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Epidermal Growth Factor VIIIRescue Septic Animals Via Milk Fat Globule Immature Dendritic Cell-Derived Exosomes

WangWang, Thanjavur S. Ravikumar, Kevin J. Tracey and PingKomura, Dhruv Amin, Youxin Ji, Zhimin Wang, Haichao Michael Miksa, Rongqian Wu, Weifeng Dong, Hidefumi

http://www.jimmunol.org/content/183/9/5983doi: 10.4049/jimmunol.0802994October 2009;

2009; 183:5983-5990; Prepublished online 7J Immunol 

Referenceshttp://www.jimmunol.org/content/183/9/5983.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Immature Dendritic Cell-Derived Exosomes Rescue SepticAnimals Via Milk Fat Globule Epidermal Growth Factor VIII

Michael Miksa,*† Rongqian Wu,*† Weifeng Dong,† Hidefumi Komura,† Dhruv Amin,†

Youxin Ji,† Zhimin Wang,† Haichao Wang,* Thanjavur S. Ravikumar,† Kevin J. Tracey,*and Ping Wang2*†

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-�,HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue,prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the activeremoval of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomesthat contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In anexperimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, whichwas associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apo-ptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats.Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was preventedby MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory re-sponse in sepsis by enhancing apoptotic cell clearance via MFGE8. The Journal of Immunology, 2009, 183: 5983–5990.

P hagocytes, including dendritic cells (DC),3 constitutivelysecrete exosomes. These are 100-nm vesicles containedand released from so-called multivesicular bodies, in-

termediates in the process of endocytosis (1). These exosomescontain both exogenic and endogenic proteins that are charac-teristic for the cells they derive from (2). Immature DCs (IDCs)secrete exosomes, that contain abundant milk fat globule EGFfactor VIII (MFGE8), or lactadherin. This protein is commonlyfound on human milk fat globules (3) and has been recentlydescribed to be necessary for the opsonization of apoptotic cellsfor phagocytosis (4). Hanayama et al. (4) found that althoughphosphatidylserine and other apoptotic “eat-me” signals can berecognized and bound by phagocytes through other anchoringproteins, MFGE8 is an indispensable factor for the completeengulfment of these dying cells.

DCs play a key role in the interface of innate and adaptive im-munity and are strategically placed throughout the body to recog-nize microbial intruders and promptly react to them (5). Under

inflammatory conditions such as an infection, however, thesephagocytes become activated and mature into inflammatory cellshelping to clear the intruding microbes (5). Although this inflam-matory response is helpful in minor infections, it becomes over-zealous in sepsis, causing more harm than good to the organism.Sepsis is a systemic inflammatory response that is often associatedwith severe infections. Despite a growing number of generallyeffective antibiotics and improved critical care, sepsis still claimsa high death toll among affected patients due to cardiovascularshock and multiple organ failure (6). So far, only activated proteinC (Xigris) has been approved as a sepsis-specific drug and it hasprovided limited success in the treatment of septic patients (7).Under septic conditions, there is a substantial neuroendocrine andimmune activation, which leads to an overstimulation of inflam-matory processes (e.g., surge in TNF-�, IL-1�, IL-6, and the nu-clear protein high-mobility group box 1 (HMGB1), acting as a lateproinflammatory cytokine) (8–11), but also to an impairment ofvital innate immune functions (e.g., phagocytosis) (12–15). Oneof the problems during sepsis is the strong induction of apoptosisof crucial immune cells, which further impairs the immune func-tion. Up-regulation of death receptors and stimulation by cyto-kines, glucocorticoids, and complement factors (especially factorC5a) lead to an early increase in activation-induced cell death (16,17). In this inflammatory environment, apoptotic cells are prone toundergoing secondary necrosis if these cells are not fast removedby phagocytes (17). Without proper clearance, these cell corpsesmay pose a potential harm to the host, as they release potentiallyharmful inflammatory and toxic mediators, further impairing theseptic condition (18–20).

As MFGE8 has been reported to be of crucial importance in theremoval of apoptotic cells, we investigated whether this proteinmay in fact play an important role in sepsis. In this study, we showthat sepsis is associated with suppressed MFGE8, causing im-paired clearance of apoptotic cells. We further investigated thebeneficial role of IDC-derived exosomes in sepsis and their ability

*The Feinstein Institute for Medical Research, and †Department of Surgery, NorthShore University Hospital and Long Island Jewish Medical Center, Manhasset,NY 11030

Received for publication September 10, 2008. Accepted for publication September2, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by Grants R01 GM057468, R01 GM053008, and R01AG028352 from National Institutes of Health (to P.W.).2 Address correspondence and reprint requests to Dr. Ping Wang, The Feinstein In-stitute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mailaddress: pwang@nshs.edu3 Abbreviations used in this paper: DC, dendritic cell; IDC, immature DC; MFGE8,milk fat globule EGF factor VIII; rmMFGE8, recombinant murine MFGE8; CLP,cecal ligation and puncture; HMGB1, high-mobility group box 1; WT, wild type.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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to restore clearance of apoptotic cells, to suppress inflammation,and to improve survival in sepsis.

Materials and MethodsExperimental sepsis using cecal ligation and puncture (CLP)

Male Sprague–Dawley rats were purchased from Charles River BreedingLaboratories. MFGE8 knockout mice (MFGE8�/� mice) were a generousgift from Dr. S. Nagata (Osaka University, Osaka, Japan). The MFGE8�/�

mice were generated by replacing exons 4–6 of MFGE8 gene with a neo-mycin cassette as described by Hanayama et al. (4). The mutant mice werebackcrossed to C57BL/6 for at least nine times. Therefore, C57BL/6 wild-type (WT) mice (Taconic) were used as a control for MFGE8�/� mice. Inmale Sprague–Dawley rats (275–325 g) or C57BL/6 WT and MFGE8�/�

mice (20–25 g), cecums were ligated and double punctured with an 18-gauge (rats) or 22-gauge (mice) needle as previously described (43, 44).Sham-operated animals underwent the same procedure without the ligationor puncture. The animals were resuscitated s.c. with 3 ml/100 g bodyweight of normal saline solution. At 5 and 10 h after surgery, the ratsreceived i.v. either 2 � 1 ml of PBS (vehicle) or 2 � 1.5 mg/kg of exo-somes based on doses used in our own titration studies. Recombinant mu-rine MFGE8 (rmMFGE8) was administered using an osmotic Alzetminipump (Durect) that was implanted s.c. and connected to right jugularvein. The pumps released 8 �l/h of rmMFGE8 over a period of 20 h (20�g/kg body weight) or the same volume PBS. For survival studies, thenecrotic cecum was excised in rats and the abdominal cavity was washedwith normal saline solution, and animals were monitored for 10 days. Thisprocedure produces a consistent mortality of about LD50. All experimentswere performed in accordance with the National Institutes of Health guide-lines for the use of experimental animals. This project was approved by theInstitutional Animal Care and Use Committee of the Feinstein Institute forMedical Research.

Quantitative real-time PCR

Quantitative PCR was conducted on cDNA samples, reverse transcribedfrom 2 �g of RNA, using the QuantiTect SYBR Green PCR kit (Qiagen),reactions were conducted in 24 �l of final volume containing 2 pmol offorward and reverse primers, 12 �l of QuantiTect Master Mix, and 1 �l ofcDNA. Amplification was performed according to the manufacturer’s rec-ommendations (Qiagen) with an ABI Prism 7700 sequence detector(PerkinElmer-Applied Biosystems). Primer sequences were as follows:MFGE8 (107 bp, Gene Bank NM_012811) sense 5�-TGA GGA ACAAGG AAC CAG-3�, antisense 5�-GGA AGG ACA CGC ACA TAG-3�;and G3PDH (100 bp, Gene Bank XM_579386) sense 5�-ATG ACT CTACCC ACG GCA AG-3�, antisense 5�-CTG GAA GAT GGT GAT GGGTT-3�. Expression of rat GAPDH mRNA was used to normalize samplesand relative expression of mRNA was calculated using the ��Ct thresholdcycle method.

Western blotting

Splenic macrophages were collected by digesting spleens with collagenaseIV, lysing RBC with ammonia-chloride-potassium (ACK) lysing buffer(0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in H2O) followed byplastic adherence and thorough washing with PBS. A total of 2 � 106 cellswere homogenized and dissolved in 1% SDS. Blood plasma was ultrafil-tered with Centricon 100 (Millipore) and the elution was concentratedtimes 30 using Centricon YM30 filters. A total of 10 �g of protein or 5 �lof plasma concentrate was fractionated on a 4–12% Bis-Tris gel and trans-ferred to 0.2-�m nitrocellulose membrane. Blots were blocked with 10%BSA in TBST and incubated with 1/100 goat anti-MFGE8 IgG (G-17;Santa Cruz Biotechnology), specific for a 17 aa sequence in the C1 domainshared by human, mouse, and rat MFGE8, washed, and incubated withHRP-labeled rabbit anti-goat IgG. For HMGB1 detection, plasma sampleswere directly denatured in Laemmli buffer containing 5% 2-ME and cellculture supernatants were concentrated 10X using YM-10 Microcons (Mil-lipore) and denatured with 2.5% SDS and 5% 2-ME. Protein was trans-ferred to polyvinylidene difluoride membranes (Invitrogen), blocked with5% BSA in TBST, and blotted with purified, polyclonal rabbit anti-HMGB1 Ab (1/1000) and HRP-conjugated anti-rabbit IgG (1/20,000) in3% BSA in TBST, incubated with ECL (Amersham Biosciences) and ex-posed on a radiograph film. The density of bands was analyzed using theBio-Rad Imaging system.

Generation of bone marrow DC-derived exosomes

DCs were generated by culturing rat bone marrow leukocytes for up to 18days in medium containing IL-4 and GM-CSF as described elsewhere (21).

Briefly, bone marrow cells were obtained from healthy rats by flushingfreshly isolated femur shafts with HBSS (CellGro; Mediatech), filteringand lysing RBC with ACK buffer. Cells were cultured for 6–18 days inDMEM (Life Technologies) containing 10% heat-inactivated exosome-free FBS (obtained by centrifuging at 100,000 � g overnight), GM-CSF(1000 U/ml; Peprotech), and IL-4 (100 U/ml; Peprotech). Generation ofbone marrow DCs was verified morphologically by visible dendrites onloosely attached cells and by flow cytometry (�95% of collected cells wereCD11b/c�/�E2 integrin-positive). Conditioned bone marrow DC mediumwas collected and gradually centrifuged to remove cells and bigger parti-cles and vesicles as described elsewhere (2). Exosomes were retrieved byultracentrifuging supernatants at 100,000 � g for 3 h followed by a washwith PBS and overnight centrifugation at 100,000 � g. Collected pelletwas washed and reconstituted in PBS and adjusted to a concentration of 1mg/ml. To confirm that the secreted MFGE8 is associated with exosomes,the purified exosomes were resolved on a sucrose gradient as describedbefore (45). Concentrated samples (2 mg/500 �l) were mixed with 2.5 ml85% (w/v) sucrose (in 10 mM Tris-HCl buffer (pH 7.5) containing 150 mMNaCl and 5 mM EDTA) and placed in centrifuge tubes. The mixtures werelayered successively with 4 ml of 60% (w/v), 3 ml of 30% (w/v), and 1 mlof 5% (w/v) sucrose, and centrifuged at 200,000 � g for 18 h at 4°C.Different fractions were collected; their refractive index assessed using arefractometer and samples directly subjected to SDS-PAGE for Westernblotting. Please note that exosomes are the extractions from cells and thecontents of MFGE8 in exosomes are different in each extraction. Accord-ing to the Western blot analysis, there is �9 �g of MFGE8 in every mil-ligram of IDC exosomes. Therefore, the amount of MFGE8 in the IDCexosomes used in this study (i.e., 3.0 mg/kg body weight) should be �27�g/kg body weight. To simplify the calculation and experimental design,we chose 20–30 �g/kg body weight rmMFGE8 to be administered to CLPanimals.

Apoptosis assay

Thymuses and spleens were homogenized and cells washed with PBS, andreconstituted in Ca2�-rich Annexin V binding buffer (BD Pharmingen) ata concentration of 107 cells/ml. A total of 100 �l of cell suspension wasstained with 2.5 �l of Annexin V-FITC or 1 �l of propidium iodide for 15min and adjusted to a total volume of 500 �l with binding buffer. Cellswere then analyzed by flow cytometry using FACSCalibur.

Phagocytosis assay

Spleens were digested for 1 h with 400 U/ml collagenase VI (Worthington)and homogenized. RBC were lysed with ACK buffer and macrophagesenriched by plastic adherence for 2 h. Cells from sham and CLP animalswere plated at a density of 5 � 105/well in a 24-well plate and PerCPanti-CD90-tagged (OX-7; BD Pharmingen) apoptotic thymocytes (inducedby 10 �M dexamethasone for 16 h; �99% of apoptotic thymocytes(CD90�) both by Annexin V/propidium iodide and TUNEL) were added ata 4:1 ratio (apoptotic cells/macrophages) for 1.5 h. Nonphagocytized thy-mocytes were removed by thorough washing with PBS. Macrophages werecollected by gentle scraping and stained with FITC-labeled anti-CD11b/c(rats) or allophycocyanin-labeled anti-CD11b (mice) (BD Pharmingen).Analysis was performed by FACSCalibur (BD Biosciences) by gating onCD11b/c� or CD11b� cells. Percentage phagocytosis was determined bythe ratio of CD90�CD11b/(c)� to total CD11b/(c)� cells. Alternatively,macrophages were also cultured on LabTek chamber slides (Nalge Nunc),incubated with apoptotic thymocytes for 1.5 h, washed three times fixedwith 4% paraformaldehyde, and stained with TUNEL for fluorescent mi-croscopy using a Nikon Eclipse E600 microscope. To determine apoptoticcell engulfment, a novel phagocytosis assay using pHrodo-labeled apopto-tic cells was used. Apoptotic thymocytes were stained with 20 ng/mlpHrodo SE for 30 min and assay performed as described.

TNF-� assay

Cytokine levels were quantified using ELISA kit (BD Pharmingen). Theassays were conducted according to the instructions provided by themanufacturer.

Cell culture

Primary rat peritoneal macrophages were cultured at a density of 106/wellin a 6-well plate and incubated with apoptotic rmMFGE8-opsonized thy-mocytes with (10 �g/ml; R&D Systems). In controls, phagocytosis of ap-optotic cells was almost entirely blocked by anti-mouse MFGE8 polyclonalAb (10 �g/ml; R&D Systems). After 90 min, macrophages were washedthree times with medium and followed by stimulation with 100 ng/ml LPS(Escherichia coli O55:B5; Difco Laboratories) for 16 h. Supernatants were

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collected for HMGB1 Western blotting. In a separate experiment, rat peri-toneal macrophages were incubated with live, necrotic or apoptotic lym-phocytes thymocytes for 90 min without LPS stimulation. Supernatantscontaining unengulfed lymphocytes were removed and cultured separately,and adherent macrophages were washed and cultured with fresh mediumfor 16 h.

Statistics

All data are expressed as mean � SEM and compared by ANOVA (one-or two-way ANOVA as indicated). The Student t test with a two-taileddistribution and equal variance was used if only two groups were present.Normal distribution of samples was verified using the Kolmogorov-Smir-nov test. The survival rate was estimated by the Kaplan-Meier method andcompared by the log-rank test. The � level for all tests was 0.05.

ResultsMFGE8 is suppressed in experimental sepsis

To investigate whether MFGE8 levels are affected in critical dis-ease, we used an experimental sepsis model in rats. At 20 h afterCLP, which produces peritonitis and sepsis in these animals, bloodMFGE8 levels decreased by 45% ( p 0.031), indicating the sys-temic scale of MFGE8 depletion under septic conditions (Fig. 1A).Using quantitative PCR, we found that under normal conditionsMFGE8 is produced in various tissues with the highest mRNAexpression levels in the spleen, followed by 34–50% of its expres-sion in the lungs, thymus, and skin. Lower mRNA levels of �13–15% of spleen levels were found in the brain, heart, and liver (datanot shown). Due to its high basal MFGE8 expression levels, spleenMFGE8 levels were analyzed after CLP. Indeed, MFGE8 mRNAlevels decreased in the septic spleen by 19.9% within the first 5 h( p 0.294) and by 49% within 20 h of CLP ( p 0.004) (Fig.1B). This decreased transcription translated into a 48% decrease of

MFGE8 protein levels in the spleen 20 h after CLP ( p 0.034)(Fig. 1C). MFGE8 Western blotting from isolated CD11b/c� mac-rophages from septic spleens revealed that they greatly contributedto the decrease of MFGE8, showing a 51% suppression vs shamlevels ( p 0.032) (Fig. 1D).

IDC-derived exosomes improve apoptotic cell clearance andsurvival in experimental sepsis

Bone marrow-generated rat IDCs secreted high amounts of exo-somes (1.91 � 0.28 mg total protein/108 DCs), with a decrease ofsecretion by over 75% to 0.45 � 0.07 mg/108 DCs upon DC mat-uration. By sucrose gradient centrifugation we found that virtuallyall secreted MFGE8 was associated with exosomes found in thefraction with a density of 1.15 g/ml (refractive index 1.375) atwhich exosomes equilibrate (1) (Fig. 2). IDCs secreted exosomesthat contained abundant MFGE8, but virtually no costimulatoryprotein B7-2, a marker for mature DCs (Fig. 3A) (21). MatureDCs, marked by the morphological change with the developmentof visible dendrites, secreted exosomes with high levels of B7-2but no MFGE8 (Fig. 3A). In septic rats, the systemic decrease inMFGE8 was associated with the accumulation and impaired clear-ance of apoptotic cells. In untreated animals, apoptotic thymocytesaccumulated over time from 4% to 5% at 0 and 5 h after CLP toup to 14% within 20 h after CLP ( p 0.001, data not shown).Phagocytosis of apoptotic cells by CD11b/c� splenic macrophageswas significantly impaired with the average phagocytotic percent-age decreasing from 81% to 67% after CLP ( p 0.021) (Fig. 2B),whereas at the same time the total amount of apoptotic cells in-creased in the spleen from 6.6% to 9.8% ( p 0.003, data notshown) and in the thymus from 6.2% to 12.5% ( p 0.001) (Fig.3C). However, IDC-derived exosomes (2 � 1.5 mg/kg) increasedthe clearance of apoptotic cells in sepsis and completely restoredthe CLP-induced suppression of phagocytic capability (iExo, Fig.3B). Concurrently, CLP-associated accumulation of apoptotic cellswas reduced by over 28% ( p 0.027) (Fig. 3C). Mature DC-derived exosomes showed no significant influence on the ability of

FIGURE 1. MFGE8 is suppressed in experimental sepsis. A, Blood wasdrawn from septic rats 20 h after the onset of CLP and MFGE8 levels werecompared with sham-operated rats by Western blotting. �, p 0.031 vssham, by the Student t test for n 5 animals. B, Time-dependent decreaseof MFGE8 mRNA expression in the spleen during experimental sepsis.Rats underwent either CLP or sham operation and 5 or 20 h later spleenswere assayed for MFGE8 mRNA expression by quantitative real-timePCR. �, p 0.04, by one-way ANOVA, Tukey’s test for n 6 rats.Decrease of MFGE8 protein levels 20 h after CLP in total spleen lysates(C) and in isolated splenic macrophages (D) using Western blot. Purity ofisolated macrophages was �95% as assessed by CD11b/c staining andFACS analysis. �, p 0.034 (total spleen); �, p 0.032 (splenic macro-phages) vs sham, by Student’s t test for n 6 rats. Representative bandsof Western blots are shown atop of each result.

FIGURE 2. Detection of MFGE8 in exosomes isolated from IDCs. A,The polyclonal goat anti-MFGE8 Ab (clone G-17) is reactive against ratMFGE8 in thioglycolate-elicited peritoneal macrophages (PM) and detectsMFGE8 in IDC-derived exosomes (Exo, day 6 of bone marrow DC culture)or standard (STD). Arrowhead indicates MFGE8. B, Sucrose-gradient cen-trifugation of exosomes. MFGE8 is detectable only in the fraction with arefractive index (1.375) corresponding to the density of exosomes. CrudeIDC-derived exosome (Exo) preparations and thioglycolate-elicited peri-toneal macrophages (PM) serve as controls.

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splenic macrophages to clear apoptotic cells (mExo, p 0.492 vsVehicle, Fig. 3B) or to alter the accumulation of apoptotic cells( p 0.436) (Fig. 3C). This experimental sepsis model caused62.5% lethality within 1–4 days when the necrotic cecum wasremoved 20 h after CLP (Fig. 3D). Only 2 of 16 rats receivingIDC-derived exosomes (2 � 1.5 mg/kg at 5 and 20 h after CLP)died within 24 h. All other animals survived the following 9-dayobservation period ( p 0.007 vs vehicle control). Neither treat-ment with 10% of the effective dose of IDC exosomes, nor treat-ment with mature DC-derived exosomes affected the outcome insepsis (Fig. 3D). As demonstrated, mature DC-derived exosomesdid not contain MFGE8 (Fig. 3A), and the beneficial effect of IDCexosomes was likely to be mediated by MFGE8.

MFGE8 reconstitutes the clearance of apoptotic cells in septicrats

The reduced detection of apoptotic cells in septic animals cannotbe explained by a direct anti-apoptotic effect of IDC-derived exo-somes. In in vitro studies, TNF-�-induced apoptosis of lympho-cytes could not be blocked by the pretreatment with exosomes inthe absence of macrophages (data not shown). Similar to IDC exo-somes, treatment of septic rats with 2 � 30 �g/kg rmMFGE8 at 5and 10 h after CLP, resulted in the reconstitution of apoptotic cellclearance in septic rats ( p 0.019) (Fig. 4A). Previous publica-tions have shown that MFGE8 is crucial for the engulfment ofapoptotic cells, an important step in the removal of apoptotic cellsadhering to the surface of macrophages (4). To investigate whetherMFGE8 leads to the engulfment of apoptotic cells under septicconditions, we used the approach of staining apoptotic cells with apH-sensitive dye (phrodo SE) that become detectable only afterthey are engulfed by a macrophage. Using this method, we foundthat the rmMFGE8 protein was able to reconstitute engulfment ofapoptotic cells in septic rats to levels observed in sham operatedanimals ( p 0.009) (Fig. 4, B–E).

IDC-derived exosomes suppress the proinflammatory response inexperimental sepsis

Sepsis is associated with a systemic inflammatory response char-acterized by increases in early (TNF-�) and late proinflammatorycytokines (HMGB1). We investigated whether the treatment withIDC-derived exosomes was able to influence the inflammatory re-sponse in experimental sepsis. IDC-derived exosomes suppressedthe CLP-induced TNF-� response by 46% ( p 0.045) (Fig. 5A),whereas mature DC-derived exosomes did not affect the TNF-�levels in septic rats (Fig. 5A). Interestingly, IDC-derived exosomesfailed to suppress TNF-� release from LPS-stimulated macro-phages in vitro in the absence of apoptotic cells (data not shown),suggesting an indirect immunosuppressive effect. Similar resultscould be found in the levels of the late cytokine HMGB1. In ve-hicle-treated septic animals, blood HMGB1 levels increased by

FIGURE 3. IDC-derived exosomes improve apoptotic cell clearanceand survival in experimental sepsis. A, Exosomes secreted from IDCs con-tain MFGE8. Exosomes were collected from the supernatant of primary ratbone marrow DC cultures (day 6 to 17 in culture) by stepwise ultracen-trifugation. Equal amounts of exosome protein were subjected to Westernblotting for MFGE8, the DC maturation marker B7-2, and the lysosomeassociated membrane protein 3 (LAMP3) as an endogenous control. B,Improved phagocytosis of apoptotic cells by splenic macrophages. Ratsunderwent CLP or sham operation and were treated with 2 � 1 ml of PBS(vehicle), 2 � 1.5 mg/kg IDC-derived exosomes (iExo) or 2 � 1.5 mg/kgmature DC-derived exosomes (mExo) 5 and 10 h after CLP. Macrophages(CD11b/c�) were isolated from spleens 20 h after CLP and analyzed forphagocytosis of CD90� apoptotic (�99% annexin V-positive) thymocytesvia FACS. Representative FACS plots are shown. �, p 0.025 vs sham;

#, p 0.012 vs vehicle; †, p 0.001 vs iExo, ANOVA and Tukey’s testfor n 6 animals. C, Reduced accumulation of apoptotic cells. Thymo-cytes were isolated from rats 20 h after CLP or sham operation and ana-lyzed by FACS for apoptosis using Annexin V and propidium iodide stain-ing (representative plots). �, p 0.001 vehicle vs Sham; �, p 0.001mExo vs sham; �, p 0.024 iExo vs sham; #, p 0.003 iExo vs vehicle,p 0.256 for mExo vs vehicle, ANOVA and Tukey’s test for n 6animals. D, Protection from sepsis-induced lethality by IDC-derived exo-somes. Rats underwent CLP and received either PBS (vehicle), exosomesderived from IDCs or mature DCs, 5 and 10 h after CLP i.v. At 20 h afterCLP the necrotic cecum was removed and rats observed for the following10 days. �, p 0.007 vs vehicle; p 0.005 vs mature DC exosomes byKaplan-Meyer log-rank test for n 16 animals.

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51% compared with sham-operated animals ( p 0.002). Thisincrease was completely suppressed by the treatment with IDCexosomes ( p 0.001), but not by mature DC exosomes ( p 0.294) (Fig. 5B). Although phagocytosis of apoptotic cells isknown to suppresses TNF-� response of macrophages, its effectson HMGB1 release remains unclear. Apoptotic cells have beenpreviously shown to increase HMGB1 release in a coculture sys-tem with macrophages (22). We therefore investigated the effect ofMFGE8-mediated clearance of apoptotic cells on HMGB1-releaseunder inflammatory conditions in vitro. Opsonization of apoptoticthymocytes with rmMFGE8 completely prevented the HMGB1release from the macrophage/apoptotic cell cocultured system( p 0.002) (Fig. 5C). Inefficient phagocytosis of apoptotic cells,conversely, resulted in the release of increasing amounts ofHMGB1 (up to 3.5 times at an apoptotic cell to macrophage ratioof 5:1, p 0.006) (Fig. 5C), depending on the amount of apoptoticcells present. HMGB1 has been shown to be actively released frominflammatory macrophages as well as passively released from ne-crotic cells (23). Analysis of the release of HMGB1 from sepa-

rately cultured macrophages and unengulfed apoptotic cells afterphagocytosis revealed that macrophages challenged with apoptoticcells released similar levels of HMGB1 as necrotic cell-challengedmacrophages, albeit to a significantly lesser degree than unen-gulfed late apoptotic cells and necrotic cells released themselves.The rmMFGE8 treatment suppressed the release of HMGB1 fromboth macrophages and late apoptotic lymphocytes (Fig. 6). Thissuggests that MFGE8-mediated clearance of apoptotic cells di-rectly attenuates the release of HMGB1 in sepsis.

FIGURE 4. MFGE8 reconstitutes phagocytosis of apoptotic cells inseptic rats. A, Rats underwent CLP to induce experimental sepsis and weretreated with 2 � 15 �g/kg rmMFGE8 i.v. at 5 and 10 h after CLP. Thy-mocyte apoptosis was assessed 20 h after CLP by Annexin V/propidiumiodide staining and FACS analysis. �, p 0.034 vs sham; #, p 0.019 vsvehicle, by ANOVA and Tukey’s test for n 5 animals. B–E, Splenicmacrophages from septic rats were isolated 20 h after CLP and challengedwith four times apoptotic thymocytes labeled with pHrodo SE, a fluores-cent dye that is detectable only after engulfment of the cells by macro-phages. After 60 min, cells were labeled with FITC-anti-CD11b/c� andanalyzed by FACS analysis. The pHrodo histogram of CD11b/c�-gatedcells (B–D). The macrophage pre-phagocytosis control is represented in theoverlay in B. E, Summary of experiments showing the percentage ofphagocytosing macrophages. �, p 0.023 vs sham, #, p 0.009 vs ve-hicle, by ANOVA and Tukey’s test for n 5 animals.

FIGURE 5. IDC-derived exosomes suppress the proinflammatory re-sponse in sepsis. A, TNF-� levels in septic rats 20 h after CLP. Rats un-derwent either sham operation or CLP with either PBS (vehicle), IDC-derived exosome (iExo), or mature DC-derived exosome (mExo)treatment. �, p 0.001 vs sham; #, p 0.041 vs vehicle, by ANOVA andStudent-Newman-Keuls test for n 6 animals. B, HMGB1 is suppressedin septic rats receiving IDC-derived exosomes. At 20 h after CLP, HMGB1was assessed by Western blot. �, p 0.002 vs sham; #, p 0.001 vsvehicle; †, p 0.001 vs iExo, by ANOVA and Tukey’s test for n 6animals. Representative bands of Western blots are shown atop the results.C, The rmMFGE8-mediated promotion of apoptotic cell clearance sup-presses late cytokine HMGB1 release in vitro. After preincubation withapoptotic cells with or without rmMFGE8 for 90 min, primary rat perito-neal macrophages were stimulated with 100 ng/ml LPS for 16 h, superna-tants were collected and assayed for HMGB1 by Western blot. �, p 0.002vs control, by ANOVA and Tukey’s test for n 3 animals. Representativeband of Western blots are shown atop graph.

FIGURE 6. Postphagocytic release of HMGB1 from macrophages andlate apoptotic cells is suppressed by rmMFGE8 treatment. Rat peritonealmacrophages were incubated with live, necrotic (necr.), or apoptotic lympho-cytes (induced by 10 �M dexomethasone for 16 h in thymus T cells) for 90min. Unengulfed lymphocytes were removed and cultured separately for 16 h,whereas macrophages were washed and cultured in fresh medium for 16 h.The initially early apoptotic (Annexin V-positive/propidium iodide-negative)cells became eventually late apoptotic after 16 h of incubation (trypan blue-possitive, total of 32 h after dexomethasone treatment) and released an equalamount of HMGB1 into the medium as the same number of necrotic cells(induced by four freeze-thaw cycles). The rmMFGE8 treatment suppressedHMGB1 release from both lymphocytes and macrophages. Incubation withlive lymphocytes served as a control.

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MFGE8-deficiency is associated with poor apoptotic cellclearance in sepsis

To address whether impaired phagocytosis due to MFGE8 defi-ciency affects apoptotic cell accumulation in sepsis, we comparedMFGE8�/� mice and their C57BL/6J WT controls 20 h after CLP.As expected, spleen macrophages from MFGE8�/� mice showeda dramatically decreased ability to phagocytose apoptotic cells un-der normal conditions (22% of WT sham, p 0.001) (Fig. 7A). Toan even stronger degree than in rats, septic WT mice showeda 74% suppression of phagocytosis of apoptotic cells 20 h afterCLP, whereas CLP had no further impact on phagocytosis inMFGE8�/� mice (Fig. 7A). Mice deficient in MFGE8 also accu-mulated higher amounts of apoptotic cells (19%) compared withWT mice at the same time point (12%, p 0.001) (Fig. 7B). Thisindicates that the clearance of apoptotic cells in sepsis is positivelyregulated by MFGE8.

MFGE8 protects from sepsis-associated mortality

Finally, we were interested in whether MFGE8 influences survivalin experimental sepsis. Indeed, MFGE8-deficient mice were moresusceptible to sepsis-mediated mortality. Although 50% of WTmice died within 10 days of CLP, 82% of MFGE8�/� mice diedin the same period ( p 0.045) (Fig. 8A). In contrast, a continuousinfusion of rmMFGE8 over 20 h protected 83% of septic rats fromdying from CLP-induced sepsis compared with 50% of rats thatdied without treatment ( p 0.042) (Fig. 8B). Thus, administrationof rmMFGE8 provided similar results as the treatment with IDC-derived exosomes (Fig. 3D).

DiscussionIDCs constitutively secrete exosomes that contain MFGE8. In ma-ture DCs, exosome and MFGE8 production and release are re-duced. In our studies, we have presented that MFGE8 is system-ically down-regulated in sepsis, which leads to a widespreadimpairment of apoptotic cell clearance. The associated proinflam-matory response in sepsis can be prevented by the administrationof exogenous exosomes from IDCs. These exosomes improve ap-

optotic cell clearance, prevent the excessive release of proinflam-matory cytokines and protect septic animals from dying. Althoughexosomes from mature DCs do not contain MFGE8 and fail to beprotective, the protein MFGE8 itself has been shown to be anindispensable factor for the prevention of accumulating apoptoticcells and mortality in sepsis.

Sepsis is marked by a systemic inflammatory response, medi-ated by innate immune cells. An increase in proinflammatory cy-tokines is normally beneficial to fight microbes in minor infections(24). In sepsis, however, this cytokine response is extensive andprolonged (25), leading to multiple organ damage and septic shock(6, 25). Systemic increases of the cytokines TNF-�, IL-1�, IL-6,and HMGB1 in sepsis have been previously associated with a highmortality rate. Being equally the source and the target of thesemediators, APCs become activated and mature, thereby shuttingdown the endocytotic machinery in favor of an immunostimulatoryresponse (26). During this maturation process, the secretion ofexosomes and the production of MFGE8 are reduced (27). A num-ber of mediators are responsible for the modulation of MFGE8production in sepsis. Endotoxin can suppress MFGE8 productionin vitro (28), and GM-CSF plus IL-4-mediated maturation of DC

FIGURE 7. MFGE8 deficiency is associated with poor clearance of ap-optotic cells in sepsis. A, Phagocytosis of apoptotic cells is severely im-paired in MFGE8-deficient mice. C57BL/6J WT or MFGE8�/� mice un-derwent CLP, and 20 h later splenic macrophages were isolated andchallenged with apoptotic thymocytes. The percentage of phagocytic mac-rophages was assessed by TUNEL staining and microscopy. �, p 0.001vs WT sham, by two-way ANOVA and Tukey’s test for n 3 animals(sham) or n 4 animals (CLP). B, Mice lacking MFGE8 accumulate moreapoptotic cells in sepsis. MFGE8�/� mice and their C57BL/6J WT controlmice underwent CLP, and 20 h later thymocytes were stained with AnnexinV and analyzed by FACS. �, p 0.001 vs respective sham; #, p 0.001vs WT, by two-way ANOVA and Tukey’s test for n 6 animals.

FIGURE 8. MFGE8 protects from sepsis-associated mortality. A,MFGE8�/� mice display dramatically reduced survival in sepsis.MFGE8�/� mice and C57BL/6J WT mice underwent CLP and were ob-served for the following 10 days. �, p 0.045 vs WT control, by Kaplan-Meyer log-rank test for n 10–11 animals. B, Treatment with rmMFGE8improves survival in septic rats. After CLP, rats were treated with eitherPBS (vehicle) or 20 �g/kg rmMFGE8, as a continuous perfusion over 20 hvia osmotic minipump and then observed for 10 days. �, p 0.042 vsvehicle, by Kaplan-Meyer log-rank test for n 18 animals.

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in vitro in the absence of bacteria or endotoxin indicates that cy-tokines may equally play a role. In this regard, GM-CSF has beenshown to induce MFGE8 expression in vivo (29). GM-CSF is re-quired for the expression of MFGE8 in APCs, and that MFGE8-mediated uptake of apoptotic cells is a key determinant of GM-CSF-triggered tolerance and immunity (29). This research isinteresting because GM-CSF has been investigated as a promisingtreatment option for septic patients due to its immune-modulatingfunction (30–33). Whether other cytokines such as TNF-� orIFN-�, also influence MFGE8 expression is unclear and needs fur-ther investigation. We have shown in this study that in sepsisCD11b/c expressing macrophages and DCs contribute to the re-duction of MFGE8 production in the spleen, which is mirrored bya decrease of this protein in the circulation.

As we have shown further, a deficiency in MFGE8 is detrimen-tal in sepsis. Mice lacking MFGE8 accumulate two to three timesas many apoptotic cells above basal level and have a 60% highermortality rate than WT mice. Similarly, the administration ofrmMFGE8 to septic rats protected the majority from sepsis-medi-ated lethality. The protective effect of MFGE8 is evident in thisacute inflammatory model of sepsis and provides further evidencethat it is a crucial part of the protective effect of exosomes derivedfrom IDCs in septic animals.

Exosomes have been previously shown to transfer cell-mediatedimmunity from one cell to another (26). Mature Ag-pulsed DCssecrete exosomes that act as cross-presenting carriers of MHC-Agcomplexes and hence confer functional immunity to the recipientcell (26). The absence of Ag-presenting and costimulatory mole-cules on exosomes from IDCs suggests that these exosomes holda minor role in directly modulating immune responses. However,IDC-derived exosomes are not at all functionally inert. As we haveshown, the administration of exosomes from IDCs, but not frommature DCs conferred protection in sepsis, highlighting the im-portant role of MFGE8 in these vesicles. Unfortunately, technicalreasons precluded conclusive experiments using exosomes fromWT and MFGE8 knockout exosomes.

MFGE8 contains two important regions to function as an opso-nin for apoptotic cells; two EGF-like domains contain an RGDmotif necessary for the binding of �v�3- or �v�5 integrins, and twocoagulation factor V/VIII like domains that bind to phosphatidyl-serine exposed on the surface of apoptotic cells (27). Binding ofMFGE8 to phosphatidylserine on apoptotic cells opsonizes themfor a complete engulfment by macrophages via �v�3 or �v�5 in-tegrins. MFGE8 has been shown to be important for the removalof apoptotic lymphocytes in the spleen and the prevention of asystemic lupus erythematosus-like disease in mice (4). In our sep-tic model, we found a similar phenomenon in an acute inflamma-tory environment.

The beneficial effect of IDC-derived exosomes is mediated bythe promotion of apoptotic cell clearance. Apoptosis is often foundin sepsis, with lymphoid CD4 T and B cells and DCs being mostcommonly affected (34–37). Particularly apoptosis of DCs maycontribute to the depletion of MFGE8 in sepsis as these are onesource of this protein. Overall, the occurrence of apoptosis hasbeen associated with poor outcome in sepsis. Targeted inhibitionof the proapoptotic Fas signaling or overexpression of anti-apoptotic proteins, such as BH4, Bcl-2, or Bcl-xL, have beenshown to prevent apoptosis and protect from associated lethality insepsis (35, 38). Historically, apoptosis has been seen as an orderlyprocess of cell suicide that, unlike necrosis, does not elicit inflam-mation (39). Recently it has become clear, however, that apoptoticcells eventually undergo secondary necrosis and stimulate an in-flammatory response if they are not removed by phagocytosis (19,20). By using the pHrodo labeling system of apoptotic cells we

have clearly demonstrated that rmMFGE8 promotes the engulf-ment of apoptotic cells also under septic conditions, which is atleast in part responsible for the decrease in apoptotic cell number.Hence, the sepsis-associated decrease of MFGE8 contributes to theaccumulation of apoptotic cells, resulting in a surge in proinflam-matory cytokines, such as TNF-� and HMGB1, which by itselfpromotes the progression and deterioration in sepsis (11, 40).

We have previously shown that the pretreatment with bone mar-row-derived DC exosomes was beneficial in septic animals andpossibly dependent on the presence of MFGE8 (41). The currentstudy shows that the clearance of apoptotic cells in septic animalsis impaired in the absence of MFGE8 and that this is detrimentalin the acute inflammatory disease model. Furthermore, we nowshow that even treatment with IDC exosomes at a later time pointis beneficial (i.e., when apoptotic cells start to accumulate in sep-sis, long after the initiation of inflammatory responses). Thepresent report also demonstrates how MFGE8-mediated internal-ization of apoptotic cells prevents the release of proinflammatorymediators, which leads to a suppression of the septic systemicinflammatory response.

The beneficial effect of IDC-derived exosomes can be particu-larly attributed to the immune suppressive effect of phagocytosis ofapoptotic cells (39). Exosomes from IDCs were neither able tosuppress TNF-� release from endotoxin-stimulated macrophages,nor to prevent TNF-�-induced apoptosis of lymphocytes in vitro(our unpublished observations). Thus the reduction in apoptosis invivo is most likely mediated via enhanced clearance of apoptoticcells.

In our experimental sepsis model, early increases in TNF-� andIL-6 (1–4 h after CLP) are followed by a significant release ofHMGB1 in the late phase of sepsis (16–24 h after CLP) (11).These cytokines play a central role in the morbidity and mortalityin experimental sepsis as well as in septic patients (42). Studiesusing inhibitors of these cytokines demonstrated increased survivalof septic mice treated with TNF-� or HMGB1 blocking Abs (8,22). We have shown in this study that IDC-derived exosomes sup-pressed both TNF-� and HMGB1 in experimental sepsis, whichwas associated with a dramatically improved survival.

Exosomes secreted from IDCs are not merely a byproduct ofexcessive endocytosis but they have, as we have shown, the abilityto suppress a once-established systemic proinflammatory response.The opsonizing protein MFGE8 plays a key role in this pro-phago-cytic and secondary immunosuppressive effect. This distinguishesthem from exosomes secreted from mature, Ag-pulsed DCs thatconfer cellular immunity by cross priming. This novel findingshould open a new option of sepsis therapy in which the clearanceof apoptotic cells is targeted. The restoration of this basic immu-nological function and the induction of the reparative phase mayultimately contribute to the attenuation of the life-threatening sys-temic inflammatory response in sepsis. It is likely that a combina-tion of apoptosis prevention and the promotion of apoptotic cellclearance can be used in conjunction to the current treatment re-gime to the benefit of critically ill patients in the future.

AcknowledgmentsMax Brenner, Herb Borrero, and Thomas McCloskey were helpful withinputs and in performing FACS analysis for the phagocytosis assay anddetection of apoptotic cells. Enesa Paric and James Mason assisted in theisolation and gradient centrifugation of exosomes. Margot Puerta, Wei Li,and Tianpen Cui were cooperative in the methodology of HMGB1 detec-tion. Maowen Hu and Yingjie Cui were instrumental in establishing thereal-time PCR for MFGE8 and detecting MFGE8 in the blood, respec-tively. Kavin Shah was instrumental in the rat sepsis model. ShigekazuNagata (Osaka University, Osaka Japan) provided us with MFGE8�/�

mice for this study, for which we are thankful.

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DisclosuresThe authors have no financial conflict of interest.

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CorrectionsMiksa, M., R. Wu, W. Dong, H. Komura, D. Amin, Y. Ji, Z. Wang, H. Wang, T. S. Ravikumar, K. J. Tracey, and P. Wang. 2009. Immaturedendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor VIII. J. Immunol. 183: 5983–5990.

The title of this article was published incorrectly. The corrected title is shown below.

Immature Dendritic Cell-Derived Exosomes Rescue Septic Animals via Milk Fat Globule Epidermal Growth Factor-Factor VIII.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0990106

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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