Ventilator-associated pneumonia (VAP) is a major public health concern due to the increasing morbidity and mortality rates among intensive care unit (ICU) patients (1). Acinetobacter baumannii (Ab) is the most common pathogen known to cause VAP (2). As antimicrobial resistance is prevalent in patients infected with Ab, options for treating this infection are limited (3). Therefore, a better understanding of the pathophysiology of Ab infection is of great clinical significance for the development of targeted novel therapies for patients with Acinetobacter baumannii ventilator-associated pneumonia (Ab-VAP).
Previous research that examined bronchoalveolar lavage fluid (BALF) found that “exosome containers” in this fluid mediate intercellular communication in respiratory diseases (4-6). Exosomes have been shown in vivo to serve as containers that carry and rapidly release selective molecules, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) (7). Recent studies have also shown that exosomal miRNAs in BALF and serum exhibit distinct features in patients with asthma, acute respiratory distress syndrome, and pulmonary infection (5,8-11). Exosomes transfer miRNAs to target cells, allowing these miRNAs to exert their effects on these targets (12). Patients with pulmonary infection, particularly that caused by Ab, often exhibit systemic inflammasome activation and metabolism dysregulation (1); however, there is a lack of evidence describing the mechanisms by which BALF exosomes modulate these patients’ circulatory metabolism and influence disease progression.
Metabolomics studies have shown that alterations occur in the circulatory metabolism of patients with pulmonary infection (13,14). Critical pulmonary infection can cause malnutrition, gut microbiota dysregulation, and changes in energy metabolism, which can result in metabolic disturbance (15-17). Due to its persistent course, the metabolic features of Ab infection are well defined. However, few studies have discussed the global metabolic profiles of patients with Ab-VAP and the mechanisms by which they are dysregulated. Recent studies have shown that adipose-derived exosomal miRNAs can modulate systemic metabolism by interfering with gene expression in distant tissues, providing a novel mechanism of cell–cell crosstalk (7). However, the effect of altered BALF exosomal miRNA profiles on serum metabolite profiles during pulmonary infection has not been explored. We therefore hypothesized that BALF exosomes released from both immune and structural lung cells carry miRNAs to target organs where they exert effects that alter the serum metabolite profiles of Ab-VAP patients.
In this study, we investigated BALF exosomal miRNA profiles and serum metabolic profiles to study the associations between lung tissue-derived exosomal miRNAs and changes in the global metabolism of Ab-VAP patients. We showed that the profile of exosomal miRNAs expressed in BALF and serum, as well as the serum metabolite profiles, were significantly different in Ab-VAP patients compared with community-acquired pneumonia (CAP) patients. These altered exosomal miRNA profiles were significantly enriched in cellular metabolic pathways and notably correlated with the metabolism of alcohols, indoles, and alkylamines. This study provides new evidence that highlights how lung tissue-derived exosomal miRNAs affect systemic metabolism. We also revealed potential therapeutic targets for the treatment of Ab infection.
Between March 2018 and September 2018, 40 patients that attended the First Affiliated Hospital of Xi’an Jiaotong University in China were enrolled in this study. Of these patients, 15 were diagnosed with VAP and 25 were diagnosed with CAP; the CAP patients were the control group. The age range of the study cohort was 50–75 years. This study was approved by the Ethics Committee of Xi’an Jiaotong University (2018G-169), and all patients gave written informed consent according to the Declaration of Helsinki (as revised in 2013). Ab was present in the BALF collected from VAP patients; it was not present in that of CAP patients. Subjects were excluded if they met any of the following criteria: they were taking or had previously taken any drugs known to influence lipid metabolism or endocrine function; they had a chronic respiratory disease, cardiac disease, or endocrine disease; they were undergoing hemodialysis for renal failure; they had acute or chronic hepatitis with increased transaminase activities; or they had a malignant tumor. Demographic information and biochemical measurements were obtained from all subjects as described in previous studies (18-20).
BALF and serum collection and isolation of exosomes
During bronchoscopy, 20 mL of BALF was collected from each patient. Serum from participants was collected after overnight fast. The collected BALF and serum were then centrifuged at 3,000 rpm for 15 min to separate cells and cell debris. The supernatant was then removed. Following the manufacturer’s instructions, exosomes were precipitated using System Biosciences’ (SBI’s) ExoQuick Kit. The precipitated exosomes were then resuspended in 100 µL 1× phosphate-buffered saline (PBS). The size and morphology of the exosomes were examined using a Hitachi HT7700 electron microscope (21). Each preparation of exosomes was based on Minimal Information for Study of Extracellular Vesicles 2018 (MISEV2018), including quantitative measures of BALF exosomes, testing for exosome abundance (protein content), and the presence of exosome-associated components. The protein concentrations of the resuspended pellet preparations were measured by detergent compatible (DC) protein assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.
Exosomal miRNA sequencing
Trizol reagent (1 mL) was added to the exosome suspension, and RNA was extracted following the Trizol reagent manual. The RNA was precipitated in 1:1 isopropanol (v/v) and 1 µL glycogen at −20 °C overnight. An exosomal miRNA library was constructed with a QIAseq miRNA library construction kit (QIAGEN) used in accordance with the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq-2500 sequencer for 50 cycles. Reads that passed the Illumina quality filters were retained for analysis. Adapters were trimmed from the reads and reads shorter than 17 nt were discarded. The remaining reads were then mapped to the miRBase human miRNA reference database using the FANSe3 algorithm on the Chi-Cloud NGS Analysis Platform (Chi-Biotech Co. Ltd., Shenzhen, China) (21).
Serum sample preparation and biochemical measurements
Serum samples were collected from both VAP and CAP patients on admission. Fasting venous blood was withdrawn the next morning and immediately centrifuged at 3,000 rpm for 10 min at 4 °C. Separated serum was stored at −80 °C and aliquots were thawed for later processing as described in previous publications (18,20). Biochemical tests, including a complete blood count, a c-reactive protein (CRP) test, a procalcitonin (PCT) test, and tests for liver and kidney function, were administered following blood collection to determine if subjects should be included or excluded in the study.
Serum metabolism profile determination
Non-targeted metabolomics profiling was performed using the XploreMET platform (Metabo-Profile Biotechnology, Shanghai, China) (18,20). The sample preparation, instrumentation, metabolic annotation, and data analysis procedures were carried out as described in previous publications (22-24) with some minor modifications. The metabolites detected by this process included alcohols, aldehydes, alkylamines, amino acids, carbohydrates, fatty acids, hormones, indoles, lipids, nucleotides, organic acids, phenols, and vitamins. The ratios between metabolites correlated with biological significance were also calculated to explore the potential of dysregulated enzymes and pathways.
RNA isolation and real-time quantitative polymerase chain reaction (PCR) analysis
Total RNA was isolated from the extracted serum exosomes. First-strand complementary DNA (cDNA) was generated from normalized RNA using random hexamer primers and the Superscript II kit (Invitrogen). Primers were designed by the Roche Universal Probe Library Assay Design Center. Two units of sensiFAST probe No-ROX Mix (Bioline) were used in each 96-well reaction plate (Axon). A Roche LightCycler® 480 was used to perform the quantitative polymerase chain reaction (qPCR) and the real-time qPCR was repeated three times for each condition (25).
Data are presented as mean ± standard error of the mean (SEM). for continuous variables and as a percentage for categorical variables. The miRNA reads were mapped to the miRBase human miRNA reference database using the FANSe3 algorithm and data were processed on the Chi-Cloud NGS Analysis Platform as described in previous publications (21). The expression levels of miRNAs in liver blood, lung blood, and peripheral blood were extracted from the Human miRNA Tissue Atlas (26). The serum metabolomics data were normalized using MetaboAnalyst before they were analyzed as described in previous publications (27-36). Enrichment analyses were performed based on metabolic pathway-associated and drug pathway-associated metabolite sets (37,38). Correlation and regression analyses of metabolites with the demographic information and biochemical measurements were calculated using Pearson’s correlation (SPSS 20.0). Heatmaps and volcano plots were created using R studio. P values of <0.05 were considered statistically significant.
BALF exosomes and serum exosomes indicated differential miRNA profiles in Ab-VAP patients compared with CAP patients
To explore whether exosomal miRNA profiles were altered in Ab-VAP patients, we collected BALF from these patients and from control CAP patients (Table 1), and performed exosome isolation using SBI kits (Figure 1A). Exosome sizes were measured by electron microscopy (Figure 1B), and we also sequenced the exosomal miRNAs. We found that miRNAs were differentially modulated in Ab-VAP patients compared with control patients. This is shown in a heatmap of the top 198 altered miRNAs (Figure 1C) and a volcano plot (Figure 1D). The top 25 downregulated miRNAs are shown in Figure 1E and the top 25 upregulated miRNAs are shown in Figure 1F. To determine if these differential miRNAs were derived from lung tissue, we searched the Human miRNA Tissue Atlas (26), as shown in Table 2. We found that, in general, these miRNAs were highly expressed in lung tissue. This further suggests that the exosomes came from lung tissue.
Significantly altered miRNA expression in BALF was correlated with serum exosome expression
A recent study identified the systemic function of exosomal miRNAs that target gene expression in liver metabolism (7). We therefore searched the Human miRNA Tissue Atlas to determine if the top 25 miRNAs with altered profiles (26), as found in our study, were expressed in liver blood and peripheral blood. The expression levels of the top 25 differentially expressed miRNAs in liver blood, lung blood, and peripheral blood are listed in Table S2. We selected miRNAs from the Human miRNA Tissue Atlas that are expressed more abundantly in the serum exosome In line with the results of our BALF analysis, qPCR analysis of serum exosomes also showed significant differential expression of miRNAs such as miR-338-3p, miR-26b-5p, miR-30e-5p, and miR-424-5p (Figure 2A). This suggests that miRNAs excreted from lung cells can enter the circulatory system and affect distant organs.
Differentially expressed miRNAs were significantly enriched in metabolic pathways
As differentially expressed miRNAs were detected in Ab-VAP patients, we sought to determine if these altered miRNA profiles played a crucial role in metabolic pathways. We used KEGG pathway analysis and Gene Ontology enrichment analysis based on the top 100 differential miRNAs found in our study. KEGG pathway analysis showed enrichment of a number of molecules related to metabolic signaling pathways, including PI3K-Akt, thyroid hormone, and AMPK; these molecules were also enriched in other cancer-related and cytoskeleton signaling pathways (Figure 3A; Table S1). Gene Ontology enrichment analysis further showed enrichment in metabolism and cell–cell crosstalk, as demonstrated by biological processes, cellular components, and molecular functions (Figure 3B; Table S2).
Serum metabolites were differentially regulated in Ab-VAP patients
Alterations in miRNA expression profiles and the results of our enrichment analysis indicated that BALF exosomal miRNA may play a role in systemic metabolism. We therefore investigated changes in the circulatory metabolic profiles of Ab-VAP patients. A total of 227 serum metabolites, including alcohols, amino acids, carbohydrates, and fatty acids, were detected in the serum of these patients. The differences in overall metabolite class values between the Ab-VAP patients and the control CAP patients are presented as a heatmap. No significant differences existed between the two groups (Figure 4A).
We then compared the metabolite levels and ratios of biological significance between CAP patients and Ab-VAP patients. A volcano plot was used to show significantly altered metabolite profiles and ratios with P values of <0.05 with fold change more than twice (Figure 4B). Of these metabolites, the profiles of hydroxypropionic acid, D-Xylose, ornithine, and caproic acid (Figure 4C) were found to be significantly altered. The ratios of biological significance between L-valine and alpha-Ketoisovaleric acid, as well as that between tryptamine and L-tryptophan (Figure 4D), were also significantly altered.
Serum metabolites were differentially regulated in metabolic pathway-associated metabolite sets and drug pathway-associated metabolite sets in Ab-VAP patients
Based on the circulatory metabolic profiles described above, we further determined whether this altered metabolite expression played a role in different metabolic pathways. Enrichment analyses were performed to identify metabolite sets grouped by involvement with the same biological processes or drug-associated pathways. The strongest associations between altered metabolite expression and different biological processes were found in the urea cycle and phenylacetate metabolism (Figure 5A; Table S3). As Ab infections have a long duration and are resistant to antibiotics, we also analyzed the relationships between altered metabolite profiles and drug-associated pathways in the two groups. Metabolites with altered expression in Ab-VAP patients were shown to be involved in a number of drug pathways (Figure 5B; Table S4). These pathways are associated with cell cycle suppression through the inhibition of purine metabolism.
Differential BALF exosomal miRNAs were correlated with serum metabolite levels
As we found alterations in both BALF exosomal miRNA profiles and serum metabolite profiles in Ab-VAP patients, we then investigated the relationship between BALF miRNA and systemic metabolism. First, we analyzed the correlations between the top 25 altered BALF exosomal miRNA profiles and classes of metabolites. All 25 miRNAs were significantly correlated with serum alcohol levels, and a majority of these miRNAs were also significantly correlated with indoles; this is consistent with the alterations found in the metabolite ratios (Figure 6A). Figure 6B shows lines drawn to connect metabolites and miRNAs with significant correlations in a network. These correlation analyses also confirmed that hydroxypropionic acid and ornithine were significantly correlated with several miRNAs, which indicates pathophysiological significance (Table S5). We can infer from this and our results that exosomes released from lung immune and structural cells may enter the circulatory system and carry miRNAs to the liver. Once in the liver, these miRNAs perform functions that affect serum metabolite profiles (Figure 6C). The exact mechanism by which these miRNAs achieve this, however, requires in vivo and in vitro investigation.
Although BALF exosomal miRNAs have been shown to regulate a wide range of inflammatory processes (4,8-11), their function in patients with Ab infection has yet to be investigated. In the present study, we found significantly altered exosomal miRNA profiles in the BALF of patients with Ab-VAP. Gene Ontology analysis further distinguished the functions of these miRNAs in systemic metabolism. We also found that the serum metabolomic profiles and ratios of biological significance were differentially regulated in patients with Ab-VAP; these changes were correlated with the differential expression of miRNAs. The differential expression pattern of exosomal miRNAs found in the lungs, liver, and circulation implies that BALF exosomes originating from immune and structural lung cells might carry miRNAs into the circulatory system to target systemic metabolism.
Although BALF has been shown to be involved in the development of asthma, lung cancer, and interstitial lung disease (4,8-11), its role in pulmonary infections remains unclear. Respiratory exosomes, nanovesicles circulating in the respiratory tract, have been shown to be involved in the progression of inflammation-related lung damage (6). The present study examined the miRNA content of lung-derived exosomes isolated from BALF in Ab-VAP patients and CAP patients. The differential regulation of exosomal miRNAs that occurs in VAP suggests that lung-derived exosomes are involved in the propagation of this disease during Ab infection. The discovery of these alterations of BALF exosomal miRNA profiles provides potential biomarkers and novel insight into the underlying mechanisms of Ab infection, as well as the development of strategies to treat this infection.
Studies on serum metabolism have provided evidence that improves the understanding of pathophysiological mechanisms of pulmonary infectious diseases. These studies have shown that many serum metabolites could be used as biomarkers to detect metabolic disorders in patients with pneumonia (13,14). Although metabolic disturbances are known to occur in pneumonia, few studies have discussed metabolic profile changes in pneumonia patients infected with Ab. Our results have shown that serum metabolism is differentially regulated during Ab infection. It should be noted that, in our study, only a few serum metabolites showed altered profiles in VAP patients compared with CAP patients. One reason for this could be our limited sample size. Another could be that, since both CAP and VAP are associated with systemic inflammation, circulatory metabolites might have undergone similar inflammatory changes. Whatever the cause, these metabolites that are differentially expressed in Ab-VAP have been shown to affect several drug pathways involved in the suppression of cell cycles by purine metabolism inhibition. This identifies several potential therapeutic targets for this disease, although their pathophysiological mechanisms require further exploration.
The primary novelty of our results is the notion that exosomal miRNAs derived from the lungs might enter blood circulation and regulate systemic metabolism. Consistent with these findings, we found that differentially expressed miRNAs carried in BALF exosomes are present in blood samples taken from the lungs, liver, and peripheral blood. We also found that these altered miRNA levels are correlated with changes in circulatory metabolism. This implies that BALF exosomes derived from both immune and structural lung cells might carry miRNAs into the circulatory system. Once in this system, the miRNAs are transported to target metabolism-related organs where they carry out functions that regulate the circulatory metabolism profile.
The primary limitation of the present study was that the sample size was relatively small; few patients between the ages of 50 and 75 with critical pulmonary infections met our study’s inclusion criteria. Finding and recruiting CAP and VAP patients admitted to the ICU due to pulmonary infection who also have no notable medical history, including severe renal or hepatic damage, is challenging. Additionally, although we can infer from our results that lung tissue-derived exosomes might be released into peripheral blood circulation and modulate metabolism in Ab-VAP patients, further in vitro and in vivo studies are necessary to confirm both this cell-cell crosstalk function and the organs targeted by BALF exosomes.
The present study identified altered BALF exosomal miRNA profiles in Ab-VAP patients. These miRNAs are enriched in metabolism-related pathways and are correlated with serum metabolism changes during Ab infection. Our data implies that lung-derived exosomal miRNAs might enter the peripheral blood circulation and regulate systemic metabolism. These metabolic changes could indicate potential novel metabolic targets for the treatment of Ab infection.
We thank all patients involved in the study.
Funding: This study was supported by National Natural Science Foundation of China (81800390), Key Research and Development Program of Shaanxi (Program No. 2020KW-049)x, the Fundamental Research Funds for the Central Universities in China (1191329724, 191329849), and the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University in China (XJTU1AF-CRF-2018-025).
Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-20-2375
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-2375). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Written informed consent was obtained according to the Declaration of Helsinki (as revised in 2013), and was approved by the ethics committee, Xi’an Jiaotong University (2018G-169).
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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