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through FoxO1 and STAT3 SignalingPolarization and Restrains Inflammation1 Promotes Alternative Macrophage Serum- and Glucocorticoid-Inducible Kinase

Shuang Liang and Huizhi WangJunling Ren, Xiao Han, Hannah Lohner, Ruqiang Liang,

ol.2001455http://www.jimmunol.org/content/early/2021/06/23/jimmun

published online 23 June 2021J Immunol 

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Serum- and Glucocorticoid-Inducible Kinase 1 PromotesAlternative Macrophage Polarization and RestrainsInflammation through FoxO1 and STAT3 Signaling

Junling Ren,* Xiao Han,* Hannah Lohner,* Ruqiang Liang,† Shuang Liang,‡

and Huizhi Wang*

Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-a, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre�mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage�priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis�induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization. The Journal of Immunology, 2021, 207: 1�13.

As a heterogeneous population of immune cells, macro-phages are essential for the initiation, maintenance, andresolution of pathogen- or tissue damage�induced inflam-

mation. Macrophages can be activated through diverse mechanismsand thus possess considerable plasticity (1, 2). In response to TLR2/TLR4 and IFNs engagement, macrophages undergo a classical acti-vation and are polarized toward classically activated (M1) macro-phages. In contrast, alternative activation of macrophages, alsocalled alternatively activated (M2) macrophage polarization, can beinduced by IL-4/IL-13 stimulation. Different populations of macro-phages have distinct functions that are phenotypically characterizedby the production of proinflammatory and anti-inflammatory cyto-kines, along with the transcription of specific genes (1). M1 macro-phages produce proinflammatory cytokines, such as TNF-a, IL-6and IL-1b, and inducible NO synthase (iNOS) that will aid in thepromotion of an antibacterial response, whereas M2 macrophages

accelerate the secretion of anti-inflammatory cytokines such as IL-10 and arginase-1, thus possessing immune-suppressive roles (3, 4).Notably, although polarized macrophages can be categorized intothese two main broad clusters with distinct phenotypes, a spectrumof polarization occurs in response to different environmental stimuli,and a plethora of transcription factors are involved in macrophagepolarization in a temporally and spatially dependent manner (5, 6).Previous studies have defined the distinct role of certain transcrip-tion factors underlying macrophage polarization (7�10). For exam-ple, activation of IFN-regulatory factor (IRF) 5, NF-kB subunit p65,and STAT1 promotes M1 polarization, whereas M2 polarization canbe induced by a predominant expression of active STAT3 andSTAT6 (7�10). However, the intracellular signaling pathwaysinvolved in macrophage polarization remain poorly defined,especially under the inflammatory milieu of many chronic infec-tious diseases.

*Department of Oral and Craniofacial Molecular Biology, VCU Philips Institute forOral Health Research, Virginia Commonwealth University, Richmond, VA; †Departmentof Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA;and ‡Department of Molecular Pathobiology, New York University College of Den-tistry, New York, NY

ORCIDs: 0000-0002-5065-4489 (X.H.); 0000-0002-3075-4554 (R.L.); 0000-0001-7692-2106 (H.W.).

Received for publication December 28, 2020. Accepted for publication April 20, 2021.

This work was supported by National Institute of Dental and Craniofacial ResearchGrant DE 026727 (H.W.).

J.R., X.H., H.L., and S.L. performed research; J.R., S.L., and R.L. analyzed the data;and H.W. designed the research, analyzed the data, and wrote the paper. All authors readand approved the final manuscript.

Address correspondence and reprint requests to Dr. Huizhi Wang, VCU Philips Institutefor Oral Health Research, Department of Oral and Craniofacial Molecular Biology,Virginia Commonwealth University, 521 N. 11th Street, Richmond, VA 23298. E-mailaddress: wangh3@vcu.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: ABC, alveolar bone crest; BMDM, bonemarrow�derived macrophage; Cat., catalog; CEJ, cemento-enamel junction; CO2,carbon dioxide; iNOS, inducible NO synthase; IRF, IFN-regulatory factor; M1,classically activated; M2, alternatively activated; qPCR, quantitative PCR; qRT-PCR,real-time qPCR; SGK, serum- and glucocorticoid-inducible kinase; siRNA, smallinterfering RNA.

Copyright©2021 byTheAmericanAssociation of Immunologists, Inc. 0022-1767/21/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2001455

The Journal of Immunology Published June 23, 2021, doi:10.4049/jimmunol.2001455

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Periodontitis is a polymicrobial infection�induced chronic inflam-matory disease characterized by severe gingival inflammation anddestruction of connective tissue surrounding the teeth. A growingbody of evidence suggests that periodontitis may enhance the riskfor several deadly conditions, including cardiovascular diseases, dia-betes, and some upper gastrointestinal cancers (11�14). The humanoral cavity has various ecological niches such as the human gingivalcrevice and periodontal pocket and is inhabited by >700 bacterialspecies as well as other types of microorganisms (15). A plethora ofvirulence factors (i.e., capsule, LPS, fimbriae, and proteinases) fromoral microorganisms continuously stimulate host immune cells toinitiate a wide range of inflammatory responses. A variety ofimmune cells, including neutrophils, macrophages, monocytes, den-dritic cells, and various lineages of T and B cells, have been shownto hierarchically orchestrate the immune�inflammatory responses inthe gingival epithelium (16, 17). Although the function of neutro-phils in oral inflammation has been widely investigated, the role ofmacrophages and their polarization under the periodontal inflamma-tory milieu has received less attention. Several recent studies haveexamined the relationship between macrophage polarization andperiodontal disease status (18�21). The quantity of proinflammatoryM1 macrophages is significantly increased in periodontitis tissues,and the increase of M2 macrophages prevents ligation-induced boneloss in murine periodontitis models (20, 21). In contrast, a recentstudy reported that there is no substantial difference in macrophagepolarization in the gingival tissues of periodontitis patients versushealthy controls (18). These controversial results suggest that moreinvestigations are required to define macrophage polarization ininflamed gingival tissue and to elucidate the underlying regulatorymechanisms involved.Serum- and glucocorticoid-inducible kinases (SGKs) are a class

of serine/threonine kinases belonging to the protein kinase A, G,and C family (22). There are three isoforms of SGK, namely SGK1,SGK2, and SGK3. SGK1 is widely expressed and rapidly respondsto a variety of stimuli, such as follicle-stimulating hormone, osmoticshock, ischemia, TGF-b, glucocorticoids, and mineralocorticoids(22). Like Akt, SGK1 can be fully activated by PI3K through phos-phorylation at T256 by 3-phosphoinositide�dependent protein kinase1 and S422 by 3-phosphoinositide�dependent protein kinase 2. Ourprevious studies have demonstrated that TLR-induced SGK1restrains the intensity and duration of inflammation in an Escheri-chia coli LPS-induced endotoxemia model (23). Moreover, SGK1has been found to facilitate Th2 cell differentiation by negativelyregulating degradation of the transcription factor JunB (24). Recentstudies have also reported that SGK1 deficiency or inhibition sup-presses M2 macrophage polarization in angiotensin II�treated mousecardiac tissue and in an experimental autoimmune encephalomyelitismodel (25, 26). All of these findings suggest the anti-inflammatoryrole of SGK1 in immune�inflammatory responses. However, SGK1was also reported to act as a proinflammatory regulator via promot-ing Th17 development, osteoclastogenesis, and enhancing proin-flammatory cytokine production in other models (27�30). Therefore,the possible regulatory role of SGK1 on macrophage polarization inthe inflammatory milieu is not well established, let alone the under-lying molecular mechanisms.In this study, we investigated the effect of SGK1 on macro-

phage polarization and revealed an undefined signaling path-way involved in using LysM-Cre�mediated SGK1-deficientmice. Moreover, we examined the effect of SGK1 on macro-phage polarization and alveolar bone loss in a Porphyromo-nas gingivalis infection�induced periodontal inflammationmodel.

Materials and MethodsMice and reagents

LysM-Cre1 sgk1fl/fl mice were generated by crossing C57BL/6 mice possess-ing LysM-driven expression of Cre (from The Jackson Laboratory) to loxP-flanked SGK1 mice (provided by Dr. Alexander; Dartmouth University).The littermates without Cre gene from this breeding were used as controls.All the mice were housed in a specific pathogen-free facility at VirginiaCommonwealth University, and the Virginia Commonwealth UniversityInstitutional Animal Care and Use Committee approved all animal protocols.All efforts were made to minimize the number of mice used and to preventanimal distress, pain, and injury. Carbon dioxide (CO2) was used for eutha-nasia of mice. Ultrapure LPS from E. coli 0111:B4 was from InvivoGen(San Diego, CA). Phospho-SGK1 Abs were from Santa Cruz Biotechnology.Total SGK1 Abs were from ProteinTech (Chicago, IL). Total iNOS and argi-nase-1 Abs were from EMD Millipore (Burlington, MA) and BD Biosciences(Franklin Lakes, NJ), respectively). Anti-FoxO1 (S256) Ab was from Abcam(Cambridge, MA). All other Abs were from Cell Signaling Technology (Dan-vers, MA). The SGK1 inhibitor EMD638683 was from MedChemExpress(Monmouth Junction, NJ) and has been characterized and shown to be spe-cific for SGK1 without discernible effects on a panel of 68 other kinases(31). All recombinant cytokines were from PeproTech (Rocky Hill, NJ). Non-targeting pools of small interfering RNA (siRNA) and a mixture of four prev-alidated siRNA duplexes specific for STAT3 (ON TARGETplus) were fromGE Healthcare Dharmacon (Pittsburgh, PA). All plasmids including STAT3-pcDNA3 (catalog [Cat.] 8706) with Flag tag and pcDNA3 Flag hemaggluti-nin (Cat. 10792), which was constructed with hemagglutinin and Flag tags,were from Addgene (Watertown, MA). Mouse IL-12/IL-23 (p40) ELISAMAX Deluxe, Mouse IL-6, and Mouse TNF-a cytokine ELISA kits werefrom BioLegend (San Diego, CA). RNeasy Mini and RNase-free DNase Setwere from QIAGEN (Hilden, Germany). High-Capacity cDNA Reverse Tran-scription Kit and TaqPath quantitative PCR (qPCR) Master Mix were fromApplied Biosystems (Foster City, CA).

Preparation of bone marrow�derived macrophages

Bone marrow�derived macrophages (BMDMs) were generated fromfemoral and tibial bone marrow cells as previously described (32).Briefly, bone marrow was flushed from the femur and tibiae of 8-wk-old LysM-Cre1 sgkfl/fl or the littermate control mice using sterile HBSS(Invitrogen, Carlsbad, CA) and homogenized by repeated passagethrough an 18.5-gauge needle. The cells were washed in PBS, centri-fuged at 1500 rpm for 5 min, and resuspended in RPMI 1640 mediumsupplemented with 10% FBS (R10) (Invitrogen), 50 mM 2-ME, 1 mMsodium pyruvate, 2 mM L-glutamine, 20 mM HEPES, 50 U/ml penicil-lin, 50 mg/ml streptomycin, and 30% L929 culture supernatant. Nonad-herent cells were collected after 24 h and cultured for 7 d in Costarultra-low attachment polystyrene culture dishes with a medium changeon day 4. On day 7, macrophages were �95% F4/801/CD11b1 asdetermined by flow cytometry and ready for further polarized stimula-tion with IFN-g/LPS or IL-4/IL-13.

siRNA and plasmid transfection, cytokine assay, and Western blots

For siRNA and plasmid transfections, BMDMs were transfected withnontargeting control siRNA, siRNA-SGK1, siRNA-STAT3, pcDNA3-STAT3, or pcDNA3 (empty vector control) using LipofectamineRNAiMAX, or Lipofectamine LTX (Invitrogen, Carlsbad, CA), fol-lowing the manufacturer’s protocol. After transfection, the cells weredirectly seeded in either 96- or 6-well plates. After 48 h, cells weretreated with IFN-g/LPS or IL-4/IL-13 for 24 h. The levels of totalSGK1 and exogenous STAT3 were assessed by Western blots. Cellswere lysed in radioimmunoprecipitation assay buffer containing phos-phatase inhibitors for Western blot assays. Images were acquiredusing the ImageQuant LAS 4100 (GE Healthcare Life Sciences, Pitts-burgh, PA). For cytokine assays, BMDMs were cultured in mediumwithout L929 culture supernatant for 24 h. Cells were then eitheruntreated or treated with IFN-g (10 ng/ml) and LPS (100 ng/ml) forM1 macrophages or IL-4 (10 ng/ml) and IL-13 (10 ng/ml) for M2macrophages. Culture supernatants were collected after 24-h stimula-tion. Cytokine concentrations were determined by ELISA followingthe manufacturer’s instructions.

RNA isolation and real-time qPCR

Total RNA was isolated using RNeasy Mini Kit with RNase-Free DNaseSet according to the manufacturer’s instructions. RNA samples were reversetranscribed using a High-Capacity cDNA Reverse Transcription Kit. Real-time qPCR (qRT-PCR) analysis was performed using specific primers formouse sgk1 (Mm00441380), Ym1/Chil3 (Mm00657889_mH), Mgl-1/Clec10a

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(Mm00546124), Arg-1 (Mm00475988_m1), iNOS (Mm00440502_m1),Fizz-1/Retnla (Mm00445109), and stat3 (Mm01219775_m1 (33) in an AppliedBiosystems 7500 system using TaqPath qPCR Master Mix (Applied Biosys-tem). Relative levels of gene expression were determined using GAPDH(Mm99999915_g1) as the control (34).

Cell staining and flow cytometry

BMDMs were either untreated or treated with IFN-g/LPS or IL-4/IL13(10 ng/ml, PeproTech) and were then washed twice with Flow Cytom-etry Staining Buffer and treated with Fc block for further flow cytome-try assays. All the reagents below for flow cytometry assay are fromeBioscience (San Diego, CA). After washing and blocking, cells wereincubated with the following fluorescently labeled anti-mouse Abs:PerCP-cy5.5�conjugated F4/80 (45-4801-82); allophycocyanin-conju-gated CD11b (17-0112-81) or FITC-conjugated CD11b (11-0112-82);and PE-conjugated CD206 (12-2061-80), following the manufacturer’sinstructions. For intracellular staining, cells were fixed with IC Fixa-tion Buffer for 30 min, followed by washing twice with permeabiliza-tion buffer and incubated with iNOS-allophycocyanin (Thermo FisherScientific, Waltham, MA; no. 17-5920-80) for 30 min. Stained cellswere then washed and analyzed on LSRFortessa flow cytometer (BDBiosciences). Data were analyzed with the FlowJo software.

P. gingivalis�induced periodontal inflammation model andimmunohistochemistry

P. gingivalis 33277 was cultured anaerobically in trypticase soy brothsupplemented with yeast extract (1 mg/ml), hemin (5 mg/ml), and men-adione (1 mg/ml) and was grown at 37�C in Anoxomat jars (SpiralBiotech) under anaerobic conditions (10% H2, 10% CO2, and 80% N2,

plus palladium catalyst). For the P. gingivalis�induced periodontalinflammation model, the endogenous oral microbiota was suppressedin 10�12-wk-old C57/B6/J mice by sulfamethoxazole (800 mg/ml) andtrimethoprim (400 mg/ml) provided ad libitum in water for 5 d. Themice then received pure drinking water for 5 d. Alveolar bone losswas induced by oral inoculation with 1 � 109 CFU of P. gingivalissuspended in 100 ml of PBS with 2% carboxymethylcellulose. Inocula-tions were performed six times at 2-d intervals. An experimental groupwas also i.p. administered EMD638683 (15 mg/kg) with each inocula-tion and every other day thereafter until euthanization. The effect ofEMD638683 on the growth of P. gingivalis in vitro was also assessed(Supplemental Fig. 1A). Sham-infected and vehicle control mice werealso established. The mice were euthanized with CO2 and cervical dis-location 42 d after the final infection. Maxillary gingiva from theupper half of each jaw was harvested for RT-PCR assay, and the otherhalf were for Western blot assay. Fresh gingival tissues were immersedin RNAlater (Ambion, Austin, TX; Cat. AM7020) or radioimmunopre-cipitation assay buffer with protease and phosphatase inhibitors (Milli-poreSigma, Burlington, MA; Cat. P8340 and P0044) (1:100) and thenstored at �20�C for further RT-PCR or Western blot assay, respec-tively. The expression of inflammatory cytokines, including TNF-a,IL-6, and IL-12P40 was determined by qRT-PCR using FastStart Uni-versal SYBR Green Master (Rox) (Roche, Basel, Switzerland), accord-ing to the manufacturer’s instructions. The primer sequences used foramplification are shown in Supplemental Fig. 1B. Gene expressionwas normalized to the GAPDH exogenous control and measured usingthe DDCT method as described in a previous study (35). Alveolar boneloss was measured in millimeters at 14 predetermined points on themaxillary molars of defleshed maxillae as the distance from thecemento-enamel junction (CEJ) to the alveolar bone crest (ABC).Bone loss was visualized by methylene blue/eosin staining and quanti-fied using a Nikon SMX 800 dissecting microscope (40�) fitted with aBoeckeler VIA-170K video image marker measurement system. Theresults were expressed as the mean with SD. The lower jaws of themice were fixed in 4% paraformaldehyde, decalcified in Immunocalsolution (StatLab Medical Products, Lewisville, TX) for 15 d, andembedded in paraffin wax for immunohistochemistry assay. The paraf-fin-embedded tissue blocks were freshly cut into 4-mm mesiodistal sec-tions for subsequent immunostaining with mouse CD68, CD163,CD206, and F4/80 Abs, followed by secondary FITC or Alexa Fluor350�conjugated Abs. PBS containing normal rabbit serum was used assham control. Images were captured using a fluorescence microscope(Nikon Eclipse E800) and processed by Neurolucida. Tissue sectionswere also stained with STAT3 Ab with visualization using 3,3'-diami-nobenzidine, and the images were captured using X-Cite FluorescenceLED Boost ZEISS microscope bright field with objective magnifica-tion of 20� and analyzed by counting positively stained cells usingImageJ software. ImageJ (National Institutes of Health) analysis was

performed as per the standard recommended algorithm (36). Imageswere imported and processed with color deconvolution, adjusted bythreshold and measurement setting, and got the percentage of posi-tive staining areas.

Statistical analyses

The statistical significance of differences among groups was evaluated withthe ANOVA and the Tukey multiple comparison test using the InStat pro-gram (GraphPad). Differences between groups were considered significant atthe level of p # 0.05.

ResultsSGK1 differentially regulates the transcription of lineage-specificgenes in macrophages

Our previous study has demonstrated that inhibition of SGK1aggravates TLRs-mediated inflammatory responses (23). Toexplore the possible role of SGK1 in macrophage polarization,resting macrophages were treated with IFN-g/E. coli LPS toinduce M1 or IL-4/IL-13 to induce M2 macrophage pheno-types. We first examined the response of SGK1 to the chal-lenge of IFN-g/LPS or IL-4/IL-13. We found that stimulationwith either IFN-g/LPS or IL-4/IL-13 leads to the activation ofSGK1, represented by the phosphorylation of NDRG, a sub-strate of SGK1 in macrophages, indicating SGK1 might beinvolved in polarization of macrophage cells (Fig. 1A, 1B).We next used EMD638683, a specific pharmacological inhibi-tor of SGK1, to investigate the effect of SGK1 on the tran-scription of specific genes in macrophage polarization. Theoptimum concentration of EMD638683 was first determinedby measuring its impact on phospho-NDRG and cell viability.We found that 10 mM EMD638683 robustly enhances the pro-duction of LPS-induced TNF-a (Fig. 1C) and reduces phosphor-ylation of NDRG1 (Fig. 1A) but does not cause substantial celldeath after 24-h treatment (Fig. 1D). We therefore used 10 mMEMD638683 throughout this study. Next, we found that inhibi-tion of SGK1 leads to a significant increase of iNOS mRNAunder M1 macrophage�inducing conditions (Fig. 1E), whichwas confirmed by flow cytometry analysis (Fig. 1H, 1I). In con-trast, under M2 macrophage�inducing conditions, inhibition ofSGK1 significantly reduces expression of arginase-1, resistin-like a (Retnla and Fizz1), chitinase-like protein 3 (Chil3 andYm-1), and macrophage galactose-type C-type lectin 1 (Mgl-1)(Fig. 1J). We further confirmed the expression of iNOS andarginase-1 by Western blots (Fig. 1F). To account for possibledifferences in protocols for the in vitro differentiation of macro-phages, we also used LPS only to polarize macrophages andtested the expression of iNOS. Similar trends of iNOS were alsoobserved (data not shown) under the M1 macrophage�inducingconditions. In addition, we also examined the expression ofCD206, a prototypical M2 macrophage molecular marker, and wefound SGK1 inhibition significantly decreases the expression ofCD206 upon the treatment with IL-4/IL-13 (Fig. 1K, 1L). Takentogether, these results suggest that SGK1 differentially regulatesthe expression of sublineage-specific genes in M1 and M2macrophages.

SGK1 suppresses the production of M1 macrophage inflammatorycytokines and promotes IL-10 in M2 macrophages

Given the distinct inflammatory cytokines produced by M1and M2 macrophages, we next investigated the possible regu-latory role of SGK1 in the production of inflammatory cyto-kines. Our results showed that inhibition of SGK1 withEMD638683 significantly elevated TNF-a, IL-6, and IL-12 atmessage and protein levels under M1 macrophage�inducing

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conditions (Fig. 2A�D). Notably, the inhibition of SGK1 alsoreduced IL-10 at the message level under M2 macrophage�primingconditions (Fig. 2E). To avoid the possible nonspecific effects of thechemical inhibitor, we used prevalidated siRNA to silence SGK1 (Fig.2F, 2G), and we found that SGK1 silencing enhances the productionof proinflammatory cytokines (TNF-a, IL-6, and IL-12) under M1macrophage�inducing conditions (Fig. 2H�J). Moreover, silencing of

SGK1 also leads to a significant increase in iNOS expression (Fig.2K) and a decrease in arginase-1 (Fig. 2L). This is consistent with ourresults using SGK1 inhibitor and consolidates the effect of SGK1 onmacrophage differentiation. The distinct effects of SGK1 on the pro-duction of M1 and M2 inflammatory cytokines and sublineage-specificgenes suggest that SGK1 suppresses M1 but promotes M2 phenotypesand thus regulates macrophage polarization.

FIGURE 1. SGK1 differentially regulates the transcription of lineage-specific genes in macrophages. BMDMs were stimulated with IFN-g/LPS or IL-4/IL-13 for 24 h in the presence or absence of 10 mM EMD638683, and then whole cell lysates were collected for analysis. (A) Western blots showing theexpression of p-NDRG and GAPDH. (B) The ratio of p-NDRG to GAPDH was determined by densitometry quantification assay. BMDMs were pretreatedwith a serial concentration of EMD638683 followed by the stimulation of E. coli LPS, and cell-free supernatants were harvested. (C) ELISA showing the pro-duction of TNF-a in LPS-stimulated BMDMs in the presence or absence of EMD638683. (D) Percentage of viable cell numbers was calculated afterBMDMs were treated with trypan blue for 24 h. (E) qRT-PCR showing the expression of M1-specific macrophage marker, iNOS. (F) Western blots showingthe dynamic expression of iNOS and arginase-1 in macrophages under M1- or M2-inducing conditions. (G�I) BMDMs were stimulated with IFN-g/LPS for24 h, and monensin was added after 6-h stimulation; then cells were analyzed for intracellular iNOS by flow cytometry. (G) Gating strategy used to obtainpure macrophage populations by selecting CD11b and F4/80 high�expressing cells. (H and I) Flow cytometry showing the influence of EMD638683 onexpression of iNOS. (J�L) BMDMs were stimulated with IL-4/IL-13 for 24 h in the presence or absence of EMD638683, and then qRT-PCR and flowcytometry were used for the next analysis. (J) qRT-PCR showing the effect of EMD638683 on expression of M2-specific macrophage markers arginase-1,Ym-1, Mgl-1, and Fizz1. (K and L) Flow cytometry showing the effect of EMD638683 on the expression of CD206. All the blots shown are representative ofthree to five biological replicates. All data represent the arithmetic mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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FIGURE 2. SGK1 suppresses the production of M1 macrophage inflammatory cytokines and promotes IL-10 in M2 macrophages. BMDMs werepretreated with 10 mM EMD638683 for 2 h or transfected with specific sgk1 siRNA for 48 h and then stimulated with IFN-g/LPS or IL-4/IL-13for 24 h. (A�D) qRT-PCR and ELISA showing the production of TNF-a, IL-6, and IL-12 under the challenge of IFN-g/LPS at message (A) andprotein levels (B�D). (E) qRT-PCR showing the effect of EMD638683 on the transcription of IL-10 under the challenge of IL-4/IL-13. (F and G)qRT-PCR and Western blots showing the expression of SGK1 at message (F) and protein (G) levels, respectively, after treatment with sgk1 siRNA.(H�J) ELISA showing the production of TNF-a, IL-6, and IL-12 in sgk1 gene silencing cells under the challenge of IFN-g/LPS. (K and L) qRT-PCR showing the effect of sgk1 gene silencing on the expression of iNOS and arginase-1 under the challenge of IFN-g/LPS or IL-4/IL-13, respec-tively. All the data were generated in the same experiment and represent the arithmetic mean ± SD of three independent experiments. **p < 0.01,***p < 0.001, ****p < 0.0001.

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FIGURE 3. Myeloid lineage-specific deletion of sgk1 leads to the enhancement of M1-like macrophage polarization. LysM-Cre1 sgk1fl/fl

mice were generated by the breeding of LysM-Cre1 mice with sgk1loxp/loxp mice. Bone marrow from SGK1 knockout and littermate controlmice were used to generate BMDMs followed by the treatment with IFN-g/LPS or IL-4/IL-13. (A) Expression of p-NDRG was measured byWestern blots to assess the knockout efficacy of SGK1 in BMDMs from LysM-Cre1 sgk1fl/fl mice. (B) qRT-PCR showing the message levelsof iNOS and the proinflammatory cytokines TNF-a, IL-6, and IL-12 under the challenge of LPS/IFN-g. (C�E) Western blots and flow cytome-try showing the expression of iNOS in the littermate control and sgk1 knockout BMDMs under the M1 macrophage�inducing conditions.(F�H) ELISA showing the production of proinflammatory cytokines TNF-a, IL-6, and IL-12 under the M1 macrophage�inducing conditions.(I) qRT-PCR showing the different levels of arginase-1, Ym-1, and macrophage galactose-type C-type lectin 1 in the littermate control andsgk1-deficient BMDMs under the M2 macrophage�inducing conditions. (J and K) Western blots showing the expression of arginase-1 in sgk1knockout BMDMs is remarkably lower than in littermate control BMDMs. All the blots shown are representative of three to five biologicalreplicates. All the data represent the arithmetic mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001,respectively.

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Myeloid lineage-specific deletion of sgk1 promotes macrophagepolarization to M1 phenotype

To avoid the possible off-target effects of sgk1 siRNA, we nextused sgk1-deficient BMDMs from LysM-Cre1 sgk1fl/fl mice,which were generated in our laboratory by crossing LysM-Cre1

mice with sgk1loxp/loxp mice and exhibit a loss of sgk1 gene inBMDMs (Supplemental Fig. 2A). As expected, phosphorylationof NDRG was abrogated in macrophages from LysM-Cre1

sgk1fl/fl mice, suggesting SGK1 protein was expunged inBMDMs (Fig. 3A; Supplemental Fig. 2A). Using BMDMs fromLysM-Cre1 sgk1fl/fl mice, we found that SGK1 deficiency leadsto a significant increase of iNOS at message levels under M1macrophage�priming conditions (Fig. 3B). The results were fur-ther confirmed by Western blots and flow cytometry assay (Fig.3C�E). Moreover, compared with the littermate control,SGK1-deficient macrophages produced a significantly higheramount of proinflammatory cytokines, including TNF-a, IL-12,and IL-6 at message and protein levels (Fig. 3F�H). In contrast,SGK1-deficient macrophages produced significantly lower lev-els of arginase-1 and two other M2 macrophage�specific genes,Fizz1 and Ym1, as compared with the littermate control underthe M2 macrophage�priming conditions (Fig. 3I). The expres-sion of arginiase-1 was also confirmed by Western blots (Fig.3J, 3K). Taken together, these results suggested that SGK1 can

indeed suppress the expression of M1 while promoting M2 phe-notypes in the process of polarization.

SGK1 differentially regulates the expression of FoxO1 and STAT3 inmacrophages

Previous studies have demonstrated that there are a plethora of tran-scription factors comprehensively controlling macrophage polariza-tion, such as M1-promoting transcription factors NF-kB, STAT1,and IRF5 and M2-promoting transcription factors IRF4, PPARg,STAT3, and STAT6 (2, 9). We next investigated the effect ofSGK1 deletion on the activity of prototypical transcription factors inmacrophage polarization. Using LysM-mediated sgk1-deficient mac-rophages from the mice described above, we found there was aslight increase of NF-kB, but we did not observe substantialchanges of STAT1, IRF5, or PPARg in sgk1�/� macrophages ascompared with the littermate control macrophages under M1 macro-phage�priming conditions (data not shown). Surprisingly, theexpression of FoxO1 was remarkably higher in sgk1�/� macro-phages than in littermate control macrophages (Fig. 4A). Moreover,SGK1 deficiency led to a decrease of FoxO1 phosphorylation underM1 macrophage�inducing conditions (Fig. 4A). Because thedecreased phosphorylation of FoxO1 has been demonstrated toresult in sequestration of FoxO1 in the nucleus and to enhance itsactivity (17, 37), we next investigated if SGK1 deficiency can

FIGURE 4. SGK1 differentially regulates the expression of FoxO1 and STAT3 in macrophages. BMDMs generated from LysM-Cre1 sgk1fl/fl mice or thelittermate control mice were stimulated with IFN-g/LPS or IL-4/IL-13 for the time indicated at day 7, and then either the whole cell lysates or the cytoplasmicand nuclear fractions were harvested for Western blot assay. (A) Western blots showing the expression of iNOS, phospho- and total FoxO1, and GAPDHunder M1 macrophage�inducing conditions. (B and C) Expression of FoxO1 upon the challenge of IFN-g/LPS was measured by Western blots, showing thetranslocation of FoxO1 from the nucleus to the cytoplasm, which restrains LPS-induced inflammatory responses. GAPDH and Histone H3 were used as cyto-plasmic and nuclear controls, respectively (B). (C) Western blots showing that SGK1 deficiency abrogates the capability of FoxO1 to translocate from thenucleus to the cytoplasm. (D) Western blots showing the expression of arginase-1, phospho- and total STAT3, phospho- and total STAT6, and GAPDH underM2 macrophage�inducing conditions. All the blots shown are representative of three to five independent experiments.

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suppress cytoplasmic translocation of FoxO1, sequester it in thenucleus, and thus enhance its activity. As shown in Fig. 4B and4C, whereas cytosolic FoxO1 in macrophages from normal litter-mates continuously increased after 6- and 24-h stimulation withLPS/IFN-g, the nuclear FoxO1 concurrently decreased during thistime (Fig. 4B), indicating FoxO1 indeed translocated from thenucleus to the cytoplasm. However, in macrophages from sgk1�/�

mice, FoxO1 translocation to the cytoplasm was abrogated, whichresulted in relatively more FoxO1 in the nucleus (Fig. 4C). Theseresults are consistent with our and other previous studies (17, 38�40)

showing that cytoplasmic translocation of FoxO1 is a key anti-inflammatory mechanism to restrain LPS-induced inflammatoryresponses. In contrast, we found that SGK1 deficiency decreasesSTAT3 expression and phosphorylation under M2 macrophage-�inducing conditions (Fig. 4D), which is also consistent with previ-ous studies showing that SGK1 regulates NF-kB and STAT3 underdifferent contexts (23, 25). Interestingly, no substantial differenceswere observed in the expression and activity of STAT6, a prototypi-cal transcription factor driving macrophage polarization toward M2phenotypes (Fig. 4D). Taken together, our results demonstrated that

FIGURE 5. FoxO1 and STAT3 regulate the expression of lineage-specific macrophage genes. BMDMs were pretreated with a FoxO1-specific inhibitor,AS1842856 (10 mM), or transfected with specific stat3 siRNA or a plasmid encoding STAT3 for 48 h and then stimulated with IFN-g/LPS or IL-4/IL-13 for24 h. (A�D) qRT-PCR showing the effect of AS1842856 on iNOS (A), TNF-a (B), IL-6 (C), and IL-12 (D) at mRNA levels, indicating that FoxO1 inhibitionsuppresses macrophage polarization toward the M1 phenotype. (E�G) qRT-PCR showing the silencing of stat3 (E) significantly decreases argniase-1, Fizz1,Ym1 (F), and IL-10 (G) at mRNA levels under M2 macrophage�inducing conditions. (H and I) Western blots and qRT-PCR showing that overexpression ofSTAT3 (H) remarkably increases the expression of arginase-1 (H) and significantly increases arginase-1, Fizz1, and Ym1 at message levels (I) under M2 mac-rophage�inducing conditions. All the data were generated in the same experiment and represent the arithmetic mean ± SD of three independent experiments.***p < 0.001, ****p < 0.0001.

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SGK1 can modulate the phosphorylation and expression of FoxO1and STAT3 in macrophages under different polarization conditions.

FoxO1 and STAT3 regulates the expression of lineage-specificmacrophage genes

Given that SGK1 deficiency leads to a remarkable alteration ofFoxO1 and STAT3, we next investigated if FoxO1 and STAT3affected the phenotypes of M1 and M2 macrophages, respectively.We found that inhibition of FoxO1 with a specific chemical inhibi-tor, AS1842856, indeed decreased SGK1 deficiency�enhancediNOS (Fig. 5A), as well as TNF-a, IL-6, and IL-12P40 under M1-inducing conditions (Fig. 5B�D). Moreover, silencing of STAT3 byprevalidated siRNAs (Fig. 5E) significantly diminished the transcrip-tion of M2-specific genes arginase-1, Fizz1, and Ym1 (Fig. 5F), aswell as IL-10 (Fig. 5G) under M2-inducing conditions. In addition,a plasmid encoding exogenous STAT3 was used to exclude the pos-sible off-target effects of the siRNA and confirm the role of STAT3in M2 macrophage polarization (Fig. 5H). We found that comparedwith control macrophages, STAT3-overexpressing macrophages hada remarkable increase in the expression of arginase-1 (Fig. 5H) andsignificantly higher levels of Fizz1 and Ym1 transcripts (Fig. 5I)under M2 macrophage�priming conditions. Taken together, theseresults clearly show that FoxO1 and STAT3 are involved in SGK1-mediated M1 and M2 macrophage polarization, respectively.

SGK1 promotes M2 macrophage polarization and protects againstalveolar bone loss in P. gingivalis�infected mice

A P. gingivalis�induced murine periodontal inflammation modelhas been widely used in periodontitis research. Inflamed gingi-val tissue is often densely colonized with microorganisms andinfiltrated with various inflammatory cytokines, such as IFN-g,IL-4, and IL-13, which forms a niche for macrophage polariza-tion (41�43). Previous studies have shown that P. gingivalisinfection leads to inflammatory responses through the activationof TLR2 and/or TLR4 on the surface of immune cells (44�46).To further investigate the potential effects of SGK1 on macro-phage phenotypes in P. gingivalis�infected gingival tissues, wefirst examined the expression of putative macrophage markers,CD68 and F4/F80, and the prototypical M2 molecular markers,CD163 and CD206. As shown in Fig. 6A�C, infection of P.gingivalis led to an increase of total macrophages in gingivaltissues, represented by the enhanced expression of CD68 andF4/F80 (Fig. 6A, 6B). Interestingly, mice pretreated with SGK1inhibitor demonstrated significantly lower expression of CD206and CD163 than the mice treated with P. gingivalis only (Fig.6A, 6C). In contrast, we observed that mice pretreated with theSGK1 inhibitor produced significantly higher amounts of IL-6mRNA and protein as compared with the mice treated with P.gingivalis only (Fig. 6D, 6E). However, there were no substantialdifferences observed at the mRNA and protein levels for TNF-aor IL-12P40 (Fig. 6E). This discrepancy between in vitro andin vivo responses to SGK1 inhibition may be due to the phase ofinflammation, differential dynamics of cytokines synthesis, andimportantly, the different sampling of cells, indicating that compli-cated regulatory mechanisms are involved in the progression ofchronic inflammation. In addition, we also examined whetherSGK1 affects inflammation-mediated alveolar bone loss, which isa characteristic of P. gingivalis�induced periodontitis. As shown inFig. 6F and 6G, P. gingivalis infection induced a significant boneloss, and the inhibition of SGK1 significantly aggravated the sever-ity of the bone loss as determined through the measurement of theCEJ�ABC distance (Fig. 6F, G). Taken together, these findingssuggest that SGK1 restrains periodontal inflammation throughfacilitating M2 macrophage polarization and curtailment of M1

polarization and protects against oral bone loss in P. gingiva-lis�infected mice.

SGK1 regulates the expression of STAT3 in mouse gingival tissues

Given that we have demonstrated STAT3 as a key transcription fac-tor involved in SGK1-mediated macrophage polarization in a cellculture model, we next examined if SGK1 inhibition affected theexpression of STAT3 in P. gingivalis�infected mouse gingival tis-sues. As shown in Fig. 7, compared with the sham control mice, P.gingivalis�infected mice had a significantly higher level of STAT3in gingival tissues (Fig. 7A, 7B), which is consistent with our resultsshowing that P. gingivalis infection enhances the expression of M2macrophage molecular markers (Fig. 6A). However, the inhibitionof SGK1 using a chemical inhibitor, EMD638683, led to a signifi-cant decrease of STAT3 in P. gingivalis�infected mouse gingivaltissues, as compared with the sham control mice or mice treatedwith EMD638683 only (Fig. 7A, 7B). These results suggest thatSGK1 is necessary to maintain the expression of STAT3, throughwhich it promotes macrophage polarization to M2 phenotypes.

DiscussionPlasticity and polarization of macrophages are key to the immuneresponses during inflammation. In this study, we investigatedwhether SGK1 is involved in macrophage polarization. We havedemonstrated that SGK1 is indeed a major factor in defining macro-phage polarization. Inhibition of SGK1 increases the characteristicgene expression of M1 macrophages and enhances proinflammatorycytokine production in BMDMs. Moreover, deficiency of SGK1leads to less secretion of IL-10 and reduces M2-specific geneexpression, indicating SGK1 is essential for the maintenance ofalternative macrophages. We also found that SGK1 suppresses M1but promotes M2 macrophage polarization and protects against alve-olar bone loss in P. gingivalis�infected mice. In addition, we dem-onstrated that FoxO1 and STAT3 are regulated by SGK1 and drivemacrophage polarization to M1 and M2, respectively. Therefore,SGK1 is key for macrophage plasticity and function, which couldbe an interventional target to manipulate the polarization of macro-phages and conversion of one subset of macrophages to the other(Fig. 8).Many transcription factors are involved in immune�inflammatory

responses of macrophages to various stimuli. In this study, althoughthe function of SGK1-mediated activation of FoxO1 and STAT3 inmacrophage polarization was demonstrated, we also found thatSGK1 inhibition or deficiency slightly enhances the expression ofNF-kBp65 (data not shown). Thus, we cannot exclude the possibil-ity that NF-kBp65 is also partially involved in macrophage polariza-tion, although it may not be a major player. In contrast, Aktsignaling is similar to SGK1 and has been reported to be key inmacrophage polarization (47�49). Therefore, it is possible that thecompensation of SGK1 signaling by Akt may impact macrophagepolarization in our models. However, we did not observe any signif-icant changes for Akt phosphorylation (data not shown), indicatingthat SGK1 could be a nonredundant regulator in the process of mac-rophage polarization. Thus, our results indicated that combinatorialexpression of various transcription factors and hierarchical activation(or repression) could be a paradigm to specify macrophage pheno-types and thus direct macrophage polarization. Further investigationsof the contribution of specific transcription factors to macrophagepolarization and their interactions are warranted to characterizeSGK1-mediated signaling and the subsequent application for thecontrol of inflammatory diseases.A delicate balance between pro- and anti-inflammatory mecha-

nisms is critical in the oral mucosa immune system to effectively

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FIGURE 6. SGK1 promotes M2 macrophage polarization and protects against alveolar bone loss in P. gingivalis�infected mice. (A and B) The 8�12-wk-old C57BL/6 mice were divided randomly into one sham control group and two experimental groups (n 5 8 per group). The sham control group was treatedwith cellulose and 0.01% DMSO. The experimental groups were treated with P. gingivalis only, P. gingivalis with SGK1 inhibitor EMD638683 (15 mg/kg),or inhibitor only. (A) Representative images showing that SGK1 inhibition by EMD638683 robustly elevates the expression of CD68 and F4/F80 (AlexaFluor 350, blue) while concurrently decreasing the expression of M2 molecular markers CD206 and CD163 (FITC, green). The arrows in the images showmacrophages being positively stained with CD68, F4/F80, CD163, or CD206. (B�E) Quantification of the expression of CD68 and (Figure legend continues)

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protect against microbial invasion and avoid excessive inflammation.Our previous study has demonstrated that SGK1 plays an essentialrole in the suppression of TLR-mediated inflammatory responses(23). In this study, we have found that the expression of bothM1 macrophage and M2 macrophage markers are increased inthe P. gingivalis�infected mice model. Given that both E. coliLPS and IL-4/IL-13 can phospho-activate SGK1, it is possiblethat the activation of SGK1 upon various external stimuli actsas a rheostat that fine-tunes inflammatory cytokine productionand macrophage polarization to maintain the homeostasis ofinflammatory responses. In contrast, our results also showedthat P. gingivalis infection leads to phosphoactivation of SGK1in BMDMs (Supplemental Fig. 2B), indicating that P. gingiva-lis may facilitate macrophage polarization toward M2 pheno-types through manipulating SGK1 activity, which is consistentwith the results from a previous study showing that P. gingiva-lis is a weak inducer for macrophage polarization (50). A veryrecent study validated this point by showing that P. gingivalisinfection indeed promotes M2 macrophage polarization andfacilitates immunoevasion of oral cancer cells (51). Given thatmacrophage polarization represents a spectrum of states throughwhich cells can transition in either direction (2), the interactionbetween P. gingivalis infection and activation of SGK1 suggests apivotal interventional potential for SGK1 signaling in the controlof macrophage polarization. Therefore, a possible paradigm is thatbacterial infection may result in the recruitment of additional mac-rophages to local inflammatory sites in which specific signaling isresponsible for the fine-tuning of macrophage polarization to pro-or anti-inflammatory sublineages. Thus, the SGK1-mediated bifur-cated signaling pathway could be a key signaling axis exploited by

P. gingivalis to manipulate macrophage polarization and benefit itssurvival in the periodontal inflammatory milieu.In this study, our results have shown a discrepancy in different

inflammatory cytokine production in in vitro versus in vivo assays.SGK1 inhibition significantly enhanced the production of IL-6, IL-12, and TNF-a in cultured cells, whereas only IL-6 was signifi-cantly increased in the gingival tissue from P. gingivalis�infectedmice. This discrepancy might be caused by multiple factors, includ-ing the phase of inflammation, variant resistance to P. gingivalis�in-duced tolerance, and importantly, the different components ofsamples. In our animal model, the gingival tissues for TNF-a andIL-12 analysis were from mice infected with P. gingivalis for 42 d,which is approximately equal to 6-y infection in humans. Comparedwith the cultured cells challenged with IFN-g and LPS for only 24h, it would not be surprising to see different levels of TNF-a andIL-12 resulting from the different phases of inflammation in the two

F4/80 (B) and CD206 and CD163 (C), represented with the mean number of positive cells per square millimeter that was approximately equal to five viewsunder 20� objective. Scale bar, 50 mM. (D) mRNA levels of TNF-a, IL-6, and IL-12P40 were determined by qRT-PCR. (E) Total lysates of gingival tissuefrom upper jaws were probed for TNF-a, IL-6, IL-12P40, and GAPDH. (F) Alveolar bone loss visualized by methylene blue/eosin staining and typical maxil-lae from sham-infected, P. gingivalis�infected, EMD638683-treated, and P. gingivalis�infected with EMD638683-treated mice are presented. (G) Quantifica-tion of alveolar bone loss represented by the distance from the CEJ to the ABC. Data are presented as the mean CEJ�ABC distance in mm ± SD. Data arepresented as the mean fluorescence density; n 5 8 mice per group. Error bars represent the SD. *p < 0.05, ***p < 0.001.

FIGURE 7. SGK1 regulates the expression of STAT3 in P. gingiva-lis�infected mouse gingival tissues. Immunohistochemical staining of serialsections of gingival tissues from experimental mice treated with P. gingiva-lis, P. gingivalis plus SGK1 inhibitor (EMD638683, 15 mg/kg), or shamcontrol mice treated with DMSO and cellulose with or withoutEMD638683, showing the expression of STAT3. (A) Representative imagesshowing that P. gingivalis infection enhances the expression of STAT3,whereas SGK1 inhibition decreases it in gingival tissue. (B) Quantificationof STAT3-positive areas using ImageJ Fiji. Data were derived from theanalysis of more than 20 microscopic fields of view in at least five serialslides. Error bars represent the SD. Data represent the arithmetic mean ±SD of three independent experiments. ***p < 0.001.

FIGURE 8. Schematic model of alternative macrophage polarization bySGK1. Activation of SGK1 by external stimuli in the inflammatory milieurestrains FoxO1 activity by facilitating its cytoplasmic translocation, thusdecreasing the amount of FoxO1 in the nucleus. Moreover, SGK1 activa-tion increases STAT3 activity, which then promotes macrophage polariza-tion toward M2 phenotypes. In contrast, the inhibition of SGK1 decreasesSTAT3 phosphorylation and reduces its activity. This concurrently abro-gates cytoplasmic translocation of FoxO1, sequestering it in the nucleus,and enhances FoxO1 activity, which drives macrophage polarization towardM1 phenotypes.

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systems. Moreover, previous studies have shown that TNF-a andIL-12 are more prone to chronic activation-mediated immune celltolerance than IL-6 (52, 53). Because the mice in our model wereinfected with P. gingivalis for 12 d, it is also reasonable to speculatethat immune cell tolerance in gingival tissue could obscure the effectof SGK1 on TNF-a and IL-12 expression. In addition, differencesof the samples we used in vitro and in vivo could also be a majorreason for this discrepancy. Unlike in vitro cultured macrophages,gingival tissue includes not only macrophages but also monocytes,neutrophils, adaptive immune cells, epithelial cells, fibroblasts, andother cells. The different regulatory functions of SGK1 in innateand adaptive immune cells, which has been extensively reported(27, 54), may counterbalance SGK1-mediated enhancement ofTNF-a and IL-12 production in macrophages. Because of the lim-ited size of murine gingival tissue, it is difficult to isolate sufficientmonocytes, macrophages, or adaptive immune cells to test the possi-ble distinct functions of SGK1 in each. Future research focusing onthe expression of polarization molecular markers and production ofinflammatory cytokines at different phases of P. gingivalis infectionand the use of single-cell sequencing from pooled tissues or cervicallymph nodes may allow for a rigorous test of this hypothesis.In this study, we found that SGK1 deficiency leads to an increase

in FoxO1 and a decrease in STAT3 in macrophages under M1- andM2-priming conditions, respectively. However, we have not demon-strated how SGK1 deficiency regulates the expression of FoxO1and STAT3 in macrophages. Although our supplementary data(Supplemental Fig. 2C) showed that SGK1 deficiency enhances themRNA levels of FoxO1, suggesting SGK1 may impact the de novosynthesis of FoxO1, it is still not clear why the expression ofSTAT3 was reduced in sgk1-deficient BMDMs. Previous studieshave shown that SGK1 activation could phosphorylate Nedd4-2(55), a member of HECT family of ubiquitin E3 ligases, and therebyreduce its activity, which would lessen the amount of ubiquitintransferred to the substrate. Thus, it is possible that SGK1 deficiencyin macrophages reduces phosphorylation of Nedd4-2, enhances itsactivity, promotes ubiquitination-mediated degradation of STAT3,and ultimately decreases STAT3 levels. Combined with thedecreased phosphorylation of STAT3, deficiency of SGK1 couldsubstantially reduce STAT3 activity and suppresses M2 macrophagepolarization. Although the decreased phosphorylation of STAT3possibly results from the diminished total STAT3 levels in SGK1knockout cells, we cannot exclude the potential influences of auto-crine signaling, such as STAT3-mediated IL-10, on STAT3 phos-phorylation. It is likely that diminished STAT3 may suppress IL-10transcription (56), which is a strong inducer of STAT3 phosphoryla-tion (57), and thereby reduce STAT3 phosphorylation in M2-primedmacrophages. Thus, SGK1-regulated expression of STAT3 andFoxO1 could be through different mechanisms. Further investigationof this point is likely to yield greater insight into macrophage polari-zation in general.In summary, we have demonstrated for the first time, to our

knowledge, that SGK1 is necessary for the maintenance of M2 mac-rophages and protects against P. gingivalis�induced alveolar boneloss in a mouse model of periodontitis. SGK1 inhibition promotesM1 polarization and restrains M2 macrophage phenotypes throughthe control of FoxO1 and STAT3, respectively. Combined with ourprevious study showing that SGK1 restrains TLR-mediated inflam-mation, our findings suggest that SGK1 could be an important inter-ventional target for manipulating the magnitude and duration ofinflammation depending on clinical necessity.

AcknowledgmentsWe thank Dr. Todd Kitten for critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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