Casein Kinase 2 Interacting Protein-1 regulates M1 and M2 inflammatory macrophage polarization

Accepted Manuscript Casein Kinase 2 Interacting Protein-1 regulates M1 and M2 inflammatory macrophage polarization

Yuhan Chen, Wen Liu, Yiwu Wang, Luo Zhang, Jun Wei, Xueli Zhang, Fuchu He, Lingqiang Zhang PII: DOI: Reference:

S0898-6568(17)30055-4 doi: 10.1016/j.cellsig.2017.02.015 CLS 8856

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

20 December 2016 6 February 2017 13 February 2017

Please cite this article as: Yuhan Chen, Wen Liu, Yiwu Wang, Luo Zhang, Jun Wei, Xueli Zhang, Fuchu He, Lingqiang Zhang , Casein Kinase 2 Interacting Protein-1 regulates M1 and M2 inflammatory macrophage polarization. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cls(2017), doi: 10.1016/j.cellsig.2017.02.015

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ACCEPTED MANUSCRIPT Casein Kinase 2 Interacting Protein-1 Regulates M1 and M2 Inflammatory Macrophage Polarization* Yuhan Chen a,e , Wen Liu a, Yiwu Wang a,b, Luo Zhang a,c, Jun Wei a,d, Xueli Zhang d,*, Fuchu He a,**, Lingqiang Zhang a,*** a

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State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing, China b Department of Infectious Diseases, Chinese PLA 532 Hospital, Huangshan, Anhui, China c 307-lvy Translational Medicine Center, Laboratory of Oncology, Chinese PLA 307 Hospital, Beijing, China, d Shanghai Fengxian Central Hospital Graduate Training base; Department of General Surgery, Fengxian Hospital Affiliated to Southern Medical University, Shanghai e Bayi Children’s hospital, Army General Hospital, Beijing, China

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Abbreviations: CKIP-1, Casein kinase 2 interacting protein 1; NF-κB, nuclear factor-κB; STAT6, Signal transducer and activator of transcription 6; qPCR, quantitative PCR; LPS, lipopolysaccharide; TPA, phorbol ester 12-O-tetradecanoylphorbol-13-acetate; BMDM, bone marrow-derived macrophage; PM, peritoneal macrophage

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*Corresponding authors at: Department of General Surgery, Fengxian Hospital Affiliated to Southern Medical University, Shanghai, 201499, China **Corresponding authors at: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing, 100850, China ***Corresponding authors at: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing, 100850, China Email address: [email protected] (X. Zhang), [email protected] (F. He), [email protected] (L. Zhang)

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Keywords: Macrophage, Inflammation, Lipopolysaccharide (LPS), CKIP-1, NF-κB transcription factor, STAT transcription factor 6

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ABSTRACT The importance of macrophage plasticity, albeit being discovered recently, has been highlighted in a broad spectrum of biological processes operative in physiological and pathological environments. Macrophage polarized activation and inactivation has profound effects on immune and inflammatory responses with several major pathways being elucidated in the past few years. However, transcriptional regulation mechanisms governing macrophage polarization is still preliminary. In this study, we identify the Casein Kinase 2 Interacting Protein 1 (CKIP-1) as a molecular toggle manipulating macrophage speciation. CKIP-1 expression was strongly induced by pro-inflammatory M1 stimuli (LPS and IFN-γ) and robustly suppressed by M2 stimuli (IL-4 and IL-13) in human and murine macrophage. Gain and loss of function studies suggest that CKIP-1 is a prerequisite for optimal LPS-induced pro-inflammatory gene activation, which exhibits its roles in a NF-κB dependent manner. Furthermore, CKIP-1 inhibits anti-inflammatory gene expression by negatively regulating JAK1-STAT6 activation in macrophages. Taken together, these data integrated CKIP-1 expression and function as a novel transcriptional regulator of macrophage polarization and identified a double feedback loop consisting of CKIP-1 and the key regulators of the M1 and M2 macrophage effectors in polarization pathway. Moreover, the inhibitory roles of CKIP-1 in LPS-mediated sepsis and TPA-mediated cutaneous provide a new target for treatments of acute inflammation.

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ACCEPTED MANUSCRIPT 1. Introduction

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In response to inflammation, monocytes emigrate from the bloodstream and gradually develop into macrophages within strategically located tissues throughout the body [1], where they play pivotal roles in acute (e.g. sepsis [2], pathogen infection [3, 4] ) as well as chronic inflammatory environments (e.g. atherosclerosis [5], chronic wounds [6], insulin resistance [7], tumorigenesis [8]). Clinical, pathological, and physiological studies supported the notion that macrophages are a special subtype of cells which display heterogeneity and plasticity in terms of both phenotype and function [9-11]. Therefore, two well-established polarized phenotypes are often classified on a continuum in which M1 macrophages represent the classically activated macrophages (CAM) on a pro-inflammatory state whereas M2 macrophages represent the alternatively activated macrophages (AAM) on a contradistinctive anti-inflammatory state [12]. M1 macrophages are generally activated by interferon-γ (IFN-γ) or other microbial products (e.g. LPS) and then produce large amounts of pro-inflammatory cytokines (e.g. IL-1α, IL-6, IL-12, TNF-α) in concomitant with high levels of major histocompatibility complex molecules to promote Th1 immune response, and can serve as potent killers of pathogens, while M2 macrophages are usually activated by the IL-4/IL-13 immune complex, IL-10, or TGF-β to exhibit an immunosuppressive phenotype with enhanced release of anti-inflammatory cytokines and the abilities to support tissue remodeling and repair, debris scavenging and angiogenesis [13]. Unlike Th1 and Th2 cells, M1 and M2 macrophages are not stably differentiated subsets. Instead, they can serve as adaptive phenotypic changes in the appropriate regulation of inflammatory response, these delicate adaptions are well orchestrated at both transcriptional and translational levels through integrated signaling pathways and key nobs that regulate target gene expression and cellular functions [14, 15]. Refined understanding of macrophage plasticity have provided evidence that except for the priming of cytokines, multiple transcriptional factors are indispensable for the process of macrophage polarization. NF-κB, IRF5, AP-1 and STAT-1 have been identified as significant regulators for pro-inflammatory M1 phenotypic response [16]. By contrast, the M2 macrophage are less sensitive to pro-inflammatory stimuli and STAT6, IRF4, JMJD3 [17], SHIP [18, 19] and PPARγ [20] have been identified as key regulators in the regulation of characteristic M2 target gene including Arg1, Mrc1, Fizz1, Ym1 [21]. More importantly, the PI3K/AKT, JAK/STAT, CREB-C/EBP axis, JNK [22], Notch-RBJ pathways [23] have been demonstrated to cooperatively participating in the well-established macrophage polarization. Although these studies have provided lines of proofs that the macrophage undergoes dramatic changes of phenotype in different circumstances, the sophisticated molecular mechanisms governing M1/M2 polarization remain incompletely unveiled. We and other studies have reported the pleckstrin homology (PH) domain-containing protein CKIP-1 (Casein Kinase 2 Interacting Protein-1, also known as PLEKHO1) plays important roles in the regulation of cell apoptosis [24, 25], cell morphology [26, 27], and cell differentiation [28] as well as tumorigenesis [29] and cardiac hypertrophy [30]. Our previous study provided the first in vivo evidence that CKIP-1 knockout mice displayed an age-dependent increase in bone formation and bone mineral density owing to increased osteoblast differentiation [31]. Furthermore, we recently found that CKIP-1 is a novel regulator of macrophage proliferation by orchestrating the initiation and termination of M-CSF signaling through interacting with TRAF6 and inhibiting TRAF6-mediated AKT ubiquitination and activation [32], and CKIP-1 is also involved in the regulation of macrophage migration [33]. However, the role of CKIP-1 in macrophage polarization is unknown. In this study, we provided multiple lines of evidence to show that CKIP-1 could be regulated by both M1 and M2 cytokines and in turn could simultaneously regulate M1 and M2 polarization through a negative feedback loop. Mechanically, CKIP-1 promotes an M1 phenotype through cooperation with NF-κB whereas inhibits the M2 targets by suppressing STAT6 expression. Physiologically, CKIP-1 is indispensable for inhibition of TPA-mediated cutaneous inflammation and LPS-mediated sepsis in vivo. Collectively, these findings identify CKIP-1 as a novel and critical molecular switch regulating macrophage polarization, and will aid in the exploration of potential therapies targeting CKIP-1 for the treatment of bacteria mediated sepsis. 2. Materials and methods 2.1 Mice Strain 2

ACCEPTED MANUSCRIPT Ckip-1−/− mice on a C57BL/6 background were prepared as previously described[31]. Ckip-1 floxed mice (Ckip-1fl/fl) and Lyz2-Cre were constructed by MARC of Nanjing University. Ckip-1-/- mice were crossed with Stat6−/− mice [34] (C57BL/6 background) to generate Ckip-1−/− and Ckip-1−/− Stat6−/− littermate controls. All mice were maintained and handled under the manufacture’s protocols approved by Chinese Academy of Military Medicine Science. 2.2 Cell Cultures

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Human primary macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml streptomycin, and 2mM glutamine in a 5% CO2 environment at 37 °C. RAW264.7 cells were cultured in RPMI-1640 medium with standard formulations, whereas mice BMDMs were cultured in DMEM conditional medium supplemented with the supernatants of L929. 2.3 Antibodies and Reagents

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Antibodies used in immunoblots were listed below: anti-CKIP-1 (Santa Cruz, sc-50225, 1:500), anti-actin (Santa Cruz, sc-1616, 1:1,000) were from Santa Cruz Biotechnologies. Anti-STAT6 (CST, No. 5397, 1:500), anti-pTyr641-STAT6 (CST, No. 5200, 1:1,000), anti-IκB-α (CST, No. 4812, 1:1,000), anti-p-Ser32-IκB-α (CST, No. 2859, 1:1,000) were all purchased from Cell Signaling Technology. Anti-GAPDH (Genetex, GT239, 1:3,000) were from Genetex. Recombinant mouse IL-4, IL-13, IFN-γ, GM-CSF were obtained by Proteintech (Chicago, USA), M-CSF were purchased from R&D Systems (Minneapolis, MN). RAW264.7 and THP-1 cell lines were kind gifts from Drs Jiang Peng, Lipopolysaccharide (Salmonella minnesota Re595), phorbolester 12-O-tetradecanoylphorbol-13- acetate (TPA), PGN, Poly (I:C) and thioglycollate broth were all purchased from Sigma, CpG were obtained by Invitrogen (California, USA), ELISA kits of IL-6, TNF-α, IL-12, IL-10, IFN-γ, IL-17 were obtained from ExCell. 2.4 CBA Protein Quantitation

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Mice were intravenously injected with 20mg/kg LPS, and blood samplings in mice eyes were conducted to collect serum at the time points of 1h and 3h, respectively. CBA protein quantification was performed according to the user’s manufacture’s instruction with the CBA kit (BD pharmingen). Samples were analysed on a FACS Calibur (BD Biosciences). Data were analysed with the FlowJo software (Treestar).

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2.5 Isolation of the Bone Marrow Derived Macrophages

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Briefly, bone marrow cells from mice with indicated genotypes were harvested from femur and tibia. These bone marrow cells were cultured in cell-culture media supplemented with mouse recombinant macrophage colonystimulating factor for 7 days. In some cases, bone marrow cells were cultured in L929-supernatant containing conditional medium, these cells were harvested and utilized for further experiments. To generate human macrophages for these studies, peripheral blood mononuclear cells (PBMC) were obtained from healthy blood donors (approved by the Chinese Academy of Military Medicine Science. Institutional Review Board). These cells were allowed to adhere onto plastic tissue culture surfaces and differentiate into macrophages. Adherent macrophages were utilized for indicated studies after non-adherent compartment being removed. 2.6 Isolation of Peritoneal Macrophage Isolation For peritoneal macrophages isolation, male C57/BL6 mice (8 to 12 weeks) were injected intraperitoneally (i.p.) with 2 ml of sterile 3% thioglycollate. Three days later peritoneal macrophages were collected by PBS intraperitoneal lavage. Cells were re-suspended in RPMI 1640 medium (Hyclone) supplemented with 10% heat-inactivated-FBS (Hyclone). After 2 h of incubation to allow macrophages to adhere, cells were seeded in culture plates. Each well was washed three times with warm Hank’s balanced salt solution medium to remove non-adherent cells. 3

ACCEPTED MANUSCRIPT 2.7 Lentivirus Packaging Mice CKIP-1 and STAT6 cDNAs were inserted into murine stem cell virus (MSCV)-IRES-GFP or (MSCV)-IRES-Puro vector for overexpression assay, CKIP-1 shRNA were inserted into U6-Puro-GFP vector for knockdown assays. CKIP-1-lentiviral shRNA (5-CCTGAGTGACTATGAGAAG-3’) and random shRNA (5′-A AGCAGGAAGAACCAAGGTTT-3′) were transfected with packing plasmids into 293Tx cells for 2 days, and virus particles were used to infect RAW264.7 cells as indicated. Selections were carried out by culturing in medium containing 2μg/ml puromycin for 2-4 days. 2.8 RNA interference

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For STAT6 reduction, the mixed siRNAs (siRNA 1, 5’-GAAUCAGUCAACGUGUUGUA-3’; siRNA 2, 5’CCAAGACAACAAUGCCAAA -3’; siRNA 3, 5’-AGACCUGUCCAUUCGCUCA -3’) were used. Control siRNA (5’-AUCUCCGAACGUGUCACGU-3’) and the on-target individual siRNAs against STAT6 were synthesized by Shanghai GenePharm. Transfection was carried out according to the manufacturer’s protocol. 2.9 Phagocytosis detection

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To assess the phagocytic capability of macrophages, opsonized Alexa Fluor 488–conjugated E. coli (strain K-12) bio-particles (Invitrogen) were incubated with macrophages and intracellular uptake was visualized by fluorescence microscopy and quantified by flow cytometry.

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2.10 RNA Isolation and Real-time PCR

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Total RNA from cultured cells, human tissues or mouse tissue samples was isolated by using TRIzol reagent (Invitrogen). In total, 0.5–1 mg RNA was reverse-transcribed using Superscript III (Invitrogen) according to the manufacturer’s instructions. Real-time PCRs were performed using 2 ml of resulting cDNA per 20-ml reaction volume containing SYBR green I master (Roche). Gene expression was normalized to GAPDH using the ΔΔCt method.

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2.11 Western blot

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Cell cultures or primary macrophages were homogenized with RIPA lysis buffer containing the protease inhibitor complex (Roche) and phosphatase inhibitors (Roche). Protein concentration was assayed using the BCA protein assay kit (Pierce), and 5 mg of the protein were resolved by 10% SDS-PAGE and then transferred to NC membrane. The blot was probed with indicated primary antibodies. Membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Jackson Laboratory). Protein bands were detected by ECL plus (Thermo scientific) and GAPDH served as an internal control for protein loading. 2.12 Statistical analysis

Data are presented as mean ± s.e.m. Statistical differences between groups were compared using Student’s t test or a one-way analysis of variance (ANOVA). P value of less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p< 0.001)

3. Results 3.1 CKIP-1 expression is upregulated by M1-stimuli and downregulated by M2-stimuli in human and mice macrophages

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As a member of PH domain containing protein, PLEKHO1, also known as CKIP-1, has been demonstrated to play prominent roles in macrophage proliferation and migration. However, the expression and function profile of CKIP-1 in polarized macrophage has not been fully elucidated. We first examine the expression of CKIP-1 among selected PH members in mouse macrophages. Our results indicate that Plekho1 is the highest expressed member of the PLEKH family both in murine PMs and BMDMs (Fig. 1A). Additionally, our survey of Plekho1 mRNA expression analysis across multiple murine tissues indicated that Plekho1 is most abundantly expressed in macrophages (Fig. 1B). We also examined the expression of CKIP-1 in human and mouse primary macrophages and corresponding cell lines (data not shown). Collectively, these results demonstrate that Ckip-1 mRNA are most robustly expressed in mouse derived macrophages among all the representative PH members. Next, to determine whether CKIP-1 is critical in macrophage polarization, we first detected the expression levels of CKIP-1 in bone marrow derived macrophages (BMDMs) that were stimulated with 100 ng/ml LPS, 20 ng/ml IFN-γ, (for M1 polarization) or 100 ng/ml IL-4, 60ng/ml IL-13 (for M2 polarization) (Fig. 1C).Unexpectedly, CKIP-1 manifested completely different variation trends with M1 versus M2 polarization stimuli, indicating it might participate in both M1 and M2 macrophage polarization, thus validating the functional significance of CKIP-1 for the regulation of macrophage polarization and balancing the M1/M2 ratio. To further evaluate the kinetics of CKIP-1 mRNA and protein level during macrophage polarization, we exposed murine macrophages cell line RAW264.7 to IFN-γ or LPS and detected induced CKIP-1 mRNA and protein expression (Fig. 1D). Similar results were seen in human primary macrophages (Fig. 1E). Next, time course studies were undertaken to examine the kinetics of LPS induced CKIP-1 mRNA and protein expression in mouse BMDMs and PMs. LPS induced CKIP-1 protein and mRNA expression in both types of the macrophage at the time point as early as 4 h, after treatment in a sustained manner, with peak at 12h (Fig. 1F and G). We also analyzed the kinetics of IFN-γ induced CKIP-1 mRNA and protein expression and similar trends were seen (Fig. 1H and 1I). Next we assessed the effect of anti-inflammatory M2 stimuli on CKIP-1 mRNA and protein expression. As shown in Fig. 2A and B, IL-4 or IL-13 stimulation strongly diminished CKIP-1 mRNA and protein expression in murine BMDMs and human primary macrophages. Accordingly, kinetic studies revealed a gradual reduction of murine (Fig.2C-F) and human (Fig.2G and H). CKIP-1 mRNA and protein levels in response to IL-4 or IL-13 exposure. Taken together, these results provided further evidence of CKIP-1 participation in both M1 and M2 polarization.

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3.2 The LPS-NF-κB pathway and the IL4/IL-13-STAT6 pathway is involved in transcriptional regulation of CKIP-1

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To gain initial insights into regulation of CKIP-1 expression during macrophage polarization, we analyzed the promoter sequences of CKIP-1 using a transcription element research system, however, the system didn’t predict any binding sites for either of the critical transcriptional factors including NF-κB, STAT6, IRF or STATs (data not shown). These results imply that these two important regulator may display its transcriptional control upon CKIP-1 in an indirect way regardless of their binding activities towards the promoters. We then systematically examined other TLR ligands besides LPS, such as CpG or poly (I: C), which can also activate the NF-κB pathway. As is shown in Fig. 3A, the p-IκBα level as well as the p-IκBα/IκBα ratio was significantly enhanced by the TLR ligands, indicating an activation of the NF-κB pathway. Time course studies indicated that introduction of the above two ligands could lead to a gradual up-regulation of CKIP-1 (Fig. 3B and C). To determine whether CKIP-1 expression is influenced by NF-κB activity, RAW264.7 cells were treated with LPS, CpG in the presence of the NF-κB inhibitor, BAY11-7082. As shown in Fig. 3D and E, inhibition of NF-κB activity blocked the up-regulation of CKIP-1 expression. Moreover, IL-4/IL-13 is capable of activating STAT6 during the M2 polarization pathway, we then aimed at unraveling the possible mechanism of STAT6 upon regulation of CKIP-1. Surprisingly, overexpression of STAT6 in the RAW264.7 cell line induced a down-regulation of CKIP-1 while knockdown of STAT6 lead to an up-regulation of CKIP-1, regardless of the status of IL-4 stimulation (Fig. 3F). Similar results were shown in response to IL-13 stimulation (Fig. 3G). Thus, we concluded that LPS inhibits CKIP-1 through the positive regulation of NF-κB and IL-4/IL-13 promote the cluster transcription through STAT6 mediated negative regulation. 3.3 CKIP-1 regulates inducible pro and anti-inflammatory cytokines in macrophages

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It’s intriguing that both M1 and M2-associated transcription factors could simultaneously regulate the expression of the same gene. Therefore, we deduced that CKIP-1 must play a crucial role in regulating macrophage polarization by modulating M1/M2 balance. As mentioned above, CKIP-1 was induced by LPS or IFN-γ. To investigate the effect of CKIP-1 on the pro-inflammatory response of M1 macrophages, RAW264.7 cells transfected with CKIP-1 shRNA were stimulated by 100ng/ml LPS. Pro-inflammatory gene expression was analyzed by qPCR. Ckip-1 depletion strongly attenuated LPS-induced pro-inflammatory genes expression of TNF-α, IL-1β, IL-6, IL-12, MCP-1, COX-2 and MIP-1α (Fig. 4A). Accordingly, RAW 264.7 cells were transfected with lentivirus packaged MSCV-vector or MSCV-CKIP-1 plasmids and stimulated with LPS. Consistently, Ckip-1 overexpression augmented the LPS-induced cytokines to various degrees (Fig. 4B). We next asked whether CKIP-1 could also participate in macrophage plasticity by influencing the IL-4-induced anti-inflammatory gene expression. Accordingly, RAW264.7 cells were transfected with control or CKIP-1 specific shRNA and induced with 10 ng/ml IL-4, expression level of anti-inflammatory genes were analyzed by qPCR. As expected, IL-4 significantly induced expression of Arg1, Ym1, Fizz1 compared with the control group (Fig. 4C). Intriguingly, overexpression of CKIP-1 in RAW264.7 cells significantly attenuated IL-4-induced anti-inflammatory gene targets (Fig. 4D). To further confirm this result, we sought out to examine the effect of Ckip-1 deficiency on LPS-induced pro-inflammatory gene expression in primary macrophages. Ckip-1+/+ and Ckip-1-/- BMDMs were stimulated with various TLR ligands of NF-κB pathway including LPS, Poly (I: C), CpG and PGN, the concentration of IL-6 and TNF-α in supernatant declined significantly due to Ckip-1 deficiency (Fig. 5A and B). In addition, TG-elicited PMs from Ckip-1 WT and KO mice were treated with 10 ng/ml LPS and detection of IL-6 and TNF-α in supernatant obtained similar results (Fig. 5C and D). Interestingly, studies utilizing CKIP-1-deficient BMDMs and TG-elicited PMs revealed enhanced expression of Arg1, Ym1, Fizz1 following IL-4 (Fig. 5E and F) or IL-13 (Fig. 5G and H). Taken together, our results indicate that CKIP-1 promotes LPS-induced M1 gene expression but abrogates IL-4 induced M2 gene expression in macrophages.

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3.4 CKIP-1-deficient mice were resistant to LPS-induced endotoxic shock

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We evaluated the in vivo physiological function of CKIP-1 in macrophage differentiation by introducing the LPS-mediated sepsis model. CKIP-1 WT and KO mouse were intraperitoneal injected with 20mg/kg LPS simultaneously and the survival ratio were counted. 80% of the Ckip-1+/+ mice developed discrepant morbidity and mortality within 2 d of LPS infection and the earliest death emerged within 8 h of the appearance of symptoms. In contrast, only approximately 20% of the infected Ckip-1-/- mice exhibited these symptoms which then died over the first 12 h with the rest of the community survived after 96 h, implying Ckip1-/- mice are insensitive to LPS mediate sepsis (Fig. 6A). Consistently, sera from Ckip-1−/− mice injected with LPS for 1h or 3 h showed significantly lower concentrations of IL-10, IL-6, IL-12p70, TNF-α and IFN-γ compared with that from the wild-type mice (Fig. 6B-F). We further investigated the role of CKIP-1 in balancing M1/M2 polarization upon LPS-induced inflammation by injecting the Ckip-1+/+ and Ckip-1-/- mice with low dose of LPS intraperitoneally. We found that the expression of pro-inflammatory cytokines including iNOS, TNF-α, IL-12 were obviously downregulated in Ckip-1-/- PMs compared to the WT control (Fig. 6G), while the expression of anti-inflammatory cytokines including Arg1, Fizz1, Ym1 were significantly upregulated, respectively (Fig. 6H). Collectively, these data suggest that Ckip-1 is essential for macrophage polarization in vivo. 3.5 CKIP-1 deficiency was dispensable neither for the GM-CSF induced pro-inflammatory phenotype of BMDM nor for the phagocytic and antigen presenting ability of PEM Previous studies have reported the distinction between M-CSF and GM-CSF stimulated macrophages in secreting inflammatory cytokines [35] ， Briefly, M-CSF favors the generation of IL-10-producing, immunosuppressive, M2-polarized macrophages, whereas GM-CSF promotes a pro-inflammatory, M1-polarized phenotype. To examine whether CKIP-1 contributes to the plasticity of macrophage polarization, we investigated the role of CKIP-1 in regulating M-CSF or GM-CSF stimulated macrophages. Treatment of CKIP-1+/+ and CKIP-1-/- BMDMs with M-CSF led to impaired production of M1 phenotypic markers including TNF-α，IL-6， 6

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IL-12，IL-10 after stimulation with LPS. Conversely, treatment of the corresponding macrophages with GM-CSF led to equal production of M2 phenotypic markers after stimulation with same amount of LPS (Fig. 7A and B). To further confirm the phenomenon, we attempted to convert one population into another by culturing M-CSF-induced M2 macrophages with GM-CSF, whereas treating GM-CSF-induced M1 macrophages with M-CSF. We demonstrated that M2 to M1 conversion of macrophage resulted in a recovery in secretion of TNF-α or IL-6, compared to the WT control. Once again, the secretion of TNF-α and IL-6 during the M1-M2 conversion of macrophage were unchanged. (Fig. 7C and D). These results implied that CKIP-1 may participate in the M-CSF induced macrophage polarization whereas indispensable for the GM-CSF induced macrophage polarization. It’s important to note that one of the hallmarks of M1 macrophage polarization is the acquisition of the antigen-presenting features, which can promote Th1 and Th17 type inflammation response and lead to efficient T H1 responses, whereas the M2 macrophage demonstrated stronger phagocytic ability. Therefore, we set out to determine the role of CKIP-1 in regulating these cellular functions of M1/M2 macrophages. Fluorescent microscopy revealed that Ckip1-/- and WT PEMs take up similar amounts of inactivated fluorescent labelled K-12 E.coli bio-particles (Fig. 7E). In addition, in splenocytes from LPS-challenged Ckip-1-/- mice cultured ex vivo for an additional 48 h, we observed similar production of IFN-γ and IL-17 (Fig. 7F), indicating the dispensable roles of CKIP-1 in regulating Th1 and Th17 type inflammation. 3.6 Generation and Characterization of a Myeloid-specific CKIP-1 deficient Murine Line

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To further investigate the functions of CKIP-1 in myeloid–derived macrophage differentiation. We generated myeloid-specific CKIP-1 null mice by crossing Ckip-1 conditional mutant mice containing loxp sites flanking exons 3 to 6 with Lyz2 Cre mice (Fig. 8A). These mice were genotyped with site-specific primers (Fig. 8B, upper panel) and further backcrossed to Lyz2 Cre mice to generate offsprings expressing two Cre and floxed Ckip-1 alleles (Fig. 8B, lower panel). qPCR analysis confirmed that the Lyz2 Cre: Ckip-1fl/fl BMDMs and thioglycollate-elicited peritoneal macrophages were defective at producing Ckip-1 mRNA (Fig. 8C). Lyz2 Cre: Ckip-1fl/fl mice were born at the Mendelian ratio and did not display any developmental abnormality (Fig. 8D). To corroborate this observation at the protein level, Lyz2cre and Lyz2 Cre: Ckip-1fl/fl BMDMs and thioglycollate-elicited peritoneal macrophage were analyzed for CKIP-1 protein expression. Our results suggest that CKIP-1 protein is absent in Lyz2 Cre: Ckip-1fl/fl thioglycollate-elicited peritoneal macrophages and BMDMs whereas the abundance of Ckip-1 in liver or heart tissue remain unaltered (Fig. 8E). We also examined the effect of myeloid Ckip-1 deficiency on adult mouse hematopoietic cell compartments. Our results revealed no significant difference in lymphocyte, neutrophil, eosinophil, and basophil populations between Lyz2 Cre and Lyz2 Cre: Ckip-1fl/fl groups (Fig. 8F). Further analysis of additional hematological parameters, including RBC and platelet count, showed no divergence between Lyz2 Cre and Lyz2 Cre: Ckip-1fl/fl groups (Fig. 8G). Collectively, our results demonstrate that myeloid-specific Ckip-1 deficiency did not significantly alter the hematopoietic cell compartment in adult mice.

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3.7 Myeloid CKIP-1 deficiency reduce cutaneous inflammation in vivo Next, we sought to determine whether myeloid CKIP-1 deficiency affects an acute inflammatory response in vivo. We employed the TPA-induced cutaneous inflammation model that is characterized by myeloid infiltration and tissue edema. Accordingly, the left ear of Lyz2 Cre: Ckip-1fl/fl mice was treated with phorbol ester TPA, and the right ear served as vehicle control. Our results demonstrate that myeloid deficiency of CKIP-1 significantly attenuated TPA-induced inflammatory edema in Lyz2 Cre: Ckip-1fl/fl mice (Fig. 9A). To examine TPA-induced myeloid cell recruitment, ear tissue extracts from Lyz2 Cre: Ckip-1fl/fl mice were evaluated for myeloperoxidase activity. As shown in Fig. 9B, myeloid-specific deficiency of Ckip-1 significantly impaired myeloid cell recruitment in Lyz2 Cre: Ckip-1fl/fl mice. Next, we examined the role of myeloid CKIP-1 in balancing pro- and anti-inflammatory gene expression following TPA treatment. Accordingly, total RNA was obtained from Lyz2 Cre: Ckip-1fl/fl mouse ears following TPA or vehicle control treatment. Quantitative analysis of major pro-inflammatory cytokines, such as IL-1β, IL-6, Mcp-1 and TNF-α, indicated that myeloid deficiency of CKIP-1 significantly attenuated expression of these cytokines following TPA exposure (Fig. 9C–F). In accordance with this observation, deficiency of myeloid CKIP-1 significantly enhanced expression of anti-inflammatory genes, including Arg1, Ym1, 7

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We then wondered how CKIP-1 participate in the balance of macrophage polarization. Ckip-1–/– mice were crossed with Stat6–/– mice, both in the C57BL/6 backgrounds, then age- and sex-matched Ckip-1–/– and Ckip-1-/-Stat6-/- mouse were intraperitoneally injected with 20mg/kg LPS simultaneously and the survival ratio was evaluated similar to Fig. 6A. As indicated in Fig. 10 A, Ckip-1-/-Stat6-/- mice exhibited reduced resistance to LPS mediated endotoxin shock, with the morality increased to a comparative level of Ckip-1+/+ mice. Consistently, sera from Ckip-1−/−Stat6−/−mice injected with LPS for 1 h or 3 h showed significantly higher concentrations of IL-10, IL-6, IL-12p70, TNF-α and IFN-γ, compared with that from Ckip-1−/− mice (Fig. 10B-F). We further investigated the relationship of Ckip-1 and Stat6 in balancing M1/M2 polarization upon LPS-induced inflammation by injecting the Ckip-1-/- and Ckip-1-/-Stat6-/- with low dose of LPS intraperitoneally. We found that the expression of pro-inflammatory cytokines including iNOS, TNF-α, IL-12 were obviously downregulated in Ckip-1-/- PMs compared to the Ckip-1-/- (Fig. 10G) , while the expression of anti-inflammatory cytokines including Arg1, Fizz1, Ym1 were significantly upregulated, respectively (Fig. 10H). Taken together, these data suggest that CKIP-1 regulates macrophage polarization in a STAT6 dependent manner. 4. Discussion

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Transcriptional control of macrophage homeostasis is currently the subject of intense investigation. To date, control of macrophage polarization has largely been attributed to the function of a small group of factors including AP-1, HIFs, NF-κB, PPARs, STATs and KLFs [36]. Our observations reinforced the notion that members of PH-like domain containing family might act as contributors to this process and, more specifically, identify CKIP-1 as critical molecular toggle in regulating M1/M2 polarization. Initially we discovered that pro-inflammatory M1 stimuli such as IFN-γ and LPS induce CKIP-1 mRNA/protein expression in human and murine macrophages whereas anti-inflammatory M2 stimuli such as IL-4 and IL-13 suppress CKIP-1 mRNA/ protein expression in human and murine macrophages (Fig. 1 and 2); Gain and loss of function studies in vitro revealed that CKIP-1 promotes LPS-induced pro-inflammatory gene expression in macrophages, in contrast it attenuates IL-4-induced anti-inflammatory gene expression in macrophages (Fig. 4); By corroborative evidence in disease models we unraveled that CKIP-1 deficiency resulted in a weakened pro-inflammatory phenotype of macrophages with reduced morality (Fig. 5 and 6). A key observation provided by our studies is that CKIP-1 can differentially affect a large repertoire of genes that characterize the M1 and M2 phenotype. With respect to M2 polarization, our studies indicate that CKIP-1 cooperates with STAT6 to induce quintessential M2 targets such as Arg-1 following IL-4/IL-13 stimulation. Our studies revealed that the negative regulation of CKIP-1 on M2 polarization is dependent on the integrity of STAT6 since double knockout of both CKIP-1 and STAT6 could counterbalance the resistance to LPS mediated sepsis caused by the single loss of CKIP-1(Fig. 6A and Fig. 10A). This type of inductive and cooperative relationship has also been observed between STAT6 and PPARγ in macrophages during M2 activation [37]. More evidences should be raised to support the possibility that CKIP-1 and STAT6 may also cooperate to augment PPARγ expression. While much of our work here has focused on M2 polarization in the context of IL-4 stimulation, a recent study suggested that alternative IL-4–independent mechanisms may also regulate the M2 phenotype [17] . Whether CKIP-1 participates in this pathway is an important issue that deserves further investigation. The induction of CKIP-1 also provides a molecular mechanism to not only inhibit the M2 phenotype but also induce the M1 pathway. The importance of CKIP-1 promotion of pro-inflammatory targets is highlighted by multiple lines of proofs. Moreover, the observation that CKIP-1 deficiency attenuates the ability of IL-4 to inhibit M1 targets suggests that CKIP-1 may also be important in suppression of pro-inflammatory genes in M2 macrophages. Our mechanistic studies demonstrate that activated NF-κB pathway exerts this pro-inflammatory effect in a CKIP-1 dependent manner since activation of IKK/IκBα by ligands of NF-κB activators moderately enhanced the transcriptional level of CKIP-1 (Fig. 3A-E). Finally, in addition to competing for coactivators, STAT6 mediated inhibition of CKIP-1 8

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likely contributes to the potent induction of M1 polarization by CKIP-1. Efforts to understand whether these two factors cooperate to stimulate classical macrophage activation is an important issue awaits further studies. Our studies also provide insights regarding discrepant observations in the literature regarding divergent effect of M-CSF and GM-CSF towards macrophage polarization (Fig. 7A-D), such phenomenon is interpretable since the idiosyncrasies of various cell lines employed (e.g., J774.1, THP-1 and RAW) and differences in the concentration (ranging from 10 ng/ml to 1,000 ng/ml) may account for this disparate results. It is also worth to note that CKIP-1 deficiency was dispensable neither for phagocytic and antigen presenting features nor for efferocytosis capacities (Fig. 7E and F) , which is quite different from other critical regulators of macrophage polarization including IRF5 [16], SCOS[38, 39] and ICAM-1[40]. CKIP-1 is a ubiquitously expressed member of the PLEKH family that has been implicated in a myriad of key cellular processes such as differentiation, development, proliferation, and programmed cell death in diverse cell types[41] .Alterations in CKIP-1 expression or function have been implicated in the pathogenesis of numerous human diseases, including cancer, cardiovascular hypertrophy, adipogenesis [42] and osteoporosis. Previous studies have also implied this factor to hematopoietic biology [43]. Since CKIP-1 knockout mice are viable, there may not be defects in erythrocyte and mixed colony formation for the normal derivations of progenitor hematopoietic cells. However, the role of CKIP-1 in adult hematopoietic cell function has not been investigated. Our approach to delete the myeloid compartment employed lyz2 cre. Although this cre is expressed at low levels during myeloid development, its activity is most significant after activation in the mature state Thus, it is not surprising that development of the myeloid and other hematopoietic lineages was unaffected (Fig.7). However, our findings do support a key role for CKIP-1 in tangling the two inflammatory phenotypic states of a macrophage. These findings prompted us for future investigations focused on the role of CKIP-1 in the function of other hematopoietic lineages. Studies from multiple laboratories highlight the importance of tight transcriptional control in macrophage polarization. The activation of stimulus-specific transcription factors integrated expression of a subset of genes that confer the functional properties of the polarized state. In some cases, distinct members within the same family might identify diametric opposite potential of polarization. For example, Scos3 enhances M1, and Scos1 promotes the M2 phenotype, similar effects have been reported for HIF-1/HIF-2, IRF5/IRF4 and KLF2/KLF4/KLF6 [44] [45]. Intriguing possibilities have been raised that in response to external cues the PLEKH members may exert differential regulation of macrophage, which might be an important event required to exact characteristic gene programs. Recent studies have linked several members of PH domain-containing protein (e.g. AKT1, AKT2, AKT3) [46, 47] to monocyte differentiation and macrophage activation. Will they resembling the PH domain containing AKT families? In summary, the in vitro, ex vivo, and in vivo observations presented here highlight the importance of CKIP-1 in macrophage polarization. As polarization has been shown to be important in various macrophage functions in acute inflammatory conditions (e.g. pathogen infections and sepsis) and chronic inflammatory conditions (e.g. obesity, insulin resistance, and atherosclerosis). Further in depth studies assessing the effect of myeloid CKIP-1 deletion on these biological processes are warranted. Confirmation would provide the requisite stimulus for efforts targeting CKIP-1 for therapeutic gain in the treatment of numerous inflammatory diseases.

Lingqiang Zhang and Fuchu He conceived the project and supervised the study; Yuhan Chen, Wen Liu, Yiwu Wang, Luo Zhang performed most of experiments, J.W. and X.Z. provided help in statistical analysis and proof reading the article ; Y.C. and L.Z. summarized the data and wrote the manuscript; all authors reviewed and revised the manuscript and approved the final version. Disclosure The authors disclose no potential conflicts of interest. All authors approved the final article. Acknowledgements 9

ACCEPTED MANUSCRIPT We thank Dr Zhengfan Jiang for kindly providing Stat6-/- mice. FengJun Xiao for FACS analysis. This work was supported by Chinese National Basic Research Programs (2013CB910803), The National Key Technologies R&D Program for New Drugs (2015ZX09J15102-001, 2014ZX09J14106-04C), the Program of International S&T Cooperation (2014DFB30020) and Chinese National Natural Science Foundation Projects (81570404, 31270912).

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Figure Legends Fig. 1. CKIP-1 expression is upregulated by M1-stimuli in human and mice macrophages. (A) Relative expression level of PH-domain containing proteins in BMDM (left panel) and PM (right panel) were determined by quantitative PCR analysis. Briefly, we assign the lowest expression of PH-containing protein in these cells as a value of 1, the relative folds of increase were indicated. (B) CKIP-1 relative expression of mRNA in multiple organs and systems including macrophages were analyzed by quantitative PCR. CKIP-1 levels in colon are assigned as value 1 and relative folds of increase are indicated. (C) qPCR analysis of the relative expression of Ckip-1 after the RAW264.7 cells were stimulated with multiple M1 or M2 stimulus. 500 ng/ml LPS, 20 ng/ml IFN-γ, 100 ng/ml IL-4 or 60 ng/ml IL-13, respectively. D-I, Macrophages from various derivations were manipulated upon different M1 stimulus with indicated times. D and E, Mouse BMDMs (D) and human primary macrophages (E) were induced with 10 ng/ml IFN-γ and 100 ng/ml LPS separately for 8h (n=4). CKIP-1 protein (upper panel) and mRNA (lower panel) expression were analyzed by Western blot and quantitative PCR analysis, respectively. Mouse BMDMs (F) and PMs (G) were induced with 100 ng/ml LPS for 0, 4, 8, 12h, while mouse BMDMs (H) and PMs (I) were induced with 20ng/ml IFN-γ for 0, 2, 4, 8h. CKIP-1 protein (upper panel) and mRNA (lower panel) expression kinetics were examined by Western blot and quantitative PCR analysis (n=4), respectively. GAPDH were used as housekeeping gene for Western blot and quantitative PCR analysis, respectively. Mean ± SEM were obtained from a minimum of three independent experiments, and p values less than 0.05 between indicated groups are considered significant. *, p < 0.05; **, p < 0.01.

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Fig. 2. CKIP-1 expression is down regulated by M2 stimuli in human and mice macrophages. A and B, Mouse BMDMs (A) and human primary macrophages (B) were induced with 10 ng/ml IL-4 and 10 ng/ml IL-13 separately for 20 h. CKIP-1 protein (upper panel) and mRNA (lower panel) expression were analyzed by Western blot and quantitative PCR analysis, respectively. C-F, Mouse BMDMs (C) and PMs (D) were induced with 10 ng/ml IL-4 for 0, 1, 2, 3 days. CKIP-1 protein (upper panel) and mRNA (lower panel) expression kinetics by Western blot and quantitative PCR analysis, respectively. Mouse BMDMs (E) and PMs (F) were induced with 10 ng/ml IL-13 for 0, 1, 2, 3 days. CKIP-1 protein (upper panel) and mRNA (lower panel) expression kinetics by Western blot and quantitative PCR analysis, respectively. F and G, human primary macrophages were induced with 10 ng/ml IL-4 (F) or 20 ng/ml IL-13 (G) for indicated times. Western blot and qPCR were done to measure protein (upper panel) and transcriptional (lower panel) levels of CKIP-1. GAPDH or Actin were used as housekeeping gene for Western blot and quantitative PCR analysis, respectively. Mean ± SEM were obtained from a minimum of three independent experiments, and p values less than 0.05 between indicated groups are considered significant. *, p < 0.05; **, p < 0.01. Fig. 3. The LPS-NF-κB pathway and the IL4/IL-13-STAT6 pathway is involved in the transcriptional regulation of CKIP-1. (A) The total IκBα and p-IκBα levels as well as their relative ratios were determined in RAW264.7 cells after stimulation with the NF- κB pathway activators, CpG OND, LPS, and Poly (I: C), normalized to GAPDH levels. B and C, Quantitative PCR analysis of the relative expression of CKIP-1 after the RAW264.7 cells were stimulated with NF-κB pathway activators including CpG (B) and Poly (I: C) (C) for indicated times. The NF-κB inhibitor, BAY11-7082, could block the down-regulation of the expression of CKIP-1 induced by LPS (D) and poly (I: C) (E). (F) Quantitative PCR analysis of the CKIP-1 expression in RAW264.7 cells transfected with the STAT6-overexpressing construct or STAT6 siRNA, following stimulation with 40 ng/ml IL-4 or PBS. (G) Quantitative PCR analysis of the CKIP-1 expression in RAW264.7 cells transfected with STAT6-overexpressing 13

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Fig. 4. CKIP-1 regulates inducible pro and anti-inflammatory cytokines in murine macrophage cell line RAW 264.7. A and B, Quantitative PCR analysis of relative expression of TNF-α, IL-1β, IL-6, IL-12, MCP-1, COX-2 and MIP-1α mRNA level in CKIP-1 depleted (A) or CKIP-1 stably over-expressing (B) RAW 264.7 cells. C and D, Quantitative PCR analysis of relative expression of Arg1, Ym1, Fizz1 mRNA level in CKIP-1 depleted (C) or CKIP-1 stably over-expressing (D) RAW 264.7 cells. Mean ± SEM were obtained from a minimum of three independent experiments, and p values less than 0.05 between indicated groups are considered significant. *, p < 0.05; **, p < 0.01. Fig. 5. CKIP-1 deficiency in murine primary macrophages regulates inducible pro and anti-inflammatory cytokines. Ckip-1+/+and Ckip-1-/- BMDMs were treated with indicated TLR ligands (PGN, 10 ng/ml, Poly (I: C), 100 ng/ml, LPS, 50 ng/ml, CpG, 2μM), after 24 hours, Concentration of IL-6 (A) and TNF-α (B) in supernatant were measured by ELISA. TG-treated PMs from Ckip-1 +/+and Ckip-1-/- mice were stimulated with 50 ng/ml LPS, Concentration of IL-6 (C) and TNF-α (D) in supernatant were measured by ELISA. qPCR showing expression level of Arg1, Ym1, Fizz1 mRNAs in Ckip-1+/+and Ckip-1-/- BMDMs (E) and PMs (F) ,Total RNAs were prepared after stimulation with 10 ng/ml IL-4. qPCR showing expression level of Arg1, Ym1, Fizz1 mRNAs in Ckip-1+/+and Ckip-1-/- BMDMs (G) and PMs (H). Total RNAs were prepared after stimulation with 40 ng/ml IL-13. Mean ± SEM were obtained from a minimum of three independent experiments, and p values less than 0.05 between indicated groups are considered significant. *, p < 0.05; **, p < 0.01.

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Fig. 6. CKIP-1-deficient mice were resistant to LPS-induced sepsis. (A) Ckip-1+/+ and Ckip-1-/- mice were intraperitoneal injected with 20 mg/kg LPS (n=16). The mortality were monitored by indicated time points (p < 0.001; Kaplan-Meier test). Serum cytokines including IL-10 (B), IL-6 (C), IL-12p70 (D), TNF-α (E), IFN-γ (F) were measured by CBA at various times after injection (n=8). Ckip-1−/− mice (n=10) and their wild-type littermates (n=10) injected intraperitoneally with sublethal dose of LPS (20 µg), peritoneal macrophages were collected and

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mRNA of certain M1 markers including iNOS, TNF-α，IL-12 (G) and M2 markers including Arg1, Ym1, Fizz1 (H) were examined. *, p < 0.05; **, p < 0.01. Student’s t-test unless noted. Data are from three independent experiments (mean and s.e.m. of eight to ten serum samples).

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Fig. 7. CKIP-1 deficiency was dispensable neither for the GM-CSF induced pro-inflammatory phenotype of BMDM nor for the phagocytic and antigen presenting ability of PEM. A and B,Ckip-1+/+ and Ckip-1-/- M-BMDMs (A) and GM-BMDMs (B) were treated with 20 ng/ml LPS respectively, after 24 hours IL-6, TNF-α, IL-12 and IL-10 in the supernatants were measured by ELISA. C and D, Ckip-1+/+ and Ckip-1-/- M-BMDMs or GM-BMDMs were cultured for additional 2 days in the presence of 50 ng/ml GM-CSF (C) or 20 ng/ml M-CSF (D) respectively as indicated, before LPS (20 ng/ml) were added. After 24 hours IL-6 (left panels) and TNF-α (right panels) in supernatant were measured by ELISA. (E) Ckip-1+/+ and Ckip-1-/- peritoneal macrophages take up similar amounts of K-12 E. coli bioparticles as revealed by fluorescent microscopy (left panel) and flow cytometry assay (right panel). (F) ELISA of IFN-γ and IL-17A in spleen cells obtained from the LPS-injected Ckip-1+/+ and Ckip-1-/- mice and cultured for 48 h in the presence of antibody to CD3. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from two independent experiments (mean and s.e.m. of four to five spleen cultures). Fig. 8. Generation and characterization of myeloid specific CKIP-1 deficient mice line. (A) Schematic representation of wild-type CKIP-1 locus shows its exons (boxes) and introns (lines). The CKIP-1 locus was disrupted by the targeting vector and the positions of the primers for genotyping are shown. The FRT sequence in loxp cassette interrupts the normal splicing of CKIP-1 gene when Cre recombinase was introduced, resulting in the conditional loss of CKIP-1 expression. (B) Genotyping of CKIP-1 myeloid specific knockout mice. Presence of upper band (~373 bp) indicates introduction of a CKIP-1 floxed site in indicated systems. WT: Lyz2 Cre, CKO: Lyz2 Cre Ckip-1fl/fl. (C) Quantitative PCR analysis of Ckip-1 mRNA abundance in BMDMs and PMs of various genotypes. GAPDH gene was used as loading control. (D) Summarization of genotypes of the offspring from the 14

ACCEPTED MANUSCRIPT breeding of Lyz2 Cre Ckip-1fl/+ mice. (E), BMDMs and PMs form Lysz2 Cre and Lyz2 Cre Ckip-1fl/fl mice were lysed and subjected to immunoblot to confirm the myeloid specific depletion of CKIP-1 (left panel), while liver and heart tissue extracts were used as parallel control (right panel). F and G, Age- and gender-matched Lyz2 Cre and Lyz2 Cre Ckip-1fl/fl mouse total blood was collected by venipuncture in heparin/EDTA-coated tubes. Samples were analyzed by MEK 6318KTM Hematology profiling unit. Data represent mean ± SEM and p value less than 0.05 between the indicated groups is considered significant.

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Fig. 9. Myeloid CKIP-1 deficiency reduce cutaneous inflammation in vivo. (A) Lyz2 Cre and Lyz2 Cre Ckip-1fl/fl mice were subjected to TPA-induced cutaneous inflammation model. Ear weight from control and experimental groups was assessed. The percentage increase in ear weight compared with control is displayed. (B) Whole amount protein extracts of ear tissue from control and TPA-treated groups from Lyz2 Cre and Lyz2 Cre Ckip-1fl/fl mice were subjected to myeloperoxidase activity assay. Data are indicated as MPO activity units/mg of total protein. Total RNA extracts from Lyz2 Cre and Lyz2 Cre Ckip-1fl/fl mouse ear tissue exposed to TPA or vehicle control was analysed for quantitative PCR analysis including IL-6 (C), IL-1ß (D), MCP-1 (E), TNF-α (F), Arg1 (G), Ym1 (H), Fizz1 (I), MRC1 (J), Retnla (K). GAPDH was used as the housekeeping gene. The combined data from three experiments are shown. Unpaired Student’s t-test unless noted. Data represent mean ± SEM and a p value less than 0.05 between the indicated groups is considered significant. *, p < 0.05; **, p < 0.01. ***, p < 0.001

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Fig. 10. CKIP-1 regulates macrophage polarization by modulating STAT6 functions. (A) Ckip-1-/- and Ckip-1-/Stat6-/- mice were intraperitoneal injected with 20 mg/kg (n=8) LPS. The mortality were monitored by indicated time points (p=0.0205; Kaplan-Meier test). Serum cytokines including IL-10 (B), IL-6 (C), IL-12p70 (D), TNF-α (E), IFN-γ (F) were measured by CBA at various times after injection (n=8). Ckip-1−/− mice (n=10) and Ckip-1-/- Stat6-/littermates (n=10) injected intraperitoneally with sublethal dose of LPS (20 µg), peritoneal macrophages were collected and mRNA of certain M1 markers including iNOS, TNF-α, IL-12 were examined (G). LPS treated peritoneal macrophages were collected in G, mRNA of certain M2 markers including Arg1, Ym1, Fizz1 were examined (H). *, p < 0.05; **, p < 0.01. Unpaired Student’s t-test unless noted. Data are from three independent experiments (mean and s.e.m. of eight to ten serum samples).

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ACCEPTED MANUSCRIPT Highlights •CKIP-1 expression was significantly induced in M1 macrophages while strongly reduced in M2 macrophages. •CKIP-1 balances inducible pro and anti-inflammatory cytokines in human and mice derived macrophages.

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•CKIP-1 deficiency impairs LPS-induced endotoxic shock in vivo. •Myeloid CKIP-1 deficiency reduces cutaneous inflammation in vivo.

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•CKIP-1 regulates M2 macrophage polarization by negatively modulating STAT6 functions

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