Reprogramming of TAM toward proimmunogenic type through regulation of MAP kinases using a redox-active copper chelate

Reprogramming of TAM toward proimmunogenic type through regulation of MAP kinases using a redox-active copper chelate
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  Reprogramming of TAM towardproimmunogenic type through regulation of MAP kinases using a redox-activecopper chelate  Paramita Chakraborty,*  ,1 Shilpak Chatterjee,*  ,1  Avishek Ganguly,* Piu Saha, †  Arghya Adhikary,  ‡ Tanya Das,  ‡  Mitali Chatterjee, †  and Soumitra Kumar Choudhuri*  ,2 *Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, India; † Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India; and  ‡ Department of Molecular Medicine, Bose Institute, Kolkata, India RECEIVED JUNE 13, 2011; REVISED DECEMBER 26, 2011; ACCEPTED DECEMBER 28, 2011. DOI: 10.1189/jlb.0611287  ABSTRACT TAMs, present in the tumor microenvironment, play animmunosuppressive role, leading to tumor progressionand metastasis. Recently, numerous attempts havebeen made to switch immunosuppressive TAMs into animmunostimulatory type. Previously, we showed that acopper chelate, viz., copper N-(2-hydroxy acetophe-none) glycinate [CuNG], can reprogram TAMs toward the proimmunogenic type to mount an antitumor im-mune response, but the underlying molecular mecha-nisms of skewing TAMs toward the proimmunogenic type remain elusive. Herein, we tried to explore the sig-naling mechanisms responsible for the reprogrammingof TAMs. We observed that CuNG-induced ROS genera- tion triggers activation of two MAPKs, i.e., p38MAPK andERK1/2, and also causes up-regulation of intracellular glu- tathione. Furthermore, activation of p38 MAPK up-regu-lated the initial IL-12 production and the activation of ERK1/2 in tandem with GSH, found responsible for IFN-   production by TAMs. This IFN-   , in turn, prolonged IL-12production and down-regulated TGF-   production and thus, plays the decisive role in CuNG-mediated repro-gramming of regulatory cytokine production by TAMs.Our work highlights that ROS-mediated activation of MAPKs can convert suppressive macrophages toward the proimmunogenic type. Thus, the present study opens the possibility of targeting TAMs by the use of redox-ac- tive compounds for designing a novel, therapeutic strat-egy against cancer.  J. Leukoc. Biol. 91:000–000;2012. Introduction Macrophages are functionally the most versatile cell popula-tion and play an indispensable role in orchestrating the im-mune system [1]. Depending on the local cytokine milieu,macrophages can adopt a diverse, functional phenotype, whichis often found to be opposite in nature, e.g., inflammatory ver-sus anti-inflammatory and tissue remodeling versus tissue de-struction [2–4]. The polarization phenotypes of macrophagesare mutually exclusive and are regulated in such a way so that macrophages can perform a plethora of balanced and harmo-nious functions [5–7].Studies with the tumor microenvironment reveal that in tu-mor stroma, a symbiotic relationship exists between a growingtumor and its associated macrophages [8–10]. Evidence sub-stantiates that the greater the macrophage infiltration in thetumor site, the worse the prognosis of patients [11]. Recent studies further reveal that tumor cells produce an array of sol-uble factors, chemokines, and cytokines, favoring the mono-cyte infiltration at the tumor site, and convert the monocytestoward tumor-associated macrophages (TAMs), which are im-munosuppressive in nature [12–15]. TAMs at the tumor sitesecrete a wide range of growth factors, proangiogenic factors,and matrix metalloproteinases providing enormous support totumor growth and metastasis [10, 16]. TAMs also secrete vari-ous immunosuppressive cytokines, such as TGF-   and IL-10, which effectively subvert the induction of proper anti-tumor Tcell response [12, 17]. Besides secretion of immunosuppressivemediators, the most important immunosuppressive strategy of TAMs at the tumor site is the generation of the regulatory Tcell (CD4  CD25  Forkhead box P3  ) population, which effec-tively suppresses the induction of other anti-tumor T cell re-sponses [18]. 1. These are joint first authors.2. Correspondence: Department of In Vitro Carcinogenesis and CellularChemotherapy, Chittaranjan National Cancer Institute, 37, S.P. Mukher- jee Rd., Kolkata-700 026, India. E-mail:  Abbreviations: 3LL  Lewis lung carcinoma, BSO  DL-buthionine sulfoxi-mine, CPCSEA   Committee for the Purpose of Control and Supervision of Experiments on Animals, CuNG  copper  N  -(2-hydroxy acetophenone) gly-cinate, Dox   doxorubicin, EAC  Ehrlich ascites carcinoma, EAC/ Dox   doxorubicin-resistant Ehrlich ascites carcinoma, IAEC  Institutional Animal Ethics Committee, PEG  polyethylene glycol, TAM   tumor-associ-ated macrophage, TCM   tumor-conditioned macrophage The online version of this paper, found at, includes supplemental information.  Article  0741-5400/12/0091-0001 © Society for Leukocyte Biology   Volume 91, April 2012  Journal of Leukocyte Biology   1   Epub ahead of print January 25, 2012 - doi:10.1189/jlb.0611287   Copyright 2012 by The Society for Leukocyte Biology.  Recent work substantiates that plasticity of TAMs may beused as a novel, therapeutic strategy to combat cancer. Reportsdisclose that redirection of the immunosuppressive phenotypesof macrophages toward the proimmunogenic types (IL-12-pro-ducing) generates a host-protective antitumor response andconsequently, reduces tumor growth and metastasis [19–21].Several attempts, so far, have been made to repolarize the sup-pressive phenotypes of macrophages toward the proimmuno-genic types through application of various proimmunogeniccytokines, such as IL-12 and IFN-    [19, 20]. Previously, we syn-thesized [22] and reported that a novel, nontoxic copper che-late, i.e., CuNG, can reprogram the functional behavior of TAMs by suppressing the production of TGF-   and simultane-ously elevating the production of IL-12 to elicit the proper an-titumor Th1 response [21], which is of tremendous impor-tance in immunotherapy of cancer [23]. However, the under-lying mechanism responsible for such conversion (up-regulation of IL-12 and down-regulation of TGF-   production)in suppressive TAMs remains obscure.Recent works suggest that activation of MAPKs critically regulates the differential cytokine production in various celltypes; e.g., defects in p38MAPK signaling significantly affect the IL-12 production by macrophages [24]. Furthermore,the production of TGF-   is regulated by the activation of different MAPKs [25, 26]. Recent studies highlight the cru-cial involvement of the intracellular redox status in regula-tion of cytokine production by DCs and macrophages. Cur-rent evidences disclose that the increase of intracellularGSH content enhances the IL-12 production in human andmurine monocytes through activating MAPKs and inversely correlates with IL-10 production [27].Our previous endeavor disclosed that skewing of the func-tional phenotype of TAMs toward the proimmunogenic typefollowing CuNG treatment was mediated through the genera-tion of a moderate level of ROS [21]. ROS generation is asso-ciated with activation of various signaling cascades, includingthose that affect the production of different inflammatory cy-tokines [28–30]. The present study describes how the preciseredox alteration by a redox-active compound, e.g., CuNG,leads to up-regulation of IL-12 and simultaneous down-regula-tion of TGF-   production and consequently, skews the behav-ior of macrophages from the suppressive to the proimmuno-genic type. The understanding of the signaling mechanismtriggered by the redox-altering compound may be helpful toreveal the intricate relationship between the oxidative stress-induced signal-transduction mechanism and alteration of thecytokine repertoire of TAMs and thus, explore the efficacy of other redox-active compounds in terms of their ability to con- vert the nature of TAMs from the immunosuppressive to theproinflammatory type. MATERIALS AND METHODS Reagents Penicillin and streptomycin were purchased from Sigma Chemical Co. (St.Louis, MO, USA). Murine IL-10, IL-12, TGF-  , IFN-   , OptEIA kit for ELISA of murine cytokines, FITC-conjugated IL-12, IFN-   - and biotin-conjugatedTGF-   mAb, and streptavidin-PE or -FITC were purchased from BD Biosci-ences/BD PharMingen (San Diego, CA, USA). FITC- or PE-conjugatedF4/80 mAb (murine) were obtained from eBioscience (San Diego, CA,USA). Antimouse phospho-p38MAPK, phospho-ERK1/2, and totalp38MAPK and ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). BSO,   -tocopherol, PEG-catalase, p38MAPK inhibitorSB203580, MEK1/2 inhibitor PD98059, and secondary antirabbit-FITC werebrought from Sigma-Aldrich (Sigma Chemical Co.). RPMI 1640, DMEM,and FBS were purchased from Gibco (Invitrogen, Carlsbad, CA, USA).  Animals Swiss albino mice, obtained from National Institute of Nutrition (Hy-derabad, India) and maintained in the institute’s animal facilities, wereused for experimental purpose with prior approval of the IAEC. The exper-imental protocols described herein were approved by the IAEC (Registra-tion No. 175/99/CPCSEA, dated 1/28/2000), in accordance with the ethi-cal guidelines laid down by the CPCSEA by the Ministry of Social Justiceand Empowerment, Government of India. Adult, female Swiss albino mice, weighing 18–20 g, were kept for a quarantine period of 1 week at a tem-perature of 25  2°C and relative humidity of 55  2%, with a photo cycleof 12 h light/12 h dark. Water and food pellets were provided ad libitum. Cell line, tumor implantation, and experimental protocol The EAC cell was maintained as an ascitic tumor in female Swiss albinomice. EAC/Dox, which is also resistant against cisplatin, cyclophosphamide,and vinblastin, was developed and maintained, according to the methodsdescribed previously [31–33]. EAC/Dox cells were maintained in a Dox-free condition for at least one passage before the start of all of the experi-ments. For checking activation status of MAPKs in TAMs, EAC/Dox-bear-ing mice (7 days following peritoneal inoculation with 1  10 6 EAC/Doxcells) were kept untreated or treated with a single dose of CuNG (5 mg/kgbody weight) [21] for different time intervals. Isolation of TAMs TAMs were isolated as described previously [21]. Briefly, total ascitic fluid was drawn and kept at a standing position in a 50-ml sterile tube for at least 2 h for settling down the tumor cells, and then, clear fluid from theupper zone was collected. TAMs were isolated from that clear fluid, first, by negative selection with anti-CD5 and anti-CD19 and then, by positive selec-tion with anti-F4/80 using the BD IMag cell separation system (BD Biosci-ences), according to the manufacturer’s protocol, and resuspended inRPMI 1640 containing 10% FBS. Flow cytometric data revealed the purity of the separated population (90%). Cell line cultures and tumor-conditioned media  preparation Tumor-conditioned media were prepared following a reported method[34]. We used three different cancer cell lines—3LL, B-16 melanoma, andEAC/Dox—in the present work. 3LL and B-16 melanoma cell lines werecultured in DMEM, and the EAC/Dox cell line was cultured in RPMI 1640,supplemented with 10% FBS. Once grown to 80% of confluence, media were changed, and flasks were rinsed twice with saline solution. Cells werethen incubated with fresh DMEM or RPMI for 24 h; the conditioned media were collected and filtered at 0.22   m. Culture of macrophages in tumor-conditioned media  In the present study, we have used in vitro-raised TCMs, resembling theTAM phenotype (IL-12 low  TGF-  high ), by culturing normal mouse perito-neal macrophages for 3 days in the presence of tumor-conditioned mediaat a 1:1 ratio with RPMI 1640, supplemented with 10% FBS in a 5% CO 2 atmosphere at 37°C. 2  Journal of Leukocyte Biology   Volume 91, April 2012  Coculture of CFSE-labeled, normal CD4  T cells withTCMs or normal macrophages For isolation of normal CD4  T cells, LNs were collected from normalSwiss albino mice, and single-cell suspension was made in RPMI 1640 con-taining 10% FBS. CD4  populations were purified by single-positive selec-tion with an anti-CD4  direct magnet (DM) particle (BD Biosciences), us-ing the BD IMag cell separation system, according to the manufacturer’sprotocol. Isolated CD4  cells were first labeled with CFSE (5   M) and thenplated (10 6 cells/ml) in the presence of normal macrophages or TCMs(CuNG-treated or untreated). In all of the experiments, the macrophageconcentration was maintained at 10 5 cells/ml. For stimulation of CD4  Tcells, anti-CD3 antibody (5   g/ml) was applied in this culture medium andincubated in a 5% CO 2  atmosphere at 37°C. After 5 days of incubation, thenonadherent population was collected, labeled with proper antibodies, andsubjected to FACS analysis. Treatment  For in vitro studies, CuNG was used at a dose of 2.5   g/ml, which was re-ported to be nontoxic [21, 23, 35]. In some experiments, the GSH deple-tor BSO, ROS scavenger   -tocopherol, p38MAPK inhibitor SB203580, andMEK1/2 inhibitor PD98059 were used at a final concentration of 50   M,50   M, 10   M, and 50   M, respectively, and incubated for 1 h before theaddition of CuNG. The doses of all of the inhibitors used were reported tobe nontoxic [21, 36, 37]. The PEG-catalase was used at a final concentra-tion of 200 U/ml and incubated for 18 h before the addition of CuNG.The neutralizing antibody for IFN-    was also used for some experiments at the concentration of 100   g/ml, along with CuNG. Treatment of normal macrophages and assay of different cytokine production by flow cytometry and ELISA  TAMs from treated or untreated groups were incubated with anti-F4/80-FITC- or -PE-conjugated mAb for 45 min at 4°C. After extensive washing,cells were fixed, permeabilized, and stained with anti-IL-12-FITC or biotin- ylated anti-TGF-   mAb. Secondary streptavidin PE was used in case of intra-cellular staining of TGF-  . To check the activation status of MAPKs inTAMs following CuNG treatment at different hours, TAMs were isolated,fixed, permeabilized, and then stained with antimouse phosphorylatedp38MAPK and ERK1/2 and then, with secondary antirabbit-FITC. Corre-sponding isotype controls were used in each case and analyzed by flow cy-tometer (FACSCalibur, BD Biosciences).Normal macrophages, cultured in tumor-conditioned media or left un-treated, and TAMs were plated (2  10 6 cells/ml) in 24-well plates in thepresence or absence of CuNG (2.5   g/ml). Supernatants were collectedafter 12, 24, 48, and 72 h of incubation and assayed in triplicate for theproduction of IL-10, IL-12, IFN-   , and TGF-  , using the OptEIA kit (ELISA kit from BD Biosciences), according to the manufacturer’s protocol. Measurement of intracellular GSH GSH was measured following the reported method [37]. Briefly, treated oruntreated cells were collected and lysed immediately with 100   l lysis buf-fer (0.1% Triton X-100, 0.1 M sodium phosphate buffer, 5 mM EDTA buf-fer, pH 7). Thereafter, 15   l HCl (0.1 N) and 15   l 50% sulfosalicylic acid were added. After centrifugation at 12,000 g for 5 min, supernatants were collected, and GSH was measured using 5,5  -Dithiobis(2-nitroben-zoic acid) (DTNB) at 412 nm. Protein was measured following the Brad-ford method [38].  Western blot analysis  Western blot analysis was done by standard protocol, described previously [39]. Briefly, treated or untreated cells were lysed with cell lysis buffer con-taining 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mMEDTA, 1 mM EGTA, 0.5 mM PMSF, 10 mM sodium orthovanadate, andprotease inhibitor cocktail. After 30 min incubation on ice, cell lysate wascentrifuged at 12,000 rpm for 15 min at 4°C. Protein concentration in ly-sates was estimated by using the Bradford reagent. The supernatants weremixed with 5  SDS-PAGE sample buffer, heated in a boiling water bath for5 min, and cooled to room temperature. Approximately 100   g protein was loaded in each lane of 12% SDS-polyacrylamide gels, electrophoresed,and then transferred onto a PVDF membrane. Immunoblotting was per-formed with appropriately diluted, specific primary antibodies, and finally,chemiluminescence was recorded with LumiGlo reagent, as described by the manufacturer. Densitometric analysis Immunoreactive bands of phospho-p38 MAPK, phospho-ERK1/2, total p38MAPK, and total ERK1/2 were scanned (Model GS800; Bio-Rad, Hercules, CA,USA), and then, images were digitized and analyzed with Bio-Rad Quantity 1software. Immunoreactive bands were quantitated and expressed as the ratioof each band density to corresponding loading control band density, and val-ues were represented after normalization to untreated control. Statistical analysis  All data reported are the arithmetic mean from three independent experi-ments performed in triplicate  sd , unless stated otherwise. The unpaired Stu-dent’s  t  -test was used to evaluate the significant differences between groups,accepting P  0.05 as a level of significance. Data analyses were performedusing the Prism software (GraphPad Software, San Diego, CA, USA). RESULTS ROS-induced activation of MAPKs critically regulatedthe cytokine production by CuNG-treated TAMs  We showed earlier that CuNG mediated ROS generation, cru-cially involved in altered IL-12 and TGF-   production by TAMs[21]. Herein, to further ascertain the involvement of CuNG-induced ROS generation in the alteration of a functional cyto-kine repertoire of TAMs, we pretreated TAMs with PEG-cata-lase (to neutralize the generation of intracellular hydrogenperoxide toward water and oxygen), and our studies, based onflow cytometry ( Fig. 1A   and  B ) and ELISA (Fig. 1C and D),revealed that pretreatment with PEG-catalase attenuated theeffect of CuNG in terms of altered cytokine production by TAMs. This result incited us to further explore the mechanis-tic details involved in CuNG-mediated modulation of the TAMphenotype.It is well documented that the generation of ROS leads tothe activation of various MAPKs regulating the production of  various pro- and anti-inflammatory cytokines in different celltypes [28–30, 37]. Therefore, we inquired whether CuNG-me-diated ROS generation could activate different MAPKs, whichactually modulated the behavior of TAMs toward the proim-munogenic type. Herein, we used pharmacological inhibitorsof all of the MAPKs and tried to construe which MAPK plays adecisive role in this signaling cascade. Interestingly, we ob-served that pretreatment of TAMs with SB203580 (p38MAPK inhibitor) significantly inhibited CuNG-mediated IL-12 produc-tion at 48 h ( Fig. 2A  ) but failed to suppress IL-12 productionat 72 h and 96 h (Fig. 2A). We further noted that SB203580treatment could not down-regulate TGF-   production (Fig.2B) by CuNG-treated TAMs. On the other hand, the inhibi-tion of activation of ERK1/2 by PD98059 could not blockCuNG-mediated production of IL-12 at 48 h; rather, it com- Chakraborty et al.  ROS-induced reprogramming of tumor-associated macrophage  Volume 91, April 2012  Journal of Leukocyte Biology   3  pletely abrogated IL-12 production at 72 h and 96 h (Fig. 2A)and could also block the CuNG-mediated down-regulation of TGF-   production by TAMs (Fig. 2B). However, our studiesrevealed that inhibition of activation of JNK bears no effect onCuNG-treated TAMs (data not shown).Thus, p38MAPK activation is involved in CuNG-mediatedup-regulation of IL-12 production at early hours (48 h) only.On the other hand, ERK1/2 activation is involved in CuNG-mediated IL-12 production at late hours (72 h and 96 h), as well as down-regulation of TGF-   production by TAMs. CuNG-induced ROS generation activated different MAPKs in TAMs  We showed in the previous section that CuNG-mediatedmodulation of functional cytokine production by TAMs wasexecuted through activation of two different MAPKs, i.e.,p38MAPK and ERK1/2. So, we were keen to check the acti- vation status of these two MAPKs in TAMs following CuNGtreatment at different hours. Flow cytometric analysisshowed that CuNG treatment caused phosphorylation of p38MAPK, which peaked at 3 h (24.79%) and remained ele- vated up to 4 h (22.87%) in TAMs ( Fig. 3A  ). Moreover,CuNG treatment induced phosphorylation of ERK1/2, whichpeaked at 2 h (69.93%) in TAMs (Fig. 3B). However, we failed todetect any modulation in phosphorylation of JNK following CuNGtreatment (data not shown). We then studied the involvement of ROS in CuNG-mediated activation of p38MAPK and ERK1/2. Weobserved that pretreatment of    -tocopherol significantly inhibitedCuNG-mediated phosphorylation of p38MAPK and ERK1/2 inTAMs (Fig. 3A and B). Figure 1. CuNG-mediated ROS generation is responsible for the alteration of a cytokine profile in TAMs.  TAMs were plated (2  10 6 cells/500   l)and divided in three parts: the first part was kept untreated; the second part was treated with CuNG (2.5   g/ml); and the third part was first pre-treated with PEG-catalase (200 U/ml) for 18 h and then treated with CuNG (2.5   g/ml) for the indicated hours. TAMs were labeled with anti-F4/80 antibodies and with intracellular TGF-   (A) or IL-12 (B) antibodies. Immunofluorescence analysis was performed by flow cytometry. Cul-ture supernatants from the above-mentioned groups were collected and analyzed for TGF-   (C) and IL-12 (D) production by ELISA. Results arepresented as mean  sd  of three independent experiments and differences between CuNG treated and CuNG  PEG catalase treated cells aresignificant at **P  0.01, ***P  0.001 by unpaired Student’s  t   test. 4  Journal of Leukocyte Biology   Volume 91, April 2012  Then, we tried to extrapolate our in vitro data in an in vivosystem. To this end, EAC/Dox-bearing mice were treated withCuNG (i.m.; 5 mg/kg body weight) or kept untreated for dif-ferent time intervals. TAMs were isolated from CuNG-treatedand untreated control mice, and the intracellular activationstatus of p38MAPK and ERK1/2 was checked by flow cytom-etry (Fig. 3C and D). The results disclosed that CuNG treat-ment for 6 days in EAC/Dox-bearing mice caused phosphory-lation of p38MAPK in TAMs compared with the untreatedcontrol. On the other hand, ERK1/2 was found to be activatedon the fourth day of CuNG treatment compared with the un-treated control.Next, we tried to confirm our data by immunoblotting, but an inadequate number of TAMs isolated from the tumor sitehindered the ability to check the activation status of MAPKsthrough immunoblotting. Therefore, based on the current rev-elations that a number of tumor cell-derived soluble factorsdictated the polarization of normal monocytes toward TAMs[12, 14, 34, 40], we generated TCMs by incubating normalmurine macrophages with the conditioned media from differ-ent tumor types (data not shown). We noted that TCMs gener-ated by incubation of normal macrophages with the condi-tioned media from EAC/Dox cells corroborated well withTAMs (Supplemental Fig. 1A–C) isolated from the tumor sitein terms of cytokine profile (IL-12 low  , TGF-  high ) and in termsof polarization of naïve T cells toward the suppressive type(Supplemental Fig. 1D and E) [12, 18]. We found that CuNG could modulate the cytokine profileof TCMs from the suppressive to proimmunogenic type (Sup-plemental Fig. 1F–H) and led to the generation of an effectiveanti-tumor T cell response (Supplemental Fig. 1D and E). Sim-ilar to our previous observation, herein, we also observed that CuNG-mediated ROS generation was responsible for the mod-ulation of the suppressive phenotype of TCMs, as   -tocopheroltreatment could abolish CuNG-mediated down-regulation of TGF-   production and up-regulation of IL-12 production by TCMs (Supplemental Fig. 1F and H). Furthermore, throughimmunoblotting, we noted the similar activation status of MAPKs in TCMs, such as TAMs, when TCMs were treated withCuNG (Supplemental Figs. 2 and 3). ERK1/2-induced IFN-    production causedCuNG-mediated alteration of a cytokine pattern in TAMs In the previous section, we described that CuNG-mediated activa-tion of ERK1/2 controlled two regulatory cytokine (IL-12 andTGF-  ) productions by TAMs. Existing literature suggests that among the MAPKs, activation of p38MAPK directly regulatesIL-12 production by transcriptional interference [24]. Other than Figure 2. Activation of p38MAPK and ERK1/2 critically regulated the cytokine production by CuNG-treated TAMs.  TAMs were treated with CuNG(2.5   g/ml) for indicated hours or left untreated. In some experiments, TAMs were incubated with SB203580 or PD98059 for 1 h prior to CuNGtreatment. TAMs were labeled with anti-F4/80 antibodies and with intracellular IL-12 (A) or TGF-   (B) antibodies. Immunofluorescence analysis was performed by flow cytometry. Representative data of three independent experiments are presented. Chakraborty et al.  ROS-induced reprogramming of tumor-associated macrophage  Volume 91, April 2012  Journal of Leukocyte Biology   5
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