Kinase AKT controls innate immune cell development and function

Abstract

The critical roles of kinase AKT in tumour cell proliferation, apoptosis and protein synthesis have been widely recognized. But AKT also plays an important role in immune modulation. Recent studies have confirmed that kinase AKT can regulate the development and functions of innate immune cells (neutrophil, macrophage and dendritic cell). Studies have shown that different isoforms of kinase AKT have different effects in regulating immunity-related diseases, mainly through the mammalian target of rapamycin-dependent or -independent pathways. The purpose of this review is to illustrate the immune modulating effects of kinase AKT on innate immune cell development, survival and function.

Introduction

The kinase AKT, also known as protein kinase B, is a serine/threonine-specific protein kinase that was originally identified as a retroviral oncogene.¹ It is a central player in the signalling pathways that regulate metabolism and cellular transformation. AKT can regulate cell growth, apoptosis and tumour-related diseases by activating a series of different downstream signalling molecules.^2–4 Hence, AKT plays an essential role in both physiological and pathological signalling mechanisms. In mammals, three different AKT isoforms exist, AKT1, AKT2 and AKT3, products of different genes. Activation of AKT proceeds downstream of phosphatidylinositol 3-kinase (PI3K) activity by bringing AKT into close proximity with its activating kinases. Many pattern recognition receptors, growth factor receptors and cytokine receptors are able to activate PI3K, and thereby activate AKT.⁵ AKT is involved in the PI3K–AKT–mammalian target of rapamycin (mTOR) pathway and other signalling pathways (Fig. 1). Recent studies have shown that AKT plays a critical role in immunity and autoimmunity. This review summarizes recent progress in understanding the role of kinase AKT in the modulation of innate immune cell development and functions.

Figure 1.

AKT-mammalian target of rapapmycin (mTOR) signalling pathway. The mTOR forms two structurally and functionally distinct complexes mTORC1 and mTORC2. Activity of mTORC1 is critically controlled by a small GTPase, Rheb, whose activity is inhibited by a GTPase-activating protein, tuberous sclerosis protein 2 (TSC2) in complex with TSC1. Many accessory molecules as well as growth factors signal via the PI3K–AKT. AKT can further promote mTORC1 activity independent of TSC1/2 by phosphorylating PRAS40, a negative regulator of mTORC1 activity. Further mTORC1 can be activated via the WNT pathway, in a signalling cascade involving glycogen synthase kinase 3 (GSK3).

The AKT signalling pathway is involved in regulating the inflammatory response. Several studies have reported the importance of PI3K–AKT signalling in inflammation-mediated diseases, such as rheumatoid arthritis,⁶ multiple sclerosis,⁷ asthma,⁸ chronic obstructive pulmonary disease,⁹ psoriasis¹⁰ and atherosclerosis.¹¹ Modulation of AKT–mTOR on adaptive immune cells has been summarized in some publications and reviews^12–19 (Fig. 2). However, there is good evidence that AKT is predominantly expressed in the innate immune cells, including neutrophils, macrophages and dendritic cells (DCs), which is critical to the inflammatory response and, more specifically, to innate immune cell development and function.

Figure 2.

AKT controls T-cell differentiation. AKT–mammalian target of rapamycin (mTOR) control CD4⁺ T-cell differentiation. The complexes mTORC1 and mTORC2 have different physiological functions. mTORC1 signalling promotes T helper type 1 (Th1) and Th17 differentiation; mTORC2 promotes Th2 differentiation; and inhibition of mTOR leads to regulatory T (Treg) cells through the transforming growth factor-β (TGF-β)-Smad3 pathway.

Modulation of neutrophil development and function by AKT

Kinase AKT has a central role in modulating neutrophil activation, chemotaxis, survival and functions.

AKT could be activated in different ways in neutrophils

There are three basic methods by which transmembrane signal transduction occurs: through cell surface receptor, i.e. G protein coupled receptor, protein phosphorylation or dephosphorylation, and ion channels. Calcium store-operated calcium influx (SOCE) is the regulation of calcium (Ca²⁺) influx into the cell and the commonest route is through the calcium store-operated calcium flow channel (SOC). Some studies^20,21 support the evidence that extracellular signal-regulated kinase 1/2 (ERK1/2), AKT phosphorylation and the control of intracellular alkalinization are dependent on capacitative Ca²⁺ entry in bovine neutrophils. Also in neutrophils, SOCE controls respiratory burst, degranulation and motility through phosphorylating AKT and ERK1/2. Research²⁰ showed that platelet-activating factor as a chemoattractant can stimulate neutrophils and increases the concentration of cytoplasmic Ca²⁺ through SOCE, then phosphorylates ERK1/2 and AKT. Activated AKT can activate different downstream signalling molecules, which help neutrophils to have an effect on immunity. Recent studies^22,23 prove that PI3K-dependent p38 kinase (p38MAPK) activation can also regulate the activity of AKT by phosphorylation of its Ser473 in human neutrophils. AKT, p38MAPK, MK2 and heatshock protein 27 (Hsp27) form a stable complex in unstimulated neutrophils (Fig. 3). Upon stimulation with chemoattractants, such as fMet-Leu-Phe (fMLP), Hsp27 performs a regulatory function, dissociates from this complex and directly phosphorylates Ser473 on AKT. Activated AKT can produce the biological effects by activating different downstream signalling molecules, so that the neutrophils can respond better to viruses and to microbial invasion.²⁴ It has also been shown²⁵ that soluble Cr(VI) can directly activate the Src family of protein tyrosine kinases and up-regulate the activation of p85, which may in turn enhance the expression of phospho-AKT. Additionally, particulate Cr(VI) can enhance the phosphorylation of AKT at serine 473 and activated AKT can promote the migration of neutrophils to the site of injury. In this way, AKT and the downstream signalling pathway are critically involved in the activation of neutrophils.

Figure 3.

AKT control innate immune cell homeostasis and activity. Dendritic cells, macrophages and neutrophils could be activated by all kinds of stimuli, lipopolysaccharide (LPS) or fMet-Leu-Phe (fMLP). The activated AKT control immune cell cytokine secretion, reactive oxygen species production, and maintain cell homeostasis by regulating the balance between apoptosis and proliferation by glycogen synthase kinase.

AKT could delay neutrophil survival during inflammation

In the circulation, neutrophils have a short lifespan, they undergo spontaneous apoptosis within 6–10 hr. However, neutrophil apoptosis is delayed after exposure to pro-inflammatory stimuli such as interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), IL-1, IL-2, interferon-γ (IFN-γ), granulocyte–macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or lipopolysaccharide (LPS). AKT, as an anti-apoptotic molecule, can regulate apoptosis by directly controlling members of the apoptotic cascades. Chemoattractant fMLP can delay neutrophil apoptosis through a mechanism involving AKT.^26,27 After stimulation of neutrophils with the chemoattractant fMLP, it binds to a G protein-coupled receptor on neutrophils and activates AKT. Kinase AKT could promote rapid phosphorylation/inactivation of the α- and β-isoforms of glycogen synthase kinase 3 (GSK-3) with phosphorylation of the α-isoform predominating.²⁸ GSK-3 could potentiate spontaneous apoptosis and induce apoptosis²⁹ (Figs 1 and 2). Hence, neutrophils could play the role of the innate immune response better. The apoptosis of neutrophils is also regulated by Bcl-2 family members and anti-apoptotic protein, besides GSK-3. Normally, upon receiving apoptotic signals, Bax, a pro-apoptotic protein of the BCL-2 family, translocates to the mitochondria where it can form oligomers and then releases cytochrome c and other substances to promote apoptosis of neutrophils.^30,31 However, anti-apoptotic stimuli lead to the activation of AKT; activated AKT can phosphorylate the Ser184 of Bax, the phosphorylated Bax loses its original function of being pro-apoptotic, so inhibiting the apoptosis of neutrophils and contributing to the balance of the number of neutrophils.³⁰ Among others, Bad and caspase-9 are regulated by AKT. Recently, it has been reported that the deactivation of AKT signalling is the mediator of spontaneous neutrophil apoptosis. This observation indicates the essential role of AKT signalling in neutrophil apoptosis. Active AKT is thought to inhibit apoptosis in a variety of ways, both upstream and downstream of mitochondrial perturbation. It can lead to inhibition of caspase-9 activity, phosphorylation of pro-apoptotic Bcl-2 family members such as Bad, or regulation of transcription factors such as cAMP-responsive element-binding protein and nuclear factor-κB (NF-κB) and members of the Forkhead family.³² Another study showed that anti-apoptotic stimuli lead to the activation of AKT and Ser184 phosphorylation of Bax.³⁰ This phosphorylation promotes its sequestration to the cytoplasm and promotes its ability to heterodimerize with the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xl, so inhibiting its pro-apoptotic abilities. In addition, several intracellular pathogens such as Anaplasma phagocytophilum, Leishmania major, Chlamydia pneumoniae and respiratory syncytial virus have developed strategies to manipulate the spontaneous apoptosis of host neutrophils.³³ Enhanced gene expression of AKT signalling in A. phagocytophilum-infected neutrophils has been reported.³³ Further studies showed that A. phagocytophilum activates the PI3K–AKT signalling pathway in primary human neutrophils. The infection prevents down-regulation and maintains the expression of Mcl-1 via the AKT signalling pathway. Infection with A. phagocytophilum also activates NF-κB in human neutrophils, which in turn results in the release of IL-8 in an autocrine fashion to delay neutrophil apoptosis. These multiple anti-apoptotic effects of AKT make it a prime kinase candidate for inducing phosphorylation of Bax and so inhibiting its pro-apoptotic abilities. However, some studies^34,35 also suggest that AKT is not sufficient to prolong neutrophil survival. Neutrophils treated with G-CSF undergo apoptosis, even in the presence of high levels of p-AKT.³⁴ In addition, inhibitors of AKT and downstream targets failed to alter neutrophil survival. In contrast, neutrophil precursors appear to be dependent on AKT signalling pathways for survival, whereas high levels of p-AKT inhibit proliferation.³⁶ These data suggest that the AKT signalling pathway, although important in G-CSF-driven myeloid differentiation, proliferation and survival of early haematopoietic progenitors, is less essential in G-CSF suppression of neutrophil apoptosis. Whereas basal AKT levels may be required for the brief life of neutrophils, further p-AKT expression is not able to extend the neutrophil lifespan in the presence of G-CSF. These results require further studies for confirmation.

AKT could promote inflammatory neutrophil recruitment

Recruitment of neutrophil granulocytes to the site of acute infection is a crucial mechanism for defence against pathogenic microorganisms.^37,38 Initially, neutrophils are captured by adhesion molecules like E-selectins and P-selectins that are expressed at the cell surface of endothelial cells after activation with pro-inflammatory cytokines like IL-1 and TNF-α.³⁸ The initial adhesion of neutrophils is followed by rolling and tight adhesion after activation by chemokines like IL-8. This enables intravascular crawling and diapedesis of the neutrophils. After leaving the blood vessels, neutrophils are guided by chemoattractants to the site of inflammation.³⁹ Depending on their origin chemoattractants can be classified in pathogen-derived end-target chemoattractants (e.g. fMLF or complement factor C5a) and host-derived intermediary chemoattractants (e.g. keratinocyte-derived cytokine; KC or IL-8).^37,39 End-target chemoattractant signalling predominantly occurs through stimulation of the p38 MAPK, whereas intermediary chemoattractant signalling uses the PI3K-AKT signalling pathway.⁴⁰

Neutrophils undergo polarization upon stimulation with chemoattractants, and this plays an important role in host defence and inflammation. The longitudinal axis of neutrophils changes, with a broad lamellipodium formed at the leading edge and a contracted tail at the posterior edge after stimulation by chemoattractants.^35,41 The neutrophils then start to move toward the chemoattractants. Neutrophil migration is initiated by this polarization, and is accompanied by asymmetric distribution of signalling molecules and asymmetric cytoskeletal organization. Studies have shown that AKT plays a vital role in cell migration.^35,42 AKT regulates actin organization through phosphorylation of actin or Girdin, an actin-binding protein. Furthermore, AKT plays a pivotal role in stabilizing actin cytoskeletal organization during cell migration, in which AKT activity is regulated not only by PI3K but also by the mTOR complex, mTORC2.⁴³ Some studies have implied that AKT is a critical component of neutrophil chemotaxis.^44,45 Recent studies of AKT knockout (KO) mice have shown that the three AKT isoforms have different roles in regulating neutrophil functions.⁴⁶ AKT2 KO neutrophils exhibited decreased cell migration. In wild-type neutrophils, AKT2 but not AKT1 translocated to the plasma membrane upon chemoattractant stimulation and translocated to the leading edge in polarized neutrophils. In the absence of AKT2, chemoattractant-induced AKT protein phosphorylation was significantly reduced. Furthermore, in a polarization study of neutrophil-like differentiated HL-60 cells (dHL-60 cells)^40,47,48 using two AKT inhibitors (AKT inhibitor and Degulin) to examine the role of AKT in cell polarization, it was found that AKT regulated the polarization of dHL-60 in response to fMLP. HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1·3% DMSO for up to 6 days. Polarization of dHL-60 cells and primary human neutrophils were measured using a Zigmond chamber. Results showed that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pre-treated with the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases were also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells. Hence, these studies demonstrate that AKT activation plays a crucial role in neutrophil polarization during inflammation.

AKT could promote neutrophil inflammatory cytokine secretions

Kinase AKT could also modulate neutrophil chemotaxis and superoxide to protect against microbial invasion.⁴⁹ At the same time, the PI3K–AKT axis also plays a central role in Toll-like receptor 2 (TLR2) -induced activation of neutrophils.^50,51 TLR2, a receptor for Gram-positive bacteria-derived peptidoglycan and lipoproteins, could be expressed in neutrophils. In the signal transduction pathways of TLR2, activation of AKT can phosphorylate the p65 subunit of NF-κB and promote NF-κB translocation to the nucleus, resulting in rapid release of pro-inflammatory cytokines and chemokines such as TNF-α and macrophage inflammatory protein 2.²³ Therefore, AKT has a central role in TLR2-associated potentiation of neutrophil responses. On the other hand, ERK1/2 and p38MAPK can also be directly activated by the stimulating action of TLR2 on neutrophils.⁵¹

Neutrophils are a critical cellular component of the innate immune response by releasing the superoxide anion (O₂⁻) free radical and its toxic metabolites against invading microorganisms.^24,52 The NADPH enzyme can release O₂⁻. In resting neutrophils, the NADPH oxidase complex consists of unassembled cytosolic and membrane components. After activation of neutrophils by chemoattractants, chemokines, complement components, p40^phox, p47^phox, p67^phox and Rac-2 as the cytosolic components translocate to plasma, then interact with flavocytochrome b558 for the activation of NADPH oxidase. Hence, activated neutrophils evoke a respiratory burst in which the oxygen consumption is increased and large amounts of O₂⁻ are generated.²⁶ AKT has been proposed to participate in p47^phox phosphorylation events (p47^phox is postulated to act as an adaptor protein that assembles the components of the functional enzyme). AKT directly interacts with and phosphorylates Ser304 and Ser328 on p47^phox, then regulates the respiratory burst activity of neutrophils.⁵² Additionally, the activation of AKT also participates in neutrophil chemotaxis.^52,53 Furthermore, a recent study⁴⁶ showed that AKT2 is responsible for the O₂⁻ production after stimulation. Using the individual AKT1 and AKT2 KO mice, AKT2 KO neutrophils exhibited decreased granule release and O₂⁻ production compared with wild-type neutrophils. Also, the decreased O₂⁻ production in AKT2 KO neutrophils was accompanied by reduced p47^phox phosphorylation and its membrane translocation, suggesting that AKT2 is important for the assembly of phagocyte NADP oxidase in wild-type neutrophils. However, the role of AKT1 will need further exploration.

In summary, activated AKT can participate in apoptosis, inflammation and chemotactic responses by phosphorylating a wide variety of downstream signalling molecules in neutrophils, so that the neutrophils can play an important role in phagocytosis, bactericidal effects and chemotaxis.

Modulation of AKT on macrophage programming and function

Macrophages play an important role in the innate immune system, and are the professional antigen-presenting cells involved in the adaptive immune system. Their main role is to clear aging cells, damaged cells, apoptotic cells, immune complexes, pathogens and other antigens from foreign bodies.⁵⁴ Studies have shown that the immune function of macrophages is closely related to AKT (Fig. 3).

AKT can promote macrophage cytokine secretion and programming

Macrophages can produce IFN and other cytokines. Using AKT KO macrophages, results showed that the AKT can regulate the production of IFN-β through two different pathways.⁵⁵ One route is that AKT can inhibit the kinase GSK3-β through phosphorylation, which should result in the accumulation of β-catenin protein (Fig. 3) and enhance the transcription of IFN-β. Another route is that AKT can directly phosphorylate the Ser552 of β-catenin, a site distinct from that targeted by GSK3-β, promote the transcriptional activity of β-catenin, then enhance the transcription of IFN-β.⁵⁶ Furthermore, AKT could promote the NO production of macrophages. When bacteria invade the body, macrophages can secrete NO to kill them, but excessive NO production leads to inflammatory responses and tissue damage.⁵⁷ Hence, a mechanism to maintain NO at normal levels is needed. Ceramide is an intracellular second messenger that can be generated by acid or neutral sphingomyelinases through cleavage of the sphingomyelin membrane. Intracellular ceramide as a signal molecule can participate in LPS-induced activation of macrophages.^58,59 Experimenting with membrane-permeable ceramide analogues such as C2-ceramide has shown that it can down-regulate the phosphorylation of the AKT signal molecule in response to LPS, then prevent the activation of NF-κB. NF-κB can bind to a major transcription factor of inducible NO synthase, which can regulate the LPS-induced production of NO.⁵⁸

AKT also contributes to the TLR4 receptor expression of macrophages.^60,61 Either treatment with pharmacological inhibitors of AKT or knockdown of AKT expression by small interfering RNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl₂.⁶² Phosphorylation of AKT by hypoxic stress preceded nuclear accumulation of HIF-1α. A PI3K inhibitor (LY294002) attenuated CoCl₂-induced nuclear accumulation and transcriptional activation of HIF-1α. In addition, HIF-1α-mediated up-regulation of TLR4 expression was blocked by LY294002.⁶² Furthermore, sulforaphane suppressed hypoxia- and CoCl₂-induced up-regulation of TLR4 mRNA and protein by inhibiting PI3K–AKT activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1α. Hence, these results demonstrate that AKT signalling contributes to hypoxic stress-induced TLR4 expression.

Activated macrophages express pro-inflammatory factors and are known as classically activated or M1 macrophages, which secrete more TNF-α, IL-6 and inducible NO synthase. However, macrophages can also undergo alternative activation to become alternatively activated macrophages or M2 macrophages, which are characterized by the induction of low levels of pro-inflammatory cytokines and in the up-regulation of arginase I and IL-10.^63,64 Recent studies^61,65 showed AKT1 KO giving rise to an M1 macrophage phenotype and AKT2 KO resulting in an M2 macrophage phenotype. Accordingly, AKT2 KO mice were more resistant to LPS-induced endotoxic shock and to dextran sulphate sodium-induced colitis than WT mice, whereas AKT1 KO mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of AKT2 KO macrophages was cell autonomous. Meanwhile, other studies also showed the differences in different isoforms of AKT on macrophage immunity. AKT1 appears not be dispensable for eNOS induction.⁶⁶ Depletion of AKT1 does not cause endotoxin tolerance to develop.⁶¹ Also, AKT1 KO protects and AKT2 KO promotes mouse mammary tumour virus (MMTV) -pyMT and MMTV ErbB2-induced mammary tumours.⁶⁷ Collectively, AKT1 KO shows a pro-inflammatory phenotype in vivo and in contrast, the AKT2 KO shows anti-inflammatory phenotypes. These results are inconsistent with neutrophil studies in inflammation,⁴⁶ so need to be clarified. Osteoclasts are a type of macrophages. Maintaining optimal bone integrity, mass and strength throughout adult life requires ongoing bone remodelling, which involves coordinated activity between the actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoporosis is a disorder of remodelling in which bone resorption outstrips deposition, leading to diminished bone mass and an increased risk of fractures. Research has identified AKT1 as a unique intermediate signal in osteoblasts that can control both osteoblast and osteoclast differentiation.⁶⁸ Targeted knockdown of AKT1 in mouse primary bone marrow stromal cells or in a mesenchymal stem cell line or genetic knockout of AKT1 stimulated osteoblast differentiation secondary to increased expression of the osteogenic transcription factor Runx2. Despite enhanced osteoblast differentiation, coupled osteoclastogenesis in AKT1 deficiency was markedly inhibited, with reduced accumulation of specific osteoclast mRNAs and proteins and impaired fusion to form multinucleated osteoclasts, defects secondary to diminished production of receptor activator of NF-κB ligand (RANKL) and M-CSF, critical osteoblast-derived osteoclast differentiation factors.⁶⁹ Delivery of recombinant lentiviruses encoding AKT1 but not AKT2 to AKT1-deficient osteoblast progenitors reversed the increased osteoblast differentiation and, by boosting accumulation of RANKL and M-CSF, restored normal osteoclastogenesis, as did the addition of recombinant RANKL to conditioned culture medium from AKT1-deficient osteoblasts.⁷⁰ Hence, these results support the idea that targeted inhibition of AKT1 could lead to therapeutically useful net bone acquisition. Collectively, these results reveal that AKT and the isoforms of AKT (AKT1 and AKT2) exert critical effects on macrophage differentiation and function during inflammation.

AKT can control macrophage survival and death

Macrophage inhibitory cytokine-1 (MIC-1) belongs to the transforming growth factor-β cytokine superfamily, which is weakly expressed in most tissues under normal circumstances but is substantially up-regulated under pathological conditions such as tissue injury and inflammation.⁷¹ Macrophage migration inhibitory factor (MIF) have three different receptors: CD74, CXCR2 and CXCR4. MIC-1 is anti-tumorigenic by promoting apoptosis and inhibiting cell proliferation in a few tumour cell lines. However, recent studies⁷² have shown that endogenous MIF can activate the AKT pathway through an autocrine mechanism or in a CD74-CXCR-dependent manner and MIF-stimulated AKT signalling is at least partially dependent on endocytosis. Activated AKT can activate a series of downstream signalling molecules and then have a biological effect in the body. In addition, MIF can enhance the expression of phosphorylated AKT, the AKT can regulate the expression of cyclin, cyclin-dependent kinase (CDK) and CDK inhibitors, such as cyclin D1 and p27^Kip1 which is an inhibitor of CDK, that have an important role in the cell cycle during the transition from G1 phase to S phase (Fig. 3). Phosphorylated AKT can up-regulate the expression of cyclin D1 at the transcriptional level and down-regulate the expression of p27^kip1 at the post-transcriptional level, then increase the proliferation of cells, such as gastric cancer cells.²⁵ Moreover, MIC-1 enhanced the phosphorylation levels of AKT, ERK1/2 and Jun N-terminal kinase, and enhanced the expression of cyclins D and E, which can increase the protein kinase activity of CDK4/6 and CDK2 through direct interaction. After retinoblastoma protein, which is one of the endogenous substrates for CDK4/6 and CDK2, is phosphorylated, it separates from the E2F transcription factor, then promoting nuclear translocation of E2F.⁷³ E2F transcription factors binds to the promoter of cyclin D1 gene, leading to up-regulation of the expression of cyclin D1, which can accelerate cell cycle progression of human endothelial cells at the G1 phase and entry into the S phase.⁷⁴

Otherwise, AKT can also inhibit macrophage autophagy and apoptosis. Autophagy could maintain cellular homeostasis by degrading the invaded pathogens and damaged organs or cells.⁷⁵ Moreover, autophagy in macrophages has an important role in innate immunity.⁷⁵ Research⁷⁶ showed that CD40 stimulation of macrophages results in killing of Toxoplasma gondii through autophagy. Other studies^74,77 suggested that AKT is an inhibitor of autophagy in macrophages. Tat, as the main transactivator of human immunodeficiency virus 1 (HIV-1), can be released from HIV-1-infected cells or dying cells. It can phosphorylate Src and FAK, which can activate AKT by phosphorylation. Activation of AKT can inhibit autophagy in bystander macrophage (non-HIV-1-infected) and monocytic cells.⁷⁴ AKT can also inhibit apoptosis of macrophages. Galectin 13, found in several normal and malignant tissues, is a protein that belongs to galectins and that has a single carbohydrate recognition domain. Human macrophages can express this protein, which promotes the process of cell death. Galectin 13 can suppress the phosphorylation of AKT and ERK1/2, then the unphosphorylated AKT will not be able to play the function of inhibiting apoptosis. Simultaneously, galectin 13 can promote the activation of JNK and p38MAPK, which can promote apoptosis. Therefore, the Galectin 13 released by macrophages can inhibit their own apoptosis through AKT signalling cytoprotective pathways.⁷⁸ Lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signalling pathways linked to cell survival.⁷⁹ The Bcl-2 family member Bad is a pro-apoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. Research showed that man-LAM stimulates Bad phosphorylation in a PI3K-AKT-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. The LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI3K (Dp85), indicating that activation of PI3K is sufficient to trigger phosphorylation of Bad by LAM. As phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase AKT, the effect of LAM on AKT kinase activity was tested. Man-LAM could activate AKT as evidenced from phosphorylation of AKT at Thr308 and by the phosphorylation of the exogenous substrate histone 2B. AKT activation was abrogated in cells transfected with Dp85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of AKT. These results establish that LAM-mediated Bad phosphorylation occurs in a PI3K–AKT-dependent manner. Collectively, these results reveal the AKT signalling is critically involved in the modulation of macrophage survival, autophagy and apoptosis.

Modulation of AKT on the development and function of DCs

Dendritic cells have the shape of a branch or dendrite and the function of being phagocytic or antigen-presenting. The DCs are also known as the most powerful cells, that can activate the resting T cells, and the only professional antigen-presenting cells in the body. They are the central link of the immune response⁸⁰ (Fig. 3).

AKT plays an important role in regulating the DC development and differentiation. The role of AKT in conventional DC (cDC) was investigated and development of functional interstitial DCs and Langerhans cells from human cord blood CD34⁺ haematopoietic progenitor cells were studied.⁸¹ The results showed that AKT should be crucial for human myeloid CD34-derived DC development in vitro and in vivo. Precursor proliferation and survival strongly depended on AKT. Although inhibition of this pathway did not affect the acquisition of a DC phenotype, DCs generated under AKT signalling inhibition were functionally impaired. In contrast to the requirement for AKT signalling during development, the survival of terminally differentiated CD34-derived myeloid DCs was not dependent on AKT signalling activity. However, this signalling module was still required for other important processes associated with DC function. AKT was investigated and contributed to human plasmacytoid DC (pDC) development and survival.⁸² In vitro pDC generation from human cord blood-derived CD34⁺ haematopoietic progenitor cells was reduced by pharmacological inhibition of AKT activity, and peripheral blood pDCs required AKT signalling to survive.⁸² Accordingly, activity of AKT in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active AKT or pharmacological inhibition of negative regulator phosphatase and tensin homologue (PTEN) resulted in increased pDC numbers in vitro and in vivo.⁸³ Furthermore, MHC class II and co-stimulatory molecule expression and production of IFN-α and TNF-α were augmented, which could be explained by enhanced interferon regulatory factor 7 and NF-κB activation. Importantly, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced AKT activity.

AKT is required for the survival of DCs by inhibiting their apoptosis. Studies found that DCs exert the biological effect related to the AKT when the body receives stimuli from microorganisms, viruses or other molecules.⁸⁴ Leptin is an adipocyte-derived hormone/cytokine that modulates immune responses and which acts as a survival factor for human DCs through the activation of an AKT signalling pathway.⁸⁵ It can phosphorylate AKT, and the phosphorylated AKT can induce the activation of NF-κB, then up-regulating the expression of Bcl-2 and BcL-xl, which have the function of inhibiting apoptosis.⁸⁵ Furthermore, the modulation of AKT1 on DC survival has been investigated. Results showed that AKT1 regulates DC survival in innate (LPS-driven) and adaptive (CD40-driven) immune responses by modulating Bcl-2, but not Bcl-xL expression.^86,87 In addition, AKT1 deficiency leads to defective DC activation and survival. In contrast, the product of a ‘functionally optimized’, constitutive AKT allele, MF-DAKT, greatly improves the efficacy of DC-based tumour vaccines by increasing maturation and lifespan of both mouse and human DCs, thereby enhancing tumour-specific T-cell responses and eradication of pre-established solid tumours in mice.⁸⁶

AKT can regulate the DC functions by promoting DCs to release cytokines. Cigarette smoke extract (CSE) can inhibit the PI3K–AKT signal pathway in pDCs, then up-regulate the production of IL-8 and down-regulate the release of TNF-α, IL-6 and IFN-α to diminish anti-viral immunity or lead to other diseases.⁹ The endocrine system and the immune system can be linked by AKT. There is also much research to clarify that the AKT signalling pathway linked the endocrine system and the immune system. Triiodothyronine (T₃) which belongs to endocrine systems can activate AKT independent of PI3K, then the phosphorylation of AKT can activate the NF-κB signal pathway, which will promote the expression of thyroid hormone receptor β₁, which acts as a target gene of NF-κB. The thyroid hormone receptor β₁ will deliver a signal to promote the DC, which belongs to the immune system, to mature and exert its function.⁸⁸ Furthermore, using poly-N-acetyl-glucosamine nanofibres as a haemostatic agent⁸⁹ to treat wound infection will increase the phosphorylation of AKT1. The p-AKT1 can promote the expression of defensins stored in neutrophils and almost all epithelial cells, which can have an effect on anti-bacterial infections.⁹⁰ The L protein, which has six conserved domains, can form the activated polymerase that plays a central regulatory role in the transcription and replication of the viral RNA together with the P protein.⁹¹ Three domains of L (LI–III) can interact with AKT1 and enhance its phosphorylation, then activated AKT1 can activate the p50, p52 and p65 subunits of NF-κB directly, and activated NF-κB will promote the expression of antiviral cytokines such as IFN-β and major pro-inflammatory cytokines such as IL-6 in innate immunity.⁹²

Concluding remarks

There are already many reports about the function of AKT related to tumours, metabolism and cellular transformation. The role of AKT in innate immune cell development and function remains a hot spot in the field of immunology. The aim of this review is to highlight the regulatory effects and mechanisms of kinase AKT in innate immune cell development and function. Although there has been striking progress in our knowledge of how AKT is connected with the immune system, much remains to be discovered, especially the mechanism of the different isoforms of AKT and the effects on newly defined cell populations. More importantly, investigation of the regulatory role of kinase AKT can also provide a target direction for clinical treatment and may provide hope for patients with inadequate function of the immune system.

Glossary

AKT

AKT8 virus oncogene cellular homologue

CDK

cyclin-dependent kinase

DCs

dendritic cells

ERK

extracellular signal-regulated kinase

fMLP

fMet-Leu-Phe

GM-CSF

granulocyte–macrophage colony-stimulating factor

GSK3

glycogen synthase kinase 3

HIV

human immunodeficiency virus

HPC

hematopoietic progenitor cells

Hsp

heatshock protein

IFN

interferon

IL-8

interleukin-8

KO

knockout

LAM

lipoarabinomannan

LPS

lipopolysaccharide

MIC-1

macrophage inhibitory cytokine-1

MIF

macrophage migration inhibitory factor

mTOR

mammalian target of rapamycin

NF-κB

nuclear factor-κB

p38MAPK

p38 mitogen-activated protein kinase

pDCs

plasmacytoid dendritic cells

PI3K

phosphatidylinositol 3-kinase

PKB

protein Kinase B

PRAS40

prolin-rich AKT substrate 40 kDa

RANKL

receptor activator of NF-κB ligand

SOCE

store-operated calcium influx

TLR

Toll-like receptor

TNF

tumour necrosis factor

TORC

mTOR complex

Disclosures

The authors declare that they have no competing financial interests.