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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Shock. 2022 Mar 1;57(3):408–416. doi: 10.1097/SHK.0000000000001846

Therapeutic Effects of Hyaluronic Acid Against Cytotoxic Extracellular Vesicles Released During Pseudomonas Aeruginosa Pneumonia

Yoshifumi Naito 1,#, Hideya Kato 1,#, Li Zhou 2, Shinji Sugita 2, Hongli He 2, Justin Zheng 2, Qi Hao 2, Teiji Sawa 1, Jae-Woo Lee 2
PMCID: PMC8840981  NIHMSID: NIHMS1730625  PMID: 34387224

Abstract

Extracellular vesicles (EVs) have now been recognized as important mediators of cellular communication during injury and repair. We previously found that plasma EVs isolated from ex vivo perfused human lungs injured with Escherichia coli bacterial pneumonia were inflammatory, and exogenous administration of high molecular weight (HMW) hyaluronic acid (HA) as therapy bound to these EVs, decreasing inflammation and injury. In the current study, we studied the role of EVs released during severe Pseudomonas aeruginosa (PA) pneumonia in mice and determined whether intravenous administration of exogenous HMW HA would have therapeutic effects against the bacterial pneumonia. EVs were collected from the bronchoalveolar lavage fluid (BALF) of mice infected with PA103 by ultracentrifugation and analyzed by NanoSight and flow cytometry. In a cytotoxicity assay, administration of EVs released from infected mice (I-EVs) decreased the viability of A549 cells compared to EV isolated from sham control mice (C-EVs). Both exogenous HMW HA or an anti-CD44 antibody, when co-incubated with I-EVs, significantly improved the viability of the A549 cells. In mice with PA103 pneumonia, administration of HMW HA improved pulmonary edema and bacterial count in the lungs and decreased TNF-α and caspase-3 levels in the supernatant of lung homogenates. In conclusion, EVs isolated from BALF of mice with Pseudomonas aeruginosa pneumonia were cytotoxic and inflammatory, and intravenous HMW HA administration was protective against Pseudomonas aeruginosa pneumonia.

Keywords: Acute Lung Injury, CD44, Extracellular vesicles, Hyaluronic acid, Pseudomonas aeruginosa

INTRODUCTION

Acute respiratory distress syndrome (ARDS) is a life-threatening condition in critically ill patients that develops most commonly following cases of bacterial or viral pneumonias, aspiration, non-pulmonary sepsis, and major trauma (1). Although much progress has been made in treating ARDS, with modalities such as lung-protective mechanical ventilation, optimal positive end expiratory pressure, prone positioning, and neuromuscular blockade, no effective pharmacological treatments have been identified (1-3). In our previous publication, we found that extracellular vesicles (EVs) isolated from plasma released during Escherichia coli pneumonia in ex vivo perfused human lungs were inflammatory and induced acute lung injury (ALI) when administered intravenously or intratracheally in naïve lungs(4). We also found that administration of high molecular weight hyaluronic acid (HMW HA) bound to these inflammatory EVs via the CD44 receptor on the EVs and decreased inflammation, improved endothelial permeability, and decreased bacterial growth in the injured alveolus(4). These results suggested that administering HMW HA could have a therapeutic effect against other bacterial pneumonias such as those caused by Pseudomonas aeruginosa (PA).

PA bacteria is a major opportunistic pathogen that causes acute pulmonary infection and sepsis, especially in critically ill or immunocompromised patients, and often progresses to ARDS(5, 6). Emergence and propagation of multidrug-resistant PA bacteria have become a serious worldwide concern because efficacious antimicrobial choices are limited(7). Hence, development of new therapeutic strategies that do not rely on the use of conventional antimicrobial agents are essential.

In the current study, we initially isolated the EVs from BALF of infected mice with PA pneumonia and examined the cytotoxicity of EVs. We next examined the therapeutic effect of HMW HA in mice injured with PA pneumonia in decreasing lung inflammation, edema, and bacterial growth.

MATERIALS AND METHODS

Isolation of EVs

At 24 h after instillation of phosphate buffered saline (PBS) or P. aeruginosa strain PA103 (5 × 104 CFU in 50 μl), bronchoalveolar lavage fluid (BALF) was collected from five mice in both groups and centrifuged twice at 3000 rpm at 4°C for 15 min, followed by 10,000 × g at 4°C for 30 min to remove cellular debris, larger particles, and bacteria. The supernatants were then ultracentrifuged at 100,000 × g at 4°C for 1 h. The pellet was resuspended and washed in PBS and then submitted to a second ultracentrifugation under the same conditions. After the second centrifugation, EVs were resuspended with PBS and stored at −80°C.

We also isolated EVs derived from the culture medium of PA103 bacteria (2.4 × 1011 CFU). PA103 bacterial cultures were grown at 37°C for 12 h in a 220 rpm shaking water incubator. The supernatants first underwent centrifugation at 3000 rpm for 15 minutes and then were instilled through a 0.22-μm filter to remove bacteria. The supernatants were ultracentrifuged twice at 100,000 × g at 4°C for 1 h to isolate EVs.

Characterization of EVs with NanoSight and Flow Cytometry

We measured the number of particles and the size distribution in the EV solutions using NanoSight NS300.

The cellular source of the EVs was analyzed by flow cytometry. I-EVs (EVs derived from BALF of infected mice) and C-EVs (EVs derived from BALF of control mice) were stained by CD9 and CD44. These EVs were analyzed by fluorescence labeling with PKH26 (PKH26GL-1KT, Sigma-Aldrich, MO, USA) and CD44 or CD9 according to the manufacturer’s protocol, followed by flow cytometric analyses.

EVs isolated from 1 ml of BALF were resuspended with 500 μl Diluent C, which was then combined with the same volume of Diluent C containing 2 μl PKH26. After 5 minutes of incubation in room temperature, 1 ml D-PBS containing 1% BSA were added to prevent further staining followed by washing with D-PBS. For flow cytometry, 1 ml of C-EVs or I-EVs were resuspended with 100 μl Staining buffer (BD Biosciences, CA, USA) containing 5 μl CD9-FITC (eBioscience, CA, USA) or 20 μl CD44-FITC (BD Biosciences) and then incubated for 30 minutes at room temperature. The EVs were washed once with D-PBS containing 1% BSA. The fluorescence expression of stained EVs was detected by a BD FACSAria™ Fusion Special Order (SORP) cell sorter (BD Biosciences) with 100 nm nozzle and ND filter 1. The threshold was set on the SSC 200. For fluorescence detection, we used a 586/15 band pass filter for PKH26 and a 525/50 band pass filter for CD9-FITC and CD44-FITC. Samples loaded were acquired for 20 seconds to be analyzed after the event rate was stable. Collected data were analyzed by Diva software (BD Biosciences).

Cytotoxicity Assay of EVs

We examined the cytotoxicity of EVs on a human epithelial cell line. A549 cells (5 × 103 cells/well) were incubated in a 96-well plate for 24 h in a CO2 incubator. Each EV group (5 × 108 particles/well), PBS, or PA103 bacteria (5 × 105 CFU/well) was added to the A549 cell culture medium, followed by the cytotoxicity assay reagent (CCK-8 kit) (Dojindo, Tokyo, Japan), and the plate was placed in a CO2 incubator. After 4 h, we analyzed absorbance at 450 nm using a spectrometer and calculated the viability of the cells.

Blocking Effect of Anti-CD44 Antibody and HMW HA on the Cytotoxicity of I-EVs

A549 cells (5 × 103/well) were incubated in a 96-well plate for 24 h in a CO2 incubator. I-EVs (5 × 108/well) with or without 10 μl/well anti-CD44 Ab (Purified Anti-Mouse CD44, BD Biosciences) or 10 μl/well control IgG (Purified Mouse IgG, BD Biosciences) were incubated with the cells for 1 h in a CO2 incubator respectively. In another series, we added 20 μl/well HMW HA (1.5 MDa, LifeCore Biomedical, MN, USA) or PBS to the I-EV solution. Each EV solution (5 × 108 particles/well) was added to the A549 cell culture medium followed by cytotoxicity assay reagent, and the plate was incubated in a CO2 incubator. Finally, after 4 h, we analyzed absorbance at 450 nm to compare the viability of the cells.

Animals, Interventions and Bacteriological and Histological Analyses

Certified pathogen-free, male C57BL/6 mice (10–11 weeks old; body weight, 25–30 g) were purchased from Jackson Laboratory. The protocols for all animal experiments were approved by The Institutional Animal Care and Use Committee at the University of California, San Francisco, prior to undertaking the experiments. The PA103 strain was used for intratracheal infection. The mice were divided into three groups and administered intravenous HMW HA (3 mg/kg) (HA 1.5M) or PBS at 2 h or 4 h after intratracheal instillation of PA103 (5 × 104 CFU) under short-term volatile anesthesia using isoflurane. The body weight change of mice was measured at 24 h, and the body temperature of the mice was monitored with rectal thermometer at 0, 4, 8, 12, and 24 h. At 24 h post-infection, mice were euthanized with intravenous ketamine and xylazine, and the blood and lungs from the mice were collected. Subsequently, WBCs and neutrophils in the blood were counted. The lungs from the mice were then homogenized for further evaluation. The lung edema index was obtained by measuring the wet to dry weight ratios of the mouse lungs. Lung homogenates were diluted and inoculated onto sheep blood agar plates and incubated at 37°C overnight. The number of bacteria in the lungs was calculated from the CFU counts on the plates. Lungs were also collected for histological analysis from the mice in both groups and non-infected mice administered PBS intratracheally. The lungs were fixed with a 10% formalin neutral-buffered solution and embedded in paraffin. The mounted sections were stained with hematoxylin and eosin.

Biochemical Assays in Mice

Concentration of cytokines such as TNF-α and IL-10 were quantified from the supernatant of lung homogenates using an enzyme-linked immunosorbent assay kit (DY410 and M1000B, R&D systems, MN, USA ELISA) according to the manufacturer's instructions. Cleaved caspase-3 was also quantified with an ELISA kit (DYC835, R&D systems) according to the manufacturer’s instructions.

Immunoblot Analysis of Caspase-3 and Toxins

We performed Western blot analyses using a caspase-3 antibody (31A1067, Novus biologicals, CO, USA) to examine apoptosis in human epithelial cells exposed to I-EVs. A549 cells (5 × 104/well) were incubated in 6 well plates for 24 h in a CO2 incubator. Subsequently I-EVs (1 × 107 particles/10 μl/well) were added to the A549 cell culture medium followed by incubation for 0 min, 30 min, 1 h, or 4 h in a CO2 incubator. The A549 cell culture medium was removed at each time point listed above, and the cells were dissolved in NP-40 cell lysis buffer (Invitrogen, CA, USA) with a protease inhibitor cocktail. The protein concentrations (1000 μg/ml) of the solutions were equalized. These solutions were mixed with sample buffer and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting onto a polyvinylidene fluoride (PVDF) membrane. The membrane was immunostained with caspase-3 antibody (diluted 1:5000) as the primary antibody and anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:5000, A16110, Invitrogen, CA, USA) as the secondary antibody. After washing the membrane, electrochemiluminescence substrate was added to the membrane and the image was acquired with a chemiluminator.

We also performed Western blot analysis using ExoU antibody to detect toxins inside EVs. Rabbit anti-ExoU and exotoxin A antibodies used were generated, validated and published in the laboratory of Dr. Sawa(8). We used the I-EV solution with or without lysis buffer and also used the EV solution added to A549 cells and subjected to SDS-PAGE following blotting onto PVDF membrane. Anti-ExoU antibody was used as a primary antibody (diluted 1:1000) and anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:5000, A16110, Invitrogen, CA, USA) was used as the secondary antibody for immunoblotting performed as above. We also used the supernatants of lung homogenates of mice to detect ExoU and exotoxin A. We used anti-ExoU antibody and anti-Pseudomonas exotoxin A antibody (diluted 1:10000, P2318, Sigma-Aldrich) as a primary antibody.

Statistical Analysis

Data are expressed as mean ± standard deviation if the data were normally distributed and median with interquartile range if not. Comparisons between two groups were made using the unpaired Student's t-test if the data were normally distributed or the Mann–Whitney test if not. Comparisons between more than two groups were made by an analysis of variance using the Tukey's or Dunnett's multiple-comparison testing if the data were normally distributed or Dunn's test following Kruskal–Wallis analysis if not. A value of P < 0.05 was considered statistically significant. All statistical analysis was performed using GraphPad Prism software version 7.03.

RESULTS

Characteristics of EVs Isolated From the Bronchoalveolar Lavage Fluid of Mice Injured with PA103 Pneumonia

We collected EVs from BALF of mice administered PBS or PA103 intratracheally and analyzed the characteristics of EVs. The average number of particles in the BALF was 6.02 × 109 ± 9.9 × 107/mouse from 5 mice. The mean diameter of EVs from non-infected mice was 126.4 ± 0.9 nm, and the mean diameter of EVs from infected mice was 133.2 ± 4.9 nm as analyzed with NanoSight (Fig. 1A). These EVs appeared as spheroids measuring between 50–200 nm by electron microscopy (Fig. 1B).

Figure 1. Characteristics of EVs Derived from BALF of Infected Mice 24 h after Infection with P. aeruginosa.

Figure 1.

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(A) By Nanosight analysis, about 28% of the C-EVs and 33% of the I-EVs were 50–100 nm, 38% of the C-EVs and 23% of the C-EVs were 100–150 nm, and 18% of the C-EV and 10% of I-EV were 150–200 nm. (B) By electron microscopy, these EVs appeared as spheroids measuring between 50–200 nm in size. (C) By flow cytometry analysis, 48 ± 23% of the C-EVs and 41 ± 16% of I-EVs were PKH26 labeled EVs, 16 ± 10% of C-EVs and 23 ± 16% of I-EVs were CD9 positive, label for exosomes, and 3.8 ± 5.4% of C-EVs and 4.3 ± 1.3% of I-EVs were CD44 positive, label for microvesicles. Data were presented as mean ± SD, n = 4. C-EV, extracellular vesicles derived from control mice; I-EV, extracellular vesicles derived from infected mice.

We analyzed the surface antigens and characteristics of the EVs by flow cytometry; 48 ± 23% of the C-EVs and 41 ± 16% of I-EVs were PKH26 label positive, demonstrating the presence of a lipid bilayer. In addition, 16 ± 10% of C-EVs and 23 ± 16% of I-EVs were CD9 positive, labeled for exosomes, and 3.8 ± 5.4% of C-EVs and 4.3 ± 1.3% of I-EVs were CD44 positive, labeled for microvesicles (N = 4) (Fig. 1C).

Cytotoxicity Assays of EVs and Induction of Apoptosis in Human Epithelial Cells

In a cytotoxicity assay, co-culture with I-EVs significantly decreased the viability of A549 cells (65%) compared to cells co-cultured with C-EVs (97%) (P < 0.01) (Fig. 2A). Surprisingly, there was no difference in viability of A549 cells when grown with either EVs released by PA bacteria (PA-EVs) (99%) and C-EVs. The viability of the A549 cells co-cultured with I-EVs + anti-CD44 antibody (85%) was significantly higher than in those with I-EVs + IgG (42%) (P < 0.05) (Fig. 2B). The viability of A549 cells co-cultured with I-EVs + HMW HA (94%) was also significantly higher than those with only I-EVs (78%) (P < 0.05) (Fig. 2C). By Western blot analyses, the signal intensity for pro-caspase-3 gradually decreased over time. Conversely, the signals for active caspase-3 at 30 min- and 1 h-intervals were stronger than those at 0 min and 4 h (Fig. 2D).

Figure 2. Cytotoxicity Assays of A549 cells with EVs Derived from BALF of Infected Mice.

Figure 2.

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(A) In the cytotoxicity assay, the viability of A549 cells co-cultured with I-EVs (65%) was significantly lower than that of cells co-cultured with C-EVs (97%). The viability of the A549 cells co-cultured with PA103 strain was lower than that of C-EVs (44%). Data were presented as mean ± SD, n = 4, *P < 0.01 and **P < 0.001. (B) The viability of the A549 cells co-cultured with I-EVs + anti-CD44 antibody (85%) was significantly higher than in those with I-EVs + IgG (42%) and PA103 strain (35%). Data were presented as mean ± SD, n = 4, *P < 0.05 and **P < 0.01. (C) The viability of I-EVs + HMW HA (94%) was significantly higher than that of A549 cells co-cultured with only I-EVs (78%). Data were presented as mean ± SD, n = 4, *P < 0.05. (D) Representative Western blot of the acute increase in active caspase-3 in A549 cells following incubation with I-EVs, n = 3. C-EV, extracellular vesicles derived from control mice; I-EV, extracellular vesicles derived from infected mice; PA, pseudomonas aeruginosa; PBS, phosphate buffered saline; Ab, antibody; IgG, immunoglobulin G; HMW, high molecular weight; HA, hyaluronic acid.

Therapeutic Effects of HMW HA in Mice Injured with PA103 Bacterial Pneumonia

The experimental animals in the HA treatment group (2 h-HA group) had significantly higher body temperature than in the injury group (PA only group) 24 h after infection (P < 0.05) (Fig. 3A). However, there was no significant difference in body weight change between the groups (Fig. 3B). Although WBC counts in the blood were not different between the groups, the number of neutrophils in 2 h-HA group significantly increased compared to those in 4 h-HA group (P < 0.05) (Fig. 3C).

Figure 3. Parameters of Infected Mice 24 h after Infection with P. aeruginosa PA103 strain.

Figure 3.

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(A) The experimental animals showed significantly higher body temperature in the HA treatment group (2 h) than in the injury group (PA only) 24 h after infection. Data were presented as mean ± SD, *P < 0.05. (B) There was no significant difference in body weight change between the groups. (C) WBC counts in the blood had no difference among the groups. The number of neutrophils in the 2 h-HA group significantly increased compared to those in the 4 h-HA group. Data were presented as mean ± SD, *P < 0.05.

BT, body temperature; BW, body weight; WBC, white blood cell; PA, pseudomonas aeruginosa; IV, intravenous; HA, hyaluronic acid.

Effect of HMW HA Treatment on Lung edema, Bacteriology, and Histology

After we euthanized the mice, we analyzed the parameters in lung homogenates. Lung edema in the PA group was severe at 24 h after PA103 infection. Administration of HMW HA 2 h after infection significantly reduced the wet/dry (W/D) ratio (P < 0.05) (Fig. 4A). Lung bacterial counts (CFU) were significantly lower in the 2 h-HA group compared to the 4 h-HA group (P < 0.05) and the PA group (P < 0.01). There was no difference between the 4 h-HA group and the PA group (Fig. 4B).

Figure 4. Effect of HMW HA Administration Against P. aeruginosa Pneumonia on Lung Parameters and Histology.

Figure 4.

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(A) Lung edema in the PA group was severe (W/D ratio) at 24 h after PA103 infection. Administration of HMW HA significantly reduced lung edema (W/D ratio in the 2 h group). Data were presented as mean ± SD, *P < 0.05. (B) Lung bacterial counts (CFU) were significantly lower in the 2 h-HA group compared to the 4 h-HA group and the PA group. There was no difference between the 4 h-HA group and the PA group. Data were presented as median ± IQR, *P < 0.01 and **P < 0.05. (C) Histological changes in the mouse lungs were evaluated at 24 h after PA103 instillation. The 2 h-HA group displayed qualitatively fewer inflammatory changes in the lung than that of the PA group. Scale bar = 50 μm. (D) The concentration of TNF-α was lower in the 2 h-HA group than in the PA group. There was no difference in the concentration of IL-10 between the groups. The concentration of cleaved caspase-3 was also significantly lower in the 2 h-HA treatment group compared to the PA group. Data were presented as mean ± SD, *,**P < 0.01. W/D, wet/dry; PA, pseudomonas aeruginosa; IV, intravenous; HA, hyaluronic acid; CFU, colony forming unit; PBS, phosphate buffered saline; TNF, tumor necrosis factor; IL, interleukin.

Histological changes in the mouse lungs were evaluated at 24 h after PA103 instillation. Enhanced neutrophil infiltration, alveolar hemorrhage, and destruction of the alveolar structure were observed in the lungs of the PA group. In contrast, the lungs from the 2 h-HA group displayed qualitatively fewer inflammatory changes than in the PA group (Fig. 4C).

Effect of HMW HA Treatment in Lung Inflammation and Apoptosis

Considering the results above, we compared the concentration of cytokines and caspase-3 in the supernatant of lung homogenates in the 2 h-HA group and the PA group. Although the concentration of TNF-α was lower in the HA group than in the PA group (P < 0.01), there was no difference in the concentration of IL-10. The concentration of cleaved caspase-3 was also significantly lower in the HA treatment group compared to the PA group (P < 0.01) (Fig. 4D).

Immunoblot Assays Using an Anti-ExoU Antibody

Because we assumed that the cytotoxicity of EVs may be due to toxins such as ExoU and exotoxin A, we attempted to detect the toxins with immunoblot analysis. Neither DAB stain nor chemiluminescence was able to detect the signal for ExoU (74 kDa) in lanes from the I-EV solution with or without lysis buffer or the I-EV solution added to A549 cells, other than that from the positive control (PA103 culture) (Fig. 5A). Furthermore, surprisingly, we could not detect either ExoU or exotoxin A from the supernatant of lung homogenates 24 h after infection (Fig. 5B, 5C).

Figure 5. Immunoblot Analyses of P. aeruginosa Toxins.

Figure 5.

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(A) We analyzed by DAB stain and Chemiluminescent to detect ExoU within I-EVs. Neither of them was able to detect the signal in lanes from the I-EV solution with or without lysis buffer or the EV solution added to A549 cells, other than that from the positive control (PA103 culture). (B) There was no signal for ExoU from the supernatant of lung homogenates either with or without HMW HA administration. (C) We couldn’t detect the exotoxin A from the supernatant of lung homogenates with or without lysis buffer. DAB, diaminobenzidine; ECL, enhanced chemiluminescence; PA, pseudomonas aeruginosa; EV, extracellular vesicle; LB, lysis buffer; IT, intratracheal; SN, supernatant.

DISCUSSION

The main findings of the current study are as follows: 1) EVs isolated from BALF of mice with P. aeruginosa pneumonia were cytotoxic, and blocking CD44 receptors reduced the cytotoxicity of I-EVs; 2) HMW HA had a beneficial effect in reducing the cytotoxicity of I-EV as well as treatment with an anti-CD44 antibody; 3) Although recent studies have shown that most bacteria release EVs that contain virulence factors, bioactive proteins, and nucleic acids(9, 10), the cytotoxicity of PA-EV was not seen in A549 cells within the time frame studied; 4) Intravenous administration of HMW HA to mice after 2 h was effective against P. aeruginosa pneumonia in reducing pulmonary edema and the total bacterial load; 5) And the cytotoxicity of I-EVs did not depend on the intra-vesicular presence of toxins such as ExoU and exotoxin A.

EVs are small phospholipid-based particles that are released by every cell into the extracellular environment in both physiological or pathophysiological conditions. They encapsulate a variety of materials including RNA, microRNA, DNA, lipids, and proteins(11). EVs often reflect the cell of origin and have emerged as important modulators of intercellular communication in diseases and maintenance of homeostasis(12-14). According to the recommendations of the International Society for Extracellular Vesicles, EVs are stratified based on their size and morphology as well as their biogenesis and mechanisms of secretion(15, 16). Exosomes are 30–150 nm in diameter and they originate from endosomes. Microvesicles are shed from the plasma membrane by outward blebbing and are larger than exosomes (100–2000 nm)(11, 17). In our study, most of the particles were less than 200 nm in both C-EV and I-EV, and some particles were CD9- and CD44-positive as analyzed by flow cytometry, suggesting that these particles were composed of both exosomes and microvesicles. Recent reports indicated that in mice, alveolar macrophages were the main sources of the EVs detected in BALF(18), but EVs derived from alveolar endothelial cells and neutrophils can also induced in ALI(19). The difference in size distribution between C-EVs and I-EVs may result from the inflammatory response against P. aeruginosa infection. Studies are on-going to determine if there are differences in the I-EVs depending on whether EVs are from the plasma or BALF such as the cellular sources of the EVs. However, due to the size of the EV, plasma and BALF EV may distribute equally between both compartments over time during ALI. For example, we previously found that administration of inflammatory EVs to naïve perfused human lungs caused a similar injury profile whether the EVs were administered intravenous or intra-trachea(4). Regardless of their origin, EVs are known to effect multiple cell types including alveolar epithelial, endothelial and immune cells(20).

Recently, EVs were found to play an important role in initiating ARDS. EVs derived from alveolar macrophages and endothelial cells induced inflammation and ALI through the initiation of a cytokine cascade(21, 22). Other studies showed that intravenous and intratracheal injection of EVs derived from blood of lipopolysaccharide-treated rats into naïve rats induced ALI(23). Our previous study also showed that intravenous and intrabronchial instillation of EVs derived from blood in E. coli pneumonia induced severe lung injury in a naïve ex vivo perfused human lung(4). In the current study, the viability of A549 cells co-cultured with I-EVs was significantly lower than that of cells co-cultured with C-EVs, demonstrating that I-EVs had a cytotoxic effect on lung epithelial cells (Fig. 2A). Additionally, the viabilities of the A549 cells co-cultured with I-EVs + anti-CD44 antibody were significantly higher than with I-EVs + IgG (Fig. 2B), suggesting that blocking the CD44 receptor reduced the cytotoxicity of I-EVs. We also analyzed the cytotoxicity of I-EVs with or without HMW HA, showing that HMW HA reduced the cytotoxicity of I-EVs (Fig. 2C). This may be partly due to the blocking of the CD44 receptor on I-EVs by HMW HA.

Caspase-3 is one of the interleukin-1-converting enzyme proteases which plays an important role in initiating apoptosis(24). Activation of caspase-3 is triggered by extrinsic stimuli through cell-surface death receptors, such as TNFα, Fas ligand receptors, or by intrinsic stimuli via the mitochondrial signaling pathway(25, 26). Caspase-3 is produced as an inactive 32-kDa proenzyme, which is cleaved at an aspartate residue to yield a 12-kDa and a 17-kDa subunit that combine to form the active caspase-3 enzyme(27). Active caspase-3 demolishes key structural proteins and is responsible for DNA fragmentation and membrane blebbing in cells during apoptosis(28). In the current immunoblot analysis, we could detect active caspase-3 from A549 cultures injured with I-EV, and peak activation of caspase-3 was seen around 1 h after incubation. These results indicated that I-EV was cytotoxic to lung epithelial cells by inducing the apoptosis of the cells through caspase activating pathways, and HMW HA protected the cells by possibly binding to CD44, inhibiting activation of the signaling pathways.

HA is a non-sulfated glycosaminoglycan component of the extracellular matrix in the lung and play a role in multiple biological functions, such as cell proliferation and migration, morphogenesis, wound healing, inflammation, angiogenesis, and tumor growth(29, 30). HA is synthesized by three hyaluronan synthases (HAS 1, 2, and 3) at the cell surface and is normally released as a high-molecular-weight polymer (>1000 kDa) into the extracellular space(31, 32). LMW HA (<500 kDa) can be produced by degradation of HMW HA by hyaluronidase, reactive oxygen species, and reactive nitrogen species, or synthesis by HAS during inflammatory processes(33). Several studies reported that LMW HA induces inflammatory responses whereas HMW HA has a protective effect both in vitro and in vivo(34-36). The differential activities of HA and its degradation products are partly due to regulation of several HA-binding proteins, including CD44(36, 37). Our previous study showed that administration of HMW HA into the perfusate of human lungs improved lung permeability and reduced inflammation following ALI induced by E. coli EV; the method of action was by reducing the release of TNF-α and IL-6 from injured monocytes through blocking CD44(4). Therefore, we focused on the possible therapeutic effect of HMW HA against PA pneumonia in mice. We initially tried to administer HMW HA intra-tracheally, but it was technically difficult because of the high viscosity of HA in such a small volume of instillate. In addition, because we previously found that intravenous administration of HMW HA increased HMW HA levels in the BALF(4), we changed to the intravenous route.

There were significantly higher body temperatures in the HMW HA treatment group than in the injury group 24 h after infection and a significantly lower W/D weight ratio in the lungs as well as TNF-α levels in the supernatant of lung homogenates than in the injury group (Fig. 3A, 4A and D). These results suggested that HMW HA was effective in restoring lung permeability and mitigating inflammation, as shown by histology (Fig. 4C), probably by inhibiting the release of factors such as cytokines in EVs via CD44 receptors as well as by repairing damaged vascular endothelium(38) and reducing the influx of immune cells(33). Although some studies showed that HMW HA ameliorated lung inflammation by promoting the production of anti-inflammatory cytokine IL-10(39, 40), there was no significant difference between the two groups, suggesting that HMW HA did not contribute to induce IL-10 secretion in our study.

In addition, the lower bacterial CFU count in the lungs in the HA treatment group than in the injury group suggested that HMW HA had bacteriostatic properties(41, 42). There are several studies reporting the antibacterial effect of HMW HA. As we showed previously, HMW HA enhanced bacterial phagocytosis by immune cells, possibly through binding to CD44 receptor and increasing phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 on the monocytes/macrophages(43). HMW HA may also compete with bacterial adhesion and prevent microbial colonization(44-46). Furthermore, the protective effect of HMW HA on the integrity of the endothelium may prevent translocation of bacteria(47).

Caspase-3 in the supernatant of lung homogenates was also significantly lower in the HA treatment group. According to our in vitro cytotoxicity assay and immunoblot analysis, HMW HA interfered with apoptosis induced by I-EVs. The therapeutic effect in the 2-h HA group was superior to that in the 4-h group, suggesting that I-EVs had already been released at 4 h after infection and that HMW HA should be administered before the inflammatory response proceeds. The peak level of activated caspase-3 in the Western blot analysis was around 1 h after administration of I-EVs (Fig. 2D), supporting the hypothesis that earlier administration of HMW HA was important to prevent ALI caused by P. aeruginosa.

Recently, most bacteria are recognized as capable of secreting EVs, which are also referred to as membrane vesicles (MV)(48, 49). MVs can carry virulence factors and increase pathogenicity(50). P. aeruginosa has multiple virulence factors such as lipopolysaccharides, flagella, exotoxin A, typeIII secretion system, biofilm formation, and quorum sensing(51). PA103 mainly secretes type III toxins, especially ExoU, which is a major pathogenic cytotoxin with phospholipase A2 activity that causes alveolar epithelial injury, macrophage necrosis(52, 53), and increased morbidity and mortality(54). Because we found that EVs from BALF of infected mice were cytotoxic, we hypothesized that the I-EVs may derive from P. aeruginosa and/or some toxins were included in the EVs. We collected EVs from cultured PA103 (PA-EV) and analyzed the cytotoxicity against A549 cells. Contrary to the EVs derived from infected mice, PA-EVs had no cytotoxicity toward A549 cells, suggesting that the cytotoxicity of EVs may not derive from PA103. Gram negative bacteria also have been reported to release vesicles in response to variety of stress such as protein accumulation in the cell envelope(55, 56). There is a possibility that PA-EV were secreted by the bacteria for removal of unwanted cellular materials, not for the purpose of transporting known PA toxins.

Although we performed immunoblot analysis to detect toxins in the I-EVs, we could not detect either ExoU and exotoxin A in the I-EVs. We were also not able to detect toxins from the supernatant of homogenized lungs. A possible explanation was that there was not enough concentration of toxins in the samples 24 h after infection and was difficult to detect with Western blot analysis. Considering these results, the cytotoxicity of I-EV may derive from host cells such as epithelial cells and immune cells(19, 20), other than PA103.

There are some limitations in the current study: (1) Lack of other surface markers that identify the origin of EVs; (2) Lack of analysis of substances such as mRNA or proteins other than toxins to identify the pro-inflammatory factors contained in EVs; (3) Difficulty collecting enough I-EVs for intratracheal instillation in the mice model and inability to evaluate the in vivo cytotoxicity of I-EVs; (4) And although CD44 is a known receptor for HMW HA on EVs released from immune cells, studies are on-going to determine if the therapeutic response is dependent on HMW HA directly binding to I-EV in vivo.

CONCLUSION

EVs isolated from BALF of mice with P. aeruginosa pneumonia are cytotoxic and induce apoptosis. Blocking the CD44 receptor plays a key role in preventing ALI caused by cytotoxic EVs. Intravenous HMW HA administration has a protective effect against P. aeruginosa pneumonia through possibly binding to the CD44 receptor and could be a new therapeutic option combined with conventional therapy such as antibiotics. Further studies are needed to determine the mechanism of cytotoxicity of EVs and to verify the therapeutic effect of HMW HA in a larger animal model.

Acknowledgment of grants:

National Institute of Health National Heart, Lung, and Blood Institute grant number HL 113022 and HL 148781 (Dr. JW Lee).

Abbreviations:

ALI

Acute lung injury

ARDS

Acute respiratory distress syndrome

BALF

Bronchoalveolar lavage fluid

CFU

Colony forming unit

DAB

Diaminobenzidine

ELISA

Enzyme-linked immunosorbent assay

EV

Extracellular vesicles

HA

Hyaluronic acid

HAS

Hyaluronan synthases

HMW

High-molecular-weight

MV

Membrane vesicles

PBS

Phosphate buffered saline

PVDF

Polyvinylidene fluoride

SDS-PAGE

Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis

WBC

White blood cell

W/D

Wet/Dry

Footnotes

Financial Disclosure and Conflicts of Interest: None for all authors.

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