GSK3b and the control of infectiousbacterial diseasesHuizhi Wang1, Akhilesh Kumar1, Richard J. Lamont1,2, and David A. Scott1,2

1 Oral Health and Systemic Disease, University of Louisville, Louisville, KY 40292, USA2 Microbiology and Immunology, University of Louisville, Louisville, KY 40292, USA

Review

Glycogen synthase kinase 3b (GSK3b) has been shownto be a crucial mediator of the intensity and direction ofthe innate immune system response to bacterial stimuli.This review focuses on: (i) the central role of GSK3b inthe regulation of pathogen-induced inflammatoryresponses through the regulation of pro- and anti-inflammatory cytokine production, (ii) the extensiveongoing efforts to exploit GSK3b for its therapeuticpotential in the control of infectious diseases, and (iii)the increasing evidence that specific pathogens targetGSK3b-related pathways for immune evasion. A betterunderstanding of complex bacteria–GSK3b interactionsis likely to lead to more effective anti-inflammatoryinterventions and novel targets to circumvent pathogencolonization and survival.

GSK3 action and isoformsGSK3 is a highly conserved, constitutively active, serine/threonine (S/T) protein kinase. GSK3 was originallynamed after its ability to phosphorylate the key rate-limit-ing metabolic enzyme, glycogen synthase, whichcontrols the final step of glycogen synthesis. However, thisname does not adequately describe the diverse substratesand multiple functions now attributed to GSK3, includingembryonic development, cell cycle control, cell differentia-tion, cell mobility, apoptosis, migration, and the focus ofthis review – as a central regulator of the inflammatoryresponse to bacterial infection and other insults [1].

In mammals, two isoforms, GSK3a and GSK3b, havebeen identified and each are widely expressed [2].Sequence analysis has revealed that although these twoisoforms are 98% homologous within their kinase domainsand 85% homologous overall [2], the final 76 C-terminusamino acids exhibit only 36% homology [2]. This mayexplain functional differences between GSK isoforms,where GSK3b deficiency is embryonic lethal in mice in amanner that cannot be compensated by GSK3a [3].

GSK3b appears to play important roles in the hostresponse to viral [4], fungal [5], and parasitic infections,including malaria [6]. However, this review will focus

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� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.01.009

Corresponding author: Scott, D.A. (dascot07@louisville.edu).Keywords: GSK3b; cytokines; immune evasion; inflammation; septic shock; TLRs.

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specifically on the significance of GSK3b in the mamma-lian immune response, therapeutic targeting of GSK3b

during bacterial infection, and exploitation of GSK3b bypathogens.

A distinct feature of GSK3 is that it displays highactivity under basal conditions. This activity is differen-tially regulated by tyrosine and S/T phosphorylation. Tyr-osine phosphorylation (Tyr279 for GSK3a and Tyr216 forGSK3b) enhances activity, whereas N-terminal serinephosphorylation (Ser21 for GSK3a and Ser9 for GSK3b)is suppressive. Other GSK3b sites, such as ERK (extra-cellular signal regulated kinase)- and p38–MAPK (mito-gen-activated protein kinase)-phosphorylated Thr43 andSer389, are also reported to influence GSK3b activity inthe brain and thymocytes [1]. The major GSK3b-regulatingevent, however, seems to be Ser9 phosphorylation. Multi-ple extracellular signals induce rapid Ser9 phosphoryla-tion, resulting in a dramatic decrease in enzymaticactivity, with the upstream activators phosphoinositide3-kinase (PI3K)–Ak thymoma/protein kinase B (Akt/PKB) being the best studied (Figure 1). Early studiesidentified the insulin-mediated signaling pathway as anactivator of PI3K and, subsequently, a phospho-inactivatorof both GSK3a (Ser21) and GSK3b (Ser9) [7]. Other mole-cules, including growth factors, phorbol esters, aminoacids, interleukin receptors, Toll-like receptors (TLRs), Tcell receptors, and CD28, as well as KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) acti-vation, have since been shown to be involved in the phos-phorylation, and thus inactivation, of GSK3 [1].

In addition to the PI3K–Akt pathway, several otherkinases, such as 90 kDa ribosomal protein S6 kinase 1(p90RSK), serum and glucocorticoid-regulated kinase 1(SGK1), and MAPK–p38, have also been shown to phos-pho-inactivate GSK3b [8–10]. Because these kinases allbelong to the protein kinase A, G, and C (AGC) family, it ispossible that GSK3b could also be phospho-inactivated byother members of this large kinase group which control amultitude of physiological processes.

Substrate recognition by GSK3b is well defined. Thekinase has a 100–1000-fold preference for phosphorylationof substrates that are pre-primed (pre-phosphorylated) ona serine or threonine located around a five amino acidconsensus sequence, Ser/Thr–X–X–X–Ser/Thr-P, wherethe first serine or threonine of this motif is the residuephosphorylated by GSK3b [11]. Frame et al. demonstratedby mutation of Arg96 to Ala96 that this is a crucial residue

AminoacidsIFN CD28

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Figure 1. Mediators of GSK3b phosphorylation. In its active state, GSK3b exhibits a 100–1000-fold increased efficiency in substrate specificity for pre-primed (pre-

phosphorylated) substrates, as compared to non-primed substrates. However, multiple agonists including growth factors, insulin, a7nAchR, TLRs, TCR, cytokines, CD28,

and amino acids phospho-activate PI3K, which results in the generation of PIP3 that allows for the recruitment of Akt via its pleckstrin homology domain. Full activation of

Akt occurs through phosphorylation (P) at threonine (T) 308 by PDK1 and serine (S) 473 by PDK2/mTORC2. Upon activation, Akt can phosphorylate GSK3b (Ser9) resulting in

GSK3b inactivation. PKC and PKA also can phospho-inactivate GSK3b. Abbreviations: Akt, Ak thymoma/protein kinase B; GF, growth factor; GSK3b; glycogen synthase

kinase 3b; IFN, interferon; mTORC2, mechanistic target of rapamycin complex 2; a7nAchR, a7 nicotinic acetylcholine receptor; PDK, pyruvate dehydrogenase kinase; PIP2,

phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PKA, protein kinase A; PKC, protein kinase C; TCR, T cell receptor; TLR, Toll-like

receptor.

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in the phosphorylation of pre-primed (pre-phosphory-lated), but not non-primed substrates [12]. The GSK3b

crystal structure has revealed that Tyr276/216 is alsoimportant conformationally, with phosphorylation of thisresidue enhancing substrate binding [13].

GSK3b is a central mediator of the immune response tobacterial infectionThe immune system recognizes and responds to bacterialchallenge primarily through pattern recognition receptors(PRRs) such as TLRs, retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide binding oligomeriza-tion domain-like receptors (NLRs). Recognition of microbe-associated molecular patterns (MAMPs) by PRRs results inthe initial activation of innate immune cells with anincrease in the expression of inflammatory cytokines, cost-imulatory molecules, and major histocompatibility com-plex (MHC) I and II molecules, all of which work together topromote subsequent activation of the adaptive immuneresponse. Activation of PRRs by various microbial compo-nents results in the recruitment of different downstreamsignaling adaptors, which confer selectivity on the reper-toire of cytokines induced. Broadly speaking, signalingpathways in innate cells can be classified as myeloid

differentiation primary response 88 (MyD88)-dependent,which initiate the expression of pro- [e.g. interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF), IL-12] and anti-inflammatory (IL-10) cytokines, or MyD88-independent,which control the production of type I interferon throughTRIF-related adaptor molecule (TRAM)–TIR-domain-con-taining adapter-inducing interferon-b (TRIF) [14,15].Although GSK3b is a central regulator of adaptive (includ-ing B cell expansion and T cell polarization and cytokineproduction) and innate immunity, we will focus on the roleof the innate arm of defense and on the increasing evidencethat GSK3b inactivation has potent therapeutic potentialin the control of bacteria-driven inflammatory diseases[1,14,16] (Figure 2).

It has been known for many years that phospho-inactivativation of GSK3b suppresses MyD88-dependentcytokine production. This leads to downregulation of proin-flammatory cytokines, but upregulation of IL-10 production,in innate cells exposed to bacterial stimuli [14,15,17].Nuclear factor-kB (NF-kB) and cAMP response element-binding protein (CREB) represent prototypical transcrip-tion factors for proinflammatory cytokines and anti-inflam-matory IL-10, respectively. Phospho-inactivation of GSK3b

enhances interactions of CREB with the co-transcriptor,

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Figure 2. GSK3b is a central player in Wnt, a7nAChR- and PI3K-mediated anti-inflammatory signaling. Microbial pathogens and their components initiate the innate

immune response through activation of multiple signaling pathways, most importantly the TLR–NF-kB axis, driving the production of multiple proinflammatory cytokines.

Unphosphorylated GSK3b is an essential and controlling mediator of this inflammatory response to bacteria. Probably to prevent an overly robust response to infection,

several endogenous mechanisms serve to dampen bacteria-driven inflammation. The cholinergic anti-inflammatory pathway, triggered upon a7nAChR engagement by

endogenous (acetylcholine) and exogenous (e.g., nicotine) agonists, is a classic example. a7nAChR-mediated activation of PI3K leads to phospho-inactivation of GSK3b at

Ser9 (through Akt and PIP3) resulting in increased activity of CREB, displacement of NF-kB p65 from the coactivator of transcription, CBP, and lowered transcriptional

activity of NF-kB p65-driven proinflammatory genes. Increased chromosomal access to CREB also promotes production of anti-inflammatory IL-10. Inhibitors of TLR

pathway molecules (JAK3; PI3K; Akt) that normally drive GSK3b Ser9 phosphorylation (white lines) exacerbate bacteria-driven inflammation, whereas promoters of GSK3b

Ser9 phosphorylation (a7nAChR pathway) or pharmaceutical GSK3b inhibitors (e.g., SB216763) suppress proinflammatory mediators while promoting anti-inflammatory

IL-10 in response to infection (red lines). Perhaps independently, activation of the canonical Wnt (Wnt/b-catenin) pathway also leads to phospho-inactivation of GSK3b and

stabilization of b-catenin which suppresses inflammation by interference with NF-kB. There is much current emphasis on the development of pharmacological inhibitors of

GSK3b that alter the magnitude of the inflammatory response through differentially regulating pro- and anti-inflammatory cytokines. Therefore, it is possible that we will

eventually be able to up- or downregulate the intensity of the innate response to bacterial infection depending on the clinical necessity. Abbreviations: CBP, CREB binding

protein; CREB, cAMP response element binding protein; IL, interleukin; JAK, Janus kinase; NF-kB, nuclear factor kB; PI3K, phosphoinositide 3-kinase; Wnt, wingless-type

MMTV integration site family; for other abbreviations see Figure 1 legend.

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CREB binding protein (CBP/CREBBP), attenuating CBPbinding to NF-kB [17]. Moreover, active GSK3b is able tophosphorylate directly NF-kB p65 and B cell lymphoma 3-encoded protein (BCL-3), a transcriptional coactivator of theNF-kB p50 homodimer [18,19]. Phosphorylation of BCL-3leads to its proteasomal degradation [18]. Thus, GSK3b

facilitates NF-kB activity by targeting phospho-activationof NF-kB p65 and limits NF-kB activation via unidentifiedBCL-3 dependent pathways. GSK3b has also been shown tophosphorylate p105 at residues Ser903 and Ser907, stabi-lizing p105 by preventing its degradation in unstimulatedcells [20,21]. However, in TNF-stimulated cells, GSK3b hasbeen found to phosphorylate p105 and prime it for subse-quent degradation [20]. Therefore, GSK3b exerts dualeffects on p105, either suppressing or augmenting NF-kB

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activity depending on the context of the signal. In addition, arecent publication has reported that deficiency of MyD88does not influence GSK3b phosphorylation levels and thatinhibition of GSK3b results in a robust increase of type Iinterferon (IFNb) production in vitro and in vivo [22]. Aug-mentation of IFNb occurs via phosphorylation of the tran-scription factor c-Jun and the enhancement of nuclear AP-1(activator protein-1)–ATF2 (activating transcription factor2) complex [22].

Apart from the classic cytokines discussed above, otherinflammatory mediators, including interleukin 1 receptorantagonist (IL-1Ra/IL1RN), chemokine (C-C motif) ligand3 (CCL3), CCL4, and nitric oxide (NO) have been reportedto be regulated by GSK3b in different contexts. Forexample, Rehani et al. have shown that GSK3b activity

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negatively regulates the level of the anti-inflammatorycytokine IL-1Ra, while concurrently increasing the levelsof IL-1b in TLR4-stimulated human monocytes. GSK3b

inhibition abrogates Rac1 [ras-related C3 botulinum toxinsubstrate 1 (rho family, small GTP binding protein)] inhi-bition at residue Ser71, increasing the activities of Rac1and its downstream target, ERK1/2, enhancing IL-1Rasecretion [23]. Jing et al. demonstrated that prostaglandinE (PGE)-induced phospho-inactivation of GSK3b leads toa robust decrease in production of CCL3/4, as well as ofseveral proinflammatory cytokines, in lipopolysaccharide(LPS)-stimulated dendritic cells, suggesting that GSK3b

may also regulate the inflammatory response to Gram-negative bacteria by modulating eukaryotic chemotaxis[24]. Expression of inducible nitric oxide synthase (iNOS)has been demonstrated to play an important role in theclearance of intracellular pathogens. Several findingsindicate that GSK3b inhibition is also capable of attenu-ating production of NO by suppressing the expression ofiNOS in TLR-stimulated cells [25]. GSK3b thus differen-tially regulates TLR-mediated production of pro- and anti-inflammatory cytokines, and such studies enhance therobustness of the original proposal by Martin et al. thatGSK3b in its active form acts as a positive regulator ofinflammation and that manipulation of GSK3b could beemployed to up- or downregulate the inflammatoryresponse, depending on the clinical necessity [17].

Our understanding of the control of inflammation byGSK3b grows ever more complex and is likely to be con-text-specific. For example, Hu et al. have demonstratedsynergy between IFNg and TLR2 in macrophages, increas-ing GSK3b activity and augmenting TNF production,while suppressing IL-10. GSK3b inhibition reversed thisphenomenon [26]. Furthermore, it appears that there iscrosstalk between the PI3K–Akt–GSK3b pathway, thetranscription factor STAT3 (signal transducer and activa-tor of transcription 3), and other endogenous signalingnetworks that control inflammation, in other words thea7 nicotinic acetylcholine receptor (a7nAChR)-initiatedcholinergic anti-inflammatory pathway and wingless-typemouse mammary tumor virus integration site (Wnt)-related cascades [1,27]. Phosphorylation of STAT3 atTyr705 is important for the production of IL-1b and IL-6in LPS-stimulated innate cells [28]. The inhibition ofGSK3b in various cell types upon stimulation with IFNg

suppresses STAT3 phosphorylation at Tyr705 [29]. Bycontrast, GSK3b inhibition enhances the production ofIL-10, a prototypic inducer of STAT3 phosphorylation[30], and leads to the later enhancement of phosphoryla-tion of STAT3. Thus, it will be interesting to determine howthese opposing effects of GSK3 inhibition on STAT3 phos-phorylation, both by directly suppressing phosphorylationof STAT3 and enhancing STAT3 phosphorylation by aug-mented IL-10, affect the overall anti-inflammatory prop-erty of GSK3 inhibition.

The PI3K–Akt–GSK3b pathway has been demon-strated as an important signaling pathway responsiblefor the acetylcholine-mediated anti-inflammatory response[27]. The cholinergic anti-inflammatory pathway is acti-vated by the interaction of neuronal or locally derived non-neuronal acetylcholine with a7nAChRs on the surface of

immune cells [27,31]. Classic studies by Tracey and col-leagues provided evidence that stimulation of the vagusnerve, and a7nAChRs agonists (acetylcholine and nico-tine), prevented cytokine release and pathological sequelaein experimental sepsis, endotoxemia, ischemia/reperfusioninjury, hemorrhagic shock, arthritis, and other inflamma-tory disorders [32]. Activation of a7nAChR by cotinine, amajor metabolite of nicotine, leads to activation of PI3Kwhich then phospho-activates Akt, which subsequentlyphospho-inactivates GSK3b. Cotinine-induced inactiva-tion of GSK3b does not affect the absolute or phosphory-lated levels of NF-kB but does increase the levels of theCREB transcription factor and by this route augments theproduction of anti-inflammatory IL-10 in monocytes sti-mulated with LPS or other surface-exposed TLR-agonists[27,33]. Such innate suppression by tobacco alkaloids maybe an important clue to the increased susceptibility ofsmokers to multiple bacterial diseases, including thosecaused by Mycobacterium tuberculosis, Legionella pneumo-phila, Moraxella catarrhalis, Neisseria meningitidis, Chla-mydia trachomatis, Porphyromonas gingivalis, andHelicobacter pylori, as well as post-surgical and nosocomialinfections [34]. Indeed, cotinine, a stable molecule thatcirculates at approximately tenfold the levels of nicotine,suppresses the innate response to bacterial stimuli atconcentrations similar to those exposed to second-handsmoke [27].

JAK2 (Janus kinase 2)-mediated signaling is alsoimportant in the cholinergic anti-inflammatory pathway.JAK2 has been reported to be recruited to a7nAChRs onthe surface of macrophages upon cholinergic anti-inflam-matory pathway engagement, leading to STAT3 activationand suppression of the innate response [35]. a7nAChRengagement promotes GSK3b phosphorylation and abro-gates TLR-induced cytokine production [27]. De Jongeet al. found that activation of JAK2 controls the anti-inflammatory potential of nicotinic agonists in macro-phages, and thus JAK2 activation may be beneficial ininfection control. JAK3 has now been established as aGSK3b-dependent negative regulator of inflammationwith suggestions, from JAK3 knockout mice, that JAK3is key in preventing bacteria-induced intestinal inflamma-tion (neutrophil infiltration, IL-17 expression, and epithe-lial damage) [36]. Thus, JAK2 and JAK3 may bedifferentially involved in the regulation of TLR-mediatedinflammatory responses.

Wnt-related signaling events have also been shown toplay an important role in the control of inflammation. Thecanonical Wnt (Wnt/b-catenin) pathway involves phospho-inactivation of GSK3b, which abrogates the degradation ofb-catenin, promoting cytosolic accumulation and nucleartranslocation of b-catenin [37]. b-Catenin stabilization isknown to suppress bacteria-induced inflammation, at leastin part, due to physical interactions with NF-kB, in asimilar manner to IKB (inhibitor of k light polypeptidegene enhancer in B cells) [38]. Wnt3a has been proposed asthe prototypic Wnt innate suppressor, partly due to itsability to suppress proinflammatory cytokine production inresponse to M. tuberculosis [39].

Furthermore, a7nAChR agonism has also been shownto augment Wnt signaling [40]. Thus, further interplay

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between endogenous pathways intended to minimize col-lateral damage consequential to infection is likely toemerge, increasing the possibility of more sophisticatedand specific therapeutic manipulations of the innateresponse to microbial stimuli.

In summary, GSK3b is essential for the generation ofMAMP-induced proinflammatory cytokines. Differentialphosphorylation of GSK3b determines the inflammatoryactivity of this crucial mediator. Ser9 phosphorylation, inparticular, suppresses GSK3b and promotes CREB–CBPinteractions, and the production of IL-10, over the forma-tion of NFkB–CBP complexes. a7nAChR agonists repre-sent efficient endogenous or exogenous GSK3b inhibitorswhich may be exploited therapeutically to alter the innateresponse to bacterial infection.

GSK3b is a promising therapeutic target for the controlof bacteria-induced inflammationAlthough GSK3b is pleiotropic, it is a crucial immunemediator with sufficient unique activities to have madeit a much sought-after therapeutic target over the pastdecade or more. Indeed, GSK3b targeting has been pro-posed as a potential strategy for the treatment of multiplediseases that are primarily inflammatory or contain animportant inflammatory component, including hyperten-sion, diabetes, inflammatory bowel disease, pancreatitis,rhinosinusitis, psoriasis, cirrhosis, hyperoxia-induced lunginjury, myocardial and brain ischemia complications, mul-tiple sclerosis, Alzheimer’s disease, neural inflammation,cystic fibrosis, and even traumatic spinal cord and braininjuries and inflammatory components underlying cancerprogression.

Novel GSK3b inhibitors continue to emerge, such asSB216763, SB-415286, AR-A014418, thiadiazolidinones[e.g., 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione(TDZD-8)], Ro3303544, CHIR98014, and GSK-silencingadenovirus. Several established plant-derived dietarycomponents with reported anti-inflammatory properties,particularly the turmeric polyphenol, curcumin, areGSK3b inhibitors [41]. Of course, lithium has long beenestablished as a potent GSK3b inhibitor. Unfortunately,current inhibitors are not as selective as hoped or are noteffective in vivo, leading to ongoing efforts to generate newones that are truly highly selective [42].

Because GSK3b is a central regulator of the inflamma-tory response, it has been extensively studied as a ther-apeutic target for sepsis, the systemic response to bacteriaand bacteria-derived toxins, and sepsis-induced multipleorgan failure and death. Inhibition of GSK3b was estab-lished as a potent downregulator of TLR-mediated inflam-matory responses in the seminal paper by Martin et al.[17]. This general phenomenon has been shown to hold truein various specific infections. In an elegant study in 2006,Dugo et al. examined the influence of several GSK3b

inhibitors (TDZD-8, SB216763, and SB415286) on LPS-or LPS plus peptidoglycan-induced mortality and organ-specific (kidney, liver, pancreas and neuromuscular) bio-markers of dysfunction. Each inhibitor effectively attenu-ated shock-induced organ damage [43]. Recently, Noh et al.have shown that, in LPS-induced toxemia, GSK3 inhibi-tion by SB415286 induces protein kinase Cd (PKCd) and,

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subsequently, ERK1/2 activation, resulting in increasedexpression of anti-inflammatory IL-10 and abrogated sep-tic shock [44].

Pulmonary and cardiac aspects of septic shock havereceived particular attention. In a murine cecal ligationand puncture sepsis model, glucan phosphate, whichattenuates cardiac dysfunction in this system, suppressessepsis-associated macrophage migration inhibitory factor(MIF) expression and cardiomyocyte apoptosis, concomi-tantly with activation of the Akt/GSK3b pathway [45]. Themanipulation of GSK3b in cardiomyocytes, through theuse of SB216763, dominant negative mutants, or adeno-viral overexpression, has also been shown to control LPS-induced TNF expression [46].

Acute respiratory distress syndrome (ARDS), associatedwith reduced lung surfactants, is the respiratory compo-nent of multiple organ failure. Acyl-CoA:lysophosphatidyl-choline acyltransferase 1 (LPCAT1) is a key enzyme in thesynthesis of dipalmitoylphosphatidylcholine, an importantsurfactant. One potential mechanism of ARDS develop-ment is that, upon LPS-stimulation, LPCAT1 is phos-phorylated by GSK3b, and is then ubiquitinated andtargeted for degradation [47]. Inhaled aerosolized insulin,known to inhibit GSK3b, has been proposed as a treatmentfor sepsis-induced ARDS [48]. In a cecal ligation andpuncture sepsis model, LiCl reduced systemic cytokine(IL-1b, IL-6, and TNF) levels. LiCl treatment also reducedpulmonary 8-iso-prostaglandin F2a (8-ISO), increasedlung superoxide dismutase (SOD) activity, and resultedin a dose-related reduction in pulmonary inflammation.Therefore, although GSK3b activity itself was not mon-itored, LiCl – the classic GSK3 antagonist – is a potentialtreatment for sepsis-induced lung injury [49].

Muscle loss is a well-established consequence of pro-longed sepsis, and LiCl or the thiadiazolidinone derivativeTDZD-8 were shown to suppress efficiently sepsis-inducedprotein breakdown [50].

The role of GSK3b in bacteria-induced bone pathologieshas also been addressed. CD40 is a costimulatory mole-cule, and interaction of CD40 with CD154 on T helper (Th)cells is required for the activation of antigen-presentingcells, such as dendritic cells and macrophages. It is nowestablished that bacterial infection with several osteo-pathogens – P. gingivalis, Staphylococcus aureus, andSalmonella enterica – promotes surface expression ofCD40 on osteoblasts, and this may then contribute toosteoinflammation by interacting with T cells and thegeneration of proinflammatory mediators [51]. Thisenhanced immunological function may well correlate withreduced osteoblastic capacity to lay down new bonematrix. Die et al. have shown that SB216763 suppressesIKBb phosphorylation and decreases the expression ofCD40 as well as the production of IL-6, TNF, and IL-1b

in P. gingivalis LPS-stimulated murine osteoblast-likecells [51]. Thus, GSK3b was identified as a potentialtherapeutic target for infection-induced bone loss. To thisend, Adamowicz et al. have recently employed the GSK3b-inhibitor, SB216763, to abrogate completely P. gingivalis-elicited alveolar bone loss in a murine model of period-ontitis [16] (Figure 3). P. gingivalis has also been asso-ciated with multiple chronic, systemic conditions. In a

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Figure 3. GSK3b inhibition abrogates pathogen-induced bone loss. B6129SF2/J mice aged 8–12 weeks were randomly divided into three control groups and two

experimental groups (n = 5 per group). The control groups were treated with cellulose (sham-infected), 0.02% dimethyl sulfoxide (DMSO), or SB216763 (10 mg/kg)

respectively. The experimental groups were orally infected with Porphyromonas gingivalis 33277 with or without pretreatment with the GSK3 inhibitor, SB216763 (10 mg/

kg). Alveolar bone loss was visualized by methylene blue/eosin staining 6 weeks later. Typical maxillae from (A) sham-infected, (B) P. gingivalis-infected, and (C) SB216763-

treated, P. gingivalis-infected mice are presented. (D) The distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured 6 weeks

postinfection at 14 predetermined maxillary buccal sites (marked by yellow diamonds) in the same three control and two experimental groups. Data are presented as mean

distance CEJ–ABC in mm �SD. Symbols: *, p<0.05 compared to the P. gingivalis-treated group; **, p<0.01 compared to the P. gingivalis-treated group. Reproduced, with

permission, from Adamowicz et al. [16].

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recent study examining P. gingivalis and diabetes, it hasbeen shown that this pathogen translocates from the oralcavity to the liver, infects hepatic cells, and decreasesglycogen synthesis in a manner likely to be driven by aninsulin receptor substrate-1(IRS-1)–Akt–GSK3b signal-ing cascade [52].

Inhibition of GSK3b has been shown to reduce inflam-mation and mortality in other infectious disease models[53–55]. In meningitis studies, GSK3b inhibition (bySB216763 or LiCl) has been shown to suppress LPS-induced proinflammatory cytokines and promote IL-10secretion in rat mixed glia-enriched cortical cultures[56]. Moreover, the anesthetic agent, propofol, suppressesLPS-induced proinflammatory cytokine production inLPS-stimulated BV2 microglia cells in a manner that couldinvolve both TLR4 downregulation and the inactivation ofGSK3b [54]. Microglia are producers of large concentra-tions of shock-inducing cytokines and others have con-firmed that GSK3b inhibition upregulates IL-10 in BV2cells as well as suppressing iNOS [57]. Most recently, it hasbeen shown that the anti-inflammatory properties of lipoicacid on LPS-stimulated BV2 cells are mediated throughGSK3b inactivation [52]. Furthermore, active (unpho-sphorylated) GSK3b is important for adhesion moleculeactivation, endothelial interaction, and monocyte crossingof the blood–brain barrier in response to bacteria and otherinflammatory stimuli [58].

Heat-inactivated S. aureus induces TNF and NO pro-duction in microglia, and the production of both inflam-matory mediators is abrogated by GSK3b inhibition byLiCl or 6-bromo-indirubin-30-oxime [59]. IL-10 is consid-ered to be particularly important in protecting against S.aureus-mediated brain abscesses and is augmented byGSK3b inhibition [59]. Francisella tularensis inducesproinflammatory cytokine production by increasingGSK3b activity. GSK3b is an attractive target for thetreatment of tularemia because LiCl suppresses inflam-matory mediators (IL-6, IL-12p40, and TNF) induced by F.tularensis upon infection of macrophages, and promotessurvival of F. tularensis-infected mice [55].

In addition to a role in controlling acute inflammatoryresponses to bacterial infection, GSK3b can also contributeto long-term vaccine responses. Age-related immunesenescence can reduce the efficacy of antibacterial vac-cines, including the pneumococcal polysaccharide vaccine.It has been established that, in aged animals stimulatedwith Streptococcus pneumoniae, the innate response issuppressed. In spleen-derived rat macrophages, this wasassociated with altered PI3K–Akt–GSK3b signal pathwayactivity and characterized by a reduced proinflammatorycytokine profile and elevated IL-10. Furthermore, inhibi-tion of PI3K restored an appropriate innate response to S.pneumoniae, as did TLR1/2-, TLR2/6-, and TLR4-specificagonists [60].

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GSK3b is thus a potentially effective therapeutic targetfor bacterial shock-induced organ damage, ARDS, bonediseases, meningitis, and tularemia. The development ofmore potent and specific GSK3b inhibitors, alongside dee-per understandings of GSK3b-related signaling events,could lead to a new paradigm of treatment for bacterialdiseases – one that aims prevents to prevent the multiplepathological consequences of robust inflammation, andwhich is likely to be employed in conjunction with con-temporary antibacterial regimens.

Exploitation of GSK3b and related molecules bypathogenic bacteriaBacteria are known to circumvent aspects of the immuneresponse by dysregulating kinase networks, as reviewedby Krachler et al. [61]. For example, the Shigella phospho-threonine lyase, OspF, inhibits ERK and, consequently,suppresses inflammation [61]; the Yersinia spp. acetyltrans-ferase, YopJ, inhibits MAPK, influencing apoptosis [61,62];Pseudomonas aeruginosa ExoS and ExoT differentiallyinfluence Rac1 GTPase activity and strongly influenceinnate cell specific invasion [63]; whereas the nucleoside-diphosphate-kinase, NDK, of P. gingivalis blocks ATP-induced reactive oxygen species (ROS) production in innatecells [64], and the serine phosphatase, SerB, dephosphor-ylates the p65 NF-kB subunit [65]. Thus, targeting ofimmune signaling molecules by bacteria is an establishedphenomenon. Although it has long been known that multi-ple viruses, including adenoviruses and influenza viruses,specifically interfere with the PI3K–Akt–GSK3b axis topromote replication [66], increasing evidence suggests thatone mechanism of immune evasion by bacterial pathogens isalso to target GSK3b-related signaling events.

The phosphoinositide phosphatase, SigD (SopB), of S.enterica phosphorylates Akt and, subsequently, GSK3b

and FoxO3a – a process thought to be important in theinvasion of enterocytes [67]. P. aeruginosa takes advantageof PI3K signaling to invade epithelial cells basolaterally.PI3K activation by P. aeruginosa results in phosphatidy-linositol (3,4,5)-trisphosphate (PIP3) generation, Aktrecruitment, and the generation of PIP3-rich membraneregions, thereby facilitating bacterial entry [68]. The role ofthe downstream regulator, GSK3b, in this invasion systemhas not been directly addressed. A similar requirement forPI3K in host cell invasion has been identified for otherpathogens, including group A streptococci [69], Helicobac-ter pylori [70], Chlamydia pneumoniae [71] and Listeriamonocytogenes [72].

Targeting GSK3b appears to increase the virulence ofsome pathogens. Bacillus anthracis influences GSK3b

phosphorylation in pulmonary epithelial cells, and boththe lethal (LeTx, a MAPK inhibitor) and edema toxins(EdTx, an adenylate cyclase) produced by this bacteriuminhibit AKT phosphorylation. Furthermore, the delivery ofwortmannin, a PI3K–AKT inhibitor, hastens death intoxin-producing B. anthracis-infected mice [73]. S. aureussimilarly exploits GSK3b for virulence, with PI3K–AKT-mediated GSK3b phosphorylation at Ser9 being a keyevent during endothelial invasion [74].

Early in macrophage infection, Yersinia enterocoliticaphosphorylates GSK3b, along with Akt and FKHRL1, in a

214

PI3K-dependent manner, and these phosphorylationevents are reversed later by virulent – but not avirulent– strains. These events are mediated by the Y. enterocoli-tica YopH protein. It has been hypothesized that one keyconsequence of such YopH activity is immunoprotectionvia the downregulation of MCP-1 and the subsequentinhibition of pathogen recruitment to lymph nodes [75].YopH also inhibited T cell proliferation and the PI3K-dependent secretion of IL-2 [75].

Group B streptococci are important etiological agents ofpneumonia, sepsis, and – particularly in newborns –meningitis. Because epithelial infection by Salmonellaenterica serovar Typhimurium (S. Typhimurium) leadsto bacterial SopB-mediated activation of Akt and suppres-sion of caspase 3-induced apoptosis [76], Burnham et al.examined the importance of GSK3b in group B strepto-coccal infection, and found that infection-elicited GSK3b

phosphorylation protected against camptothecin-inducedepithelial apoptosis for at least 18 h [77]. Presumably, thisis a mechanism that promotes bacterial persistence insidetarget eukaryotic cells. Inhibitors of the Akt–GSK3b sig-naling cascade, which is also important in actin dynamics,have been used to block effectively bacterial invasion of,but not attachment to, epithelial cells [77]. Similar datahave been found with group A streptococci [53].

Direct or indirect inactivation of GSK3b by bacteriapresumably suppresses the innate response, thereby pro-viding immune protection to the infecting microbe. Indeed,non-typeable Haemophilus influenza has been shown toactivate the PI3K–Akt pathway in epithelial cells, leadingto p38 MAP kinase inhibition and downregulation of TLR2[78]. S. Typhimurium infection leads to reduced phosphor-ylation, and thus enhanced activity, of GSK3b and degra-dation of b-catenin, thus augmenting the production ofproinflammatory cytokines IL-6 and IL-8 and colonicinflammation [38].

Virulent strains of H. pylori, that are associatedwith gastric ulcers and cancer, secrete high amounts ofvacuolating toxin, VacA – a promising H. pylori vaccinecomponent. VacA, which exerts an influence onmultiple eukaryotic signaling events, inactivates GSK3b

in a PI3K-dependent manner [79,80]. H. pylori isolatesfrom subjects exhibiting regression of gastric intestinalmetaplasia following H. pylori eradication, more effi-ciently phosphorylate GSK3b in gastric adenocarcinoma(AGS) cells than do strains from patients with persistentintestinal metaplasia [81]. Furthermore, the Chlamydo-monas-derived peptide, H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser), has been shown to suppress H. pylori-inducedhyperproliferation and migration of AGS cells by amechanism that involves epidermal growth factor recep-tor (EGFR) activation of the PI3K–Akt pathway andGSK3b inactivation [82]. Thus, GSK3b represents apotential therapeutic target for the prevention of H.pylori-driven gastric cancer.

In mice, pulmonary infection with Klebsiella pneumoniaalso leads to GSK3b phosphorylation, together with multi-ple other effects on eukaryotic kinases, that may well berelevant to immune suppression and avoidance [83].

Finally, there may be scope to exploit bacterial GSK-targeting products therapeutically. For example, the

Box 1. Outstanding questions

� Can more specific, pharmacologically desirable GSK3b inhibitors

be developed?

� What pro- and anti-inflammatory signaling pathways crosstalk

with GSK3b?

� Will conjunctive antibacterial (antibiotics) and anti-inflammatory

(GSK3b) therapies prove more efficacious against shock than

individual therapies?

� Is targeting of GSK3b a common immune evasion strategy of

pathogenic bacteria?

Review Trends in Microbiology April 2014, Vol. 22, No. 4

Streptomyces albus-derived livestock antibiotic, salinomy-cin, that is being developed as an anticancer drug, clearlyphosphorylates Akt, GSK3b, and mammalian target ofrapamycin (mTOR) in head and neck squamous cell carci-noma stem cells [84]. In a similar vein, S632A3, a newglutarimide antibiotic isolated from Streptomyces hygro-scopicus, is a potent inhibitor of inflammation in LPS-stimulated macrophage-like RAW cells. This inflammatorysuppression occurs via inactivation of GSK3b and thedifferential promotion of CREB–CBP interactions overNF-kB which, subsequently, leads to increased IL-10 pro-duction [85]. Several other antibiotics have also beenshown to influence GSK3b activity [86]. There may alsobe occasion to employ GSK3b inhibitors as an adjunct toantibiotic treatment. Streptomyces verticillus-derived bleo-mycin, used in the treatment of various cancers, can inducelung injury. In a murine model, bleomycin-induced pul-monary inflammation, characterized by neutrophil infil-tration, edema, and the accumulation of inflammatorymediators (myeloperoxidase, iNOS, TNF and IL-1b), isdramatically reduced upon GSK3b inhibition by TDZD8[87].

We are beginning to understand that several bacteriaexploit GSK3b and related signaling molecules to subvertthe immune response. It is not yet clear whether or not thisis a widespread strategy among human pathogens.Furthermore, the molecular mechanisms of GSK3b target-ing by microbes remain to be elucidated more fully. Under-standing such pathogen–GSK3b interactions could lead tonovel antibacterial therapeutics.

Concluding remarksGSK3b plays a crucial role in the regulation of pathogen-induced inflammatory responses through the regulation ofpro- and anti-inflammatory cytokine production. GSK3b

has also been aggressively pursued as a therapeutic targetfor the control of infectious diseases. Furthermore, GSK3b

is exploited by pathogenic bacteria seeking to avoid theimmune system. Further molecular understanding of com-plex GSK3b signaling events (Box 1), combined with theemergence of better and more-specific inhibitors, is likelyto lead to more effective interventions.

AcknowledgmentsThe authors acknowledge the support of NIDCR grants DE023633 (HW),DE017921 (RJL) and DE017680, DE019826 (DAS).

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