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    Paso ote this ac in prossas: mani 2019), tock de org/10.10767. muni 2018.08.89 ‘quoredo ofa, Antracycines leo DNA Damage Response-Masated Protection agains! Sovre Sopes, Fm Immunity Anthracyclines Induce DNA Damage Response-Mediated Protection against Severe Sepsis Nuno Figueiredo, '-:* Angelo Chora, Helena Raquel ** Nadia Pejanovie,’ Pedro Pereira,’ Bjém Hartleben,” ‘Ana Neves-Costa,’ Catarina Moita,’ Dora Pedroso,” Andreia Pinto,’ Sofia Marques,” Hafeez Faridi,® Paulo Costa,* Raffaella Gozzelino,’ Jimmy L. Zhao, Miguel P. Soares,’ Margarida Gama-Carvalho,” Jennifer Martinez, ° Qingshuo Zhang," Gerd Déring,"*"* Markus Grompe,"’ J. Pedro Simas,' Tobias B. Huber,” David Baltimore, Gupta,” Douglas R. Green,"° Joao A. Ferreira," and Luis F. Moita’-" ‘instute de Medicina Molecular, Faculdade de Medicina, Unversidade de Lisboa, 1649-028 Lisboa, Portugal 2Cnica Unverstria de Cirugia |, Centro Hospitala Lisboa Noy EE, 1849-028 Lisboa, Portugal Gulbenktan Programme for Advances Medical Education, 2780-158 Ociras, Portugal “Champalmaud Foundation, 100-088 Lsbea, Portugal ‘Ronal Division, University Hospital Freiburg, 78106 Freiburg, Germany “Department of Internal Medicine, Rush University Medical Center, Chicago,IL 60612, USA “ipsttsto Gulbenkian de Ciéncia, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal *Duision of Biology, Calfomia Insitute of Technology, Pasadena, CA 91725, USA, ‘centro de Biocversidade, Gendmica Funcionale Integrativa (BioFIG), Faculdade de Ciéncias, Universidade de Lisboa, 1749-016 Lisboa, Portugal *2Department of Immunology, St Jude Children's R earch Hospital, Memphis, TN 88105, USA “oregon Stor Cell Center. Department of Pecatics, Oregon Heath & Science University, Porland, OR 87239, USA Institut ur Meizinsche Mikrobiologie und Hygiene, Unversity of Tubingen, 72076 Tubingen, Germany Clinical Re These authors contributed equaly to this work ‘Deceased July 2, 2013, “Correspondence: imotatim ulpt pid doi org/*0.1016).immuni.2013.08.039 ‘SUMMARY Severe sepsis remains a poorly understood systemic. inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeuti cally at alow dose regimen to confer robust protec- tion against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect, of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA dam- ‘age responses, including the ATM and Fancony Ane- mia pathways, are important modulators of immune responses and might be exploited to confer protec- tion to inflammation-driven conditions, including severe sepsis. INTRODUCTION Sepsis isa life-threatening condition that arises as a systemic, inflammatory response to an infection (Bone et l., 1992; Levy rch Center of The Lisbon Academic Medical Center, 1649-026 Lisboa, Port ‘ot al, 2003) Includes 2 continuum of clinical severity ranging from systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, to septic shock (Suitrecin) andi Munford, 2071). t is the leading cause of death in intensive care urits and the third cause of overall hospital mortality (angus anc Wax, 2001 Ulloa and Tracey, 2005). In spite of substantial improvernent in lagnosis and support measures, the global annual mortality rate fs ~28% (Holchk’ss anc Karl, 2003), ranging trom less than 10% in SIRS to up to 70% in septic shock (Angus and Wax, 2001; Annane eta, 2003). The pathophysiology of sepsis, remains poorly understood. AS a result, the basic elements of tueatment—early antibiotics, prompt control of the source of infection, and organ support—have not changed substantially inthe last 50 years, and attempts to translate basic research re- sults into effective new interventions have been met with limited fF no success (Suredini and Munford, 2011). In the same Period, the incidence of sepsis and Its economic burden has increased by 1% each year (Varin eta, 2003; Ulloa and Tracey, 2005), inciting the urgent need for novel therapeutic options. Inflammation isa response to harmful stimu that limits tissue damage and aims at restoring homeostasis (Medzhtov, 2008). Pathogen-associated molecular patterns (PAMPs) on microor- ganisms and damage-associated molecular patterns (DAMPs) ‘originating trom dying cells are sensed by the host through germ- line-encoded pattern-recognition receptors (PRRs) that recog- rize conserved signature structures in nonself and self (Janeway land Medzhitov, 002). These sensors are presentin both profes- sional including neutrophils, macrophages, and dendritic cels) and nonprofessional immune cells and their activation initiates intracellular signaling cascades leading to the transcriptional Iremunity 39, 1-11, November 14, 2013 9201S Elsevier Ine. 1 ie Pease cloths aren arose as Figunvodo a, Aehvacyeines Indice ONA Derage Hespanse-Midaled Protection aginst Sovore Sepa muni 201}, nipex.do,org10.+016/ er. 2018.08,089 ‘expression of inflammatory mediators, such as cytokines and chemokines. Inflammation needs to be effectively terminated after removal of the original tigger for repair of damaged tissue ‘to.occur Inthe susceptiole host, overproduction of nfiammatory mediators or an exaggerated response to their presence can lead to septic shock, tissue destruction, or permanent loss of function (Takeuchi and Akira, 2010), ‘There are two evolutionarly conserved defense strategies ‘against infection that can limit host disease severity, One relies, ‘onreducing pathogen load, e., resistance to infection, whereas, the other provides host tissue damage control, limiting dis- ease severity respectively of pathogen load, ie, tolerance to Infection (Riberg et al, 2008; Schneider and Ayres, 2008). AS demonstrated originally for plants and thereafter in Drosophila, tolerance to infection also operates in mammals, as revealed {or Plasmodium (Raberg et al, 2007; Seixas et al, 2009) and ppolymicrobial infections in severe sepsis (Larsen e! a. 2070). Here we show in an experimental mouse model that anthracy- lines confer strong protection against sepsis by increasing disease tolerance to infection; that is, acting inespectvely of pathogen burden. We further show that ATM (ataxia telangiecta- ‘sia mutated) kinase and the induction of autophagy are strictly required forthe in vivo protection against sepsis. These molec- Ula pathways provide strong damage control in tissues, specit- ically in the lung RESULTS: Anthracye Sepsis In an in vito chemical screen using ~2.820 compounds, we identified several lead candidates capable of inhibiting inftam- matory cytokine production in response to E.coli challenge by the THP-1 macrophage line (see Fguro Stand Tablo St avai. able online). This inhibitory effect was dissociated trom cytotox- leity of the compounds tested on THP-1 cells (Foure SB}, ‘Among these, we found three representatives of the anthracy- cline family of chemotherapeutic agents namely epirubicin, doxorubicin, and daunorubicin, and validated their inhibitory activity on cytokine production (Figure SIC) We then used the cecal gation and puncture (CLP) mouse ‘model of experimental sepsis to investigate the in vivo effects of epirubicin (Ritirsch esl, 2008), In CLP, sepsis results from ‘8 polymicrobial infection of abdominal origin, leading to bacter- jemia and a systemic inflammatory response (Ritirsch etl. 2008). We adjusted CLP severity to a high-grade sepsis, where at least 80% of C57BL/6 mice succumbed within 48 br after the intial procedure, Under these conditions, epirubicin adminis- ‘tered intraperitoneally (ip atthe time of CLP and again 24 nrlater inatotalof 1.2 ug/g of body weight reproducibly and significantly (p< 0.001) increased the survival of C57BL/6 mice subjected 10 CLP by neatly 80%, without the use of antibiaties (Figure 1A) A similar protective etfect was observed in epitubicin-treated animals with the same dose and schedule but administered iv. (Figure 81D). This appeared to be a general property of the anthracycline family because other representative members of this family of drugs identified in the initial chemical screen conferred a similar degree of protection aganst CLP (Figure 'B). ‘The protective effect of anthracyclines was not dependent on 105 Confer Strong Protection against Severe 2. Immunity 39, 1-11, November 14, 2018 ©2019 Elsevier ne. Immunity Anthracyclines Induce Tolerance to Sepsis the mouse strain as outbread NMRI mice were similar pro- tected by epirubicin (Figure 10). Epiubicin was equally effective against another clinically relevant pathogen causing sepsis, IK. pneumoniae administered intranasally (Figure 1D), arguing that epirubicin can be effective in the treatment of sepsis of different origins in adltion to peritoneal sepsis. Mice previously ‘subjected to CLP and treated with epirubicin were not immuno- ‘compromised because they could clear a secondary ntvanasal viralinfection similar to controlmice (igure 1B) Takentogether, these results indicate that low doses of the antnracyctine family ff chemotherapeutic agents confer strong protection against ‘vere sepsis, without causing host immunosuppression Epirubicin Acts Therapeutically to Promote Disease Tolerance to Severe Sepsis \We found that in epirubicin-treated mice subjected to CLP, the bacterial load in blood and target organs of seps's, eg., ung, liver, kidney, and spleen 24 hr post-CLP did not differ trom that fof untreated controls (Figure 2A). Although at 48 hr post-CLP, we noticed a trend toward a lower bacterial load in the target ‘organs of epirubicintreated animals, the differences were not Statistically significant, even if most untreated cortrol animals die between 24 and 48 hr after the CLP procedure. These results raised the possibilty thatthe protective effect of epirubicin in vivo isrelated to disease tolerance without directly affecting the path- ‘ogen burden (Wviedzhitov etal, 2012). This idea was supported by the observation that the serum concentrations of several markers of tissue damage such as LOH (ung and general cellular damage), CK (muscle), ALT (ver), and urea (kidney) wore sub- stantially reduced to almost basal levels in epitubicin-treated mice, 24 hr after CLP, compared to untreated mice (Figure 2B). In addition, we observed a substantial reduction in the levels of, inflammatory mediators including tumor necrosis factor (TNF), interloukin-1f (L-1f), IL-6, and HMGB1 compared tonontreated CLP mice {Figure 26). We have also observed improvement of histological lesions in the lung, liver, and kidney after CLP by treatment with epirubicin (Figure S2}. To explore this further in the absence of bacteria, we found that the drug protected (C57BL/6 mice from lethal septic shock caused by lipopolysac- ccharide (LPS, endotoxin) (igure 20) Large spectrum antbioties such as meropenem are very ‘effective at lowering bacteremia and are standard drugs used in sepsis (Russell, 2008), We tested the efficacy of meropenem in CLP in comparison to epirubicin and found that although mer- ‘openem delayed the death rate of CLP-subjacted mice, didnot prevent mortality (Figure 26, in spite of a strong impact on bac- terial burden (Figure 2F). This was in sharp contrast tothe action ‘of epirubicin, which did not interfere with bacteremia (Figure 2F) but prevented CLP-induced mortality (Fgure 2E}, again arguing for a role of epirubicin in conferring disease tolerance against severe sepsis (Larsen etal, 2010; Medzhiov et al, 2012), Both epirubicin and meropenem decreased the amounts ofIL- 1. TNF, and HMB! inthe serum of mice subjected to CLP (Fic- ure 2G), This indicates that whereas decreased circulating levels of inflammatory mediators might contribute to confer protection ‘against severe seps's inhibition of L-1, TNF, and HMGBI isnot sufficient per se to explain the protective etfect of epirubicin, Which isin accordance with what ls observed for other therapeu= tic approaches in the clinical setting (Hotchkiss arc Kel, 2009), Paso ote rmaniy 20% 16 ain poss as: tps del ag/T0.1076). mmr 2013.08.088 ‘quoredo ota, Antracycines lice DNA Damage Response-Mesated Protection agains! Severe Sap, Immunity ‘Anthracyclines Induce Tolerance to Sepsis a) zn gn gr a yo Te 1 > D Pas, — root ‘rop000 Figure 1, Epinubicin Afords Protection agtinst Severe Sepsis (a) SunevalofC579LI6WT animale sbjoctedto CLP teseduith ca (8) Sunval of CS7BL/6 WT anmals subjected to CLP tes achadde and doses a in A (P85) or oprubicin Ep (6 9/a body weight athe tne of procedure and 24 ater. rt wth carer PBS) bein (Ep, dexorbcn[Dexe), oF daunorusien (Daune). Treen {G) Suvvalot NURI mmce subjected to CLP and trealed wit aie (PAS) or epubicin Ep asin W. {0} Survalof CS7BL8 WT animals flowing intranasal nol of Keil preureige and vested with caer PBS) ar piruicn (pn (N {© Quantticaton of ifectous val MufV~t partes nung of CS7BLI8 WT animal revoul subjected to mack CLP (Smock CLP bested with epubicin ‘set or CLP rested wth eprabiln(O¥8 Epruben veatent dose and schedule as nice were iver assly nected vith! 000 PFU of Wut-en day 3 os-CLP anal partlesquantedby plaque assy tds 6 and 12 afer valivecton Each creer [tthe means SEM to two Indapendert assays, The dished horaorta ne reprevets thei ot detection othe aaay. ns, nt wget p< 0.05) “p< 001;""p. 0.00 fog-rarMarteLCox| test fr Ato and Mann-Whitney tet for). See ao ite 8) and (abi = Taken together, these data suggest that epirubicin acts through ‘an additional altemative mechanism to eytokine inhibition to confer tolerance to sepsis. Epirubicin Protection a atm Next, in order to explore the molecular mechanism behind the protective effects of anthracyclines, we used our in vivo assay ‘system to perform a short hairpin RNA (shRNA}-based screen in THP-1 cells, focusing on kinases and phosphatases and with IL-1 and TNF secretion as assay readouts. While ourin vivo results suggested the possibilty that anthracyclines ameliorate the lethal effects of sepsis by a mechanism affecting tissue toler ‘ance, we reasoned that our in vito assay would be useful forthe identification of candidate pathways mediating the antwracyctine ‘effects. We found several negatve regulators ofl-1 production in response to E.coli challenge, including the genes encoding |ATM, checkpoint kinase 1 (CHEK1) and ataxia telangiectasia and Rad3 related (ATR) (Pigure $3; Table $2), These findings ‘suggest that DNA damage response (DDR) components are negative regulators of IL-1 secretion, By using a phospho- ‘specific antibody against the activated form of ATM, we found that although E. col! alone was a poor but reproducible ATM activator (Figure $2), epirubicin alone or in combination with E, coll tiggered a robust ATM activation (Figure $3). This was ‘confirmed with immunoblotting (Figure 89) st Sepsis Is Mediated by ATMs amasterregulatorofthe DDR (Ciccla anc Elledge, 2010) and is known to be activated by anthracyclines and other DNA- damaging agents (Siu etal, 2004). Therefore, we used ATM-def- cient mice to test the contribution of the DOR to the protective effect of antnracyclines against severe sepsis. ATV-deficient (At) ice were not protected by epirubicin against CLP and died with similar kinetic to those of wild-type (WT, Atm”) ani rmals that were treated with PBS alone (Figure 2A). We conclude that ATM expression is necessary to meciate the protective effect of epirubicin in sepsis. In stking contrast to WT mice (Fig~ lures 2B and 20), in the absence of ATM, epirubicin no longer ormalized the serologic markers of organ lesion (Figure 28) ot decreased the levels of iniammatory mediators (gure 30). How- ‘over, in mice subjected to CLP and treated with etoposide (after in vivo tration to find the best and most etfective dose), an agent known to cause DNA double-strand oreaks and to activate ATM- dependent pathways (Viontecucea ans Biamonti 2007), mortality induced by CLP was onlypattily rescued (igure 3D), suggesting that ATM Is necessary but not sufficient for the protection, conferred by anthracyclines against sepsis. In addition to double-strand breaks (repaired in an ATM- dependent manner}, anthracyclines also cause ONA interstrand crosslinks, a ONA lesion known to be repaired by the Fanconi Anemia (FA) pathway (Ciccia and Elledge, 2010), Interestingly, FA patients were reported to spontaneously overproduce TNF (Griot et al, 2008; Vanderwert et al, 2009), possibly because Iremunity 39, 1-11, November 14, 2013 2018 Elsevier Ine. 3 ie Paso cit this arco m press as: Fguotodo ota, Anthvacytnes Iniuce DNA Damage Hesponso-Medited Protection against Severe Sepsis, I rmunty 2013), psec. erg/T0.1016) er 2078.08.039, Immunity ‘Anthracyclines Induce Tolerance to Sepsis © 2. Epirubicin Prometes Disease Tolerance to Severo Sopsis (Polymer oa CFs} n blood, ung, Iver ey. ane pean at aeatd te pons, of C5TBLIS animals undergoing GLP and este wih PBS (0+F) or episien (6+) 08 y9/a body weight at he ime of procedure and 24 er. Each etl epresenis vidual annals. Horzoril Ines ncaeartete means + SM 1B) and) Epiubich counteracts tisase damage and ifanmation assorted with CLP as assessed by (9) LOM, CK, ALT, wea, and(6) TNF, L-1, L6, an HMB plasma concerratons in CS7OLI6WT armals 24 rafter mock CP (S} (n= 2)0°CLPfolowedby restment wih POS 64P} f= 5} 2" epinbein CC) tn 7) a8 (A Results shown represent arihmete means = SEM tom duplste (8) or traleate (0) reading pr ani (Dj Suvi ot U8 WT armas folowing thal LPS nection and resiment wih carer (PS) or epruien (ED an {© Sunil of CS7BL/6 WT animale sete to CLP teste vith carer PS), metapene 1019/8 body Wegh/day or eprunien Ep) a ini FICFUsin Blood, bested ie, of S78LI animal urcergeng mock CL? S}or CLP flowed Lestment ith PBS (C+?) epraein (CVE, or meropenem (Cit a nA. Each cele represents vidual animal, Honzoral nes ate athmete means + SEM (G)ILTP. THF, nd MGB plasma concentrators in OSTBLIBWT alals 2 ater CLP folbwed by treatment wth PAS (CoP) (04) plublen (C8 f=), ormeropenem (C+ n=) asin}. n. net sigean"p <0.08:"p<0.07;""p <0.001Jogvank Mante-Coy estar DandE Mann WAtney testa 2 fd unpated eter 8, Cand). See also Figue S the FA protein FANCD2 can directly inhibit TNF promoter activ- ity (Matsushita ot al, 2011). In THP-1 cells, we observed that FANCD? is activated in an ATV-Independent manner upon epl- rubicin treatment, as shown by its monoubiqutinaton (Fic Lure 3E}, These findings support the independence of signaling events initiated by the generation of DNA double-strand breaks land DNA interstrand crosslinks. We examined the contribution ofthis pathway for epirubicin protection of CLP and found that Fancd2"" mice were slightly but significantly (p < 0.05) impaired {or the protective effects (F gure SF). 4 Immunity 9, 1-11, November 14,2013 2013 Elsevier ne. These results suggest that activation of DOR is protective ‘against sepsis. To further test this hypothesis, we have used whole-body sublethal y irradiation. We found a significant Increase in the number of cells with yH2AX-positve foc! (p < (0,001), @ surrogate marker of ATM activation (Ciscia anc Flecge, 2070), in the lungs of whole-body sublethal y-rad ‘ated mice as compared to controls (Figure 3G). Mice subjected to CLP that were iadiated showed a signlicant increased survival (p < 0.001) as compared to noniradiated mice (Fig- ure SH). We conclude that the protective phenotype induced Paso ote rmaniy 20% Immunity ‘Anthracyclines Induce Tolerance to Sepsis 16a in poss a: Figuorega ola, Antracylines luce DNA Damage Resbonse-Mecat Proecton aganst Swvere Son, tps del ag/T0.1076). mmr 2013.08.088 ye i aim iif > sane ae s Ho gress = io ind Be i . Figure 3. The Protection Aforded by Epirubicin | Sunira tA" and Atm ~~ CS7BLIS animal subjected to CLP and ested wih PBS or pirbicin Ep wih sare schedule and dove ain igure {B)LDH, CX. ALT, wea. anc) TNE, -19, and IL-8 plasma concentrations in Atm" OS7BLI6 arial 24 afer meck CLP (8) n= 2) or CLP followed by ‘veatment wih 88S (2-7) f= 8 oF eprubcn(C-E) n= Basin. Resuls shown represetartimete means + SEM tom pete readings pe anima {Self Bb aes or wee Ep eees WT CHL wea aero CLP. pred we 2! be eT {© FANCO2 and Ub-FANCD? protein evls by mmunoblting In THP-1 call lowing Ecol challenge ae a preincubation (1 with cari, epibicin or KU55009 a8 nates {F Sunmal of Faned2” ana Faned2~~ animal subjected to CLP ad rested with PBS or pic Eph with same schedule and dose 8 i. {G) Representative sections of H2AX staring ana percentage of yHZAK" calls pr fla ht pane in args slate 1 hr ater mic wor subjoctetto whole body radaton (Gy, Rests shown rpresortactmete means = SD trom ves Sole (H Suvial of C57BLI6 WT animals subjected to CL? folowing whle-bodyy imaiaton {4G or treated with carer PBS) or epubicin {Ep a n A 1, nolsigiieats"p< 005; "p «O07; < 0.00% fog [Manton] Wea or A.D, Fan H ard unpsied tex for 9, C, and G). See slo "ure 5) by epirubicin is dependent on the activation of multiple path- ways downstream of a DDR. The activation of the ATM Autophagy Pathway pathway is the main contributor, but the full protection requires. Although itis possible that the dominant ATM-mediated protec- tion of aditional DDR pathways, including the FA tion against sepsis might rely on ROS scavenging (Cosentino lal, 2011), on the induction of apoptosis of inflammatory cells Iremunity 39, 1-11, November 14, 2013 2018 Elsevier Ine. 5 ie Pease cloths aren arose as Figunvodo a, Aehvacyeines Indice ONA Derage Hespanse-Midaled Protection aginst Sovore Sepa muni 201}, nipex.do,org10.+016/ er. 2018.08,089 Immunity Anthracyclines Induce Tolerance to Sepsis 14, The ATM-Dependent Protection of Epirubicin against Sever ahaa ta fl “ell lll ll Sepsis Relies onthe Induction of Autophagy 1 Survalf Lea” and Le36~ animals subjected to CLP aa veated wth PBS or epubcin (Ep with same schedo and dose a8 mF. (B)LDH, CX. ALT, ra, and 6) TNF L-1P, aI plasma concenratinsinLe3b-/ animals 24hr ator mock LP} 0 CLP flea by eatmant with 3S {0-P] f= 4) epruoiin(C+5) n= 7) asm 2B. Rests Shaw ropresentattimete meas = SEM tom tila eadgs por animal ns. ot signet ‘p<005:"p.<.01;™p.< 001 fg-rark Mantal.Co teat for, empire est for Bans C}. See aga 5.70 Ss (Garrison et al, 2010), on the preservation of genomic stabilty (Westbrook ana Schest, 2070), of on the biogenesis of ant-in- flammatory microRNAs such as miR-146a (Zhang etal, 2071), we found no significant contribution for any of these processes (Figure $4) We therefore explored a possible role for autophagy inthis process, given that ATM is @ negative regulator of mTOR, Which is an inhibitor of autophagy (Alexander et al., 20102: ‘Alexander ea, 20100). By using autophagy-defectve Le3b-"") mice, we found that the autophagy pathway is required for the in vivo effect of epirubicin (Figure 4A). Similarly to Atm“ mice (Figures SB and 8C), epirubicin was not able to decrease ‘the serologic markers associated with organ lesion (Figure 48) oF to normalize cytokine levels in autophagy-defective mice (Figure 0). We then used LC2b-GFP mice to study the contribution of the autophagy pathway in the protection conferred by enirubi- cin, While FACS analysis shows that CLP alone induces LC3b aggregation in different splenocyte populations, namely mono- yes and neutrophils, epirubicin treatment did not increase ‘the autophagy pathway in these critical players in sepsis (Fia- lure 5A), We then tested the impact of epirubicin on the survival ‘of a conditional depletion of Atg? specifically in neutrophils and monocytes upon CLP, with Atg?™"™ Lys! GFP-LC3D animals. Strkingly, these animals were equally protected by epirubicin as compared to control mice (igure SB), suggesting that the autophagy pathway is not required in the myeloid compartment for the protective effects of epirubicin against sepsis. ‘Autophagy can be effectively monitored by the conversion and immoblization of LC3 (Kabeya etal, 2006}. Because the auto- phagy pathway was not required in the myeloid compartment {or protection against sepsis by epirubicin, we then looked at target organs of sepsis (ung, Iver, and kidney) by using immuno~ bioting to identity Iipidation of LCb as indicative of activation of 5 Immunity 39, 1-11, November 14, 2018 ©2019 Elsevier ne. the autophagy pathway, We found that epirubicin specifically induced lipidation of LC3b inthe lung at 6 hr, but not inthe liver fr kcney (Figure SC). Although LC3 was transiently lipidated ‘after CLP inthe lver at 6 and 24 hr a previously reported (Chien ‘ea, 2017), levels of LC3 were not altered by epirubicin veat- ment (Figure 8C). We have further confirmed that autophagy ‘was induced in the lung as shown by the increase of LC3b positive vesicles in lung sections at 6 nr and 24 hr comparing ‘epirubicin treated and nontreated mice (Ficure 5D). We then deleted Atg7 specifically in the lung (Figure $8) by using an adenovirus-expressing CRE (Ad) to intranasally infect Aig?" mice (Komatsu et al., 2005). When subjected to CLP, these mice were no longer protected trom CLP by ‘epirubicin treatment (Figure SE}. In contrast to control mice, in tg?" 3°" mice, the treatment with epirubicin does not improve the levels of circulating markers of organ lesion (Fig ure 5F) Accordingly, we observed a significant protective effect in survival after overexpression of ATG in the lung wth adeno- virus (Figure 56). By assessing the levels of yH2AX in the lungs of control or ‘epirubicin-treated CLP-subjected mice, we found a significant increase inthe numberof cells with yH2AX-postve foc inlungs. of epirubicin-treated mice (Figure SH).To test whether ATM acti- vation was also required in the lung, we used Atm?" mice and Ad‘ to delete ATM specifically in the lung. Upon ‘Ad"-mediated ATM deletion in the lung, mice were ne longer protected against sepsis by treatment with epirubicin ("igure 5). We therefore conclude thatthe protective effect of epirubicin in ‘sepsisis, at leastin part, dependent onthe activation of ATM and ‘of autophagy in target organs, namely the lung. These concli- sions are further supported by intranasal delvery of epirubicin fr etoposide to the lung (Figure 54) because the protective cffects as measured by survival are similarto thelp. administra~ tion of those drugs (Figue SY). Paso ote rmaniy 20% 16 ain poss as: tps del ag/T0.1076). mmr 2013.08.088 ‘quoredo ota, Antracycines lice DNA Damage Response-Mesated Protection agains! Severe Sap, Immunity ‘Anthracyclines Induce Tolerance to Sepsis “TE Ea esenesins F =. |: =} —— z7 z : ye Figure 5. The Protective Etfect of Epirubicin Ie Dependent onthe Activation of ATM and the Autophagy Pathway in the Lung INGEP expression in blood monocytes and rutopi, ol (By Suvivatot Alger an Aig?" aA" mic subjeted to CLP and ested wih PBS tom transgenic LCSb-GFP animals, 2 hr afler mice were subjected fo mock CLP (Sor CLP {ollwedby trestnet with PBS (Par eprubicin (C15) 09/9 Body weg athe reo piri Ep wits sare schedule and dove an {G)LC36-t nd LC36-11potentevels by immunoboting uing specie aiibody agains! LCG in ung, Wer ane ey, oats at he nate ies of ie (05731/6 anima (0) or mice subjected lo mock CLP (or CLP followed by reatmert with PBS (+) or epraniin (018) a8 A, {O Representative sectors of Bb staring nlung,solted the inate mes of me subjected tL? oloued by reatmnt wth PSS (C+P1 or epkubiin (pana, {Seal of 1 36 and Atg7™ naltion of aaenovia ctor encocig Cre (LDH. OK ALT and ureaplasma concoratensin WT (BS Ad") and ty" A animals 24h ater mock GLP ‘resiment wih PBS (C-P) n= 501 36 Aa ana n= 2 for Ag?" Rg reprint) snialssjcted to CLP and trealea wih PBS or apie wth sane seb and dove a8 (2) Says ater for B6Ad™)orCLP tolowed by fr ata" A) a8 or B6 Ad ara {G) Survival of WT (8) animals subjected to CLP 4 days afer whan of adenoviral vector encoding GFP (Ad*™) or Ata fe), (H Representative sc {ollwed by teatro with PBS CP) {9 Sul of WTB aA of yHOAK staring and pereetage of /H2AX" cell per ld (ght panel ngs, ole ‘spirsien (C1€) an Results shown represent sheote ms Shinals subjected lo CLP and ented with PBS or eprsbcn Ep wih same schedule an dose a8 nS days ater hr ater mice were subjects 9 CLP rs + 5D om ten fel {) Survnal of CS7BL/G WT animal subjected to CLP tested wit canier PBS), etoposide ua/9 body weight or eprbicin Ep (2.6 vala body weigh) 920003 fog-ank Marie Cox) eso", €,6, land, Mann= \Wintney ea for A. ane unpared eat for Fan Hight pane Se ako "our 55. Epirubiein Has a 24 hr Therapeutic Window to Protect against Sepsis Finally, we studied the therapeutic window of epirubicin in mice When given alone, epirubicin conferred strong protection atthe time of the procedure or unt 3 hr after the initiation of CLP (F9- ure 6A), When adminstered only 6 hr after CLP, epirubicin, Quickly lost its protective effect (Figure 6A). However, if given in combination with meropenem, even when this antibiote is ‘only administered 12h after CLP, low-dose epirubicin conferred complete protection until at least 24 hr after the intial procedure (ioutes 6B and 60), These results suggest that anthracyclines ‘can be used not only to prevent sepsis but also that they can act therapeutically when their administration is combined with a large-spectrum antibiotic DISCUSSION Here we report that epirubicin, and more generally the group of anthracyclines, are very effective at conferring protection against severe sepsis in mice, even when used up to 24 hr after the onse of infection. This therapeutic window is ikely to be sufficient to ‘make these drugs good candidates as useful therapeutic options Iremunity 39, 1-11, November 14, 2013 2018 Elsevier Ine. 7 ie Pease cloths aren arose as Figunvodo a, Aehvacyeines Indice ONA Derage Hespanse-Midaled Protection aginst Sovore Sepa muni 201}, nipex.do,org10.+016/ er. 2018.08,089 Immunity Anthracyclines Induce Tolerance to Sepsis Pei og 1 6. Eprubicin Confers Protection agsinst Severe S ( Sunival of 0578167 animals subjctea to OLPvoa'ed win PBS or opin same dose an ainda times ithe absence 0! merepenem: [Bl wth admnistration of meropenem 0 rs, not sigan p< 0.08: "p.< 0.01: p< 0.00 fog-rank Mane.Coy, In the clinic to reduce the mortalty of sepsis in most patients that are eitherin the hospital or seek medical attention within the frst ‘ew hours of symptoms inition ‘Athough we began our investigation of the use of anthracy- lines in sepsis by virtue oftheir effects in inhibiting inflammatory ytokine expression in myeloid cells in vivo, our studies have Idontied a mode of protection that seems to be much stronger and perhaps completely independent of such effects, and rather manifests atte level of DNA damage response and autophagy- induced protection in the lung. Thus, our findings uncover an Unexpected role for these pathways in tissue (ung) tolerance to the pathological consequences of infection. These findings are especially relevant given that agents discovered in studies, ‘over the last few years targeting various proinflammatory cyto- kines have had limited success in humans. Our studies suggest a crltical role for protecting host tissues thereby conferring pro- tection against sepsis. Recent studies have highlighted the role of tssue tolerance to infection as an important aspect of host, pathology (Medzhitov et al, 2012) Interestingly, the protective effect of epirubicin seems to act iespectively of the host pathogen burden, revealing that it Confers disease tolerance to polymicrobial infection (Larson et al, 2010; Raberg et al, 2008; Schneider and Ayres, 2008) ‘his finding reveals that pharmacologic agents that provide tissue damage cortrol can limit disease severty inespectively ‘of pathogen load and represent a promising therapeutic strategy against sepsis. Moreover, based on our identification of ATM. as a major mediator of epirubicin effects, we propose that this, protein and other components of the DNA damage response machinery constitute regulators of tolerance, without affecting pathogen resistance mechanisms. Recent reports make our findings counterinultive because doxorubicin and daunorubicin have been shown to induce acute Inflammation when injected inthe abdomen, where they induce cytokine secretion (

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