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

Abstract

Background: 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.

Methods: Neonatal sepsis was induced in 5- to 7-day-old male and female C57BL6 mice using an intraperitoneal injection of cecal slurry. At 1 hour after sepsis induction, a single dose of 40 μg/kg recombinant mouse MFG-E8 or vehicle was administered via retro-orbital injection. All neonates were returned to their mothers as a group. At 10 hours 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.

Results: Serum lactate dehydrogenase, IL-1β, and IL-6 were significantly increased 10 hours 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.

Conclusion: 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.

Figure 1. Effect of rmMFG-E8 on organ injury and inflammation after neonatal sepsis

Male and female C57BL/6 pups were subjected to neonatal sepsis by IP CS injection and treated with PBS (Vehicle) or rmMFG-E8 (40 μg/kg body weight) at 1 h after CS. Serum samples collected at 10 h after CS were measured for (A) lactate dehydrogenase (LDH), (B) IL-1β and (C) IL-6. Serum samples from neonatal mice with neither CS injection nor treatments were included as controls (Control) for the experiment. Data shown as mean ± SE and compared by one-way ANOVA and SNK. *P< 0.05 vs. Control; ^#P< 0.05 vs. Vehicle.

Figure 2. Effect of rmMFG-E8 on lung injury after neonatal sepsis

Lung tissues from Control, Vehicle, and rmMFG-E8 treatment groups harvested at 10 h after CS were sectioned, stained with hematoxylin and eosin, and examined under light microscopy. (A) Representative images at 200× magnification. (B) The histological lung injury scores quantified as described in “Materials and Methods”. Data shown as mean ± SE and compared by one-way ANOVA and SNK. *P< 0.05 vs. Control; ^#P< 0.05 vs. Vehicle.

Figure 3. Effect of rm-MFG-E8 on lung inflammation and neutrophil infiltration after neonatal sepsis

Lung tissue lysates were quantified for (A) IL-6 and (B) IL-1β proteins using enzyme-linked immunosorbent assay. Lung tissue sections were stained with granulocyte-differentiation antigen-1 (Gr-1) and examined under light microscopy. (C) Representative images display brown Gr 1-positive staining cells at 200× magnification. (D) The number of neutrophils or Gr 1-positive staining cells quantified from immunohistochemistry. Data shown as mean ± SE and compared by one-way ANOVA and SNK. *P< 0.05 vs. Control; ^#P< 0.05 vs. Vehicle.

Figure 3. Effect of rm-MFG-E8 on lung inflammation and neutrophil infiltration after neonatal sepsis

Lung tissue lysates were quantified for (A) IL-6 and (B) IL-1β proteins using enzyme-linked immunosorbent assay. Lung tissue sections were stained with granulocyte-differentiation antigen-1 (Gr-1) and examined under light microscopy. (C) Representative images display brown Gr 1-positive staining cells at 200× magnification. (D) The number of neutrophils or Gr 1-positive staining cells quantified from immunohistochemistry. Data shown as mean ± SE and compared by one-way ANOVA and SNK. *P< 0.05 vs. Control; ^#P< 0.05 vs. Vehicle.

Figure 4. Effect of rmMFG-E8 on lung apoptosis after neonatal sepsis

Lung tissue sections were stained with terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) and examined under fluorescent microscopy. (A) Representative images display green TUNEL-positive cells and blue nuclei (DAPI staining) at 200× magnification. (B) The number of TUNEL-positive cells averaged over 10 visual fields/section. Data shown as mean ± SE and compared by one-way ANOVA and SNK. *P< 0.05 vs. Control; ^#P< 0.05 vs. Vehicle.