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 provide a bridge for translating experimental therapies to human studies^4,5. Commonly used models include local installation of oleic acid, bacteria, LPS, and bleomycin. Other approaches include ischemia-reperfusion, cecal ligation puncture, mechanical ventilation-induced stretch injury, hyperoxia or systemic administration of bacteria and LPS⁵. These models provide a useful biological system to test clinical hypotheses and for the development of potential therapies. To simulate human ARDS, animal models should reproduce inflammation and acute injury to the epithelial and endothelial cells with defects in barrier function in the lungs.

Exosomes are membrane vesicles with 20 - 200 nm in diameter, whose molecular content contains proteins, DNA, RNA, and lipids, and facilitate inter-cellular communications in tissue microenvironment via molecular composition transfer. Exosomes are secreted by multiple types of cells, such as endothelial cells, epithelial cells, smooth muscle cells and tumor cells, and exist in human body fluid. Studies indicate that exosomes regulate cross-talk between immune cells and stromal cells during infectious and sterile inflammatory diseases, and their abnormal release appears to be regulated by various natural and experimental stimuli during physiological and pathological processes⁶. Such communication network may play an important role in the pathogenesis of lung diseases and may affect pathophysiological progression^7,8. As 18 - 22 nucleotide non-coding RNAs, miRNAs exist in both tissue and body fluids, plasma, sera, and modulate mRNA expression at post-translational level^9,10.

Packaged miRNAs in exosomes influence differentiation and function of multiple types of cells, and excessive levels are associated with a variety of diseases, including cancer, lung diseases, obesity, diabetes, and cardiovascular disease^{11,12,13,14,15,16}. Entry into the recipient cells and shuttling of exosomal miRNAs facilitate intercellular communications modifying the hemostasis of microenvironment^17,18. Acute lung-injury is a complex processes, involving multiple cell types with extensive intercellular communications through exosomes⁸. miR-155 and miR-146a share the common transcriptional regulatory mechanism and contribute to the inflammatory response and the immune tolerance^19,20. Recent studies indicate that both modulate inflammatory response via exosomal miRNAs shuttling between immune cells²¹. However, the molecular mechanisms underlying modulatory effects of exosomal miRNAs on alveolar response to endotoxin remain unclear, undoubtedly the potential clinical relevance and translational implication merit further investigation.

Co-culture models are being employed to define the interaction of the specific cell types in the complex environment, such as inflammation and cancer^22,23. These platforms provide an alternative strategy to interrogate cross talk between cell types particularly for immune and structural cells.

Intra-tracheal, aerosolized, intraperitoneal or systemic administration of LPS are widely used to induce experimental lung injury^24,25,26, and have shown to induce epithelial and endothelial permeability defects. Here we use intraperitoneal LPS to induce a septic model of acute lung injury in mice. Within 24 h of intraperitoneal administration of LPS, permeability defects are induced in the lungs with recruitment of inflammatory cells. Further, we show that exosomes from BAL contain miRNA-155 and miR-146a, and exosomes from BAL fluid induce pro-inflammatory cytokines expression in recipient epithelial cells, including IL-6 and TNF-α. These data are the first to show that exosomal miRNAs are secreted in BAL in this model of acute septic lung injury.

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 distribute mice into experimental and control group. Restrain mice on a surgical board. Treat mice with Escherichia coli lipopolysaccharide (LPS, 15 mg/kg) by intraperitoneal route. According to the individual body weight, dilute appropriate LPS dosages to 0.1 mL PBS per mouse, then inject LPS solution using a 1 mL syringe with 27 G needle and inject an equivalent volume of PBS to control animals.   
3. Keep mice in the animal BSL2 facility, which consists of clean cages at appropriate room temperature.
4. 24 h later, euthanize mice with CO₂ inhalation.
5. Select the surgical area in the center of mouse neck. Make a small incision (approximately 5 mm), and gently separate muscles for the access to the trachea using blunt forceps until the trachea is exposed.
6. Withdraw mouse BAL fluid with 10 mL sterile syringe with a 27 G needle. First, gently, insert the needle into the trachea and inject 1 mL of sterile PBS slowly into the trachea, then draw BALF into a 3 mL disposable syringe with a 27 G needle. Upon completion of the procedure, dispose of syringes and needles in biohazard sharps container.
7. Treat surgical instruments with chlorine dioxide, as described previously by Chauret et al.²⁷, and then sterilize all surgical instruments by autoclaving for 20 min at 121 °C.

2.  Exosomes Isolation-serial Ultra-centrifugation and Characterization

1. Collect mouse BAL fluid in 15 mL conical tubes. To harvest alveolar cells, centrifuge samples for 10 min at 1,000 g, at 4 °C.
2. Transfer the supernatant to new 15 mL conical tubes, then centrifuge samples for 30 min at 4,000 x g at 4 °C to remove cell debris.
3. Collect the supernatant in ultracentrifuge tubes and centrifuge samples for 60 min at 10,000 x g at 4 °C to remove larger cellular particles.
4. Transfer the supernatant to new conical tubes, then filter the samples through 0.2 µm syringe-filter to remove remaining larger particles.
5. Collect samples in ultracentrifuge tubes, and balance tubes. Centrifuge samples for 2 h at 140,000 x g at 4°C to pellet the exosomes.
   Note: The size of exosomes varies 20 - 200 nm. 0.2 µm filter is used for removing larger particles that are more than 200 nm in size.
6. Discard the supernatants gently. Dissolve the pellet with 100 µL PBS or lysis solution, and transfer exosomes samples to 1.5 mL tubes, and store at -80 °C until use.
   Note: Transmission electron microscope is recommended to verify isolated exosomes.
7. Follow the standard TEM sample processing protocol²⁸. Analyze exosomes samples using transmission electron microscopy, and take images, scale bar 200 nm.

3. Preparation of miRNA and Performing miRNA- q-RT-PCR

Note: miRNA-q-RT-PCR is recommended to analyze expression level of miRNAs.

1. Follow standard miRNAs isolation protocol and make sure to use an equal amount of purified RNAs in single-strand reverse transcription²⁴.
2. Follow the standard real-time PCR protocol to detect exosomal microRNA level in BAL fluid. Use specific real-time PCR primers against miR-155, miR-146a, and U6 SnRNA. Run PCR reaction with the quantitative PCR apparatus and normalize the expression values to internal control U6 snRNA.
   Note: Each group consists of triplicate samples²⁴.

4. Exosomes Labeling and Purification

1. Suspend exosomes pellet with 100 µL suspension solution in a 1.5 mL polypropylene tube, then add 0.4 µL of the exosomes dye into the other tube with 100 µL of the suspension solution.
2. Quickly add exosomes in the solution to the dye solution and mix it thoroughly by pipetting. Keep samples for 5 min at room temperature, protect from light.
3. Put one exosome spin column in a 1.5 mL elution tube. Transfer the solution mixture to the center of the exosome spin column.
4. Spin the column for 2 min at 800 x g at room temperature.
5. Discard the column and store the eluted sample at -20 °C until use.

5. Validation of Exosomes Up Taken by Recipient Cells

1. Seed 1 x 10⁴ MLE 12 /cell in an 8-well chamber slide, and culture cells at 37 °C, 5% CO₂.
2. Add 10 µL fluorescent-labeled exosomes to the cells. Incubate for 1 h at 37 °C.
3. Remove culture media and wash cells with PBS, then fix cells with 1% paraformaldehyde for 10 mins at room temperature.
4. Mount the slide in mount medium with DAPI.
5. Analyze samples through a fluorescence microscope and take images, 40X magnification.

6. Verification of Interaction between Exosomes and Recipient Cells

1. Seed 2 x 10⁵ MLE12/well in a 12-well culture plate containing appropriate culture medium, and culture cells at 37 °C, 5% CO₂.
2. Add 20 µg exosomes from mouse BAL fluid to MLE12 cells and set up replicates per experimental group.
3. 24 h later, wash cells with PBS and add 600 µL lysis buffer into well. Gently shake the plate and collect lysis samples in 1.5 mL tube with the pipette.
4. Follow the standard total RNA isolation protocol and cDNA reverse transcription protocol, complete the single-strand cDNA reverse transcription²⁹.
5. Follow the standard real-time PCR protocol to detect mRNA expression level in recipient cells²⁹. Use specific real-time PCR primers against TNF-α, IL-6, and ZO-1. Run PCR reactions with the quantitative PCR apparatus and normalize the expression values to the internal control GAPDH.
   Note: Each group consists of triplicate samples.

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

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