Milk fat globule-epidermal growth factor-factor 8 attenuates neutrophil infiltration in acute lung injury via modulation of CXCR2.
ABSTRACT: 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-?, 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-?(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.
Project description:Abnormal features of the systemic lupus erythematosus (SLE)-derived neutrophils, promoted aberrant immune response, have inspired new studies of the induction of autoimmunity and the development of organ damage in SLE. In this study, we explore the effect of milk fat globule-EGF factor 8 (MFG-E8) on the aberrant nitrification features in pristane-induced lupus. SLE patients and mice with pristane-induced lupus develop autoantibodies associated with MFG-E8 overproduction. However, the deletion of MFG-E8 leads to uncontrolled early pulmonary and peritoneal inflammation and tissue damage in mice with pristane-induced lupus. Consistent with these findings, MFG-E8-deficient mice that are exposed to pristane show enhanced neutrophil accumulation and increased neutrophil death, including apoptosis, necrosis and NETosis, as well as impaired phagocytosis of macrophages. The consequences are the expansion of diffuse pulmonary hemorrhage, increased anti-nuclear antibody, anti-dsDNA antibody and anti-neutrophil cytoplasmic antibody levels, and enhanced immune complexes deposition and neutrophil extracellular traps (NETs) formation in the lung and kidney tissues of MFG-E8-deficient mice exposed to pristane. In patients with SLE and mice with pristane-induced lupus, neutrophil accumulation is elevated, which depends on higher expression of the surface receptor CXCR2. After pretreatment with recombinant MFG-E8, the surface expression of CXCR2 on neutrophil is downregulated, and the MFG-E8 deletion increase CXCR2 expression by ~40%. These studies indicate that MFG-E8 reduces neutrophil migration and NETosis via downregulating surface CXCR2 expression in parallel with its role in the phagocytosis of apoptotic neutrophils, suggesting that MFG-E8 may serve as a therapeutic agent for attenuating the early inflammatory responses of SLE and protect patients from lupus-related damage.
Project description:Sustained activation of NLRP3 inflammasome and release of neutrophil extracellular traps (NETs) impair wound healing of diabetic foot ulcers (DFUs). Our previous study reported that milk fat globule epidermal growth factor VIII (MFG-E8) attenuates tissue damage in systemic lupus erythematosus. However, the functional effect of MFG-E8 on "NLRP3 inflammasome-NETs" inflammatory loop in wound healing of diabetes is not completely elucidated. In this study, neutrophils from DFU patients are susceptible to undergo NETosis, releasing more NETs. The circulating levels of NET components neutrophil elastase and proteinase 3 and inflammatory cytokines IL-1? and IL-18 were significantly elevated in DFU patients compared with healthy controls or diabetic patients, in spite of higher levels of MFG-E8 in DFU patients. In Mfge8 -/- diabetic mice, skin wound displayed exaggerated inflammatory response, including leukocyte infiltration, excessive activation of NLRP3 inflammasome (release of higher IL-1?, IL-18, and TNF-?), largely lodged NETs, resulting in poor angiogenesis and wound closure. When stimulated with high-dose glucose or IL-18, MFG-E8-deficient neutrophils release more NETs than WT neutrophils. After administration of recombinant MFG-E8, IL-18-primed NETosis of WT or Mfge8 -/- neutrophils was significantly inhibited. Furthermore, NET and mCRAMP (component of NETs, the murine equivalent of cathelicidin LL-37 in human)-mediated activation of NLRP3 inflammasome and production of IL-1?/IL-18 were significantly elevated in Mfge8 -/- macrophages compared with WT macrophages, which were also significantly dampened by the administration of rmMFG-E8. Therefore, our study demonstrated that as inhibitor of the "NLRP3 inflammasome-NETs" inflammatory loop, exogenous rMFG-E8 improves angiogenesis and accelerates wound healing, highlighting possible therapeutic potential for DFUs.
Project description:Apoptotic cells must be rapidly cleared, as defects in this process can lead to autoimmunity. Milk fat globule EGF factor 8 (MFG-E8) binds to apoptotic cells and facilitates their removal through interaction with phagocytes. Mice deficient in MFG-E8 develop lupus-like autoimmunity associated with accumulation of apoptotic cells in vivo. Here, we have shown that MFG-E8 controls phagocytic ingestion of cell fragments as well as their intracellular processing into MHC-antigen complexes. Older Mfge8-/- mice spontaneously developed dermatitis associated with CD8+ T cell infiltration and striking activation of effector memory CD8+ T cells. CD8+ T cell responses to both exogenous and endogenous apoptotic cell-associated antigens were enhanced in Mfge8-/- mice. MFG-E8 deficiency accelerated the onset of disease in a mouse model of autoimmune diabetes. Enhanced CD8+ T cell responses were attributed to increased cross-presentation by DCs along with increased detection of antigen-MHCI complexes. Intracellular trafficking analysis revealed that intact apoptotic cells ingested by wild-type DCs rapidly fused with lysosomes, whereas smaller fragments persisted in Mfge8-/- DC endosomal compartments for 24 hours. These observations suggest that MFG-E8 deficiency promotes immune responses to self antigens not only by delaying the clearance of dying cells but also by altering intracellular processing, leading to enhanced self-antigen presentation.
Project description: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-1?, IL-6, and TNF-?) 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.
Project description:The glycoprotein milk fat globule-epidermal growth factor factor 8 (MFG-E8) is expressed in several tissues and mediates diverse homeostatic functions. However, whether it plays a role in bone homeostasis has not been established. In this study, we show for the first time, to our knowledge, that osteoclasts express and are regulated by MFG-E8. Bone marrow-derived osteoclast precursors from MFG-E8-deficient (Mfge8(-/-)) mice underwent increased receptor activator of NF-?B ligand-induced osteoclastogenesis, leading to enhanced resorption pit formation compared with wild-type controls. Consistently, exogenously added MFG-E8 inhibited receptor activator of NF-?B ligand-induced osteoclastogenesis from mouse or human osteoclast precursors. Upon induction of experimental periodontitis, an oral inflammatory disease characterized by loss of bone support of the dentition, Mfge8(-/-) mice exhibited higher numbers of osteoclasts and more bone loss than did wild-type controls. Accordingly, local microinjection of anti-MFG-E8 mAb exacerbated periodontal bone loss in wild-type mice. Conversely, microinjection of MFG-E8 inhibited bone loss in experimental mouse periodontitis. In comparison with wild-type controls, Mfge8(-/-) mice also experienced >60% more naturally occurring chronic periodontal bone loss. In conclusion, MFG-E8 is a novel homeostatic regulator of osteoclasts that could be exploited therapeutically to treat periodontitis and perhaps other immunological disorders associated with inflammatory bone loss.
Project description:Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in inflammation. In sepsis, interleukin (IL)-17 acts as a proinflammatory cytokine to exaggerate systemic inflammation. We hypothesize that MFG-E8 downregulates IL-17 expression in sepsis.Sepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Recombinant mouse MFG-E8 (rmMFG-E8) at a dosage of 20 ?g/kg body weight or phosphate-buffered saline was concurrently injected. After 10 hours, blood and spleen samples were harvested for analysis. For in vitro studies, splenocytes isolated from healthy mice pretreated with rmMFG-E8 and splenocytes from MFG-E8 knockout (mfge8(-/-)) mice were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, followed by measurement of IL-17 expression with either quantitative PCR or enzyme-linked immunosorbent assay.At 10 hours after CLP, rmMFG-E8 inhibited the elevated levels of IL-17 protein in serum by 31%, compared with the vehicle. In the spleen, rmMFG-E8 reduced the upregulated IL-17 mRNA and protein levels by 81% and 51%, respectively. This correlated with a significant reduction in organ injury markers AST and ALT in sepsis after administration of rmMFG-E8. In vitro treatment of splenocytes isolated from healthy mice with rmMFG-E8 showed significant downregulation in PMA/ionomycin-induced IL-17 expression. In contrast, CD4 T-cells from mfge8(-/-) mice showed significant upregulation of IL-17 compared with wild-type mice. The phosphorylated level of signal transducer and activator of transcription 3 (STAT3) was downregulated in spleen tissue of septic mice treated with rmMFG-E8. Conversely, mfge8(-/-) mice showed increased phosphorylated STAT3 compared with wild-type mice after sepsis.Our findings demonstrate MFG-E8-mediated downregulation of IL-17 expression, implicating its potential as a novel therapeutic agent against sepsis.
Project description:Acute lung injury (ALI) remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability, but no small molecule CXCR2 inhibitor has been demonstrated to be effective in ALI or animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its effects in two models of ALI, induced by either lipopolysaccharide (LPS) inhalation or acid instillation.In two ALI models in mice, we measured vascular permeability by Evans blue and evaluated neutrophil recruitment into the lung vasculature, interstitium and airspace by flow cytometry.Pharmacological inhibition of CXCR2 by Reparixin reduced CXCL1-induced leukocyte arrest in the microcirculation of the cremaster muscle, but did not influence arrest in response to leukotriene B4 (LTB4) demonstrating specificity. Reparixin (15 microg g(-1)) reduced neutrophil recruitment in the lung by approximately 50% in a model of LPS-induced ALI. A higher dose did not provide additional reduction of neutrophil recruitment. This dose also reduced accumulation of neutrophils in the interstitial compartment and vascular permeability in LPS-induced ALI. Furthermore, both prophylactic and therapeutic application of Reparixin improved gas exchange, and reduced neutrophil recruitment and vascular permeability in a clinically relevant model of acid-induced ALI.Reparixin, a non-competitive allosteric CXCR2 inhibitor attenuates ALI by reducing neutrophil recruitment and vascular permeability.
Project description:A prolonged increase in proinflammatory cytokines is associated with osteoporotic and autoimmune bone loss and, conversely, anti-inflammatory pathways are associated with protection against bone loss. Milk fat globule-epidermal growth factor (MFG-E)-8 is a glycoprotein that is proresolving, regulates apoptotic cell clearance, and has been linked to autoimmune disease and skeletal homeostasis. The role of MFG-E8 in the young vs. adult skeleton was determined in mice deficient in MFG-E8 (KO). In vivo, trabecular bone was similar in MFG-E8KO and wild-type (WT) mice at 6 and 16 wk, whereas 22 wk adult MFG-E8KO mice displayed significantly reduced trabecular BV/TV. The number of osteoclasts per bone surface was increased in 22-wk MFG-E8 KO vs. WT mice, and recombinant murine MFG-E8 decreased the number and size of osteoclasts in vitro. Adult MFG-E8KO spleen weight:body weight was increased compared with WT, and flow cytometric analysis showed significantly increased myeloid-derived suppressor cells (CD11bhiGR-1+) and neutrophils (CD11bhiLy6G+) in MFG-E8KO bone marrow, suggesting an inflammatory phenotype. PTH-treated MFG-E8KO mice showed a greater anabolic response (+124% BV/TV) than observed in PTH-treated WT mice (+64% BV/TV). These data give insight into the role of MFG-E8 in the adult skeleton and suggest that anabolic PTH may be a valuable therapeutic approach for autoimmune-associated skeletal disease.-Michalski, M. N., Seydel, A. L., Siismets, E. M., Zweifler, L. E., Koh, A. J., Sinder, B. P., Aguirre, J. I., Atabai, K., Roca, H., McCauley, L. K. Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.
Project description:Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and enhances the engulfment of apoptotic cells by macrophages. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in the MFG-E8-deficient (MFG-E8(-/-)) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus. We found that the MFG-E8 deficiency was accompanied by the increased production of immunoglobulins. Further Western blot and ELISA analyses validated the increase in the IgM levels in the MFG-E8(-/-) mice. It was also revealed that the sera from the MFG-E8(-/-) mice cross-reacted with oxidation-specific epitopes generated upon incubation of serum albumin with the peroxidized lipids. Among the modified proteins with several unsaturated aldehydes of chain lengths varying from three to nine carbons, the MFG-E8(-/-) mice sera exclusively cross-reacted with the protein-bound 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ?6 polyunsaturated fatty acids. In addition, the IgM monoclonal antibodies (mAbs) that selectively cross-reacted with the ONE-modified proteins were generated from the MFG-E8(-/-) mice. A subset of the ONE-specific IgM mAbs significantly recognized the late apoptotic and necrotic cells and enhanced the phagocytosis by macrophages. These data demonstrate that the impairment of the phagocytic clearance of apoptotic cells through MFG-E8 can lead to the generation of natural antibodies, which may play a critical role in removing multiple damage-associated molecules, including oxidation-specific epitopes and late apoptotic/necrotic cells.
Project description: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.