Epidermal Growth Factor VIII Rescue Septic Animals Via Milk Fat ...

The Journal of Immunology

Immature Dendritic Cell-Derived Exosomes Rescue Septic Animals 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 active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8deficient 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 prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8. The Journal of Immunology, 2009, 183: 5983–5990.

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hagocytes, including dendritic cells (DC),3 constitutively secrete exosomes. These are 100-nm vesicles contained and released from so-called multivesicular bodies, intermediates in the process of endocytosis (1). These exosomes contain both exogenic and endogenic proteins that are characteristic for the cells they derive from (2). Immature DCs (IDCs) secrete exosomes, that contain abundant milk fat globule EGF factor VIII (MFGE8), or lactadherin. This protein is commonly found on human milk fat globules (3) and has been recently described to be necessary for the opsonization of apoptotic cells for phagocytosis (4). Hanayama et al. (4) found that although phosphatidylserine and other apoptotic “eat-me” signals can be recognized and bound by phagocytes through other anchoring proteins, MFGE8 is an indispensable factor for the complete engulfment of these dying cells. DCs play a key role in the interface of innate and adaptive immunity and are strategically placed throughout the body to recognize microbial intruders and promptly react to them (5). Under

*The Feinstein Institute for Medical Research, and †Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 Received for publication September 10, 2008. Accepted for publication September 2, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This study was supported by Grants R01 GM057468, R01 GM053008, and R01 AG028352 from National Institutes of Health (to P.W.).

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Address correspondence and reprint requests to Dr. Ping Wang, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mail address: [email protected]

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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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802994

inflammatory conditions such as an infection, however, these phagocytes become activated and mature into inflammatory cells helping to clear the intruding microbes (5). Although this inflammatory response is helpful in minor infections, it becomes overzealous in sepsis, causing more harm than good to the organism. Sepsis is a systemic inflammatory response that is often associated with severe infections. Despite a growing number of generally effective antibiotics and improved critical care, sepsis still claims a high death toll among affected patients due to cardiovascular shock and multiple organ failure (6). So far, only activated protein C (Xigris) has been approved as a sepsis-specific drug and it has provided limited success in the treatment of septic patients (7). Under septic conditions, there is a substantial neuroendocrine and immune activation, which leads to an overstimulation of inflammatory processes (e.g., surge in TNF-␣, IL-1␤, IL-6, and the nuclear protein high-mobility group box 1 (HMGB1), acting as a late proinflammatory cytokine) (8 –11), but also to an impairment of vital innate immune functions (e.g., phagocytosis) (12–15). One of the problems during sepsis is the strong induction of apoptosis of crucial immune cells, which further impairs the immune function. Up-regulation of death receptors and stimulation by cytokines, glucocorticoids, and complement factors (especially factor C5a) lead to an early increase in activation-induced cell death (16, 17). In this inflammatory environment, apoptotic cells are prone to undergoing secondary necrosis if these cells are not fast removed by phagocytes (17). Without proper clearance, these cell corpses may pose a potential harm to the host, as they release potentially harmful inflammatory and toxic mediators, further impairing the septic condition (18 –20). As MFGE8 has been reported to be of crucial importance in the removal of apoptotic cells, we investigated whether this protein may in fact play an important role in sepsis. In this study, we show that sepsis is associated with suppressed MFGE8, causing impaired clearance of apoptotic cells. We further investigated the beneficial role of IDC-derived exosomes in sepsis and their ability

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

Briefly, bone marrow cells were obtained from healthy rats by flushing freshly isolated femur shafts with HBSS (CellGro; Mediatech), filtering and lysing RBC with ACK buffer. Cells were cultured for 6 –18 days in DMEM (Life Technologies) containing 10% heat-inactivated exosomefree FBS (obtained by centrifuging at 100,000 ⫻ g overnight), GM-CSF (1000 U/ml; Peprotech), and IL-4 (100 U/ml; Peprotech). Generation of bone marrow DCs was verified morphologically by visible dendrites on loosely attached cells and by flow cytometry (⬎95% of collected cells were CD11b/c⫹/␣E2 integrin-positive). Conditioned bone marrow DC medium was collected and gradually centrifuged to remove cells and bigger particles and vesicles as described elsewhere (2). Exosomes were retrieved by ultracentrifuging supernatants at 100,000 ⫻ g for 3 h followed by a wash with PBS and overnight centrifugation at 100,000 ⫻ g. Collected pellet was washed and reconstituted in PBS and adjusted to a concentration of 1 mg/ml. To confirm that the secreted MFGE8 is associated with exosomes, the purified exosomes were resolved on a sucrose gradient as described before (45). Concentrated samples (2 mg/500 ␮l) were mixed with 2.5 ml 85% (w/v) sucrose (in 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 5 mM EDTA) and placed in centrifuge tubes. The mixtures were layered successively with 4 ml of 60% (w/v), 3 ml of 30% (w/v), and 1 ml of 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 a refractometer and samples directly subjected to SDS-PAGE for Western blotting. Please note that exosomes are the extractions from cells and the contents of MFGE8 in exosomes are different in each extraction. According to the Western blot analysis, there is ⬃9 ␮g of MFGE8 in every milligram of IDC exosomes. Therefore, the amount of MFGE8 in the IDC exosomes 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 CLP animals.

Materials and Methods Experimental sepsis using cecal ligation and puncture (CLP) Male Sprague–Dawley rats were purchased from Charles River Breeding Laboratories. MFGE8 knockout mice (MFGE8⫺/⫺ mice) were a generous gift from Dr. S. Nagata (Osaka University, Osaka, Japan). The MFGE8⫺/⫺ mice were generated by replacing exons 4 – 6 of MFGE8 gene with a neomycin cassette as described by Hanayama et al. (4). The mutant mice were backcrossed to C57BL/6 for at least nine times. Therefore, C57BL/6 wildtype (WT) mice (Taconic) were used as a control for MFGE8⫺/⫺ mice. In male Sprague–Dawley rats (275–325 g) or C57BL/6 WT and MFGE8⫺/⫺ mice (20 –25 g), cecums were ligated and double punctured with an 18gauge (rats) or 22-gauge (mice) needle as previously described (43, 44). Sham-operated animals underwent the same procedure without the ligation or puncture. The animals were resuscitated s.c. with 3 ml/100 g body weight of normal saline solution. At 5 and 10 h after surgery, the rats received i.v. either 2 ⫻ 1 ml of PBS (vehicle) or 2 ⫻ 1.5 mg/kg of exosomes based on doses used in our own titration studies. Recombinant murine MFGE8 (rmMFGE8) was administered using an osmotic Alzet minipump (Durect) that was implanted s.c. and connected to right jugular vein. 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, the necrotic cecum was excised in rats and the abdominal cavity was washed with normal saline solution, and animals were monitored for 10 days. This procedure produces a consistent mortality of about LD50. All experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. This project was approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research.

Quantitative real-time PCR Quantitative PCR was conducted on cDNA samples, reverse transcribed from 2 ␮g of RNA, using the QuantiTect SYBR Green PCR kit (Qiagen), reactions were conducted in 24 ␮l of final volume containing 2 pmol of forward and reverse primers, 12 ␮l of QuantiTect Master Mix, and 1 ␮l of cDNA. Amplification was performed according to the manufacturer’s recommendations (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 ACA AGG AAC CAG-3⬘, antisense 5⬘-GGA AGG ACA CGC ACA TAG-3⬘; and G3PDH (100 bp, Gene Bank XM_579386) sense 5⬘-ATG ACT CTA CCC ACG GCA AG-3⬘, antisense 5⬘-CTG GAA GAT GGT GAT GGG TT-3⬘. Expression of rat GAPDH mRNA was used to normalize samples and relative expression of mRNA was calculated using the ⌬⌬Ct threshold cycle method.

Western blotting Splenic macrophages were collected by digesting spleens with collagenase IV, lysing RBC with ammonia-chloride-potassium (ACK) lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in H2O) followed by plastic adherence and thorough washing with PBS. A total of 2 ⫻ 106 cells were homogenized and dissolved in 1% SDS. Blood plasma was ultrafiltered with Centricon 100 (Millipore) and the elution was concentrated times 30 using Centricon YM30 filters. A total of 10 ␮g of protein or 5 ␮l of plasma concentrate was fractionated on a 4 –12% Bis-Tris gel and transferred 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 domain shared by human, mouse, and rat MFGE8, washed, and incubated with HRP-labeled rabbit anti-goat IgG. For HMGB1 detection, plasma samples were directly denatured in Laemmli buffer containing 5% 2-ME and cell culture supernatants were concentrated 10X using YM-10 Microcons (Millipore) and denatured with 2.5% SDS and 5% 2-ME. Protein was transferred to polyvinylidene difluoride membranes (Invitrogen), blocked with 5% BSA in TBST, and blotted with purified, polyclonal rabbit antiHMGB1 Ab (1/1000) and HRP-conjugated anti-rabbit IgG (1/20,000) in 3% BSA in TBST, incubated with ECL (Amersham Biosciences) and exposed on a radiograph film. The density of bands was analyzed using the Bio-Rad Imaging system.

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

Apoptosis assay Thymuses and spleens were homogenized and cells washed with PBS, and reconstituted in Ca2⫹-rich Annexin V binding buffer (BD Pharmingen) at a concentration of 107 cells/ml. A total of 100 ␮l of cell suspension was stained with 2.5 ␮l of Annexin V-FITC or 1 ␮l of propidium iodide for 15 min and adjusted to a total volume of 500 ␮l with binding buffer. Cells were 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 macrophages enriched by plastic adherence for 2 h. Cells from sham and CLP animals were plated at a density of 5 ⫻ 105/well in a 24-well plate and PerCP anti-CD90-tagged (OX-7; BD Pharmingen) apoptotic thymocytes (induced by 10 ␮M dexamethasone for 16 h; ⱖ99% of apoptotic thymocytes (CD90⫹) both by Annexin V/propidium iodide and TUNEL) were added at a 4:1 ratio (apoptotic cells/macrophages) for 1.5 h. Nonphagocytized thymocytes were removed by thorough washing with PBS. Macrophages were collected 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 on CD11b/c⫹ or CD11b⫹ cells. Percentage phagocytosis was determined by the 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 fixed with 4% paraformaldehyde, and stained with TUNEL for fluorescent microscopy using a Nikon Eclipse E600 microscope. To determine apoptotic cell engulfment, a novel phagocytosis assay using pHrodo-labeled apoptotic cells was used. Apoptotic thymocytes were stained with 20 ng/ml pHrodo SE for 30 min and assay performed as described.

TNF-␣ assay Cytokine levels were quantified using ELISA kit (BD Pharmingen). The assays were conducted according to the instructions provided by the manufacturer.

Cell culture Primary rat peritoneal macrophages were cultured at a density of 106/well in a 6-well plate and incubated with apoptotic rmMFGE8-opsonized thymocytes with (10 ␮g/ml; R&D Systems). In controls, phagocytosis of apoptotic cells was almost entirely blocked by anti-mouse MFGE8 polyclonal Ab (10 ␮g/ml; R&D Systems). After 90 min, macrophages were washed three times with medium and followed by stimulation with 100 ng/ml LPS (Escherichia coli O55:B5; Difco Laboratories) for 16 h. Supernatants were


FIGURE 1. MFGE8 is suppressed in experimental sepsis. A, Blood was drawn from septic rats 20 h after the onset of CLP and MFGE8 levels were compared with sham-operated rats by Western blotting. ⴱ, p ⫽ 0.031 vs sham, by the Student t test for n ⫽ 5 animals. B, Time-dependent decrease of MFGE8 mRNA expression in the spleen during experimental sepsis. Rats underwent either CLP or sham operation and 5 or 20 h later spleens were assayed for MFGE8 mRNA expression by quantitative real-time PCR. ⴱ, 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 of isolated macrophages was ⬎95% as assessed by CD11b/c staining and FACS analysis. ⴱ, p ⫽ 0.034 (total spleen); ⴱ, p ⫽ 0.032 (splenic macrophages) vs sham, by Student’s t test for n ⫽ 6 rats. Representative bands of Western blots are shown atop of each result.

collected for HMGB1 Western blotting. In a separate experiment, rat peritoneal macrophages were incubated with live, necrotic or apoptotic lymphocytes thymocytes for 90 min without LPS stimulation. Supernatants containing unengulfed lymphocytes were removed and cultured separately, and adherent macrophages were washed and cultured with fresh medium for 16 h.

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

Results MFGE8 is suppressed in experimental sepsis To investigate whether MFGE8 levels are affected in critical disease, we used an experimental sepsis model in rats. At 20 h after CLP, which produces peritonitis and sepsis in these animals, blood MFGE8 levels decreased by 45% ( p ⫽ 0.031), indicating the systemic scale of MFGE8 depletion under septic conditions (Fig. 1A). Using quantitative PCR, we found that under normal conditions MFGE8 is produced in various tissues with the highest mRNA expression levels in the spleen, followed by 34 –50% of its expression in the lungs, thymus, and skin. Lower mRNA levels of ⬃13– 15% of spleen levels were found in the brain, heart, and liver (data not shown). Due to its high basal MFGE8 expression levels, spleen MFGE8 levels were analyzed after CLP. Indeed, MFGE8 mRNA levels 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

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

MFGE8 protein levels in the spleen 20 h after CLP ( p ⫽ 0.034) (Fig. 1C). MFGE8 Western blotting from isolated CD11b/c⫹ macrophages from septic spleens revealed that they greatly contributed to the decrease of MFGE8, showing a 51% suppression vs sham levels ( p ⫽ 0.032) (Fig. 1D). IDC-derived exosomes improve apoptotic cell clearance and survival in experimental sepsis Bone marrow-generated rat IDCs secreted high amounts of exosomes (1.91 ⫾ 0.28 mg total protein/108 DCs), with a decrease of secretion by over 75% to 0.45 ⫾ 0.07 mg/108 DCs upon DC maturation. By sucrose gradient centrifugation we found that virtually all secreted MFGE8 was associated with exosomes found in the fraction with a density of 1.15 g/ml (refractive index ⫽ 1.375) at which exosomes equilibrate (1) (Fig. 2). IDCs secreted exosomes that contained abundant MFGE8, but virtually no costimulatory protein B7-2, a marker for mature DCs (Fig. 3A) (21). Mature DCs, marked by the morphological change with the development of visible dendrites, secreted exosomes with high levels of B7-2 but no MFGE8 (Fig. 3A). In septic rats, the systemic decrease in MFGE8 was associated with the accumulation and impaired clearance of apoptotic cells. In untreated animals, apoptotic thymocytes accumulated over time from 4% to 5% at 0 and 5 h after CLP to up to 14% within 20 h after CLP ( p ⬍ 0.001, data not shown). Phagocytosis of apoptotic cells by CD11b/c⫹ splenic macrophages was significantly impaired with the average phagocytotic percentage decreasing from 81% to 67% after CLP ( p ⫽ 0.021) (Fig. 2B), whereas at the same time the total amount of apoptotic cells increased in the spleen from 6.6% to 9.8% ( p ⫽ 0.003, data not shown) and in the thymus from 6.2% to 12.5% ( p ⬍ 0.001) (Fig. 3C). However, IDC-derived exosomes (2 ⫻ 1.5 mg/kg) increased the clearance of apoptotic cells in sepsis and completely restored the CLP-induced suppression of phagocytic capability (iExo, Fig. 3B). Concurrently, CLP-associated accumulation of apoptotic cells was reduced by over 28% ( p ⫽ 0.027) (Fig. 3C). Mature DCderived exosomes showed no significant influence on the ability of

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IMMATURE EXOSOMES PREVENT LETHALITY IN SEPSIS splenic macrophages to clear apoptotic cells (mExo, p ⫽ 0.492 vs Vehicle, Fig. 3B) or to alter the accumulation of apoptotic cells ( p ⫽ 0.436) (Fig. 3C). This experimental sepsis model caused 62.5% lethality within 1– 4 days when the necrotic cecum was removed 20 h after CLP (Fig. 3D). Only 2 of 16 rats receiving IDC-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-day observation period ( p ⫽ 0.007 vs vehicle control). Neither treatment with 10% of the effective dose of IDC exosomes, nor treatment with mature DC-derived exosomes affected the outcome in sepsis (Fig. 3D). As demonstrated, mature DC-derived exosomes did not contain MFGE8 (Fig. 3A), and the beneficial effect of IDC exosomes was likely to be mediated by MFGE8. MFGE8 reconstitutes the clearance of apoptotic cells in septic rats The reduced detection of apoptotic cells in septic animals cannot be explained by a direct anti-apoptotic effect of IDC-derived exosomes. In in vitro studies, TNF-␣-induced apoptosis of lymphocytes could not be blocked by the pretreatment with exosomes in the absence of macrophages (data not shown). Similar to IDC exosomes, treatment of septic rats with 2 ⫻ 30 ␮g/kg rmMFGE8 at 5 and 10 h after CLP, resulted in the reconstitution of apoptotic cell clearance in septic rats ( p ⫽ 0.019) (Fig. 4A). Previous publications have shown that MFGE8 is crucial for the engulfment of apoptotic cells, an important step in the removal of apoptotic cells adhering to the surface of macrophages (4). To investigate whether MFGE8 leads to the engulfment of apoptotic cells under septic conditions, we used the approach of staining apoptotic cells with a pH-sensitive dye (phrodo SE) that become detectable only after they are engulfed by a macrophage. Using this method, we found that the rmMFGE8 protein was able to reconstitute engulfment of apoptotic cells in septic rats to levels observed in sham operated animals ( p ⫽ 0.009) (Fig. 4, B–E). IDC-derived exosomes suppress the proinflammatory response in experimental sepsis Sepsis is associated with a systemic inflammatory response characterized by increases in early (TNF-␣) and late proinflammatory cytokines (HMGB1). We investigated whether the treatment with IDC-derived exosomes was able to influence the inflammatory response in experimental sepsis. IDC-derived exosomes suppressed the 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 exosomes failed to suppress TNF-␣ release from LPS-stimulated macrophages in vitro in the absence of apoptotic cells (data not shown), suggesting an indirect immunosuppressive effect. Similar results could be found in the levels of the late cytokine HMGB1. In vehicle-treated septic animals, blood HMGB1 levels increased by

FIGURE 3. IDC-derived exosomes improve apoptotic cell clearance and survival in experimental sepsis. A, Exosomes secreted from IDCs contain MFGE8. Exosomes were collected from the supernatant of primary rat bone marrow DC cultures (day 6 to 17 in culture) by stepwise ultracentrifugation. Equal amounts of exosome protein were subjected to Western blotting for MFGE8, the DC maturation marker B7-2, and the lysosome associated membrane protein 3 (LAMP3) as an endogenous control. B, Improved phagocytosis of apoptotic cells by splenic macrophages. Rats underwent 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/kg mature DC-derived exosomes (mExo) 5 and 10 h after CLP. Macrophages (CD11b/c⫹) were isolated from spleens 20 h after CLP and analyzed for phagocytosis of CD90⫹ apoptotic (⬎99% annexin V-positive) thymocytes via FACS. Representative FACS plots are shown. ⴱ, p ⫽ 0.025 vs sham;

#, p ⫽ 0.012 vs vehicle; †, p ⫽ 0.001 vs iExo, ANOVA and Tukey’s test for n ⫽ 6 animals. C, Reduced accumulation of apoptotic cells. Thymocytes were isolated from rats 20 h after CLP or sham operation and analyzed by FACS for apoptosis using Annexin V and propidium iodide staining (representative plots). ⴱ, p ⬍ 0.001 vehicle vs Sham; ⴱ, p ⬍ 0.001 mExo vs sham; ⴱ, 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 ⫽ 6 animals. D, Protection from sepsis-induced lethality by IDC-derived exosomes. Rats underwent CLP and received either PBS (vehicle), exosomes derived from IDCs or mature DCs, 5 and 10 h after CLP i.v. At 20 h after CLP the necrotic cecum was removed and rats observed for the following 10 days. ⴱ, p ⫽ 0.007 vs vehicle; p ⫽ 0.005 vs mature DC exosomes by Kaplan-Meyer log-rank test for n ⫽ 16 animals.


FIGURE 4. MFGE8 reconstitutes phagocytosis of apoptotic cells in septic rats. A, Rats underwent CLP to induce experimental sepsis and were treated with 2 ⫻ 15 ␮g/kg rmMFGE8 i.v. at 5 and 10 h after CLP. Thymocyte apoptosis was assessed 20 h after CLP by Annexin V/propidium iodide staining and FACS analysis. ⴱ, p ⫽ 0.034 vs sham; #, p ⫽ 0.019 vs vehicle, by ANOVA and Tukey’s test for n ⫽ 5 animals. B–E, Splenic macrophages from septic rats were isolated 20 h after CLP and challenged with four times apoptotic thymocytes labeled with pHrodo SE, a fluorescent dye that is detectable only after engulfment of the cells by macrophages. After 60 min, cells were labeled with FITC-anti-CD11b/c⫹ and analyzed by FACS analysis. The pHrodo histogram of CD11b/c⫹-gated cells (B–D). The macrophage pre-phagocytosis control is represented in the overlay in B. E, Summary of experiments showing the percentage of phagocytosing macrophages. ⴱ, p ⫽ 0.023 vs sham, #, p ⫽ 0.009 vs vehicle, by ANOVA and Tukey’s test for n ⫽ 5 animals.

51% compared with sham-operated animals ( p ⫽ 0.002). This increase was completely suppressed by the treatment with IDC exosomes ( p ⬍ 0.001), but not by mature DC exosomes ( p ⫽ 0.294) (Fig. 5B). Although phagocytosis of apoptotic cells is known to suppresses TNF-␣ response of macrophages, its effects on HMGB1 release remains unclear. Apoptotic cells have been previously shown to increase HMGB1 release in a coculture system with macrophages (22). We therefore investigated the effect of MFGE8-mediated clearance of apoptotic cells on HMGB1-release under inflammatory conditions in vitro. Opsonization of apoptotic thymocytes with rmMFGE8 completely prevented the HMGB1 release 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 of HMGB1 (up to 3.5 times at an apoptotic cell to macrophage ratio of 5:1, p ⫽ 0.006) (Fig. 5C), depending on the amount of apoptotic cells present. HMGB1 has been shown to be actively released from inflammatory macrophages as well as passively released from necrotic cells (23). Analysis of the release of HMGB1 from sepa-

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FIGURE 5. IDC-derived exosomes suppress the proinflammatory response in sepsis. A, TNF-␣ levels in septic rats 20 h after CLP. Rats underwent either sham operation or CLP with either PBS (vehicle), IDCderived exosome (iExo), or mature DC-derived exosome (mExo) treatment. ⴱ, p ⬍ 0.001 vs sham; #, p ⫽ 0.041 vs vehicle, by ANOVA and Student-Newman-Keuls test for n ⫽ 6 animals. B, HMGB1 is suppressed in septic rats receiving IDC-derived exosomes. At 20 h after CLP, HMGB1 was assessed by Western blot. ⴱ, p ⫽ 0.002 vs sham; #, p ⬍ 0.001 vs vehicle; †, p ⬍ 0.001 vs iExo, by ANOVA and Tukey’s test for n ⫽ 6 animals. Representative bands of Western blots are shown atop the results. C, The rmMFGE8-mediated promotion of apoptotic cell clearance suppresses late cytokine HMGB1 release in vitro. After preincubation with apoptotic cells with or without rmMFGE8 for 90 min, primary rat peritoneal macrophages were stimulated with 100 ng/ml LPS for 16 h, supernatants were collected and assayed for HMGB1 by Western blot. ⴱ, p ⫽ 0.002 vs control, by ANOVA and Tukey’s test for n ⫽ 3 animals. Representative band of Western blots are shown atop graph.

rately cultured macrophages and unengulfed apoptotic cells after phagocytosis revealed that macrophages challenged with apoptotic cells released similar levels of HMGB1 as necrotic cell-challenged macrophages, albeit to a significantly lesser degree than unengulfed late apoptotic cells and necrotic cells released themselves. The rmMFGE8 treatment suppressed the release of HMGB1 from both macrophages and late apoptotic lymphocytes (Fig. 6). This suggests that MFGE8-mediated clearance of apoptotic cells directly attenuates the release of HMGB1 in sepsis.

FIGURE 6. Postphagocytic release of HMGB1 from macrophages and late apoptotic cells is suppressed by rmMFGE8 treatment. Rat peritoneal macrophages were incubated with live, necrotic (necr.), or apoptotic lymphocytes (induced by 10 ␮M dexomethasone for 16 h in thymus T cells) for 90 min. 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 bluepossitive, total of 32 h after dexomethasone treatment) and released an equal amount of HMGB1 into the medium as the same number of necrotic cells (induced by four freeze-thaw cycles). The rmMFGE8 treatment suppressed HMGB1 release from both lymphocytes and macrophages. Incubation with live lymphocytes served as a control.

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FIGURE 7. MFGE8 deficiency is associated with poor clearance of apoptotic cells in sepsis. A, Phagocytosis of apoptotic cells is severely impaired in MFGE8-deficient mice. C57BL/6J WT or MFGE8⫺/⫺ mice underwent CLP, and 20 h later splenic macrophages were isolated and challenged with apoptotic thymocytes. The percentage of phagocytic macrophages was assessed by TUNEL staining and microscopy. ⴱ, p ⬍ 0.001 vs 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 more apoptotic cells in sepsis. MFGE8⫺/⫺ mice and their C57BL/6J WT control mice underwent CLP, and 20 h later thymocytes were stained with Annexin V and analyzed by FACS. ⴱ, p ⬍ 0.001 vs respective sham; #, p ⬍ 0.001 vs WT, by two-way ANOVA and Tukey’s test for n ⫽ 6 animals.

MFGE8-deficiency is associated with poor apoptotic cell clearance in sepsis To address whether impaired phagocytosis due to MFGE8 deficiency affects apoptotic cell accumulation in sepsis, we compared MFGE8⫺/⫺ mice and their C57BL/6J WT controls 20 h after CLP. As expected, spleen macrophages from MFGE8⫺/⫺ mice showed a dramatically decreased ability to phagocytose apoptotic cells under normal conditions (22% of WT sham, p ⬍ 0.001) (Fig. 7A). To an even stronger degree than in rats, septic WT mice showed a 74% suppression of phagocytosis of apoptotic cells 20 h after CLP, whereas CLP had no further impact on phagocytosis in MFGE8⫺/⫺ mice (Fig. 7A). Mice deficient in MFGE8 also accumulated higher amounts of apoptotic cells (19%) compared with WT mice at the same time point (12%, p ⬍ 0.001) (Fig. 7B). This indicates that the clearance of apoptotic cells in sepsis is positively regulated by MFGE8. MFGE8 protects from sepsis-associated mortality Finally, we were interested in whether MFGE8 influences survival in experimental sepsis. Indeed, MFGE8-deficient mice were more susceptible to sepsis-mediated mortality. Although 50% of WT mice died within 10 days of CLP, 82% of MFGE8⫺/⫺ mice died in the same period ( p ⫽ 0.045) (Fig. 8A). In contrast, a continuous infusion of rmMFGE8 over 20 h protected 83% of septic rats from dying from CLP-induced sepsis compared with 50% of rats that died without treatment ( p ⫽ 0.042) (Fig. 8B). Thus, administration of rmMFGE8 provided similar results as the treatment with IDCderived exosomes (Fig. 3D).

Discussion IDCs constitutively secrete exosomes that contain MFGE8. In mature DCs, exosome and MFGE8 production and release are reduced. In our studies, we have presented that MFGE8 is systemically down-regulated in sepsis, which leads to a widespread impairment of apoptotic cell clearance. The associated proinflammatory response in sepsis can be prevented by the administration of exogenous exosomes from IDCs. These exosomes improve ap-

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 observed for the following 10 days. ⴱ, p ⫽ 0.045 vs WT control, by KaplanMeyer log-rank test for n ⫽ 10 –11 animals. B, Treatment with rmMFGE8 improves survival in septic rats. After CLP, rats were treated with either PBS (vehicle) or 20 ␮g/kg rmMFGE8, as a continuous perfusion over 20 h via osmotic minipump and then observed for 10 days. ⴱ, p ⫽ 0.042 vs vehicle, by Kaplan-Meyer log-rank test for n ⫽ 18 animals.

optotic cell clearance, prevent the excessive release of proinflammatory cytokines and protect septic animals from dying. Although exosomes from mature DCs do not contain MFGE8 and fail to be protective, the protein MFGE8 itself has been shown to be an indispensable factor for the prevention of accumulating apoptotic cells and mortality in sepsis. Sepsis is marked by a systemic inflammatory response, mediated by innate immune cells. An increase in proinflammatory cytokines is normally beneficial to fight microbes in minor infections (24). In sepsis, however, this cytokine response is extensive and prolonged (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 high mortality rate. Being equally the source and the target of these mediators, APCs become activated and mature, thereby shutting down the endocytotic machinery in favor of an immunostimulatory response (26). During this maturation process, the secretion of exosomes and the production of MFGE8 are reduced (27). A number of mediators are responsible for the modulation of MFGE8 production in sepsis. Endotoxin can suppress MFGE8 production in vitro (28), and GM-CSF plus IL-4-mediated maturation of DC

The Journal of Immunology in vitro in the absence of bacteria or endotoxin indicates that cytokines may equally play a role. In this regard, GM-CSF has been shown to induce MFGE8 expression in vivo (29). GM-CSF is required for the expression of MFGE8 in APCs, and that MFGE8mediated uptake of apoptotic cells is a key determinant of GMCSF-triggered tolerance and immunity (29). This research is interesting because GM-CSF has been investigated as a promising treatment option for septic patients due to its immune-modulating function (30 –33). Whether other cytokines such as TNF-␣ or IFN-␥, also influence MFGE8 expression is unclear and needs further investigation. We have shown in this study that in sepsis CD11b/c expressing macrophages and DCs contribute to the reduction of MFGE8 production in the spleen, which is mirrored by a decrease of this protein in the circulation. As we have shown further, a deficiency in MFGE8 is detrimental in sepsis. Mice lacking MFGE8 accumulate two to three times as many apoptotic cells above basal level and have a 60% higher mortality rate than WT mice. Similarly, the administration of rmMFGE8 to septic rats protected the majority from sepsis-mediated lethality. The protective effect of MFGE8 is evident in this acute inflammatory model of sepsis and provides further evidence that it is a crucial part of the protective effect of exosomes derived from IDCs in septic animals. Exosomes have been previously shown to transfer cell-mediated immunity from one cell to another (26). Mature Ag-pulsed DCs secrete exosomes that act as cross-presenting carriers of MHC-Ag complexes and hence confer functional immunity to the recipient cell (26). The absence of Ag-presenting and costimulatory molecules on exosomes from IDCs suggests that these exosomes hold a minor role in directly modulating immune responses. However, IDC-derived exosomes are not at all functionally inert. As we have shown, the administration of exosomes from IDCs, but not from mature DCs conferred protection in sepsis, highlighting the important role of MFGE8 in these vesicles. Unfortunately, technical reasons precluded conclusive experiments using exosomes from WT and MFGE8 knockout exosomes. MFGE8 contains two important regions to function as an opsonin for apoptotic cells; two EGF-like domains contain an RGD motif necessary for the binding of ␣v␤3- or ␣v␤5 integrins, and two coagulation factor V/VIII like domains that bind to phosphatidylserine exposed on the surface of apoptotic cells (27). Binding of MFGE8 to phosphatidylserine on apoptotic cells opsonizes them for a complete engulfment by macrophages via ␣v␤3 or ␣v␤5 integrins. MFGE8 has been shown to be important for the removal of apoptotic lymphocytes in the spleen and the prevention of a systemic lupus erythematosus-like disease in mice (4). In our septic model, we found a similar phenomenon in an acute inflammatory environment. The beneficial effect of IDC-derived exosomes is mediated by the promotion of apoptotic cell clearance. Apoptosis is often found in sepsis, with lymphoid CD4 T and B cells and DCs being most commonly affected (34 –37). Particularly apoptosis of DCs may contribute to the depletion of MFGE8 in sepsis as these are one source of this protein. Overall, the occurrence of apoptosis has been associated with poor outcome in sepsis. Targeted inhibition of the proapoptotic Fas signaling or overexpression of antiapoptotic proteins, such as BH4, Bcl-2, or Bcl-xL, have been shown to prevent apoptosis and protect from associated lethality in sepsis (35, 38). Historically, apoptosis has been seen as an orderly process of cell suicide that, unlike necrosis, does not elicit inflammation (39). Recently it has become clear, however, that apoptotic cells eventually undergo secondary necrosis and stimulate an inflammatory response if they are not removed by phagocytosis (19, 20). By using the pHrodo labeling system of apoptotic cells we

5989 have clearly demonstrated that rmMFGE8 promotes the engulfment of apoptotic cells also under septic conditions, which is at least in part responsible for the decrease in apoptotic cell number. Hence, the sepsis-associated decrease of MFGE8 contributes to the accumulation of apoptotic cells, resulting in a surge in proinflammatory cytokines, such as TNF-␣ and HMGB1, which by itself promotes the progression and deterioration in sepsis (11, 40). We have previously shown that the pretreatment with bone marrow-derived DC exosomes was beneficial in septic animals and possibly dependent on the presence of MFGE8 (41). The current study shows that the clearance of apoptotic cells in septic animals is impaired in the absence of MFGE8 and that this is detrimental in the acute inflammatory disease model. Furthermore, we now show that even treatment with IDC exosomes at a later time point is beneficial (i.e., when apoptotic cells start to accumulate in sepsis, long after the initiation of inflammatory responses). The present report also demonstrates how MFGE8-mediated internalization of apoptotic cells prevents the release of proinflammatory mediators, which leads to a suppression of the septic systemic inflammatory response. The beneficial effect of IDC-derived exosomes can be particularly attributed to the immune suppressive effect of phagocytosis of apoptotic cells (39). Exosomes from IDCs were neither able to suppress TNF-␣ release from endotoxin-stimulated macrophages, nor to prevent TNF-␣-induced apoptosis of lymphocytes in vitro (our unpublished observations). Thus the reduction in apoptosis in vivo is most likely mediated via enhanced clearance of apoptotic cells. In our experimental sepsis model, early increases in TNF-␣ and IL-6 (1– 4 h after CLP) are followed by a significant release of HMGB1 in the late phase of sepsis (16 –24 h after CLP) (11). These cytokines play a central role in the morbidity and mortality in experimental sepsis as well as in septic patients (42). Studies using inhibitors of these cytokines demonstrated increased survival of septic mice treated with TNF-␣ or HMGB1 blocking Abs (8, 22). We have shown in this study that IDC-derived exosomes suppressed both TNF-␣ and HMGB1 in experimental sepsis, which was associated with a dramatically improved survival. Exosomes secreted from IDCs are not merely a byproduct of excessive endocytosis but they have, as we have shown, the ability to suppress a once-established systemic proinflammatory response. The opsonizing protein MFGE8 plays a key role in this pro-phagocytic and secondary immunosuppressive effect. This distinguishes them from exosomes secreted from mature, Ag-pulsed DCs that confer cellular immunity by cross priming. This novel finding should open a new option of sepsis therapy in which the clearance of apoptotic cells is targeted. The restoration of this basic immunological function and the induction of the reparative phase may ultimately contribute to the attenuation of the life-threatening systemic inflammatory response in sepsis. It is likely that a combination of apoptosis prevention and the promotion of apoptotic cell clearance can be used in conjunction to the current treatment regime to the benefit of critically ill patients in the future.

Acknowledgments Max Brenner, Herb Borrero, and Thomas McCloskey were helpful with inputs and in performing FACS analysis for the phagocytosis assay and detection of apoptotic cells. Enesa Paric and James Mason assisted in the isolation and gradient centrifugation of exosomes. Margot Puerta, Wei Li, and Tianpen Cui were cooperative in the methodology of HMGB1 detection. Maowen Hu and Yingjie Cui were instrumental in establishing the real-time PCR for MFGE8 and detecting MFGE8 in the blood, respectively. Kavin Shah was instrumental in the rat sepsis model. Shigekazu Nagata (Osaka University, Osaka Japan) provided us with MFGE8⫺/⫺ mice for this study, for which we are thankful.

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Disclosures

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The authors have no financial conflict of interest.

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