CREB pathway links PGE2 signaling withmacrophage polarizationBing Luana,b,1, Young-Sil Yoona, John Le Layc, Klaus H. Kaestnerc, Susan Hedricka, and Marc Montminya,1

aPeptide Biology Laboratories, The Salk Institute, La Jolla, CA 92037-1002; bDepartment of Endocrinology, Shanghai Tenth People’s Hospital, School ofMedicine, Tongji University, Shanghai 200092, China; and cDepartment of Genetics, Institute for Diabetes, Obesity and Metabolism, University ofPennsylvania School of Medicine, Philadelphia, PA 19104-6145

Contributed by Marc Montminy, October 8, 2015 (sent for review September 3, 2015; reviewed by Seung-Hoi Koo and Ling Qi)

Obesity is thought to promote insulin resistance in part via activationof the innate immune system. Increases in proinflammatory cytokineproduction by M1 macrophages inhibit insulin signaling in whiteadipose tissue. In contrast, M2 macrophages have been found toenhance insulin sensitivity in part by reducing adipose tissueinflammation. The paracrine hormone prostaglandin E2 (PGE2)enhances M2 polarization in part through activation of the cAMPpathway, although the underlying mechanism is unclear. Here weshow that PGE2 stimulates M2 polarization via the cyclic AMP-responsive element binding (CREB)-mediated induction of Krupple-like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulatedtranscriptional coactivators 2 and 3 (CRTC2/3) in macrophages down-regulated M2 marker gene expression and promoted insulin re-sistance in the context of high-fat diet feeding. As re-expressionof KLF4 rescued M2 marker gene expression in CREB-depleted cells,our results demonstrate the importance of the CREB/CRTC pathwayin maintaining insulin sensitivity in white adipose tissue via itseffects on the innate immune system.

cAMP | CREB/CRTC | M2 macrophage | insulin resistance

Under obese conditions, macrophage infiltration and activa-tion in adipose tissue leads to a chronic inflammatory state

with increased secretion of proinflammatory cytokines (1). Theactivation of IkB and Jun N-terminal kinases impairs insulinsignaling in metabolic tissues and thereby contributes to insulinresistance (2–4). Classically activated M1 macrophages secreteproinflammatory cytokines, such as TNF-α and IL-12, whichpromote insulin resistance. Alternatively activated M2 macro-phages are thought to protect adipocytes from the developmentof insulin resistance in response to IL-4 signaling (5, 6). In-creases in STAT6 activity stimulate the expression of Krupple-like factor 4 (KLF4), which in turn promotes expression of theM2 program. Obesity causes an M2-to-M1 shift in adiposetissue that leads to insulin resistance (7).The eicosanoid prostaglandin E2 (PGE2) has been found to

promote M2 macrophage polarization in part via induction ofthe cAMP pathway. Indeed, circulating catecholamines also ex-ert potent anti-inflammatory effects on macrophage function viacAMP signaling (8). In this regard, a number of bacteria appearto evade the innate immune system by producing toxins thatenhance cAMP production. cAMP stimulates the expression ofcellular genes in part via the phosphorylation of CREB at Ser133and via the dephosphorylation of the cAMP regulated tran-scriptional coactivators (CRTC) family of coactivators (9). Fol-lowing its activation, the cyclic AMP-responsive element binding(CREB) pathway appears to block M1 macrophage function inpart via the induction of the anti-inflammatory cytokine IL-10(10, 11). Superimposed on these effects, cAMP also inhibits theexpression of proinflammatory cytokines via the induction ofclass IIa histone deacetylases (HDACs) and subsequent de-acetylation of NF-κB.Here we explore the potential roles of the class IIa HDAC and

CREB/CRTC pathways in M2 macrophages. We found that,although PGE2 stimulates both pathways, only one of these is

required for M2 macrophage polarization. As disruption of thispathway increases insulin resistance in the setting of dietaryobesity, our results suggest that small molecules that enhanceits activity in macrophages may provide therapeutic benefit todiabetic individuals.

ResultsWe evaluated the effect of cAMP signaling on the expression ofM2 macrophage marker genes, including arginase-1 (Arg1), themannose receptor (Mrc1), resistin-like α (Fizz1, Retnla), andchitinase 3-like 3 (Ym1, Chi3l3) in cultured bone marrow mac-rophages (BMMs) (12, 13). Exposure to PGE2, potentiated IL-4–induced increases in M2 marker gene expression (Fig. 1 A andB). We observed similar effects using other cAMP agonists, in-cluding the β2 adrenergic receptor agonist, isoproterenol, as wellas a number of bacterial toxins (Fig. S1A).Overnutrition triggers leptin-mediated increases in catechol-

amine signaling to white and brown adipose tissues (WAT andBAT) that promote fat burning. Indeed, leptin injection into ob/obmice up-regulated circulating concentrations of norepinephrine,leading to elevations in intracellular cAMP (14, 15). In keepingwith these effects, leptin administration augmented M2 markerexpression in WAT from leptin-sensitive ob/ob mice; these ef-fects were blocked by pretreatment with β-adrenergic receptorantagonist propranolol (Fig. 1 C and D).In contrast with acute effects of overnutrition, chronic high-fat

diet (HFD) feeding causes leptin resistance and attenuatessympathetic catecholamine signaling to WAT (14). As a result,M2 marker gene expression was down-regulated in WAT fromHFD mice; these effects were reversed by administering thephospho-diesterase 4 (PDE4) inhibitor Rolipram (5 mg/kg) (Fig.1 E and F) and increasing intracellular concentrations of cAMP.Taken together, these results show that increases in cAMP signalingpromote M2 macrophage polarization.

Significance

The second messenger cAMP exerts potent immunosuppres-sive effects on the innate immune system, in part by activatingclass IIa histone deacetylases, and thereby inhibiting NF-κB–dependent transcription. We found that cAMP also promotesM2 macrophage polarization by stimulating the cyclic AMP-responsive element binding (CREB)/cAMP-regulated transcriptionalcoactivators TORC1 pathway. In the setting of acute overnutrition,macrophage CREB inhibits the production of inflammatory medi-ators and contributes to the maintenance of insulin sensitivity.

Author contributions: B.L. and M.M. designed research; B.L., Y.-S.Y., J.L.L., K.H.K., and S.H.performed research; B.L., Y.-S.Y., J.L.L., K.H.K., S.H., and M.M. analyzed data; and B.L.wrote the paper.

Reviewers: S.-H.K., Korea University; and L.Q., Cornell University.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: bluan@tongji.edu.cn or montminy@salk.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1519644112/-/DCSupplemental.

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cAMP triggers the activation of the CREB/CRTC and class IIaHDAC pathways (9, 16, 17) (Fig. S1B). Of the three members of theclass IIa HDAC family (HDAC4, -5, and -7), HDAC4 is expressed athighest levels in macrophages, prompting us to use cells from micewith a macrophage-specific knockout of HDAC4 (HDAC4 MKO)to evaluate the importance of this pathway for M2 polarization.Although exposure of BMMs to PGE2 plus IL-4 promoted HDAC4dephosphorylation and activation (Fig. 1G), M2 marker geneexpression was up-regulated comparably between wild-type andHDAC4MKOBMMs (Figs. S2A and S3 A and B), arguing againsta significant role for the HDAC4 pathway in this setting.Next we tested the importance of the CREB pathway for M2

polarization using homozygous CREB floxed (CREB fl/fl) miceexpressing a macrophage-specific LysM-cre transgene. Exposureof wild-type BMMs to PGE2 increased CREB Ser133 phos-phorylation and augmented CRTC2 and CRTC3 dephosphorylation(Fig. 1G) (14). Exposure to IL-4 and PGE2 increased M2 markergene expression cooperatively in wild-type cells, and these effectswere potently disrupted in CREB mutant BMMs (Fig. 2 A and B,and Fig. S2B).We further evaluated the role of CREB in M2 polarization by

expressing the dominant-negative CREB inhibitor ACREB, asynthetic polypeptide that selectively heterodimerizes with and

disrupts the DNA binding activity of all CREB family members(CREB1, CREM, and ATF1). Similar to CREB1 knockout cells,ACREB expression in BMMs disrupted M2 marker gene ex-pression following exposure to IL-4 plus PGE2 (Fig. S4 A and B).CRTC proteins are potent coactivators of CREB. Sequestered

in the cytoplasm under basal conditions through phosphorylation-dependent interactions with 14-3-3 proteins, CRTCs shuttle to thenucleus following their dephosphorylation in response to cAMP,where they bind to CREB over relevant promoters (9). Of the threefamily members, CRTC2 and CRTC3 proteins, but not CRTC1,were detected in cultured BMMs by immunoblot assay (Fig. S2C).Based on their overlapping effects on CREB target gene

expression, we evaluated the importance of both CRTC2 andCRTC3 (CRTC2/3 DKO) for macrophage polarization. M2marker expression was reduced in CRTC2/3 DKO BMMs exposedto IL-4 plus PGE2 (Fig. 2 C and D, and Fig. S2C). Taken together,these results demonstrate that the CREB/CRTC pathway is re-quired for cAMP induction of M2 marker genes (Fig. 2D).Although CREB and CRTC2/3 appeared important for induc-

tion of M2 marker genes by PGE2, the absence of conservedCREB binding sites on these promoters pointed to the involvementof additional transcriptional activators in mediating these effects.In this regard, KLF4 has been shown to cooperate with STAT6 in

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Fig. 1. cAMP promotes M2 macrophage polarization. (A and B) Effect of PGE2 exposure on M2 macrophage marker mRNA amounts (A) (Arg1, Mrc1, Fizz1,Ym1) and Arg1 protein level (B) in BMMs treated with IL-4. (C and D) Effects of leptin on M2 macrophage marker mRNA (C) and Arg1 protein amounts (D) infat pads of ob/ob mice. Effect of β-adrenergic antagonist (Propanolol; pro) shown. Body weights indicated (n = 4 per group). (E and F) Effect of normal chow(NC) or HFD feeding on M2 macrophage marker mRNA amounts (E) and on Arg1 protein levels (F) in WAT. Coinjection of phospho-diesterase inhibitorRolipram (5 mg/kg) in HFD mice indicated. (n = 4 per group). (G) Effect of PGE2 exposure on amounts of phosphorylated CREB, CRTC2/3, or HDAC4 in wild-type BMMs. *P < 0.05 and **P < 0.01.

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promoting M2 macrophage polarization; deletion of the KLF4gene in macrophages disrupts M2 function and increases proin-flammatory gene expression (13). Moreover, the mouse KLF4promoter contains CREB binding sites at −269 and −232 relativeto the transcription start site. Correspondingly, exposure to FSKup-regulated KLF4-luciferase reporter activity in HEK293Tcells. CRTC2 overexpression further enhanced KLF4 promoteractivity, whereas A-CREB blocked it (Fig. 3A). Consistent with adirect effect on KLF4 expression, exposure to PGE2 increasedphospho-CREB and CRTC2 occupancy over the KLF4 promoterin BMMs (Fig. 3B). Exposure of wild-type BMMs to PGE2 in-creased endogenous KLF4 mRNA amounts maximally after 4 h;KLF4 expression was substantially down-regulated in CREB MKOas well as CRTC2/3 DKO BMMs (Fig. 3C).We tested whether the induction of KLF4 is required for the up-

regulation of M2 marker genes in response to PGE2. Supportingthis idea, IL-4/PGE2-dependent induction of M2 marker geneswere down-regulated in CREB and CRTC2/3 mutant cells relativeto control; these effects were rescued following lentiviral expressionof KLF4 (Fig. 3D). We detected similar changes in Arg1 proteinexpression following KLF4 expression (Fig. 3E). Taken to-gether, these results indicate that the CREB/CRTC pathway pro-motes M2 polarization via the up-regulation of KLF4 in responseto IL-4/PGE2 signaling.M2 macrophages have been shown to maintain insulin

sensitivity in adipose tissue by reducing proinflammatory cy-tokine expression (18). The loss of M2 macrophages leads toreciprocal increases in M1 polarized macrophages in adiposetissue that promote insulin resistance. Based on its effects onM2 marker genes, we speculated that the CREB/CRTC pathway

may protect against the development of insulin resistance inthis setting. To test this notion, we evaluated effects of HFDfeeding on insulin sensitivity in CREB MKO versus wild-typelittermates. Although body weights, adiposity, and leptin levelswere comparable between the two groups (Fig. 4 A and B andFig. S5A), CREB MKO mice exhibited higher fasting glucoseand insulin concentrations in the context of HFD feedingcompared with wild-type (Fig. 4C and Fig. S5B); they were alsomore glucose intolerant and had reduced glucose clearance rela-tive to controls by glucose and Insulin tolerance testing (Fig. 4Dand Fig. S5C). Indeed, macrophage infiltrates in WAT were morepronounced in CREB MKO mice compared with wild-type (Fig.4E). M1 macrophage markers were significantly elevated in CREBmutants, whereas M2 markers were reduced (Fig. 4F). Takentogether, these results demonstrate that the macrophage CREB/CRTC pathway promotes M2 polarization that protects adiposetissue from insulin resistance in the setting of obesity.

DiscussionIn addition to its role as a starvation state signal, the secondmessenger cAMP exerts potent anti-inflammatory effectsthrough the inhibition of macrophage function. cAMP attenuatesproinflammatory M1 macrophage gene expression, for example, viainduction of the anti-inflammatory cytokine IL-10 (10, 11) and viathe induction of HDAC4. Superimposed on these effects, wefound that cAMP promotes anti-inflammatory M2 macrophagegene expression through up-regulation of the CREB/CRTCpathway (Fig. 4G). Both CREB and class IIa HDAC pathwaysinhibit the production of inflammatory mediators and contribute tothe maintenance of insulin sensitivity in the setting of overnutrition.

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Fig. 2. PGE2 stimulates M2 polarization via the CREB pathway. (A and B) Effect of IL-4 and PGE2 on M2 macrophage marker mRNA (A) and Arg1 proteinamounts (B) in in BMMs from mice with a deletion of the CREB gene in macrophages (CREB MKO) or control littermates. (C and D) Effect of IL-4 and PGE2 onM2 macrophage marker mRNA (C) and Arg1 protein amounts (D) in wild-type and CRTC2/3 DKO BMMs. *P < 0.05 and **P < 0.01.

15644 | www.pnas.org/cgi/doi/10.1073/pnas.1519644112 Luan et al.

In addition to KLF4, a number of other CREB target genes,most notably CEBPβ and SOCS3, have been shown to promotemacrophage polarization (19, 20). Future studies should providefurther insight into these and other mechanisms by which cAMPsignaling modulates innate immunity.

Experimental ProceduresCells. HEK293T cells were maintained in DMEM and exposed to FSK (10 μM)for transient transfection assays. BMMs were prepared as described pre-viously (21). Cells were treated with IL-4 (10 ng/mL) and PGE2 (10 nm),isoproterenol (10 μm), pertusis toxin (1 μg/mL), cholera toxin (1 μg/mL),edema factor (0.1 μg/mL) + protective antigen (0.3 μg/mL) for 16 h, andmRNA or protein was extracted for examination.

Mice. C57BL/6J, ob/ob, and LysMcre mice were purchased from The JacksonLaboratory. CREB1 fl/fl mice were a generous gift of E. Nestler (Mount SinaiHospital, New York, NY). Macrophage-specific knockout of CREB wasobtained by a two-step cross of CREB1 fl/fl mice with LysMcre mice accordingto The Jackson Laboratory protocol. CRTC2 fl/fl mice were as describedpreviously (22). CRTC3 fl/fl mice were generated with loxP sites flankingexon 4 (ES cell clone obtained from KOMP). Floxed CRTC2/3 mice were

obtain by cross of CRTC2 fl/fl with CRTC3 fl/fl. In studies with CRTC2/3 DKOmacrophages, BMMs from floxed CRTC2/3 mice were infected with cre-expressing or control lacz-expressing lentivirus. For studies with KO mice,age-matched wild-type littermates were used as controls. For HFD studies,6-wk-old mice were transferred to a 60% HFD (Research Diets, D12492) for12 wk. MRI scans for fat and lean mass were performed using an Echo MRI-100 instrument according to the manufacturer’s instructions. All mice werehoused in colony cages with a 12-h light/dark cycle in a temperature-controlledenvironment. Animal studies were approved by the Institutional Animal Care andUse Committee at the Salk Institute.

Glucose Tolerance Testing and Insulin Tolerance Testing. For glucose tolerancetesting, mice were fasted for 16 h and then injected intraperitoneally withglucose (1.5 mg/kg). For insulin tolerance testing, mice were fasted 2 h andinjected intraperitoneally with insulin (Humulin; 1 U/kg). Blood was collectedfrom the tail vein at indicated times and glucose levels were measured with aOne Touch Ultra Glucometer (Johnson & Johnson).

ChIP and Quantitative PCR. BMMs were plated in 150-mm plates and exposedto PGE2 (100 nM) for 1 h. ChIP assays were performed as described previously(23). BMMs were stimulated for indicated times. RNA was isolated by RNeasy

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Fig. 3. The CREB/CRTC pathway promotes M2 function via induction of KLF4. (A) Transient assay of HEK293T cells showing effects of FSK on KLF4 reporter activity.Cotransfection with ACREB or CRTC2 indicated. (B) ChIP (26) assay of pCREB and CRTC2 recruitment to the KLF4 promoter in BMMs exposed to PGE2. (C) Effect ofPGE2 exposure for various times on KLF4 mRNA amounts in BMMs from wild-type, CREB MKO, and CRTC2/3 DKO mice. (D and E) Effect of lentiviral KLF4 expressionon M2 macrophage marker mRNA amounts (D) and Arg1 protein level (E) in wild-type and CREB MKO BMMs exposed to IL-4 and PGE2. *P < 0.05 and **P < 0.01.

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kit (Qiagen). Quantitative PCR was carried out with SYBR Green, as describedpreviously (23).

Blotting and Immunostaining. Immunoblot and immunostaining assays were per-formed as described previously (24). Quantitated results for Arg1 protein level areshown in Fig. S6. Mouse adipose tissues were fixed and paraffin-embedded. Sec-tions (5 μm) were used for immunostaining with F4/80 antibody.

Luciferase Reporter Assay. HEK293T cells were transfected with KLF4-Lucreporter, RSV-βgal, and indicated plasmids for 48 h, and luciferase assayswere performed as described previously (25).

Statistical Analyses. All studies were performed on at least three independentoccasions. Results are reported as mean ± SEM. The comparison of differentgroups was carried out using two-tailed unpaired Student’s t test or two-way ANOVA test. Differences were considered statistically significant at *P <0.05 and **P < 0.001.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth Grants R37DK083834 (to M.M.) and P01-DK049210 (to K.H.K.); theClayton Foundation for Medical Research; the Kieckhefer Foundation; andthe Leona M. and Harry B. Helmsley Charitable Trust Grant #2012-PG-MED002 (to M.M.).

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Fig. 4. Loss of M2macrophages and insulin resistance in CREBMKOmice. (A and B) Relative weight gain (A) (n= 8 per group) as well as fat mass (n= 4 per group) andcirculating leptin levels (B) (n= 8 per group) in CREBMKO and control littermates under HFD. (C) Circulating glucose and insulin concentrations in CREBMKO comparedwith control littermates maintained on a HFD for 12 wk (n = 8 per group). (D) Glucose tolerance (Left) and insulin tolerance (Right) testing of CREB MKO and controllittermates maintained on HFD (n = 12 per group). (E) Relative macrophage infiltration in WAT by immunohistochemical (Left) and quantitative PCR (Right) analyses.(Scale bar, 50 μm.) (F) Expression of M1 and M2 marker genes in WAT by quantitative PCR analyses. (G) Schematic of proposed mechanism. *P < 0.05 and **P < 0.01.

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