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

Zhimin Wang,Weng-Lang Yang,Shingo Matsuo,Archna Sharma,Fangming Zhang,Ping Wang,Haichao Wang

doi:10.1186/s13054-015-1094-3

Abstract

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.

Highlights

Sepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and death
MFG-E8-derived short peptide 68 (MSP68) effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice
Cell adhesion assay The Differentiated Human promyelocytic leukemia cell line (HL-60) cells (dHL-60) cells were labeled with calcein AM (Life Technologies, Grand Island, NY, USA). 1.5 × 105 labeled dHL-60 cells were added to 96-well plates coated with 10 μg/ml fibronectin (Life Technologies) or 10,000 Pulmonary artery endothelial cells (PAEC)/well in the presence of phosphate-buffered saline (PBS) or various concentrations (0.5 and 5 μg/ml) of synthesized small peptides derived from human Milk fat globule epidermal growth factor-factor 8 (MFG-E8) (GenScript, Piscataway, NJ, USA)

Summary

Introduction

Sepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and death. Sepsis is defined as an infection-associated initial hyperinflammatory and subsequent immune-suppressive response, which leads to multiple organ dysfunction, shock, secondary infections and lethality [1,2,3]. It is one of the most prevalent diseases and accounts for 20 % of all admission to intensive care units [4]. These activated neutrophils release proteolytic enzymes and reactive oxygen species These released molecules help kill invading pathogens in sepsis, their excessive production disrupts the endothelial barrier and causes extravascular tissue damage, contributing significantly to multiple organ failure (MOF) and lethality [12,13,14,15]. A therapeutic strategy designed to attenuate neutrophil infiltration has the potential to prevent organ injury and reduce mortality in sepsis [17, 18]

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