Fat Globule-epidermal Growth Factor-factor Research Articles

Tag-free glycosylated rhMFG-E8 as a therapy for acute kidney injury.

Acute kidney injury (AKI) can result from renal ischemia and reperfusion (I/R) and often occurs during surgical procedures in cardiac, liver, kidney transplantation and trauma-hemorrhage. Milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. Since MFG-E8 promotes clearance of apoptotic cells, we have explored its therapeutic potential in various organ injury conditions. To develop human MFG-E8 as a potential therapy, we have generated a human cell-expressed, and thus glycosylated, tag-free recombinant human (rh) MFG-E8 and tested its safety and biological activity in vitro. We hypothesize that the tag-free glycosylated rhMFG-E8 is protective in I/R-induced AKI and it can be developed as an effective therapy for AKI. To assess the pharmacokinetic properties of the tag-free rhMFG-E8, Sprague Dawley rats were either untreated or treated with a bolus dose of the tag-free rhMFG-E8, blood collected at various time points and the recovery of human MFG-E8 in the blood were measured by ELISA. Adult male C57BL6 mice underwent bilateral renal ischemia for 30 min and immediately upon reperfusion, mice were treated intraperitoneally with either normal saline (vehicle) or 20 μg/kg human cell expressed, glycosylated tag-free rhMFG-E8. At either 24 h or 48 h after I/R, blood and kidneys were harvested for further analysis. In separate cohorts of mice after I/R and treatment, mice were observed for 10 days, and survival recorded. AKI rats treated with the tag-free rhMFG-E8 had similar half-life as those in the treated control rats. At 48 h after I/R-induced AKI, renal function markers, BUN and creatinine were increased and treatment with the tag-free rhMFG-E8 significantly decreased these markers. At both 24 h and 48 h after AKI, inflammatory cytokines, TNF-α, IL-6 and IL-1β were increased and treatment decreased these levels. The kidney mRNA expressions of these cytokines were also increased at 24 h after AKI and treatment significantly decreased those mRNA expressions. Histologically, at 48 h after AKI, tubular damage, and the number of TUNEL staining cells were increased and treatment markedly decreased these measurements. Administration of tag-free rhMFG-E8 at the time of reperfusion improved survival in a 10-day survival study. Our new human cell-expressed tag-free rhMFG-E8 is protective in I/R-induced AKI and it may have the potential to be further developed as a safe and effective therapy for AKI.

Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.

Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart.

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Recombinant human milk fat globule-EGF factor VIII (rhMFG-E8) as a therapy for sepsis after acute exposure to alcohol

BackgroundAlcohol intake predisposes to infections and sepsis. Alcohol and sepsis inhibit the expression of milk fat globule epidermal growth factor-factor VIII (MFG-E8), a glycoprotein essential for optimal efferocytosis, resulting in the release of proinflammatory molecules and increased sepsis severity. We previously reported that recombinant mouse (rm) MFG-E8 attenuates sepsis-induced organ injury in rats with acute alcohol intoxication. In order to develop a therapy that can be safely used in humans, we have produced recombinant human (rh) MFG-E8 and evaluated its efficacy to ameliorate sepsis after acute exposure to alcohol.MethodsWe induced acute alcohol intoxication with a bolus injection of alcohol (1.75 g/kg BW) followed by an intravenous infusion of 300 mg/kg/h alcohol for 10 h. Sepsis was then induced by cecal ligation and puncture (CLP). At -10, 0, and 10 h relative to CLP, rats received MFG-E8 or vehicle (albumin) intravenously. Animals were euthanized at 20 h after CLP for blood and tissue collection. Additional groups of animals were used for a survival study.ResultsCompared to vehicle, rhMFG-E8 treatment ameliorated blood levels of proinflammatory cytokines (% improvement: TNF-α 49.8%, IL-6 34.7%) and endotoxin (61.7%), as well as of transaminases (AST 36.2%, ALT 40.1%) and lactate (18.4%). Rats treated with rhMFG-E8 also had a significant histological attenuation of the acute lung injury, as well as a reduction in the number of apoptotic cells in the thymus (43.4%) and cleaved caspase 3 (38.7%) in the spleen. In addition, rhMFG-E8 improved the 10-day sepsis survival rate from 45 to 80%ConclusionrhMFG-E8 significantly ameliorated sepsis in rats with acute alcohol exposure, demonstrating rhMFG-E8’s potential to be developed as an effective therapy for sepsis in alcohol abusers.

Treatment with milk fat globule epidermal growth factor-factor 8 (MFG-E8) reduces inflammation and lung injury in neonatal sepsis

Sepsis remains one of the leading causes of infant death worldwide. It is characterized by uncontrolled inflammatory responses due to proven bacterial infection. Despite improvement in supportive care and the availability of effective antibiotics, no specific therapy targeting the dysregulated inflammatory response is available for neonatal sepsis. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory glycoprotein abundantly present in human milk. MFG-E8 suppresses the systemic inflammatory responses in adult murine injury models by improving the clearance of dying cells. We hypothesized that exogenous administration of recombinant mouse MFG-E8 could inhibit the exaggerated inflammatory response and lung injury in a murine model of neonatal sepsis. Neonatal sepsis was induced in 5- to 7-day-old male and female C57BL6 mice using an intraperitoneal injection of cecal slurry. At 1hour after sepsis induction, a single dose of 40μg/kg recombinant mouse MFG-E8 or vehicle was administered via retro-orbital injection. All neonates were returned to their mothers as a group. At 10hours after cecal slurry injection, pups were killed and blood and lung tissues were collected. Control mice underwent a similar procedure with the exception of cecal slurry intraperitoneal injection. Serum lactate dehydrogenase, IL-1β, and IL-6 were significantly increased 10hours after cecal slurry injection. Treatment with recombinant mouse MFG-E8 decreased these levels by 30%, 56%, and 37%, respectively. Lung morphology was significantly compromised in the vehicle group after cecal slurry injection, whereas the recombinant mouse MFG-E8-treated groups demonstrated a 48% improvement in the lung injury score. Lung IL-6 and MIP-2 protein levels were significantly reduced with recombinant mouse MFG-E8 treatment. Lung neutrophil infiltration as observed by Gr-1 staining and, TUNEL-positive cells were also significantly reduced with recombinant mouse MFG-E8 treatment. Treatment with recombinant mouse MFG-E8 attenuated inflammation and lung injury in murine neonatal sepsis. Thus, MFG-E8 could be developed as a possible therapy for neonatal sepsis.

Correction of MFG-E8 Resolves Inflammation and Promotes Cutaneous Wound Healing in Diabetes

Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8(-/-) mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8(-/-) bone marrow to MFG-E8(+/+) mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8(-/-) mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8-directed therapeutics in diabetic wound care.

Milk fat globule epidermal growth factor-factor 8-derived peptide attenuates organ injury and improves survival in sepsis.

IntroductionSepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and ultimately death. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation results in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis.MethodsThe differentiated human neutrophil-like HL-60 cells (dHL60) were incubated with a series of peptides flanking the RGD motif of human MFG-E8 for a cell adhesion assay to fibronectin or human pulmonary artery endothelial cells (PAECs). For the induction of sepsis, male C57BL/6 mice (20–25 g) were subjected to cecal ligation and puncture (CLP). Peptide MSP68 (1 mg/kg body weight) or normal saline (vehicle) was injected intravenously at 2 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements.ResultsAfter screening, peptide MSP68 (VRGDV) had the highest inhibition of dHL-60 cell adhesion to fibronectin by 55.8 % and to PAEC by 67.7 %. MSP68 treatment significantly decreased plasma levels of organ injury marker AST by 37.1 % and the proinflammatory cytokines IL-6 and TNF-α by 61.9 % and 22.1 %, respectively after CLP. MSP68 improved the integrity of microscopic architectures, decreased IL-6 levels in the lungs by 85.1 %, and reduced apoptosis. MSP68 treatment also significantly reduced the total number of neutrophil infiltration by 61.9 % and 48.3 % as well as MPO activity by 40.8 % and 47.3 % in the lungs and liver, respectively, after CLP. Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57 % with MSP68 treatment. Finally, the 10-day survival rate was increased from 26 % in the vehicle group to 58 % in the MSP68-treated group.ConclusionsMSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis.

A protective effect of milk fat globule EGF factor VIII (MFG-E8) on the spontaneous fusion of milk fat globules in breast milk.

Lipid droplets synthesized in mammary epithelial cells are secreted into breast milk by the budding-off mechanism. The milk lipids, termed mik fat globules (MFGs), are surrounded with the cell plasma membrane and contain various membrane proteins, including milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8), on their surface. We report here that the MFGs in the milk of MFG-E8-deficient mice fused each other and turned into abnormally large size of lipid droplets within ∼48 h after being secreted into mammary alveolar lumen in situ or being incubated at 37°C in vitro. This biophysical degeneration of MFGs in the MFG-E8-deficient milk was efficiently rescued in vitro by adding the milk serum of wild-type mice, isolated MFG-E8 or annexin V. Moreover, addition of ethylenediaminetetraacetic acid (30 mM) also protected the MFG fusion remarkably in vitro. In addition, bovine MFGs also fused each other when isolated from milk serum, and the fusion was inhibited by adding isolated MFG-E8 or mouse milk serum, but not the milk serum of MFG-E8-deficient mice. MFG-E8 in breast milk may mask the phosphatidylserine exposed on the surface of MFGs with time after secretion and thereby suppress the membrane fusion among MFGs resulting in the enlargement of MFGs in the breast milk.

Milk Fat Globule-Epidermal Growth Factor-Factor 8 Attenuates Neutrophil Infiltration in Acute Lung Injury via Modulation of CXCR2

Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factor-factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8(-/-) mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8(-/-) mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-α, MIP-2, and myeloperoxidase. An increased number of apoptotic cells was trapped into the lungs of Mfge8(-/-) mice compared with WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2-mediated chemotaxis revealed higher migration of neutrophils of Mfge8(-/-) mice than those of WT mice via increased surface exposures to CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-α(v) integrin Abs. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.

Milk fat globule epidermal growth factor-factor 8 mitigates inflammation and tissue injury after hemorrhagic shock in experimental animals

Insufficient clearance of apoptotic cells leads to increased inflammation and exaggerated organ injury. The opsonizing protein, milk fat globule epidermal growth factor-factor 8 (MFG-E8), upregulates apoptotic cell clearance. The purpose of this study was to determine the degree of apoptotic cell clearance, and whether inflammation, organ injury, and survival are improved after treatment with recombinant human MFG-E8 (rhMFG-E8) after hemorrhagic shock. Male mice underwent a pressure-controlled (25 mm Hg ± 5 mm Hg) model of hemorrhagic shock for 90 minutes. They were resuscitated with normal saline with or without recombinant human MFG-E8 (rhMFG-E8) over 30 minutes. At 3.5-hour postresuscitation, blood and tissue were collected. MFG-E8 levels in the plasma, lungs, and spleen were measured. Apoptotic cell clearance was measured by cleaved caspase-3 levels and TUNEL staining. Neutrophil infiltration was assessed using myeloperoxidase activity in the lungs and spleen. Plasma and tissue levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were measured by ELISA. Finally, a seven-day survival study was also conducted. MFG-E8 levels in the plasma, lungs, and spleen significantly decreased by 33%, 44%, and 55%, respectively, at 3.5 hour after hemorrhage and resuscitation with rhMFG-E8. Treatment with rhMFG-E8 significantly improved apoptosis, by reducing TUNEL+ cells after treatment and restoring cleaved caspase-3 expression back to baseline. Neutrophil infiltration was blunted by 29% and 41% in the lungs and spleen, respectively. Cytokine expression was also reduced significantly, by 64% to 73% in plasma, 24% to 58% in the lungs, and 49% to 76% in the spleen. Finally, animals demonstrated a superior survival rate over 7 days after treatment with rhMFG-E8. The administration of rhMFG-E8 is a potent treatment in animals after hemorrhagic shock.

Milk Fat Globule EGF Factor 8 Attenuates Sepsis-Induced Apoptosis and Organ Injury in Alcohol-Intoxicated Rats

Despite advances in our understanding of excessive alcohol-intake-related tissue injury and modernization of the management of septic patients, high morbidity and mortality caused by infectious diseases in alcohol abusers remain a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The purpose of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis. Acute alcohol intoxication was induced in male adult rats by a bolus injection of intravenous alcohol at 1.75 g/kg BW, followed by an intravenous infusion of 300 mg/kg BW/h of alcohol for 10 hours. Sepsis was induced at the end of 10-hour alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 or vehicle (normal saline) was administered intravenously 3 times (i.e., at the beginning of alcohol injection, the beginning of CLP, and 10 hours post-CLP) at a dose of 20 microg/kg BW each. Blood and tissue samples were collected 20 hours after CLP in alcoholic animals for various measurements. Acute alcohol exposure per se did not affect the production of MFG-E8; however, it primed the animal and enhanced sepsis-induced MFG-E8 downregulation in the spleen. Administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in the spleen, lungs, and liver. In addition, administration of rmMFG-E8 after alcohol exposure and subsequent sepsis decreases circulating levels of TNF-alpha and interleukin-6 and attenuates organ injury. rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats.

Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor-factor VIII [corrected

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, 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 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 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.

Targeting Tumor Antigens to Secreted Membrane VesiclesIn vivoInduces Efficient Antitumor Immune Responses

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Fractalkine-Induced MFG-E8 Leads to Enhanced Apoptotic Cell Clearance by Macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.

DENDRITIC CELL-DERIVED EXOSOMES CONTAINING MILK FAT GLOBULE EPIDERMAL GROWTH FACTOR-FACTOR VIII ATTENUATE PROINFLAMMATORY RESPONSES IN SEPSIS

In sepsis, several cell types (e.g., lymphocytes) undergo apoptosis and have the potential to harm the host if not cleared by professional phagocytes. Apoptotic cells display "eat me" signals such as phosphatidylserine that can be readily recognized by phagocytes. For full engulfment of these cells, binding to integrin alpha(v)beta(3), mediated by the bridging protein, milk fat globule epidermal growth factor-factor VIII (MFG-E8), is necessary. We hypothesized that, in sepsis, phagocytosis of apoptotic cells is impaired due to decreased MFG-E8 expression and that adoptive transfer of exosomes containing MFG-E8 is beneficial. Sepsis was induced in rats by cecal ligation and puncture (CLP) and MFG-E8 expression assessed by Western blot 20 h later. Dendritic cells were generated from bone marrow cells, and secreted exosomes were collected and injected into CLP animals. Plasma cytokines (enzyme-linked immunosorbent assay) and thymocyte apoptosis (TC-Ao, annexin V) were assessed. The ability of peritoneal macrophages from septic animals to engulf apoptotic cells was determined in an ex vivo phagocytosis assay. A 10-day survival study was conducted. Cecal ligation and puncture reduced MFG-E8 protein levels in the spleen and liver by 48% and 70%, respectively, and increased TC-Ao by 1.6-fold. Injection of MFG-E8-containing exosomes, however, led to a 33% reduced detection of TC-Ao, without directly inhibiting apoptosis. In fact, peritoneal macrophages from exosome-treated rats displayed a 2.8-fold increased ability to phagocytose apoptotic thymocytes. Inhibition of MFG-E8 before injection of exosomes completely abrogated the enhanced phagocytosis. Treatment with bone marrow dendritic cell-derived exosomes also reduced plasma tumor necrosis factor alpha and interleukin (IL)-6 levels and improved survival from 44% to 81%. We conclude that, by providing the indispensable factor MFG-E8 for complete engulfment of apoptotic cells, these exosomes lead to an attenuation of the systemic inflammatory response and overall beneficial effect in sepsis.

FRACTALKINE INCREASES APOPTOTIC CELL CLEARANCE BY UPREGULATING MFG-E8 IN MACROPHAGES

Clearance of apoptotic cells is crucial in sepsis. We have previously shown that administration of immature dendritic cellderived exosomes containing milk fat globule epidermal growth factor-factor VIII (MFG-E8) to septic animals promoted phagocytosis of apoptotic cells and improved survival. MFG-E8 is an opsonin for apoptotic cells and acts as a bridging protein between phosphatidylserine on apoptotic cells and integrins on phagocytes. Fractalkine (Fr) is a CX3C-chemokine (both a chemoatractant and an adhesion molecule) that is upregulated by proinflammatory cytokines and has been proposed to play a role under inflammatory conditions. A recent study has shown that Fr increased MFG-E8 gene transcription in microglia. We hypothezised that Fr upregulates MFG-E8 in peritoneal macrophages and promotes apoptotic cell clearance without inducing inflammation. To study this, rat peritoneal macrophages (pMφ) were isolated and cultured. MFG-E8 expression was assessed by Western blot after incubation with either LPS or Fr (without LPS). The ability of macrophages to engulf apoptotic thymocytes (A0TC) was determined by TUNEL. Cytokine secretion was assessed by ELISA in cultured cell supernatant. Our results have shown that recombinant MFG-E8 (1 μg/ml) doubles phagocytosis of A0TC by pMφ. Further results are shown in the table below (PI = phagocytosis index): We found similar results in the murine RAW 264.7 cell line, indicating that fractalkine is an important prophagocytic factor. We conclude that fractalkine enhances clearance of apoptotic cells by induction of MFG-E8 in macrophages. Since it does not induce a proinflammatory response in vitro by itself, fractalkine may be safe for the use in animal models of sepsis (NIH R01 GM057468).Table