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NOX2-dependent ATM kinase activation dictatespro-inflammatory macrophage phenotype andimproves effectiveness to radiation therapyQiuji Wu1,2,3,4,5,6,Awatef Allouch1,2,3,4,Audrey Paoletti1,2,3,4,Celine Leteur2,3,4,Celine Mirjolet7,Isabelle Martins1,2,3,4,Laurent Voisin1,2,3,4,Frdric Law1,2,3,4,Haithem Dakhli1,2,3,4,Elodie Mintet1,2,3,4,Maxime Thoreau1,2,3,4,Zeinaf Muradova1,2,3,4,Mlanie Gauthier7,Olivier Caron3,Fabien Milliat8,David M Ojcius9,Filippo Rosselli10,Eric Solary11,Nazanine Modjtahedi2,3,4,Eric Deutsch2,3,4,12and Jean-Luc Perfettini*,1,2,3,4,12Although tumor-associated macrophages have been extensively studied in the control of response to radiotherapy,the molecularmechanisms involved in the ionizing radiation-mediated activation of macrophages remain elusive.Here we show that ionizingradiation induces the expression of interferon regulatory factor 5(IRF5)promoting thus macrophage activation toward a pro-inflammatory phenotype.We reveal that the activation of the ataxia telangiectasia mutated(ATM)kinase is required for ionizingradiation-elicited macrophage activation,but also for macrophage reprogramming after treatments with-interferon,lipopolysaccharide or chemotherapeutic agent(such as cisplatin),underscoring the fact that the kinase ATM plays a centralrole during macrophage phenotypic switching toward a pro-inflammatory phenotype through the regulation of mRNA level andpost-translational modifications of IRF5.We further demonstrate that NADPH oxidase 2(NOX2)-dependent ROS production isupstream to ATM activation and is essential during this process.We also report that the inhibition of any component of thissignalingpathway(NOX2,ROSandATM)impairspro-inflammatoryactivationofmacrophagesandpredictsapoor tumorresponseto preoperative radiotherapy in locally advanced rectal cancer.Altogether,our results identify a novel signaling pathway involvedin macrophage activation that may enhance the effectiveness of radiotherapy through the reprogramming of tumor-infiltratingmacrophages.Cell Death and Differentiation advance online publication,2 June 2017;doi:10.1038/cdd.2017.91Approximately,half of all cancer patients are treated withradiotherapy alone or in combination with chemotherapy.Although ionizing radiation(IR)directly causes senescenceand death of tumor cells through the generation of reactiveoxygen species(ROS)and DNA damage,1recent studiesunderscore the fact that IR can also modulate immune cellfunctions and favor consequently the development of antic-ancer immunity.2,3IR can induce the exposure of eat-mesignals(suchas calreticulin)and the release of danger signals(such as ATP and HMGB1)by the irradiated dying tumor cells(also referred as immunogenic cell death),4thus contributingto specific T-cell response by increasing the tumor antigencross-presentation to dendritic cells5and/or modifying theimmunosuppressive microenvironment of tumors.6,7IR canalso control tumor immune response through the directmodulation of innate immune cell functions.Treatment withIR can modulate Langerhans cell functions and induce theaccumulation of regulatory T cells into tumors.8In addition,IR controls macrophage plasticity and programs tumor-associated macrophages(TAMs)toward a pro-inflammatoryphenotypethatorchestratesspecifictumorimmuneresponse.9However,microenvironment factors such ashypoxia may affect this IR-responsive macrophage activationprogram by favoring a pro-tumorigenic activation phenotypethat is associated with tumor resistance.10Nevertheless,themolecular mechanisms underlying IR-induced macrophageactivation remain elusive.TAMs represent a major cellular component of the tumormicroenvironment.11These macrophages derive from bloodmonocytes that,after their recruitment into tumors,differentiateand are activated in response to different environmental signals.Macrophages can be broadly classified as classically activatedpro-inflammatory macrophages and alternatively activated pro-tumorigenic macrophages.12The interferon regulatory factor 51Cell Death and Aging Team,Gustave Roussy,114 rue Edouard Vaillant,Villejuif F-94805,France;2Laboratory of Molecular Radiotherapy,INSERM U1030,GustaveRoussy,114 rue Edouard Vaillant,Villejuif F-94805,France;3Gustave Roussy,114rue Edouard Vaillant,Villejuif F-94805,France;4Universit ParisSud-Paris Saclay,114rue Edouard Vaillant,Villejuif F-94805,France;5Department of Radiation and Medical Oncology,Zhongnan Hospital,Wuhan University,169 Dong Hu Road,Wuhan430071,China;6Hubei KeyLaboratoryof Tumor Biological Behaviors,Zhongnan Hospital,Wuhan University,169 Dong Hu Road,Wuhan 430071,China;7Centre GeorgesFranois Leclerc,1 rue du Pr Marion,Dijon F-21079,France;8Laboratoire de Recherche en Radiobiologie et radiopathologie,Institut de Radioprotection et de SuretNuclaire,Fontenay-aux-Roses F-92262,France;9Department of Molecular Cell Biology,Health Sciences Research Institute,University of California,Merced,CA 95343,USA;10Laboratoire Stabilit Gntique etOncogense,CNRS-UMR 8200,114 rue Edouard Vaillant,Villejuif F-94805,France and11INSERM U1170,Gustave Roussy,114 rue Edouard Vaillant,Villejuif F-94805,France*Corresponding author:J-L Perfettini,Cell death and Aging Team,Gustave Roussy,114 rue Edouard Vaillant,Villejuif F-94805,France.Tel:+33 1 42115424;Fax:+33 1 42116665;E-mail:perfettiniorange.fr12ED and J-LP share senior coauthorship.Received 31.8.16;revised 19.4.17;accepted 02.5.17;Edited by M PiacentiniCell Death and Differentiation(2017),113&2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved 1350-9047/ demonstrated to determine the pro-inflammatorymacrophagephenotype1315alongwithothertranscriptionfactors(such as STAT1 and NF-B),whereas IRF4,16STAT617andKLF418are key transcription factors required for the pro-tumorigenic macrophage phenotype.Interferon gamma pluslipopolysaccharide(LPS)or tumor necrosis factor alone areknown to induce classical macrophage activation that ischaracterized by an increased secretion of pro-inflammatorycytokines and chemokines(such as interleukin(IL)-1),anaugmentedproductionofinduciblenitricoxidesynthase(iNOS)19and of ROS.12Conversely,glucocorticoids,IL-4/IL-13and IL-10 drive macrophages toward the alternative activationprogramwithareducedproductionofpro-inflammatorycytokinesbut a higher level of anti-inflammatory IL-10,TGF-,arginase,membranescavengerandmannosereceptors.Pro-inflammatorymacrophages possess bactericidal and anti-tumoral activities,while pro-tumorigenic macrophages are associated with immuneregulatory and tissue repair activities,highlighting the diversity ofmacrophage functions.In response to tumor microenvironmentsignals(such as hypoxia and tumor-derived lactate20,21),TAMsmainly adopt a pro-tumorigenic phenotype that contributes totumor progression by promoting tumor growth,metastasis,angiogenesis and by suppressing immune responses.2224Inaddition,several studies have demonstrated that TAMs are alsoassociated with treatment resistance and poor clinical outcomesin various cancer settings(such as lymphoma,melanoma,sarcomas and lung cancer2528),making them attractive targetsfor the development of new anticancer strategies.29,30In thiscontext,a better understanding of the molecular basis of the IR-mediated macrophage activation is needed for the improvementof the efficacy of radiotherapy.In this study,we explored themolecular mechanisms involved in IR-induced macrophagereprogramming.ResultsCell-autonomous activation of macrophages after ioniz-ing radiation.Considering that immune cells may influencethe functional reprogramming of macrophages,29we firstanalyzed IR-mediated macrophage activation using humancolon tumor xenografts in immunodeficient mice.HCT116cells were subcutaneously inoculated into the right flanks ofathymic nude mice.Seven days after inoculation,theirradiation of the palpable tumor mass with a single dose of20 Gy resulted in significant tumor growth inhibition,ascompared to the controls(Figure 1a).After 29 days,theresidual irradiated tumors did not show any increase of thedensity of CD11b+macrophages(Supplementary Figure 1a),but revealed an increased frequency of CD11b+macro-phages that expressed iNOS(iNOS+CD11b+)(Figure 1b).We detected a significant accumulation of iNOS+CD11b+macrophages in irradiated tumors as compared with non-irradiatedones(Figure1c).TheaccumulationofiNOS+CD11b+TAMspositivelycorrelatedwithtumorresponse to IR,confirming as previously published that thepresenceofiNOS+/pro-inflammatoryphenotypemacro-phages in irradiated tumors is required for the modificationof tumor microenvironment and tumor regression.Of note,previous reports characterized this process in conditions ofrelatively low-dose IR(2 Gy)exposure.6,9Our observationthat TAMs exhibited an increased phosphorylation of thehistone variant H2AX(also known as-H2AX)on serine 139(Figures 1d and e)underlined an unsuspected link betweenDNA damage response and macrophage activation.To checkthe possibility whether IR can directly target and activatemacrophages,we irradiated human THP1 macrophages witha single dose of 2 Gy and analyzed,by fluorescencemicroscopy and flow cytometry,the expression levels ofiNOS and-H2AX.The increase in iNOS and-H2AXexpressionlevelsinirradiatedTHP1macrophages(Figures 1fh,Supplementary Figures 1b and 1c)revealedthat IR could directly target macrophages to promote theiractivation toward a pro-inflammatory phenotype.We thenanalyzedbyimmunoblottheexpressionofacentraltranscription factor involved in macrophage activation,theIRF515and observed that after single radiation doses of 2and 4 Gy,phorbol-12-myristate-13-acetate(PMA)-differen-tiated human THP1 macrophages(Figure 1i),human primarymonocyte-derived macrophages(hMDM)(Figure 1j)andFigure 1Irradiation activates macrophages toward pro-inflammatory phenotype.(a)Colorectal HCT116 cells were injected subcutaneously(4106cells per mouse)intoimmunodeficient mice and tumor growth was monitored.Results are expressed as mean valueS.E.M.P-value(Po0.01)was calculated by means of two-way ANOVA test.(be)RepresentativeconfocalmicrographsandfrequenciesofiNOS+CD11b+(b,c)or-H2AX+CD11b+(d,e)tumor-associatedmacrophagesdetectedinabsenceorafter20 Gysingle-dose irradiation are shown(scale bar,20 m).Representative iNOS+CD11b+or-H2AX+CD11b+macrophages are shown in inserts(scale bar,5 m).Results areexpressed as mean valueS.E.M.P-value(*Po0.05)was calculated using MannWhitney U-test.(fh)Representative confocal micrographs and frequencies of phorbol-12-myristate-13-acetate(PMA)-differentiated human THP1 macrophages showing-H2AX+nuclear foci(f,g)or expressing iNOS(iNOS+)(f,h)in control cells or 24 h after 2 Gyirradiationareshown(scalebar,20 m).Representative-H2AX+nuclearfocioriNOSexpressingmacrophagesareshownininserts(scalebar,5 m).Resultsareexpressedasmean valueS.E.M.P-values(*Po0.001,*Po0.0001)were calculated using unpaired Students t-test.(ik)IRF5 expression after,respectively,96,96 and 6 h culture ofPMA-differentiated human THP1 macrophages(i),hMDM(j)or murine RAW264.7 macrophages(k)that have been irradiated(or not)with indicated doses.Representativeimmunoblots are shown.GAPDH is used as loading control.(l)Murine RAW264.7 macrophages that have been irradiated(or not)with 2 Gy were immunoprecipitated 6 h postirradiation for IRF5 and phopsho-serine(pSer),and analyzed for IRF5 and pSer expressions.Inputs were analyzed for IRF5,pSer,ATMS1981*,ATM and GAPDH.(m,n)Detection of IL-1 and IL-8 release in the supernatants of hMDM(m)or murine RAW264.7 macrophages(n)that have been irradiated(or not)with indicated doses.Representative immunoblots are shown.(or)TNF,IFN,IL-6 and IL-23 mRNA expressions on PMA-differentiated THP1 macrophages that have been irradiated(or not)with2 Gy were determined by quantitative real-time PCR.Results are expressed as mean valueS.E.M.and represented as fold change as compared to controls.P-values(*Po0.05,*Po0.01)were calculated using unpaired Students t-test.(s,t)Detection of cytokine secretion in the supernatants of hMDMs that have been treated(or not)with4 Gy irradiation.Array images were captured following 110 min exposures to peroxidase substrate(s).Relative levels of cytokines detected in the supernatants of irradiatedmacrophages as compared to those detected in non-irradiated macrophages are revealed as fold change ofarbitrary units.Pro-inflammatory and pro-tumorigenic cytokines andchemokines are indicated(t).Data are representative of three independent experiments performed with primary human macrophages obtained from three healthyrepresentative donorsATM activation during macrophage reprogrammingQ Wu et al2Cell Death and Differentiationmurine RAW264.7 macrophages(Figure 1k)exhibited anenhanced expression of IRF5(Figures 1ik).In addition,through immunoprecipitation experiments,we observed thatafter 2 Gy irradiation of RAW264.7 macrophages the pro-inflammatory transcription factor IRF5 was phosphorylatedon serine(Figure 1l).We also demonstrated that after,respectively,96 and 12 h of irradiation,hMDM(Figure 1m)and murine RAW264.7 macrophages(Figure 1n)releasedtwo pro-inflammatory cytokines IL-1 and IL-8.To furthercomplete the cytokine profile analysis,we determined thetranscription levels of pro-and anti-inflammatory cytokinesandobservedincreasedmRNAexpressionofpro-inflammatory cytokines(such as TNF-,IFN-,IL-6 or IL-23)in 2 Gy-irradiated PMA-differentiated human THP1 macro-phages(Figures 1or)and detected an increased secretionof pro-inflammatory cytokines by 4 Gy-irradiated hMDMATM activation during macrophage reprogrammingQ Wu et al3Cell Death and Differentiation(including TNF-,IFN-,IL-6 and IL-8)as compared withcontrols(Figures 1s,t and Supplementary Figures 1d1g).Altogether,these results indicate that IR can promote acell-autonomousactivationofmacrophagestowardapro-inflammatory phenotype.ATM-mediated DNA damage response regulates thetranscription of IRF5 in response to ionizing radiation.To further characterize the molecular mechanisms involved inIR-elicitedmacrophageactivation,wefirststudiedtheinduction of DNA damage-associated signaling pathways.Fifteen minutes after single radiation dose of 2 Gy,PMA-differentiated human THP1 macrophages(Figures 2ac)exhibited a strong nuclear accumulation of-H2AX+foci(Figures 2a and b)and of 53BP1+foci(Figures 2a and c)thatcould still be detected 6 h after exposure(Figures 2b and c),revealing the fact that DNA double-strand breaks areproduced in response to IR.One hour after single radiationdose of 2 Gy,murine RAW264.7 macrophages also displayedincreased-H2AX+foci(Figures 2d and e).Considering thatthe kinase ATM(mutated in the inherited recessive autosomaldisease ataxia telangiectasia)is the major kinase involved inthe phosphorylation of H2AX(on serine 139),31we evaluatedthe role of ATM in the activation of macrophages in responseto IR.We observed that the vast majority of 2 Gy-irradiatedmurine RAW264.7 macrophages exhibited the activatingauto-phosphorylation of ATM on serine 1981(ATMS1918*)1-h post irradiation(Figures 2d and f).T