflammation-related diseases, that converts glucose into sorbitol using NADPH as a cofactor (Petrash, 2004); (Figure 3). The  Importantly some AR inhibitors have gone through Phase-3 clinical studies (Pandey, Srivastava, & Ramana, 2012) and Tolrestat has been approved to prevent diabetic complications (Rosa & Dias, 2014).

| CONCLUSIONS
A large variety of receptors are present in microglia, whose activation by immune signals, neurotransmitters, neuropeptides, hormones, lipid messengers, metabolites, and even alimentary components drives microglia toward beneficial functions. Their signaling pathways converge on common anti-inflammatory and pro-regenerative molecular programs. While the nuclear receptor PPAR-γ, that promotes glucose and fatty acid metabolism in mitochondria, is consistently induced, the inflammatory pathways dependent on NF-κB, p38-MAPK, iNOS, COX-2 and NOX2 are commonly inhibited. Importantly, iNOS, NOX2 and NF-κB activities are strictly connected to glucose metabolism through glycolysis and the PPP, implicating that a change in microglia bioenergetics would shape the microglia phenotype. Specifically switching from aerobic glycolysis to oxidative phosphorylation would dampen microglia pro-inflammatory activity.
A set of miRNAs with anti-inflammatory action has been identified. Among their validated target genes, we here highlighted many metabolic enzymes involved in glucose uptake, glycolysis, lactate production, pentose phosphate shunt, suggesting a connection between miRNA-induced transition toward protective phenotype and changes in energy metabolism.
Finally stem cells, (at least NPCs), which redirect microglia toward protective function, induce a profound rearrangement of metabolic and synthetic microglial processes, further supporting the key involvement of bioenergetics in microglia reprogramming toward an alternative activation state.
From this scenario, targeting cell metabolism emerges as a new potential therapeutic approach to redirect microglia from detrimental to pro-regenerative phenotype. In this respect, two drugs shifting glucose metabolism toward the TCA cycle/oxidative phosphorylation (DCA and DMF) have already been approved for the treatment of MS, the prototypical neuroinflammatory disease characterized by microglia inflammation and drugs approved to treat metabolic disorders (metformin and thiazolidinediones) have been proven to dampen excessive microglial activation in brain diseases. Francesco Bifari (Università di Milano) for useful discussion.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS
All authors: conception of idea, review of the literature, manuscript writing and editing.