Th eJournal of Brief Reviews Immunology Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization Eleni Vergadi,*,† Eleftheria Ieronymaki,* Konstantina Lyroni,* Katerina Vaporidi,† and Christos Tsatsanis* Macrophages become activated initiating innate immune The different stages of macrophage activation are broadly responses. Depending on the signals, macrophages obtain described as M1 and M2 polarization, both consisting of an an array of activation phenotypes, described by the broad array of states that depend on converging signals from in- termsofM1orM2phenotype.ThePI3K/Akt/mTOR flammatory stimuli and the cellular environment (1). M1 and pathway mediates signals from multiple receptors includ- M2 status can be defined according to the activation stimulus. ing insulin receptors, pathogen-associated molecular pat- The M1 spectrum includes M(IFN-g), M(LPS+IFN-g), and tern receptors, cytokine receptors, adipokine receptors, M(LPS) depending on whether the signal is IFN-g or LPS, and hormones. As a result, the Akt pathway converges whereas the M2 spectrum includes M(IL-4), M(Ic), M(IL- inflammatory and metabolic signals to regulate macro- 10), M(GC+TGF-b), and M(GC) when the stimulus is phage responses modulating their activation phenotype. from IL-4, immune complexes (Ic), or glucocorticoids (GC) b Akt is a family of three serine-threonine kinases, Akt1, in combination or not with TGF- (1). These phenotypes describe the different states of macrophages and their func- Akt2, and Akt3. Generation of mice lacking individual tions, which range from elimination of diverse pathogens to Akt, PI3K, or mTOR isoforms and utilization of RNA phagocytosis of apoptotic cells and tissue remodeling. Ac- interference technology have revealed that Akt signaling cordingly, M(IFN-g) macrophages are associated with Th1- pathway components have distinct and isoform-specific related pathologies where IFN-g is abundant, and M(LPS) as roles in macrophage biology and inflammatory disease well as M(LPS+IFN-g) macrophages are found in Gram- regulation, by controlling inflammatory cytokines, miR- negative bacterial infections before and after production of NAs, and functions including phagocytosis, autophagy, IFN-g from Th1 cells. The M2 spectrum of macrophages is and cell metabolism. Herein, we review the current broader and includes macrophages that contribute to parasitic knowledge on the role of the Akt signaling pathway in infections or other pathologies with abundant IL-4 such as macrophages, focusing on M1/M2 polarization and asthma (2, 3), activated by immunocomplexes, termed M(Ic), highlighting Akt isoform–specific functions. The Jour- such as those found in rheumatoid arthritis (4) and sclero- nal of Immunology, 2017, 198: 1006–1014. derma (5) patients, activated by IL-10, termed M(IL-10), found in the tumor stroma or in inflamed tissues (6, 7), or acrophages respond to pathogens and other nox- induced by abundant glucocorticoids, termed M(GC), such as ious stimuli, such as apoptotic or necroptotic cell late stages of sepsis (8, 9) or in cancer (9, 10). Macrophage M debris, and initiate inflammatory responses. Acti- activation is a tightly regulated phenomenon, determined by vation of macrophages is mediated by signaling cascades several signaling cascades elicited by receptor stimulation or downstream of TLR and cytokine receptors. Upon activation, intracellular regulatory proteins. Many of the aforementioned transcriptional and epigenetic changes lead to production of activation signals use common and distinct signaling cascades, proinflammatory cytokines and chemokines, migration, and some of which are used for defining macrophage phenotype. elimination of the insult. At later stages of inflammation, Among the different signaling cascades, the PI3K/Akt path- macrophages contribute to the resolution of inflammation, way and its downstream targets have emerged as central reg- and prohibit prolonged inflammatory responses that may lead ulators of activation phenotype in macrophages. to tissue injury. The diminished response following pro- longed activation has been described as endotoxin tolerance PI3K/Akt signaling regulates macrophage activation when the inflammatory stimulus derives from Gram-negative Since the discovery of Akt 25 y ago (11) and identification of bacterial LPS. PI3K as its upstream regulator (12), the contribution of Akt *Laboratory of Clinical Chemistry, School of Medicine, University of Crete, Heraklion Address correspondence and reprint requests to Prof. Christos Tsatsanis, Laboratory of 71003, Greece; and †Laboratory of Intensive Care Medicine, School of Medicine, Uni- Clinical Chemistry, School of Medicine, University of Crete, PO Box 2208, Heraklion versity of Crete, Heraklion 71003, Greece 71003, Crete, Greece. E-mail address: [email protected] ORCIDs: 0000-0003-4026-0328 (E.V.); 0000-0002-8441-298X (E.I.); 0000-0001- Abbreviations used in this article: ApoE, apolipoprotein E; DSS, dextran sodium sulfate; 7446-0999 (K.L.); 0000-0003-1214-4151 (C.T.). EAE, experimental autoimmune encephalomyelitis; LDLR, low-density lipoprotein re- ceptor; mTORC, mTOR complex; RA, rheumatoid arthritis; TAM, tumor-associated Received for publication August 30, 2016. Accepted for publication September 26, macrophage; TSC, tuberous sclerosis complex. 2016. This work was supported by Greek national and European Union funds under the Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Aristeia Grant (2071). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601515 The Journal of Immunology 1007 in multiple cell types and functions has been widely recog- IL-1 receptor–associated kinase M (IRAK-M), a suppressor of nized. The PI3K/Akt pathway regulates macrophage survival, TLR4 signaling via TRAF6 inactivation (33). Activated Akt migration, and proliferation but also orchestrates the response also phosphorylates and inactivates the transcription factor to different metabolic and inflammatory signals in macro- FOXO1 (34), resulting in suppression of its target genes that phages (13). The PI3K/Akt pathway is activated by TLR4 and include TLR4 (35). Akt also regulates let-7e, which in turn other pathogen recognition receptors, cytokine and chemo- suppresses TLR4 expression (36) and miR-146a, which sup- kine, and Fc receptors (14–17), modulating downstream sig- presses the TLR signaling mediator TRAF6 (32). nals that control cytokine production. Activated PI3K type I In most studies, the PI3K/Akt/mTOR pathway was studied phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) as a single entity, and the effect of different isoforms of PI3K or to generate phosphatidylinositol 3,4,5-triphosphate (PIP3) at Akt kinases was not defined. However, studies on knockout the plasma membrane; PIP3 further recruits Akt and the mice and studies using small interfering RNAs and chemical mechanistic target of rapamycin complex (mTORC) 2, and inhibitors provided evidence on the differential function of facilitates Akt activation by mTORC2 (13, 14). Activated PI3K/Akt kinase isoforms on macrophage activation. The Akt (phosphorylated) Akt subsequently phosphorylates and inac- family of serine-threonine protein kinases consists of three tivates the tuberous sclerosis complex (TSC) 1/2. TSC1/2 isoforms expressed from independent genes (Akt1/PKBa, inhibition by Akt leads to mTORC1 activation, via Ras ho- Akt2/PKBb and Akt3/PKBg) (12, 37). Akts are activated by molog enriched in brain (termed Rheb) suppression (14, 16). PI(3,4,5)P3 and to a lesser extent by PI(3,4)P2 that are generated Activation of the PI3K/Akt pathway is critical in restricting by PI3K class I activation (12, 37, 38). Class IA PI3K includes proinflammatory and promoting anti-inflammatory responses three members, namely, p110a, p110b,andp110d,whichin- in TLR-stimulated macrophages (18), and has been consid- teract with an SH2 domain–containing p85 regulatory subunit ered as a negative regulator of TLR and NF-kB signaling in (31, 39). PI3Kg belongs to class IB PI3K with its catalytic macrophages (15, 16) (Fig. 1). Activation or overexpression of subunit p110g coupled with either the p101 or the p84 regu- PI3K or Akt kinases resulted in reduced macrophage activa- latory subunit. Among PI3K members, PI3Kd and PI3Kg are tion by LPS, whereas nonspecific chemical inhibition of PI3K the predominant isoforms expressed in cells of the hematopoietic signaling in TLR-activated cells augments NF-kB activation lineage (39). Whether individual Akt isoforms are activated by and inducible NO synthase expression (19–21) promoting specific PI3K members in macrophages remains to be elucidated. M1-type macrophage responses. PI3K activation has been Nevertheless, isoform-specific effects on macrophage function reported as an essential step toward M2 activation of mac- have been reported both for Akt and PI3K. rophages in response to surfactant protein A or IL-4 (22, 23). Akt upstream signals in macrophage polarization: the role of PI3K A crosstalk between the STAT6 and PI3K activation is re- isoforms, PTEN, and PDK-1. PI3K, the upstream regulator of quired for IL-4–induced M2 macrophage activation in SHIP- Akt, is shown to mediate M2 macrophage phenotypes (19, 23, deficient macrophages (22). Akt activation is required for M2 40). Accordingly, induction of Arg1 activity and M2 phe- activation, because Akt inhibition abrogates the upregulation notype in SHIP-deficient macrophages depends on PI3K of M2 genes (14, 24, 25). Several signals such as TGF-b, IL- activity, and specifically on class IA PI3K p110d