Glycogen Synthase Kinase 3β Modulates the Inflammatory Response Activated by Bacteria, Viruses, and Parasites

Knowledge of glycogen synthase kinase 3β (GSK3β) activity and the molecules identified that regulate its function in infections caused by pathogenic microorganisms is crucial to understanding how the intensity of the inflammatory response can be controlled in the course of infections. In recent years many reports have described small molecular weight synthetic and natural compounds, proteins, and interference RNA with the potential to regulate the GSK3β activity and reduce the deleterious effects of the inflammatory response. Our goal in this review is to summarize the most recent advances on the role of GSK3β in the inflammatory response caused by bacteria, bacterial virulence factors (i.e. LPS and others), viruses, and parasites and how the regulation of its activity, mainly its inhibition by different type of molecules, modulates the inflammation.


INTRODUCTION
Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved eukaryotic Ser/Thr kinase that regulates a broad range of substrates, which to date includes more than 100 proteins (1) with diverse function such as receptors, structural proteins, signaling molecules, and transcriptional factors, making GSK3 one of the most versatile kinases in the cell (2). This enzyme plays an important role in glycogen metabolism, cell cycle control, apoptosis, embryonic development, cell differentiation, cell motility, microtubule function, cell adhesion and inflammation (3,4). The main isoforms of GSK3, GSK3a and GSK3b (5), are encoded by two different genes gsk3a and gsk3b. The isoforms share an identity of approximately 98% within their kinase domains and 100% similarity, being able to phosphorylate the same substrates (2). GSK3b is activated by phosphorylation at Tyr216 and it is inactivated by phosphorylation at Ser9. Given its involvement as repressors of several pathways such as apoptosis, insulin, phosphoinositide 3-kinase (PI3K), wingless and int-1 (Wnt)/b-catenin, hedgehog, and notch, this enzyme is involved in essentially every major process in the cell (3). Besides, GSK3b regulates many components of the innate and adaptive immune systems due to the modulation of a number of important transcription factors (6)(7)(8).
Activation of the canonical Wnt signaling pathway has previously been shown to recruit Dishevelled (Dvl/Dsh) protein and enhances activation of GSK3b, thus influencing b-catenin degradation and toll-like receptor (TLR)/NF-kB signaling activation (16,17). Also, Wnt5a/receptor tyrosine kinase-like orphan receptor 1 (ROR1)/Dvl2/Akt signaling activates NF-kB promoting the secretion of the cytokines, Interleukin (IL) 6 (IL-6), IL-11, and IL-18 (18). However, the regulatory effects of Wnt/bcatenin signaling are controversial because they depend on different contexts. For example, Wnt3a-frizzeled protein 1(Fzd1) interaction induces b-catenin accumulation and suppresses TLR/ NFkB-dependent pro-inflammatory cytokine production (17). Recently, it was demonstrated in primary monocytes stimulated with lipopolysaccharide (LPS) and in a mice endotoxin model that Wnt3a/Dvl3 signaling functions as a negative regulator of TLR4-mediated inflammation through an increase of GSK3b phosphorylation at Ser9, the accumulation of b-catenin, and a subsequent suppression of NF-kB activity (10) (Figure 1).
TNF receptor (TNFR)-associated intracellular signaling has been established as a pivotal activator of NF-kB and mitogenactivated protein kinases (MAPK) pathways. In addition, TNF signaling serves as negative regulator of noncanonical NF-kB and proinflammatory toll-like receptor (TLR) pathways (19). TNF-induced signaling promotes nuclear accumulation of GSK3b, which promotes anti-inflammatory response by mediating sustained expression of the signaling inhibitor A20 and IkBa synthesis that rapidly terminates TLR4-induced canonical NF-kB signaling, and by suppressing chromatin remodeling (20) (Figure 1). Therefore, activation of TLRs, Frizzleds, and TNFR may lead to the active and inactive state of NF-kB, in the same way the active state of GSK3b leads to the active and inactive state of NF-kB ( Figure 1). All these different and complex responses make it hard to predict the end response of this transcriptional factor in the presence of an inflammatory stimulus. This is an open question and an exciting area for future research.
In order to update and discuss the most recent data published on modulation of the inflammatory response by GSK3b we searched for articles from 2012-2021 in the PubMed database that contained one of the following combination of keywords: GSK3beta inflammation bacterial infections (12 articles), GSK3beta inflammation lipopolysaccharide (104 articles), GSK3beta inflammation peptidoglycan (PGN) (6 articles), GSK3beta inflammation virus infection (25 articles), GSK3beta inflammation parasites infection (6 articles). In case of articles describing the role of GSK3b on inflammation caused by viruses and parasites we decided to include all articles published before and after 2012. Our first selection criteria were mainly based on articles containing original data obtained from cells or whole animal models infected with pathogenic bacteria, viruses and parasites or stimulated with purified PAMPs. Among these articles we only considered those with measurements of inflammatory molecules [e.g. IL-12 subunit p40 (IL-12p40), IL-1a/b, IL-6, tumor necrosis factor-a (TNF-a), nitric oxide (NO)] and, in some cases, those containing measurements of the anti-inflammatory molecules, such as IL-10. We have also included articles in which inflammation was reduced by modulation of GSK3b activity during infection with bacteria, viruses and parasites or PAMPs stimulation. It is worth to mention that this updated review does not include articles already cited in our previous article on the topic (21).

GSK3b in the Inflammatory Response Activated by Bacteria Infection and LPS
In an animal model of keratitis it was observed that infection of corneal cells with Pseudomonas aeruginosa promoted GSK3b activation by decreasing its phosphorylation at Ser9, while the inhibition of GSK3b with SB216763, before P. aeruginosa infection, reduced the cornea inflammation by reducing the expression of IL-6 and IL-1b and by reducing the bacterial load (22). The traditional method for identifying Escherichia coli strains uses antibodies to test for surface antigens: The Opolysaccharide antigens, a component of LPS, flagellar Hantigens, and capsular K-antigens. There are currently ∼186 different E. coli O-groups and 53 H-types (23). Infection of HAEC HA549 with Mycobacerium bovis Bacillus Calmette-Guerin (BCG), a Gram-positive bacillus, or stimulation with LPS from unspecified origin (UO) exhibited TLR2/6 signaling activation and Wnt/b-catenin activity inhibition due to an increase in Axin and GSK3b expression. This combined effect led to a potent inflammatory response characterized by a robust expression of NF-kB and over-regulation of IL-6, IL-1a, IL-2, IL-8, and TNF-a (24). In MMC BV2 cell line activated by LPS (UO), it was observed an important induction of nitric oxide synthase (iNOS) protein expression and NO synthesis, which were reduced by LiCl and SB216763 (25). Moreover, based on the rat septic myocardial injury model, it was found that LPS (UO) induces GSK3b phosphorylation at its active site (Y216) and upregulates FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK3b inhibitors. In vivo, GSK3b suppression consistently improved FIGURE 1 | GSK3b transcriptionally regulates pro-and anti-inflammatory responses. (1). All TLR and TNFR1-associated intracellular signaling promote transcriptional activation of NF-kB. (2a-c) Wnt3/5/Dvl2/Akt signaling also promotes activation of NF-kB. (3) The active state of phosphorylated GSK3b at Tyr216 (GSK3b pTyr216) promotes the activation of NF-kB and at the same time it promotes the inhibition of AP1, CREB, STAT1/3, Nrf2, and b-catenin, leading to proinflammatory cytokines production. (4a, b) During viral infection, TLR3/TRIF/GSK3b signaling pathway positively regulates both pro-inflammatory cytokines and IFN-b production through NF-kB and AP-1 activation and IRF3 activation, respectively. (5a, b) Activation of the canonical Wnt signaling pathway enhances activation of GSK3b, thus influencing b-catenin degradation and NF-kB activation (6). Overexpression of GSK3b inhibits NF-kB transcriptional activity. (7) Upon the activation of TLR (2, 4, 5, or 9)/MyD88/PI3K/Akt signaling, GSK3b is inhibited by phosphorylation at Ser9 (pSer9), leading to an anti-inflammatory response. GSK3b is also phosphoinhibited by S6K, PKA/C, and Dvl3 proteins. (8) TNF induces an increase in the nuclear expression of GSK3b and GSK3b promotes anti-inflammatory responses by mediating expression of the signaling inhibitors that terminate TLR4-induced NF-kB signaling, and by suppressing chromatin remodeling. Lines in red denote a proinflammatory response, while lines in blue denote an anti-inflammatory response. AP-1, activator protein 1; IRF3, interferon regulatory factor 3; CREB, cAMP-response element binding protein; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor erythroid 2-related factor 2; PI3K, phosphoinositide 3-kinase; PKA/C, protein kinase A/C; PKB, protein kinase B, also known as Akt; STAT1-3, signal transducers and activators of transcription 1-3; S6K, ribosomal protein 6 kinase; TLR2, 4, 5, 9, Toll-like receptor 2, 4, 5 and 9; TNFR, tumor necrosis factor receptor; Wnt5, wingless-related integration site member 5:: dishevelled segment polarity protein 1, 2 and 3 (Dvl1/2/3). Lipoproteins (TLR2 ligand), LPS (TLR4 ligand), flagellin (TLR5 ligand), bacterial CpG DNA (TLR9 ligand) and viral double-stranded RNA (TLR3 ligand). cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines IL6, IL1b and TNFa in LPS-induced model was also blocked by inhibition of GSK3b, which curbed both ERK and NF-kB pathways, and suppressed cardiomyocyte apoptosis via activating the AMPactivated protein kinase (AMPK) (26). Long term exposure of male mice C57BL/6 to E. coli O26:B6 LPS in the neonatal period showed memory impairment and increased levels of TNFa and IL-1b as well as increased expression of GSK3b and Tau proteins in the hippocampus and cortex (27) ( Table 1).
In contrast, human stem cells from the apical papilla (SCAP) stimulated with LPS from Porphyromonas gingivalis, a Gramnegative coccobacillus, showed an increase in phosphorylated GSK3b at Ser9 (phospho-GSK3b-Ser9), a residue recognized to inhibit the catalytic activity of the enzyme when it is phosphorylated, that resulted in the expression of IL-1b and TNFa (28). LPS (UO) triggered TLR4/PI3K/Akt signaling pathway that resulted in phospho-GSK3b-Ser9 in MCF7 and MDA-MB-231 breast cancer cell lines. In addition, LPS promoted NF-kB p65 and p50 subunits nuclear translocation, suggesting an increase in the proinflammatory response (29). Also, a study in mice macrophages demonstrated that intracellular osteopontin, an inflammatory cytokine, negatively regulated the inflammatory response caused by LPS from E. coli O111:B4 via inhibition of GSK3b phosphorylation at Ser9 and activation of 4EBP1phosphorylation at Thr37/46 (30) ( Table 1).
It is important to note that the source of LPS used in some studies previously described was not specified. Probably, the LPS used come from different E. coli serotypes having different lipid A-or O-antigens. Therefore, we speculate that opposing inflammatory response observed when GSK3b is inhibited by phosphorylation at Ser9 may depend on the type of cell stimulated and/or the origin and composition of LPS, specifically the type of lipid A and/or O-antigen components.

GSK3b in the Inflammatory Response Activated by Viruses
The transcription factor that binds to the purine-rich (PU)-box (PU.1) is a member of the E-twenty-six (ETS) family expressed exclusively in B lymphocytes, macrophages, and all hematopoietic lineages, except T-cell lines and mature T-lymphocytes (36). Interaction of the regulatory protein p30 from HTLV-I with PU.1 inhibited its DNA binding and transcription activity in THP-1 monocytes. This gave rise to a down-regulation of TLR4 number, an increase in phospho-GSK3b-Ser9, and a reduction of the pro-inflammatory cytokines TNF-a and IL-8, and monocyte chemoattractant protein-1 (MCP-1). HTLV-I p30 also stimulated the release of IL-10, an anti-inflammatory cytokine, after THP-1 monocytes down-regulation of TLR4 (31) ( Table 1).
Infection of triple-transgenic mouse brain model 3xTg-AD with MHV caused a decrease in phospho-GSK3b-Ser9 that correlated with a strong increase in GSK3b activity, an increase in the number of cells expressing the major histocompatibility complex II (MHCII + ), the cluster of differentiation (CD) 4 (CD4 + ), and 8 (CD8 + ), and an increase in the pro-inflammatory cytokines TNF-a, IL-1b, and IL-6 (32). The same pro-inflammatory response was observed when mice 3xTg-AD were treated with LPS from E. coli O55:B5. Altogether, these data suggest that viralor bacterial-mediated infections trigger central nervous system inflammation that in turn may play a comorbidity factor for Alzheimer's disease.
In mouse ESC and EFC cell lines infected with SeV GSK3a and GSK3b activated the antiviral innate immune response by phosphorylation of the b-catenin phosphodegron motif, which subsequently regulates interferon regulatory factor 3 (IRF3)-DNA binding and interferon b (IFN-b) gene expression (33). As GSK3b is considered a negative regulator of b-catenin, this study sheds light to this apparent paradox by demonstrating a different role of b-catenin phosphorylation by GSK3b in retinoic acid-inducible gene 1-like receptor (RLR) signaling. Furthermore, it also confirms that the GSK3b isoform activity alone is not sufficient for the antiviral response (33) ( Table 1). Injection of GSK3b inhibitors in mice caused an in vivo increase in CD8 + cytotoxic T lymphocyte (CTL) function. It was also observed a clearance of MHV68 and LCMV clone 13 and blocking of T cell exhaustion (37). This indicates that beneficial effects of GSK3b inhibitors in viral infections could be due to the activation of the immune system cells.
In humans, the aspartate transaminase (AST) and alanine transaminase (ALT) are specific indicators of liver inflammation and disease severity in a number of chronic liver diseases such as alcoholic and non-alcoholic liver disease, autoimmune liver disease and hepatitis infection (38). Interestingly, it was concluded that HBV infection enhanced b-catenin expression by activating the Akt/GSK3b signaling, which led to a marked increase in phospho-GSK3b-Ser9. Moreover, serum b-catenin levels correlated with elevated levels of ALT and AST but not with viral load, supporting the notion that serum b-catenin may be a useful tool for assessing HBV-related liver diseases (34) ( Table 1). It is likely that during viral infection the inhibited or activated state of GSK3b may promote inflammation, a similar situation to those observed in bacterial infections.

GSK3b in the Inflammatory Response Activated by Parasites
Leishmania spp. is a parasite injected in the human blood as promastigotes by an insect that are phagocytized by macrophages (39). Studies in RAW264.7 murine macrophages and bone marrow-derived monocytes (BMDM) from mice infected with Leishmania donovani have demonstrated that GSK3b is phosphorylated and inhibited by Akt. As a consequence, GSK3b is no longer able to phosphorylates bcatenin and regulates the activation of forkhead box protein O1 (FOXO-1), a pro-apoptotic transcriptional regulator limiting both proinflammatory response and macrophage apoptosis. Macrophages transfected with a constitutively active GSK3b mutant and infected with L. donovani showed a decrease in parasite survival, reduction of IL-10 expression, and stimulation of IL-12 production. Collectively, these findings revealed that the intra-macrophage survival and multiplication of L. donovani is the result of host cell apoptosis and immune response inhibition (35) ( Table 1). A similar situation, in which the inflammatory response is reduced by the GSK3b inhibited state was found in a rat sepsis model induced by either intravenous E. coli LPS or LPS plus Staphylococcus aureus peptidoglycan (PGN) (40,41), when macrophages were stimulated with TLR2, 4, 5, 9 microbial agonists (12) and in other cellular types stimulated with PAMP or infected with bacteria (21). According to these published reports there still remain many open questions and it is evident that more studies are necessary to get a deeper insight on the function of GSK3b in the inflammatory response during parasite infection.

STRATEGIES TO INHIBIT THE INFLAMMATORY RESPONSE BY GSK3b INHIBITION
The GSK3b inhibitors so far designed for the treatment of infectious and non-infectious diseases such as diabetes, cancer, and neurodegenerative disorders have not been fully successful mainly because GSK3b is embedded in multi-protein complexes, which makes the access of inhibitory compounds difficult. Another problem concerns the almost structurally identical active sites of GSK3a and GSK3b. The lack of selectivity of the organic compounds designed against both GSK3 isoforms have excluded a range of promising GSK3 inhibitors from their journey into clinical trial phases (42). Nevertheless, a broad spectrum of GSK3b inhibitors as well as other inhibitors have been reported to inhibit GSK3b and inflammation during infection with bacteria, viruses and parasites as well as LPS and PGN cell stimulation. These inhibitors are classified within the following categories.

ATP-Competitive and ATP-Noncompetitive Inhibitors of GSK3b
Among many competitive inhibitors against GSK3 (42), SB216763 is a potent, selective, and ATP-competitive GSK3a/b inhibitor with an IC 50 of 34.3 nM for both isoforms (43). In a murine model of periodontal bone loss infected with Porphyromonas gingivalis and an in vitro study in MOLC MC3T3-E1 treated with LPS from P. gingivalis the inhibition of GSK3b with SB216763 before infection or LPS stimulation, induced the inhibition of the pro inflammatory cytokines IL-12p40, TNF-a, IL-1b, IL-6, and IL-17 expression (44,45) (Table  S1). Interestingly, in mice treated with LPS from E. coli the compound SB216763 attenuated the NF-kB-mediated expression of IL-6 but not TNF-a (46). Moreover, in a murine acute liver failure (ALF) model induced by D-Galactosamine (D-GalN)/LPS (UO) the inhibition of GSK3b by SB216763 resulted in downregulation of TNF-a, IL-1b, and IL-12p40 expression (47,48) (Table S1).
The small molecule BIO is a highly potent, selective, and reversible ATP-competitive inhibitor of GSK3a/b with IC 50 values around 5 nM. BIO maintains self-renewal in human and mouse ESC, regulates cell mass proliferation, and keeps

Pseudomonas aerugiosa
Animal model of keratitis the undifferentiated state of pancreatic MSC (49). Inhibition of GSK3a/b activity with SB216763 or BIO caused a delay in IkBa degradation and diminished the expression of TNFa in LPS (UO) stimulated neutrophils and macrophages. In vivo inhibition of GSK3a/b with SB216763 or BIO induced a decrease in the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNFa and macrophage inflammatory protein 2 (MIP-2), and release of the alarmins high-mobility group protein 1 (HMGB1) and histone H3 in lungs (50). In the context of viral infection, human hepatic CSCs (Huh7, JFH-1-Huh7, Huh7.5, and MH14C) treated with BIO and infected with HCV suffered an impairment of IFN signaling via inhibition of signal transducer and activator of transcription 1 (STAT1) phosphorylation and degradation (51). Also, a BIO analog 6BIGOE inhibited LPS (UO)-induced release of the pro inflammatory cytokines IL-1b, IL-6, and TNF-a, chemokine IL-8, and prostaglandin (PG) in human primary monocytes while increasing b-catenin and IL-10 levels via intracellular inhibition of GSK3 (52) (Table S1).
Compound CHIR99021 is another cell-permeable, ATPcompetitive inhibitor of GSK3 with IC 50 values of 10 nM and 6.7 nM for GSK3a and GSK3b, respectively (53). CHIR99021 or SB216763 strongly reduced gene expression and secretion of the pro inflammatory cytokines TNF-a, IL-1b and IL-6, the chemokines IL-8 and MCP-1, the intercellular adhesion molecule 1 (ICAM-1), and the vascular cell adhesion molecule 1 (VCAM-1) in adipose tissue and skeletal muscle from women with gestational diabetes stimulated with LPS from E. coli 026:B6 (54) (Table S1). In rats, the non-ATP competitive inhibitor thiadiazolidinone 8 (TDZD-8) or insulin treatment similarly reduced the plasma level of IL-1b and the organ injury/dysfunction caused by LPS from E. coli (O127:B8) plus PGN from S. aureus administration (55). Others non-ATP competitive inhibitors of GSK3b such as benzothiazepinones derivatives 3j and 6j highly attenuate in vivo the LPS (E. coli O55:B5)-induced acute lung injury (ALI) and diminish inflammation response in mice by inhibiting the IL-1b and IL-6 expression (56). Data from this study indicate that 3j and 6j might be potential candidates for further development of inflammation pharmacotherapy in LPS-induced ALI (Table S1).

Plant Bioactive Compounds
Crude methanolic extracts (CME) from Gleichenia truncata used in malarial and melioidosis infection models showed antimalarial and anti-inflammatory effects that were mediated in part by increased GSK3b phosphorylation at Ser9 (57). Apigenin is one of the most widespread flavonoids in plants (58). Apigenin from Matricaria chamomilla suppressed LPS (UO)-induced TNF-a, IL-1b, and IL-6 production in BV2 microglia via activating GSK3b/Nrf2 signaling pathway and suppressing NFkB activation (59). Also, Gastrodin, a natural phenol from Gastrodia elata BI, mediated anti-inflammatory and antiproliferation effects in LPS (E coli O111:B4)-stimulated BV-2 or in primary microglia by modulating the Wnt/GSK3b/b-catenin signaling pathway (60). Phytochemicals such as ISO from Inula helenium, trigonoreidon B from Rigonostemon reidioides, betulin from the bark of birch trees, and xanthohumol from Humululus lupulus were able to induce inhibition of the inflammatory response by phospho-inhibition of GSK3b in BV2 and Raw 264.7 cells, and mice lung, respectively, treated with LPS from E. coli O55:B5 or LPS (UO) (61,(62)(63)(64)(65). The phytopigments curcumin and anthocyanins exhibited anti-inflammatory activity in mice infected with the protozoan parasite Plasmodium berghei and also decreased the inflammatory response induced by LPS (UO) stimulation through an increase in phospho-GSK3b-Ser9 (66,67) (Table S1).

Proteins
Administration of the bioactive protein EPO to mice treated with LPS (UO) inhibited GSK3b and NF-kB activity, caused the reduction of the inflammatory cytokine IL-1b, and enhanced the formation of NO, which in turn caused local vasodilation, inhibited adhesion of platelets and neutrophils, and regulated angiogenesis (68,69). Inhibition of GSK3b by recombinant human vaspin in HPMEC stimulated with E. coli LPS 0111:B4 promoted the reduction of mRNA expression levels of TNF-a, IL-6, VCAM, and E-selectin. In addition, mice subjected to systemic administration of adenoviral vector expressing vaspin were protected against LPS-induced acute respiratory distress syndrome by alleviating the pulmonary inflammatory response and pulmonary endothelial barrier dysfunction, which was accompanied by activation of the Akt/GSK3b pathway, leading to the phospho-inactivation of GSK3b (70) (Table S1).
On the other hand, absence of active GSK3b and reduction of inflammation was observed in the myocardium of Tg mice overexpressing heat Shock 70 kDa protein 12B protein (HSPA12B) that were treated with LPS from E. coli O111:B4 (71). By manipulating the triggering receptor expressed on myeloid cells 2 (TREM2) levels with a lentiviral-mediated strategy, it was demonstrated in microglia that TREM2overexpression following LPS (UO) stimulation led to a markedly reduction in GSK3b activity and tau hyperphosphorylation via suppression of the inflammatory response (72). In a mouse model of LPS from E. coli O55:B55-induced neuroinflammation, both gene deletion and pharmacological inhibition of the calciumactivated potassium channel KCa3.1, which is active in the phenotypic switch that occurs during astrogliosis in Alzheimer's disease and ischemic stroke decreased CNS glia inflammation, including reactive astrogliosis and microglial activation via the Akt/GSK3b signaling pathway (73). Mice with deletion of ZNRF1 (another inflammation model) in their hematopoietic cells displayed an increased resistance to endotoxic and polymicrobial septic shock due to attenuated inflammation via the Akt-GSK3b pathway (74). On the other hand, Syk protein deficiency in MDC resulted in the suppression of LPS (E. coli O111:B4)-induced TNFa and IL-6 but enhancement of IFNb and IL-10 due to GSK3b inactivation (75) (Table S1).
Interestingly, the rLrp of Mycobacterium tuberculosis inhibited proinflammatory cytokine production and downregulated APC function in mouse macrophages via a TLR2-mediated PI3K/Akt pathway activation-dependent mechanism (76). Also it was identified that the protein GRA18 from Toxoplasma gondii, once released in Raw 264.7 macrophages cytoplasm, induced b-catenin up-regulation and the expression of a specific set of genes that are commonly associated with an anti-inflammatory response mediated by the chemokine C-C motif (CC) ligand(L)-17 (CCL-17) and CCL-22 by interacting with GSK3b/PP2A-B56 (77) (Table S1).

Compounds Designed to Selectively Inhibit Proteins Structurally Unrelated to GSK3b
Reduction of inflammatory markers has also been observed by compounds designed to selectively inhibit proteins structurally unrelated to GSK3b in cells stimulated with LPS from E. coli O111: B4, O55:B5, and LPS (UO). Among these types of compounds, it is worth mentioning the cell permeable small molecule AMBMP that is a recognized canonical Wnt/b-catenin activator, the g-secretase and notch inhibitor DAPT, and the novel benzoxazole derivative K313 with immunosuppressive activity toward T cell proliferation. They all inhibit the activity of GSK3b by increasing the relative abundance of phospho-GSK3b-Ser9 (82)(83)(84). (Table S1), suggesting that these molecules may be used as effective GSK3b inhibitors of LPS-induced inflammation.
There are several FDA-approved compounds that indirectly modulate the inflammatory response in cell cultures stimulated with LPS from E. coli (UO) or PGN from S. aureus by increasing the relative abundance of phospho-GSK3b-Ser9. Some of these compounds are: S632A3, a glutarimide antibiotic; fluoxetine, an anti-depressive drug that selectively inhibits the serotonin reuptake; propofol, an enhancer of anesthesia used in surgical trauma; ephedrine hydrochloride, a vasopressor used to treat anesthesia-induced hypotension, sympathectomy or hypotension conditions derived from overdose of antihypertensive drugs; lithium, an element used to treat bipolar disorder that also acts as a tumor suppressor, and dexmedetomidina, that has sedative and analgesic properties (85)(86)(87)(88)(89)(90)(91)(92) (Table S1). Interestingly, it was demonstrated that progressively weighted ladder climbing as a rodent model of resistance-exercise training (RT) ameliorated LPS(UO)-induced cognitive impairment, a forerunner to neuroinflammatory diseases. These improvements in cognitive function occurred in concert with RT-induced IGF-1R/Akt/ GSK3b signaling (93).

GSK3b-GENE KNOCKDOWNS BY siRNAs AND miRNAs
Small interfering RNA (siRNA) and microRNA (miRNA) are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting specific messenger RNAs (mRNAs). However, their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former is highly specific to only one mRNA target, whereas the latter have multiple targets. Clinical trials of siRNA and miRNA-based drugs are already underway (94).
GSK3b-siRNA knockdown diminished expression of TNF-a in LPS(UO)-stimulated RAW 264.7 macrophages (50), inhibited NF-kB activation, enhanced CREB activation in LPS(UO)stimulated acute monocytic leukemia THP-1 cells (95), and diminished expression of IL-1b, IL-6, and TNF-a in WNV infected HGC U251 cells (96). Silencing of phosphatase and tensin homolog (PTEN) and GSK3b with miR-21 induced macrophage efferocytosis and modulated LPS(UO)-induced inflammatory response (97). Recently, it was also shown that GSK3b-gene knockdown with miR-199b caused attenuation of the inflammatory response in THP-1 monocytes treated with LPS (E. coli O26:B6) (98) (Table S1). Furthermore, IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3a and GSK3b in BVE-E6E7 endothelial cells stimulated with PGN from S. aureus. Interestingly, GSK3a-gene silencing resulted in a marked increase in IL-12p40 while GSK3b-gene silencing had an opposite effect (99) (Table S1). These data indicate that regulation of the inflammatory response by GSK3a or GSK3b may depend on the spatio-temporal regulation of both isoforms and the predominance of molecular mechanisms controlling their activity in each type of cell.

STRATEGIES TO INHIBIT THE INFLAMMATORY RESPONSE BY GSK3b ACTIVATION
The phytochemicals salidroside and sappanone A induced inhibition of inflammation in mice stimulated with LPS (UO) through overexpression of GSK3b and reduction of the relative abundance of phospho-GSK3b-Ser9, respectively (100,101). Isoproterenol inhibition of resistin or its siRNA-gene silencing in PDLC stimulated with nicotine and LPS from P. gingivalis had anti-inflammatory effects associated with activation of GSK3b and inactivation of b-catenin (102). The lipid derivative Mar-1 relieved inflammation in PDLC stimulated with LPS from P. gingivalis by GSK-3b activation and b-catenin expression inhibition (103). Interestingly, Nrf2 knockdown in RAW264.7 along with LPS (UO) stimulation caused an increase in the protein level of the glucose transporter type 4 and reduction of Akt and GSK3b phosphorylation. Nrf2 knockdown also induced a high-level secretion of IL6 and IL10. These results demonstrate that Nrf2 regulates inflammation and glucose metabolism besides its classic function in redox regulation (104) ( Table 2). Table 1 show that LPS from P. gingivalis and other type of LPS from different origin promoted opposing inflammatory responses on different cell types (HAEC A-549, MMC BV2, MCF7, MDA-MB-231, and Human SCAP) when GSK3b activity was inhibited. This variability may depend on the cell stimulated and/or the origin and composition of LPS used in these studies, specifically the lipid A and O-antigen components. The same phenotype was observed with viral stimuli. Human or murine cells stimulated with the viral PAMP, p30 protein or infected with viruses MHV or SeV, clearly shows that inhibition of GSK3b suppressed the inflammation. In contrast, data from a clinical study in humans infected with HBV indicate that inhibition of GSK3b also promoted inflammation (Table 1 and Figure 2). In regard to parasites, inhibition of GSK3b suppressed the inflammatory response in murine cells infected with Leishmania donovani (Table 1 and Figure 1). Further studies with different parasites and cells will allow to draw solid conclusions on the role of GSK3b in the inflammatory response.

Data included in
Some of the studies included in Table S1 and Table 2 indicate that stimulation of human adipose tissue and skeletal muscle, RAW 264.7, and BV2 with LPS from E. coli serotype O55:B5 or stimulation of HPEC, mouse macrophages, mice BMDM RAW264.7, MDC, BV2, and primary human monocytes with LPS from E. coli serotype O111:B4 elicited a pro inflammatory response, which was suppressed through the inhibition of GSK3b with different molecules. In contrast to LPS from E. coli serotype 055:B5 and 0111:B4, LPS from P. gingivalis induced a proinflammatory response in human SCAP after inhibition of GSK3b (Table 1). Accordingly, LPS from P. gingivalis elicited a proinflammatory response in HPLC, which was suppressed through the activation of GSK3b with MaR1, isoprotenerol or resistin-siRNA (Table 2). These apparently contradictory results indicate that inhibition of GSK3b may lead to inhibition or activation of the inflammatory response as it is the case when cells are stimulated with LPS from different sources (Figure 2). Molecules with different structural nature were able to control inflammation through GSK3b inhibition (Table S1). However, we also observed that inflammation was inhibited by GSK3b overexpression (i.e. salidroside and maresin-1) or by decreasing the relative abundance of phospho-GSK3b-Ser9 (i.e. sappanone A, isoprotenerol or resistin-siRNA) ( Table 2). GSK3b signaling promoted by infection, activated NF-kB-mediated expression of proinflammatory molecules and inhibited the activity of bcatenin, Nrf2, CREB, STAT1/3, and cJun-AP1, except in MCF7 and MDA-MB-231 stimulated with LPS (Tables 1, S1, 2 and Figure 2). A related situation was found in human periodontal ligament cells stimulated with LPS from P. gingivalis and nicotine, and treated with isoprotenerol or resistin-siRNA. In  Table 2.
Finally, data discussed in this review indicate that inhibition of GSK3b can induce a proinflammatory or anti-inflammatory response during infection, depending mainly on the microbial stimulus. Also, reduction of the inflammatory response does not always lead to GSK3b inhibition. Consequently, GSK3b should be considered as a switch to modulate inflammation. This is important when choosing the type of anti-inflammatory molecule required in each particular case and provides the basis to design new inhibitors.

AUTHOR CONTRIBUTIONS
RC-V and VB-A conceived the idea and wrote the manuscript. OS-G, AG-G, SG-C, and C Á-A carefully and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.