Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Summary

Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury.

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high morbidity and mortality. The molecular pathogenesis of ALI is being better defined; however, because of the complex nature of the disease molecular therapies have yet to be developed. Here we use a lipopolysaccharide (LPS) induced mouse model of acute septic lung injury to delineate the role of exosomes in the inflammatory response. Using this model, we were able to show that mice that are exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid from the lungs that are packaged with miRNA and cytokines which regulate inflammatory response. Further using a co-culture model system, we show that exosomes released from macrophages disrupt expression of tight junction proteins in bronchial epithelial cells. These results suggest that 1) cross talk between innate immune and structural cells through the exosomal shuttling contribute to the inflammatory response and disruption of the structural barrier and 2) targeting these miRNAs may provide a novel platform to treat ALI and ARDS.

Introduction

ALI and ARDS are the life-threatening forms of respiratory failure with severe hypoxemia caused by non-cardiogenic pulmonary edema which affects approximately 1 million people worldwide annually¹. The etiology of ARDS includes direct injury to the lungs from infections or aspiration and a variety of indirect insults. Over the last decade there has been an increased understanding of the molecular pathogenesis of ARDS, however, specific targeted treatments for ARDS are yet to be developed^2,3.

Several animal models of acute lung injury have been developed which ....

Protocol

The overall protocol requires 2 days, including the first day of sepsis induction and isolation of BAL fluid from the animal, and the second day for exosomes isolation from the mouse BALF. All procedures have been reviewed and approved by the Institutional Animal Care and Use Committee at Atlanta VA medical center.

1. Mouse Acute Septic Lung Injury Model

1. Use 6 - 8 weeks- old male wild-type C57BL/6J mice (weight 20 - 22 g) for the animal model, and perform all procedures using aseptic techniques and instruments. Autoclave all surgical instruments, including forceps and scissors for 20 min at 121 °C.  
2. Randomly....

Results

To induce septic lung injury, mice were treated with intraperitoneal LPS (15 mg/kg). Within 24 h of LPS administration, neutrophilic influx was seen in the lungs, as shown in Figure 1A. Mouse BAL fluid was drawn, followed by isolation and purification of exosomes. Morphology of BALF exosomes was confirmed by electron transmission microscopy (Figure 1B).

Expression of se.......

Discussion

Mouse models of diseases are commonly used to evaluate the physiological function of specific genes and to reduce the cost of experimentation². The acute septic lung injury described here mimics inflammatory response seen in humans with ARDS. This model is relevant to investigate the molecular pathogenesis, development of biomarkers and to test potential new therapies⁵.

Co-culture systems are relevant for in vivo studies in the context o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
lipopolysaccharide	Sigma-Aldrich	Escherichia coli 055:B5
PBS pH7.2(1X)	Life technologies	20012-027
mirVana miRNA isolation kit	Thermo Fisher	449774
TaqMan Gene Expression Master Mix	Thermo Fisher	4369016
mmu-miR-155 (002571) primer	Thermo Fisher	4427975
has-miR-146a (000468) primer	Thermo Fisher	4427975
U6snRNA primer	Thermo Fisher	4427975
TNF-α(Mm00443258_m1 ) primer	Thermo Fisher	4331182
IL-6 (Mm00446190_m1   )primer	Thermo Fisher	4331182
ZO-1 (Mm00493699_m1  )primer	Thermo Fisher	4331182
minimal essential medium (MEM)	Thermo Fisher	11095-072
Trysin-EDTA solution(0.05%)	Thermo Fisher	25300054
Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit	Sigma-Aldrich	MINI67-1KT
Exosome Spin Columns (MW3000)	Thermo Fisher	4484449
Beckman Coulter Optima L-100XP ultracentrifuge	Beckman Coulter	L-100XP
Ultra-clear centrifuge tubes	Beckman Coulter	344058
Applied Biosystems 7500 Fast Real-Time PCR System	Thermo Fisher
transmission electron microscopes (TEM)	JEOL Ltd	120 kV TEM
Olympus BX41 microscope	Olympus	BX41