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Published in final edited form as: J Trauma Acute Care Surg. 2024 Nov 29;98(1):55–63. doi: 10.1097/TA.0000000000004499

Lung Contusion Complicated by Pneumonia Worsens Lung Injury via the Inflammatory Effect of Alveolar Small Extracellular Vesicles on Macrophages and Epithelial Cells

Keita Nakatsutsumi 1, Wooil Choi 1, William Johnston 1, Katie Pool 1, Dong Jun Park 1, Jessica L Weaver 1, Raul Coimbra 1, Brian Eliceiri 1, ToddW Costantini 1
PMCID: PMC12978313  NIHMSID: NIHMS2136429  PMID: 39621452

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Lung contusion complicated by pneumonia worsens lung injury via the inflammatory effect of small extracellular vesicles in the alveolar space on macrophages and epithelial cells.

Keywords: #Trauma, #Lung contusion, #Pneumonia, #Extracellular vesicles, # Acute lung injury

BACKGROUND

Lung contusion (LC) after blunt chest trauma is associated with high mortality (1, 2). LC primes an exaggerated local and systemic inflammatory response and places the patient at risk for subsequent bacterial pneumonia (3, 4). Pneumonia following LC contributes to the development of Acute Lung Injury (ALI) and multiple organ dysfunction, which leads to higher mortality and longer length of intensive care unit stay (5–7). Currently, there are no treatments to prevent the pulmonary dysfunction associated with a second hit after trauma. Thus, understanding the molecular mechanism that drives the lung inflammatory response after LC complicated by pneumonia remains a key area of research.

Extracellular vesicles (EVs), lipid-bilayer particles (<1000 nm) derived from cells, play a crucial role in cell-to-cell communication by transferring intracellular products and can contribute to proinflammatory immune processes in disease development (8, 9). EVs in the alveolar space are released from multiple cellular sources in the lung, including epithelial cells, endothelial cells, and resident immune cells, including alveolar macrophages (10). These alveolar EVs transfer molecular cargo, including proteins, cytokines, lipids, and microRNAs to other pulmonary cells and trigger their immune response, resulting in the production of proinflammatory mediators (11). This cellular cross-talk can serve as a key factor in airway inflammation (10, 11). Small EVs (sEVs) (<200nm), also known as exosomes, are the focus of our studies, as they are well-defined nanocarriers of biologically active nucleic acids and proteins (12, 13). Alveolar sEVs play an essential role in the pathogenesis of airway inflammatory diseases such as asthma by regulating pulmonary immune cell function (14). Moreover, alveolar sEVs can increase the release of inflammatory mediators and disrupt the integrity of alveolar epithelial barriers, which results in the development of ALI (15). Also, we have recently shown the increased expression of CD44 on alveolar sEVs relevant to immune-epithelial cross-talk in a burn-induced ALI mouse model and injured patients (16). Thus, understanding how sEVs may contribute to post-injury ALI may provide valuable insights into the pathology of LC complicated by pneumonia.

The present study aims to evaluate the ability of alveolar sEVs to contribute to the lung inflammatory response of macrophages and epithelial cells in a model of Pseudomonas aeruginosa (P. aeruginosa) pneumonia following LC. We hypothesized that LC complicated by pneumonia increases the pro-inflammatory effect of alveolar sEVs on macrophages and increases the cytotoxicity of alveolar sEVs to pulmonary epithelial cells, thus worsening the severity of ALI.

METHODS

Animal Models

All animal experiments were performed based on protocols approved by our Institutional Animal Care and Use Committee. 8-10 week-old C57BL/6J mice (#JAX000664) were housed in cages under a 12-hour light/dark cycle and provided with standard laboratory chow and tap water ad libitum during the acclimation and experimental periods.

All mice were simultaneously randomized to four groups: Sham, LC, Pneumonia (Pneu), and LC+Pneu (Fig 1A). The LC and LC+Pneu groups were anesthetized with 2% inhaled isoflurane. The right chest was clipped using an electric hair shaver. LC was induced with a cortical contusion impactor (CCI) (Leica Microsystems, Deerfield, IL). The mice were set to the left lateral position. The CCI struck their right chest along the posterior axillary line, 1 cm above the costal margin, using a 5mm probe. The settings were: velocity of 6.0 m/s, depth of penetration of 10 mm, dwell time of 2 sec (17). After the procedure, Ethiqa XR (Fidelis Animal Health, North Brunswick) 3.25 mg/kg was subcutaneously injected into the mice for analgesia. The Sham and Pneu groups were shaved under the same anesthetic and analgesia regimens, but did not receive LC with the CCI. P. aeruginosa (strain PA103, ATCC # 29260) was cultured overnight in Tryptic Soy Broth media (T8907, Sigma-Aldrich, St Luis, MO) at 37 °C. Twenty-four hours after the injury, pneumonia was induced in the Pneu and LC+Pneu groups by intratracheal instillation of 1 x 105 CFU (30 μL) of P. aeruginosa under anesthesia with 2% inhaled isoflurane (18–20). Sham and LC groups were intratracheally injected with 30 μL of PBS as a control. Triplicate biological samples from each group were utilized for every experiment. Figure legends indicate the number of samples per experimental condition. Experimental details are reported using the Animal Research: Reporting of In Vivo Experiments guidelines (ARRIVE).

Figure 1. Lung contusion complicated by pneumonia severely injured lungs and increased susceptibility to P.aeruginosa.

Figure 1.

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(A) LC was created in the LC and LC+Pneu groups. Twenty-four hours after the injury, P.aeruginosa was intratracheally injected into lungs in the Pneu and LC+Pneu groups. Samples were harvested 24 hours later. (B) LC and pneumonia increased neutrophil infiltration and debris in the alveolar space and thickened the alveolar septum. Arrowheads indicate neutrophil infiltration. The LC+Pneu group increased the histological ALI score. (C) LC increased the concentration of P.aeruginosa in BAL, homogenized lungs, and blood. Data were presented as mean ± standard deviation (n=3 for each group). ALI: Acute lung injury, BAL: Bronchoalveolar lavage fluid, IT: Intratracheal instillation, LC: Lung contusion, Pneu: Pneumonia, P. aeruginosa: Pseudomonas aeruginosa. *p < 0.05, **p < 0.01, ***p < 0.001.

Histopathology

Mice were euthanized by cervical dislocation 24 hours after infection. Left lungs not directly contused were harvested from each group, after inflation with 10 % formalin via a 23 G needle inserted into the trachea. Lungs were fixed in 10 % buffered formalin, embedded in paraffin and sectioned at 5 μm thickness for staining with hematoxylin and eosin (H & E) by our institutional Tissue Technology Shared Resource (21). Twenty high-power fields (400x total magnification) were randomly acquired from each sample with a microscope (IX70, Olympus) then evaluated by two independent observers blinded to the study group assignment to calculate the acute lung injury score as previously described (22). Briefly, this scoring method evaluates neutrophil counts in the alveolar and interstitial spaces, proteinaceous debris in the air space, and alveolar septal thickening.

Collection of Bronchoalveolar Lavage Fluid

A 27G needle was inserted into a 0.38 mm inner diameter polyethylene plastic tube to make a catheter. At 24 hours post-infection, mice were placed under general anesthesia, and a tracheotomy was performed for terminal collection of the bronchoalveolar lavage fluid (BAL). The catheter was inserted and tied to the trachea. 1 mL of PBS was gently injected into the lung via the catheter with a 1 mL syringe. Subsequently, BAL was harvested and placed on ice in 1.5 mL microcentrifuge tubes. This procedure was repeated two times per mouse, and the collections were pooled (23). Of the ~2 mL BAL collected from each mouse, 100 μL of harvested BAL was used to measure evaluate bacterial concentration. The rest of the samples were centrifuged at 3,000 ×g for 15 minutes at 4°C to remove cellular components. The supernatants were collected and centrifuged again at 10,000 ×g for 15 minutes at 4°C to further remove debris. Protein concentration in the supernatants was measured using the BCA Protein Assay Kit (Thermo Fisher, Carlsbad, CA) to evaluate lung epithelial barrier injury according to the manufacturer’s recommendations. The supernatants were stored at −80°C for future sEV analysis and Enzyme-linked immunosorbent assay (ELISA).

Bacteria Concentration in BAL, Blood, and Lung

Bacteria concentration in BAL, blood, and homogenized lungs was analyzed to evaluate susceptibility to bacteria in the Pneu and LC+Pneu groups. 100 μL of harvested BAL was used for this assay. In a separate mice cohort, blood was harvested via cardiac puncture, and both lungs were harvested and homogenized in 500 μL of PBS. Serial 10-fold dilutions of the samples were prepared in PBS, and 100 μL of the samples were plated on 5% sheep blood agar plates (Cat# R02053, Thermo Fisher Scientific, Carlsbad, CA). The plates were incubated at 37 °C overnight, and CFU was counted.

sEVs Analysis with Single Vesicle Flow Cytometry

sEVs analysis was conducted based on the methodological recommendations of the Minimal Information for Studies of Extracellular Vesicles 2023 (24). Size exclusion chromatography was performed to isolate sEVs from the BAL supernatants of each group with a qEV1 column (Cat# ICI-70, IZON Science LTD, Portland, OR), according to the manufacturer’s recommendations (12, 25).

Single vesicle flow cytometry (vFC) using a commercial assay based on a fluorescent lipophilic membrane dye, vFRed (Cellarcus Biosciences Inc., La Jolla, CA), with a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) was utilized to analyze sEVs concentration and size as previously described (16, 26). Fluorescent intensity standard beads (nanoRainbow, Cellarcus Biosciences Inc., La Jolla, CA) and antibody capture beads (nanoCal, Cellarcus Biosciences Inc., La Jolla, CA) were used to conduct calibration (27, 28). The flow cytometer could detect a diameter limit of 80 nm. After size exclusion, samples were incubated with lipophilic fluorescent dye vFRed for one hour at room temperature, and sEVs were subjected to a 1,000-fold dilution to reduce swarm effects in the flow cytometer. Subsequently, 120 μL of sample were measured on the flow cytometer at a 60 μL/min flow rate. Liposomes (80-120 nm) (Lipo100, Cat# CBS1, Cellarcus Biosciences Inc., La Jolla, CA) were utilized as a negative control, while mouse platelet-derived EVs (PLT, Cat# CBS2, Cellarcus Biosciences Inc., La Jolla, CA) were utilized as a positive control. All flow cytometry data was analyzed using FCS Express (Version 7, De Novo Software, Pasadena, CA).

Western Blots

Isolated sEV and whole cell lysate samples were diluted with NuPAGE™ LDS Sample Buffer (Cat# NP0008, Thermo Fisher, Carlsbad, CA) and 1 % of fresh 1 M DTT was added to the samples for protein reduction. After heating at 90°C for 5 minutes, samples were loaded onto a 1 mm pre-cast 12% Bis-Tris Mini Gel (Cat# NP0342BOX, Thermo Fisher) and subjected to electrophoresis at 200 V for 42 minutes. Proteins were transferred to a PVDF membrane (Cat# LC2005, 0.45 μm, 8.3 x 7.3 cm, Thermo Fisher) with electrophoresis at 20 V for 60 minutes. The membranes were blocked for one hour using 5% Nonfat Dry Milk (Cat# 9999, CST, MA, USA) in 1X Tris-buffered saline (Cat# 9997, CST) with 0.05% Tween 20. The membranes were incubated overnight at a dilution of 1:1,000 with the primary antibodies anti-CD9 (Cat# 98327, CST, Danvers, MA) at a dilution of 1:500 anti-CD81 (Cat# 10037, CST Danvers, MA) for isolated sEV at a dilution of 1:2,000 with the primary antibodies anti-ICAM-1 (Cat# AF796, R&D Systems, Minneapolis, MN) or at a dilution of 1:2,000 β-actin (Cat# 3700, CST, Danvers, MA) for whole cell lysates. Subsequently, membranes were incubated with the secondary antibody, anti-rabbit IgG HRP-linked antibody (Cat# 7074, CST Danvers, MA), anti-goat IgG HRP-linked antibody (Cat# A15999, Thermo Fisher, Carlsbad, CA), or anti-mouse IgG HRP-linked antibody (Cat# 7076, CST, Danvers, MA) for 1 hour at room temperature. SignalFireTM ECL Reagent (#12757, CST Danvers, MA) was used to image the blots. The Xenogen IVIS-Lumina imager (Caliper Life Science Inc., Hopkinton, MA) was utilized for chemiluminescent detection of immunoblots.

Cell Culture

A mouse macrophage cell line (RAW264.7) (ATCC# TIB-71, ATCC, Manassas, VA) and a mouse lung epithelial cell line (MLE 12) (ATCC# CRL-2110, ATCC, Manassas, VA) were purchased and used for sEV treatment assay. Cells were maintained in Dulbecco’s modified eagle medium (DMEM; Cat# 12430054, Gibco, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS; Cat# F0926, Sigma-Aldrich, St.Louis, MO) and 1 × antibiotic-antimycotic (AA; Cat# 15240062, Gibco, Carlsbad, CA) in a humidified chamber supplemented with 5% CO2 at 37°C. Cell counting was performed with a hemocytometer with dead cells being stained with 0.4% trypan blue (Cat# T10282, Thermo Fisher, Carlsbad, CA).

sEV Treatment of Macrophages

A total of 1 x 105 RAW 264.7 cells were seeded onto wells of a 12-well plate and cultured for 24 hours. 100 μL of sEVs (5.0 × 108 in 100 μL) harvested from each group was added to each well. Twenty-four hours post-treatment, the cell supernatant was collected and centrifuged at 500 ×g for 5 minutes from which cell lysates were made in RIPA cell lysis buffer (Cat# 89901, Thermo Fisher, Carlsbad, CA) supplemented with 1 × protease & phosphatase inhibitor cocktail (HaltTM protease & phosphatase single-use inhibitor cocktail (100X), PIC, Cat# 78442, Thermo Fisher, Carlsbad, CA). The supernatants and whole cell lysates were stored at −80°C for ELISA and western blots.

Enzyme-linked immunosorbent assay

ELISAs were conducted to evaluate the cytokine concentration in BAL and the cytokine release from macrophages incubated with sEVs. Commercially available ELISA kits were utilized to evaluate Macrophage inflammatory protein 1-alpha (MIP1 α) and Intercellular Adhesion Molecule 1 (ICAM-1) in BAL, cell supernatant samples, and sEV lysates from each group, based on the manufacturer’s protocol (Cat# DY450-05, DY796, R&D Systems, Minneapolis, MN). Furthermore, Caspase-3 ELISA for lysate RAW 264.7 cells after sEV treatment was performed to evaluate cell death in macrophages (Cat# DYC835-2, R&D Systems, Minneapolis, MN). As a pilot study, we also comprehensively assessed several cytokine concentrations in RAW 264.7 cells incubated with sEVs to pick up targeted cytokines, using the Proteome Profiler Mouse Cytokine Array Kit based on the manufacturer’s protocol (Cat# ARY006, R&D Systems, Minneapolis, MN).

Epithelial Cytotoxicity Assay

The cytotoxicity of sEVs to MLE 12 epithelial cells was evaluated with a commercially available kit, cell counting kit-8, based on the manufacturer’s protocol (Cat# CK04, Donjindo, Japan). A total of 5 × 103 MLE 12 cells were seeded on a 96-well plate and cultured for 24 hours. 10 μL of sEVs (5.0 × 107 in 10μL) harvested from each group was added per well. Twenty-four hours after treatment, the cytotoxicity assay reagent was added to the wells. The absorbance at 450 nm was analyzed with a spectrometer, and the viability of the cells was calculated.

Epithelial Apoptosis Assay

Epithelial cell apoptosis caused by sEVs was analyzed using a commercially available assay kit (Click-iT Plus TUNEL Assay, Cat# C10617, Life Technologies, Carlsbad, CA). A total of 1 × 105 MLE 12 cells were seeded on wells of a 12-well plate (black frame 12-well plate with glass-like polymer bottom, Cat# P12-1.5P, Cellvis, Mountain View, CA) and cultured for 24 hours. 100 μL of the sEVs (5.0 × 108 in 100 μL) harvested from each group was added to each well. Twenty-four hours after treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and incubated with TUNEL working solutions based on the manufacturer’s protocol. The nuclei were stained with DAPI. Fluorescence signals were detected with a confocal microscope (AXR, Nikon, Tokyo, Japan) and quantified by ImageJ v1.54i software .

Statistical Analysis

Data are presented as mean ± standard deviation. A comparison of the two groups was performed using unpaired t-tests. One-way Analysis of variance (ANOVA) with Tukey’s test was performed to compare multiple groups. Differences were considered statistically significant at p-values less than 0.05. We performed sample size estimation referring to the paper comparing bacteria concentrations in lungs of Pneu and LC + Pneu mice (3). Assuming an effect size of 6, a significance of 5%, and a statistical power of 80%, n = 3 per group was enough to detect a difference. All statistical analyses were performed using GraphPad Prism software, version 10. 2. 3 and R 4.4.1 (The R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Pneumonia after Lung Contusion Increased Lung Injury Severity

To evaluate the difference in the severity of indirect lung injury, we performed a histological evaluation of the contralateral lung that was not directly injured by LC. In the LC+Pneu group, there were increased numbers of neutrophils in the alveolar and interstitial space and increased proteinaceous debris in the alveolar space compared to other groups. Blinded histological evaluation demonstrated increased lung injury score in the LC+Pneu group (p<0.01) (Fig. 1B) (Supplementary Table 1 (sTable 1)). These results demonstrated that LC complicated by pneumonia worsened histologic lung injury.

Next, we evaluated changes in bacterial load between groups. The bacteria concentration in BAL, blood, and homogenized lungs increased in the LC+Pneu group compared to the Pneu group (BAL, p=0.02; Blood, p<0.01; Lungs, p=0.05) (Fig. 1C) (sTable 1), demonstrating that LC increased the susceptibility to P. aeruginosa infection.

Small EV Purification

BAL was collected from each group 24 hours after infection. Serial centrifugation and size exclusion chromatography were performed to separate sEVs from cells and free protein (Fig 2A). The sEVs-most-enriched fraction in each sample was utilized for further analysis (16, 26). sEVs were quantified, and their diameter was calculated by staining with a fluorescent membrane dye, vFRED. The number and size of sEVs did not show a difference between groups (Number, p=0.69; Size, p=0.12) (Fig. 2B, C) (sTable 1). The expression of sEV markers, CD9 and CD81 in each group was determined by Western blotting (Fig. 2D).

Figure 2. Alveolar sEVs were purified with size-exclusion chromatography.

Figure 2.

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(A) sEVs were isolated by serial centrifugation and size exclusion chromatography. (B, C) There was no difference in the number and size of alveolar sEVs between each group. The representative histogram from each group showed the sEVs size distribution with median and 25-75 percentile. (D) Western blotting demonstrated the expression of CD9 and CD81 in alveolar sEVs from each group. Data were presented as mean ± SD (n = 3 for each group). LC: Lung contusion, Pneu: Pneumonia; sEVs: Small extracellular vesicles.

Alveolar sEVs from LC with Pneumonia Increased Cytokine Expression in Macrophages

As a pilot study, we performed a survey of changes in several cytokines from RAW 264.7 cells incubated with sEVs, using the Proteome Profiler Mouse Cytokine Array Kit. This demonstrated increases in MIP1α and ICAM-1 and led to further evaluation of these cytokines in subsequent experiments.

In vivo, the concentration of MIP1α and soluble ICAM-1 increased in BAL of the LC+Pneu group (MIP1α, p<0.01; ICAM-1, p=0.01) (Fig 3A) (sTable 1). Thes results suggest that LC complicated by pneumonia was associated with increased lung inflammation in vivo.

Figure 3. Alveolar sEVs from the LC+Pneu group increased cytokine expression in macrophages.

Figure 3.

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(A) Increased concentration of MIP1α and ICAM-1 in BAL in the LC+Pneu group. (B) Increased concentration of MIP1α and ICAM-1 in cell supernatants from RAW264.7 cells incubated with alveolar sEVs of the LC+Pneu group. (C) Increased expression of ICAM-1 on macrophages in the LC+pneu group. β-actin was utilized as a loading control. Data were presented as mean ± standard deviation (n = 3 for each group). BAL: Bronchoalveolar lavage fluid, ICAM-1: Intercellular Adhesion Molecule 1, LC: Lung contusion, MIP1α: Macrophage inflammatory protein 1-alpha, Pneu: Pneumonia; sEVs: Small extracellular vesicles. *p < 0.05, **p < 0.01, ****p < 0.0001.

Next, to clarify the contribution of alveolar sEVs to the macrophage inflammatory response, we evaluated MIP1α and soluble ICAM-1 concentration in cell supernatants from macrophages incubated with alveolar sEVs of each group in vitro. The concentration increased in the LC+Pneu group (MIP1α, p=0.02; ICAM-1, p<0.01) (Fig 3B) (sTable 1). In addition to soluble ICAM-1 release, we evaluated the ICAM-1 expression on macrophages incubated with alveolar sEVs by western blotting. The LC+Pneu group demonstrated higher expression of ICAM-1 (Fig. 3C). On the other hand, caspase-3 expression in macrophages did not demonstrate any difference between the groups, suggesting that sEVs did not affect cell death in macrophages (sFig. 1A). We also assessed the MIP1α and ICAM-1 concentration in sEV lysates from each group to evaluate if sEVs were the source of cytokines found in BAL and found no difference between the groups (sFig. 1B). Therefore, alveolar sEVs from the LC+Pneu group activated macrophages and contributed to lung inflammation.

Alveolar sEVs from LC with Pneumonia Injured Lung Epithelial Cells

The protein concentration in BAL was increased in the LC + Pneu group (p<0.01) (Fig 4A) (sTable 1). We then focused on the effect of alveolar sEVs on lung epithelial cells. sEVs from the LC+Pneu group caused increased cytotoxicity to lung epithelial cells and decreased cell viability (p<0.01) (Fig 4B) (sTable 1). Moreover, an apoptosis assay for lung epithelial cells incubated with alveolar sEVs demonstrated an increased ratio of apoptotic cells in the LC+pneu group (p<0.01) (Fig 4C, D) (sTable 1). Therefore, we concluded that alveolar sEVs collected after LC+Pneu directly injured the lung epithelium.

Figure 4. Alveolar sEVs from the LC+Pneu group had higher cytotoxicity and increased epithelial cell apoptosis.

Figure 4.

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(A) The protein concentration in BAL increased in the LC+Pneu group, suggesting epithelial barrier disruption. (B) Alveolar sEVs from the LC+Pneu group demonstrated higher cytotoxicity to lung epithelial cells. (C,D) The TUNEL assay demonstrated that lung epithelial cell apoptosis increased by LC complicated by pneumonia. Data were presented as mean ± standard deviation (n = 3 for each group). BAL: Bronchoalveolar lavage fluid, LC: Lung contusion, Pneu: Pneumonia; sEVs: Small extracellular vesicles. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

DISCUSSION

LC increases the susceptibility to bacterial infection, and pneumonia following LC contributes to the development of ALI and multiple organ dysfunction after trauma. This study demonstrated that sEVs released into the alveolar space after LC complicated by pneumonia increased cytokine expression in macrophages and directly injured lung epithelial cells. These results contribute to clarifying the pathology of LC complicated by pneumonia by defining a role for sEVs and guide the identification of potential therapeutic targets.

Alveolar sEVs have previously been reported to contribute to the development of ALI. For instance, BAL collected from patients with ALI has demonstrated increased concentrations of sEVs composed of various proteins associated with autophagy, necroptosis, and apoptosis (29). Alveolar sEVs in animal models of ALI have the ability to increase macrophage recruitment in BAL and cytokine release from macrophages (30). Another study showed that alveolar sEVs in a mouse ALI model decreased the expression of tight junction proteins in lung epithelial cells, disrupting the epithelial barrier (31). The present study also demonstrated that alveolar sEVs may contribute to the development of trauma-related ALI after LC complicated by pneumonia by increasing the pro-inflammatory response of pulmonary macrophages and causing damage to lung epithelial cells.

Alveolar sEVs cause a pro-inflammatory response from macrophages, which are well-known to be integral to the lung inflammatory response. Macrophages are activated by cellular cross-talk with other pulmonary cells and cause inflammation in the lungs, resulting in the development of ALI (32, 33). Prior studies have demonstrated that lung epithelial cell-derived EVs from an ALI model increased cytokine release from macrophages and contributed to the development of ALI (30, 34). In another study, ALI altered the cargo of alveolar sEVs, which exacerbated nucleotide-binding oligomerization like receptor 3 inflammasome in macrophages (35). Similarly, the present study demonstrated the possibility that alveolar sEVs contribute to the development of trauma-related ALI after LC complicated by pneumonia by enhancing the pro-inflammatory effect of macrophages.

MIP1α and ICAM-1 released from macrophages in BAL are associated with lung inflammation and increased severity of ALI. MIP1α in the alveolar space increases neutrophil recruitment and pro-inflammatory cytokine expression, promoting lung injury (36, 37). ICAM-1 was previously shown to be highly expressed in injured lungs, and soluble ICAM-1 in the alveolar space was associated with increased lung inflammation and injury (38–40). Furthermore, ICAM-1 was highly expressed on macrophages during pro-inflammatory conditions, and its expression was correlated with the severity of inflammation, with enhanced macrophage phagocytosis and efferocytosis.(41, 42). As the present study demonstrated, alveolar sEVs from LC complicated by pneumonia increased the expression of these cytokines in macrophages, which was associated with cytokine increase in BAL, contributing to lung inflammation.

Alveolar sEVs collected after LC complicated by pneumonia injured lung epithelial cells. Epithelial cell dysfunction is essential to ALI progression and is associated with decreased surfactant production and disruption of the alveolar barrier (43). A prior study demonstrated that alveolar sEVs collected in a mouse model of pneumonia caused increased cytotoxicity to lung epithelium by inducing apoptosis of epithelial cells through caspase-activating pathways (44). Apoptosis pathways are activated in the lung during ALI, and the extent of lung epithelial cell apoptosis has been associated with the severity of ALI (45, 46). Furthermore, sEVs have been reported as a regulator of cell death, including apoptosis pathways under lung inflammatory conditions. (47). Here, we have demonstrated that LC complicated by pneumonia can increase the cytotoxicity of alveolar sEVs and cause lung epithelial cell apoptosis, which is associated with the development of trauma-related ALI.

There are some limitations in the present study. We demonstrated that LC complicated by pneumonia altered the activity of alveolar sEVs to pulmonary cells, and alveolar sEVs can play an important role in the second hit context; however, we did not comprehensively evaluate how LC and pneumonia alter sEV cargo. As the number and size of alveolar sEVs did not change after LC and pneumonia, the alteration of sEV cargo is assumed to be the driver of the inflammatory response. To further elucidate the mechanism and to find a new treatment approach via sEVs, further studies focusing on sEV cargo will be essential. Also, we did not evaluate the cellular origin of alveolar sEVs. Using transgenic mice to label cell-specific EVs will be important to define the cellular origin of pro-inflammatory sEVs in future studies (48). Another limitation is the time course. We decided on the exposure period of bacteria or sEVs based on past papers (3, 6, 49) and performed single-time-course experiments; however, the exposure time difference can possibly change the activity of sEVs.

In conclusion, LC complicated by pneumonia increased the pro-inflammatory effect of alveolar sEVs on macrophages and the cytotoxicity of alveolar sEVs to pulmonary epithelial cells, which was associated with increased severity of ALI. These results demonstrate the potential importance of alveolar sEVs in lung inflammation following a bacterial infection after trauma.

Supplementary Material

Supplemental Figure 1
Supplemental Table 1
ARRIVE Checklist

Funding information:

Research supported by a grant from the National Institutes of Health (5R35GM149345-02, TWC).

Footnotes

Meetings information: This study will be presented as an Quickshot presentation at the 83rd Annual Meeting of the AAST and Clinical Congress of Acute Care Surgery, September 14th,2024, Las Vegas, NV.

Conflicts of interest: The authors declare no conflict of interest in association with the present study. JTACS COI Disclosure forms for all authors have been supplied and are provided as supplemental digital content.

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Associated Data

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Supplementary Materials

Supplemental Figure 1
NIHMS2136429-supplement-Supplemental_Figure_1.tiff (3.7MB, tiff)
Supplemental Table 1
NIHMS2136429-supplement-Supplemental_Table_1.docx (21.8KB, docx)
ARRIVE Checklist
NIHMS2136429-supplement-ARRIVE_Checklist.pdf (116.2KB, pdf)

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