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. 2023 Mar 29;18(3):e0283702. doi: 10.1371/journal.pone.0283702

Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model

Atsushi Senda 1, Mitsuaki Kojima 1,2,*, Arisa Watanabe 3, Tetsuyuki Kobayashi 3, Koji Morishita 1, Junichi Aiboshi 4, Yasuhiro Otomo 1
Editor: Jon M Jacobs5
PMCID: PMC10058167  PMID: 36989330

Abstract

Intestinal ischemia-reperfusion injury leads to proinflammatory responses via gut-derived mediators, and accumulating evidence suggests that exosomes secreted by intestinal epithelial cells are involved in the development of systemic inflammation. Studies have reported changes in protein, lipid, and microRNA (miRNA) expression; however, considering the different experimental conditions, information on the relationships among these biomolecules remains insufficient. The aim of this study was to elucidate the multiple changes that simultaneously occur in exosomes after ischemic stimulation. Here, differentiated human intestinal Caco-2 cells were exposed to 95% air (normoxia group) or 5% O2 (hypoxia group) for 6 h. Cells in each group were subsequently incubated for 24 h in an atmosphere of 5% CO2 plus 95% air. The conditioned medium of each group was collected for isolating intestinal epithelial cell-derived exosomes. Together with proteome analyses, lipid analyses, and miRNA quantification, biological functional assays were performed using monocytic NF-κB reporter cells. Lipid metabolism-related protein expression was upregulated, miRNA levels were slightly altered, and unsaturated fatty acid-containing lysophosphatidylcholine concentration increased after hypoxia and reoxygenation injury; this suggested that the changes in exosomal components associated with ischemia-reperfusion injury activates inflammation, including the NF-κB pathway. This study elucidated the multiple changes that co-occur in exosomes after ischemic stimulation and partially clarified the mechanism underlying exosome-mediated inflammation after intestinal ischemic recanalization.

Introduction

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions, including trauma, mesenteric ischemia, septic shock, and bowel inflammatory diseases. It is known to cause multiple organ failure after intestinal ischemia-reperfusion injury [1], with dysregulated inflammatory response considered the main cause [2, 3]. This response can be prevented by the ligation of the mesenteric lymph (ML) duct [4]; therefore, ML is considered to have a pathologically crucial role [4–7]. Various ML components have been identified as potential causative agents of this response but have not yet been confirmed. In recent years, an increasing number of studies have suggested that exosomes are the primary causative agent.

Exosomes are nanosized (30–150 nm) extracellular vesicles [8] that carry microRNAs (miRNAs), proteins, and lipids [9]. They have been demonstrated to upregulate monocyte nuclear factor (NF)-κB expression and increase macrophage intra-cellular TNF-α levels, which subsequently cause lung injury [10, 11]. A few studies have identified the exosome cargo responsible for this reaction. One in vivo study identified that polyunsaturated fatty acids containing lysophosphatidylcholine (LPC) upregulate and induce NF-κB expression [12]. Another in vivo study showed changes in the protein composition of exosomes following ischemic stimulation [13]. These results suggest that ischemic stimuli induce alterations in various exosome components that contribute to the subsequent inflammatory response. Therefore, for integrated understanding of the pathogenesis of systemic inflammation, it is necessary to simultaneously capture the changes in multiple components. To achieve this purpose, we intended to establish an in vitro intestinal ischemia-reperfusion injury model as it is currently difficult to harvest a sufficient amount of exosomes from ischemia-reperfusion small-animal models.

Given that ML exosomes mainly originate from intestinal epithelial cells (IECs) [11], we developed an in vitro experimental system that mimics ischemia-reperfusion injury. Corresponding to the ischemic state, cultured IECs were first placed under hypoxic conditions. Subsequently, they were returned to an oxygenated environment corresponding to the reperfusion state. Accordingly, this hypoxia reoxygenation model has been used to simulate ischemia-reperfusion injury in an in vitro setting in previous studies [14, 15]. This facilitated integrated understanding of the changes that occur in the exosome following ischemic stimulation.

Materials and methods

Study design

Differentiated human intestinal Caco-2 (ATCC HTB37) cells were exposed to either 5% CO2 and 95% air (normoxia group) or 5% O2, 5% CO2, and 90% N2 (hypoxia group) for 6 h. Then, the culture medium was changed. To place the cells in an environment analogous to the reperfusion period during hemorrhagic shock, each group was subsequently incubated in a normoxic environment (5% CO2 and 95% air) for 24 h. The conditioned medium of each group was collected for isolating IEC-derived exosomes. Subsequently, biological function assays were performed using monocytic NF-κB reporter cells, proteome analysis was conducted using high-performance liquid chromatography–mass spectrometry (HPLC–MS), and miRNA quantification was conducted using qPCR (S1 Fig).

Cell culture

Caco-2 is a carcinoma epithelial cell line of colonic origin with small intestinal enterocyte-like features [16]. Cells were cultured in Dulbecco’s modified Eagle medium (Nakaraitesk, Kyoto, Japan) supplemented with 2 mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) fetal bovine serum (FBS). THP-1 is a monocytic cell line engineered for monitoring NF-κB activation by determining the activity of secreted embryonic alkaline phosphatase. The growth medium consisted of RPMI-1640 supplemented with 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated FBS, 100 μg/mL normocin, and 100 U/mL penicillin-100 μg/mL streptomycin. Both cell types were transferred to FBS-free medium 72 h before the experiments to avoid contamination [17].

Isolation of exosomes

To avoid contamination, exosomes were isolated from serum-free conditioned medium (Opti-MEM) of IECs according to the method described previously, with some modifications [18]. First, the conditioned medium was centrifuged at 3,000 × g for 15 min; then, the supernatant was passed through a 0.2-μm filter. Next, the medium was centrifuged using the Amicon Ultra-15 (Sigma-Aldrich, St. Louis, MO, USA) Centrifugal Filter Unit with a 100 kDa cutoff at 4,000 × g for 20 min at 25°C for concentration [18]. Through these concentration methods, 80 mL of each media was concentrated into 5 mL. Subsequently, 1 mL of the ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) was added to each sample, and the mixture was incubated overnight for exosomal extraction following the manufacturer’s instructions.

Immunoblotting of exosomal surface proteins

The isolated exosomes were verified via immunoblotting with the Exo-Check Antibody Array (System Biosciences), containing pre-printed spots of eight antibodies against known exosome markers (FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, TSG101, and ANXA5). Samples containing 50 μg of exosome proteins were prepared. The exosome proteins were quantified using the Qubit fluorometer. The samples were subsequently diluted and reacted with the labeling reagent and the blocking buffer according to the manufacturer’s instructions. The labeled exosome lysate/blocking buffer mixture was incubated with the membrane containing pre-printed spots of the eight antibodies at 5°C overnight on a rocker. After removing the lysate/blocking buffer mixture, washing was performed using a washing buffer. Then, the membrane was incubated for 30 min with detection buffer. Finally, a picture of the membrane was captured using an EOS 80D camera (Canon, Tokyo, Japan). All reagents mentioned above were included in the Exo-Check Antibody Array (System Biosciences) and the experimental conditions were maintained following the manufacturer’s instructions. The blot/gel image data are original and have not been cropped or adjusted.

Transmission electron microscopy (TEM)

Exosomal specimens for TEM were prepared by the personnel of the Instrumental Analysis Division at the Research Center of Tokyo Medical and Dental University. The exosomes were immersed in Karnovsky’s fixative (pH 7.42, 2.5% glutaraldehyde, and 2% paraformaldehyde in 0.15 M sodium cacodylate buffer) for 4 h, post-fixed in 0.15 M cacodylate buffer and 1% osmium tetroxide for 60 min, and stained in 2% uranyl acetate for 60 min. The samples were treated with pure ethanol for dehydration, resin-embedded, and sectioned to 50–60-nm thickness using the Leica EM UC7 Ultramicrotome (Leica Microsystems, Wetzlar, Germany). Subsequently, 27 grids were viewed using an H7100 Transmission Electron Microscope (Hitachi High-Technologies, Tokyo, Japan), equipped with an XR81 (8 megapixels) charge-coupled device camera (AMT Imaging Systems, Woburn, MA, USA).

Nanoparticle tracking analysis (NTA)

NanoSight LM10 supplied with fast video capture and NTA software version 2.3 (both Malvern Instruments, Malvern, UK) were used for particle detection, counting, and sizing. The experiment was performed following the manufacturer’s instructions.

Monocyte NF-κB reporter assay

A THP-1 Blue NF-κB reporter monocyte cell suspension of an approximate concentration of 5 × 105 cells/mL was incubated with 20 μL (approximately 1.0 × 108 exosomes) of each sample at 37°C for 18 h. After incubation, 20 μL of sample from each well was extracted and mixed with 180 μL QUANTI-Blue (InvivoGen). The plate was incubated at 37°C for 4 h, and secreted embryonic alkaline phosphatase levels were quantified using a spectrophotometric microplate reader (VersaMax Microplate Reader, Tokyo, Japan) at 620 nm and SoftMax Pro 5.3 software (Molecular Devices, Silicon Valley, CA, USA).

Shotgun protein analysis

For protein analysis, a combination of Nano-HPLC Chromatography System and timsTOF Pro (Bruker Daltonics Billerica, MA, USA) was used. To ensure reproducibility, two identical experiments were conducted using separate samples (H1 and H2; samples under hypoxic conditions; N1 and N2, samples under normoxic conditions). The mass spectrometer was operated in the parallel accumulation serial fragmentation mode. Protein identification was conducted using the Mascot software (Matrix Science, Chicago, IL, USA; version 1.7) [19].

RNA/miRNA isolation and cDNA synthesis

Total RNA was isolated from IEC-derived exosomes using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration of the isolated RNA was determined by measuring its absorbance at 260 nm using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted RNA was then reverse-transcribed to cDNA using the miScript II RT Kit (Qiagen).

miRNA PCR array

The gene expression profile of 84 miRNAs was examined using a qRT-PCR assay with the miScript miRNA PCR Array (Human miFinder in 96-well, Qiagen, Germany; 331221-MIHS-001ZD-2) according to the manufacturer’s instructions. The relative expression of each miRNA was calculated using the 2–ΔΔCt method. Six housekeeping genes (snoRNAs and the RNU6B snRNA) were used for normalization (S1 Methods).

HPLC/ESI–MS/MS-based lipidomic analysis

Lipids in the exosomes were extracted using the Bligh and Dyer technique [20]. HPLC/ESI (electrospray ionization)–MS/MS lipidomic analyses were performed using a combination of the QTRAP5500 triple quadrupole-linear ion trap mass spectrometer (AB SCIEX, Foster, CA, USA) and Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific). LC separations were achieved using a Develosil C30-UG-3 column (150 × 1.0 mm I.D., 3 μm pore-diameter; Nomura Chemical, Tokyo, Japan). The analyses were performed in the negative ion mode, using multiple reaction monitoring approaches to determine each PC and LPC species. The Analyst Software (AB SCIEX) was used to integrate the LC chromatogram peaks.

Statistical analysis

Student’s t-test was performed to assess the differences between samples with and without ischemic stimulation. Volcano plots [21] were also generated for miRNA and protein analysis results. All statistical analyses were performed using R (version 3.6.0; R Foundation for Statistical Computing, Vienna, Austria).

Bioinformatics analysis

Proteins were analyzed using the Gene Ontology (GO) term enrichment analysis with g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). Protein–protein interaction network functional enrichment analysis was performed using the STRING v11.3 database (https://string-db.org/).

Results

Identification and quantification of the collected exosomes

The isolation of exosomes was confirmed using immunoblotting with the Exo-Check Antibody Array, western blotting, TEM, and NTA. Biochemical analysis revealed the presence of exosomes in IECs based on positive staining for ICAM, ALIX, CD81, ANXA5, TSG101, which are common exosomal protein markers (Fig 1A) [22]. The isolated particles exhibited the typical cup-shaped morphology, and the size profiles of exosomes were confirmed using TEM (Fig 1B). Exosomal size was determined to be 89 ± 69 nm (mean ± standard deviation), which is consistent with the expected size of exosomes. The size distribution of the exosomes is shown in Fig 1C.

Fig 1. Identification and morphological characterization of exosomes.

Fig 1

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Exosome isolation was confirmed via immunoblotting using the Exo-Check Antibody Array, a transmission electron microscope, and nanoparticle tracking analysis. (A) The exosome compartments of the samples were placed in the pre-printed spots of eight antibodies against known exosome markers (FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, TSG101, and ANXA5), in the positive control, and in the blank (negative control) to examine the presence of exosome-specific proteins. (B) Representative images from transmission electron microscopy showing typical exosome cup-shaped morphology. (C) The size distribution (black line) and cumulative distribution (gray line) of the exosomes were measured using the nanoparticle tracking analysis.

Changes in the biological activity of exosomes under hypoxic stimulation

As shown in Fig 2, the production of NF-κB increased in the hypoxia group compared with that in the untreated and normoxia groups, indicating that hypoxic stimulation induced the production of exosomes, which activate monocytes.

Fig 2. Effect of intestinal epithelial cell-derived exosomes on NF-κB reporter activation in THP-1 monocytic cells.

Fig 2

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The supernatants of the normoxia and hypoxia groups were harvested, and the exosome compartment was extracted. THP-1 Blue NF-κB cells were identically stimulated by the obtained exosomes. The black bar indicates the exosomes obtained from the normoxia group, the red bar indicates the exosomes obtained from the hypoxia group, and the gray bar indicates exosomes obtained from the negative control using 10% (v/v) sterile PBS. Results are expressed as mean ± standard deviation of three experiments. NF-κB, nuclear factor-kappa B.

Proteomic changes in exosomes derived under hypoxic stimulation

The number of detected proteins in the N1, N2, H1, and H2 samples was 3519, 3407, 3290, and 3127, respectively. Fig 3A presents the changes in protein expression between groups (H1/H2 and N1/N2). Samples in the same group showed almost identical expression patterns. Different patterns were observed between groups, indicating high reproducibility and high experimental precision. Among these identified proteins, 110 showed a >2-fold increase, and 75 showed a <2-fold decrease compared with those in the normoxia group (Fig 3B). Proteins specifically associated with inflammation are labeled in Fig 3B. Their annotations are listed in S1 Table. The proteins whose concentrations largely changed (i.e., >2-fold increase/decrease with p < 0.01) are shown in S2 Fig. Among them, proteins associated with inflammation are presented in Fig 4. The protein network analysis showed increased levels of the proteins involved in ubiquitination and subsequent protein degradation (DDX3X, PSMD3, PSMC2, CAND1, and UBR4), and metabolism (C3, ACLY, AGL, PYGL, and NAMPT). Moreover, it showed that they interacted with each other (Fig 3C).

Fig 3. Proteomic analysis and GO term enrichment analysis of the isolated exosomes.

Fig 3

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The expression of proteins in the exosomes was quantified and compared between the normoxia and hypoxia groups using high-performance liquid chromatography-mass spectrometry. The proteins were analyzed using GO term enrichment analysis. (A) Heatmap showing protein expression in each group (N1, N2: normoxia; H1, H2: hypoxia). Red indicates high protein expression, whereas green indicates low expression. (B) Volcano plot showing the proteins analyzed using mass spectrometry. Red labels indicate proteins involved in inflammation. The description of each protein is given in S1 Table. (C) Results of protein network analysis conducted using the STRING v11.3 database. This figure was obtained using STRING v11.3 web-based software (https://string-db.org/). (D) Enrichment scores of GO analysis: biological processes. GO, Gene ontology.

Fig 4. Heatmap showing the changes in inflammation-related protein expression after ischemic stimulation.

Fig 4

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The protein contents of exosomes were extracted and quantified using high-performance liquid chromatography-mass spectrometry. Inflammation-related proteins that showed >2-fold increase/decrease and p < 0.01 (based on Student’s t-test) between the normoxia and hypoxia groups. The changes in the expression of all proteins identified in the experiment are provided in S2 Fig. Values increase progressively from blue to red on the color scale. N1 and N2, normoxia samples; H1 and H2, hypoxia samples.

As shown in Fig 3D, the results of the GO term enrichment analysis showed that these proteins were related to the activation (any process that initiates an immune response) of several components in white blood cells (granulocyte activation, p = 4.15 × 10−3; neutrophil activation, p = 3.52 × 10−3; myeloid leukocyte activation, p = 1.86 × 10−2).

Changes in miRNA expression in exosomes derived under hypoxic stimulation

Fig 5A presents a volcano plot of the miRNAs obtained from qPCR analysis. Although the absolute change between the normoxia and hypoxia groups was small, a few proteins showed notably low p-values. The most prominent miRNAs that showed low p-values were hsa-miR-21-5p (26.75 ± 0.73 normoxia vs. 33.98 ± 0.82 hypoxia, p = 6.2 × 10−5), hsa-miR-23a-3p (29.61 ± 0.64 normoxia vs. 33.77 ± 0.85 hypoxia, p = 6.2 × 10−3), hsa-miR-124-3p (34.31 ± 0.29 normoxia vs. 33.10 ± 0.45 hypoxia, p = 3.5 × 10−3), and hsa-miR-30d-5p (32.81 ± 0.10 normoxia vs. 33.70 ± 0.03 hypoxia, p = 2.8 × 10−2; all results are presented as mean ± standard deviation, and p-values are based on Student’s t-test).

Fig 5. Changes in the expression of miRNAs and lipids after hypoxic stimulation.

Fig 5

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The expression levels of miRNAs extracted from exosomes were measured using a qPCR array and calculated using the 2–ΔΔCt method (three replicates). The lipid compartment in the exosome was extracted using Bligh and Dyer’s method. Analyses were performed using high-performance liquid chromatography-mass spectrometry. (A) Volcano plot showing miRNA expression plotted using the miScript miRNA PCR Array Data Analysis software. (B, C) Changes in the levels of unsaturated lysophosphatidylcholine (B) and free fatty acids (C) between the normoxia group (black) and hypoxia group (gray). The results are expressed as mean ± standard deviation of three experiments. (D) Volcano plot representing unsaturated lysophosphatidylcholine and free fatty acids. LPC, lysophosphatidylcholine; FFA, free fatty acid; AA, arachidonic acid; DHA, docosahexaenoic acid; LA, linoleic acid; SA, stearic acid; miRNA, microRNA.

Changes in the lipid profile in exosomes following hypoxic stimulation

The changes in the lipid profile following ischemic stimulation are shown in Fig 5B–5D. The amount of unsaturated LPCs and free fatty acids tended to increase and decrease, respectively, following ischemic stimulation. Fig 5D is a volcano plot of the results representing the increase in the levels of LPC20:1 and LPC16:1 and the decrease in the levels of arachidonic acid.

Discussion

In this study, using protein, lipid, and miRNA analyses under the same conditions, we gained comprehensive understanding of the changes in exosomal cargo upon ischemic stimulation. The increase in NF-κB activity confirmed that biological activity is induced by hypoxic stimulation. Our results demonstrate that (1) the expression of lipid metabolism-related proteins was upregulated, (2) the levels of miRNAs were slightly altered, and (3) the concentration of LPCs containing unsaturated fatty acids increased following hypoxia.

The results of our protein analysis were largely different from those of a previous study [13]. In the previous study, the expression of 35 proteins was found to change following ischemia. However, using p < 0.05 as the criterion similar to the previous study, our study found the expression of 1632 proteins to be altered [13]. As too many proteins in the present study met the p < 0.05 criterion, the 185 proteins that met the p < 0.01 and >2-fold increase/decrease criteria were listed for comparison with the proteins identified in the previous study. Of these, only one protein, prosaposin, was common in both studies. This may be due to the high sensitivity of our mass spectrometry analysis. Although previous studies with bioinformatic analyses have identified proteins related to cell survival and metabolism, in this study, we could identify trace levels of proteins related to inflammation and thoroughly examine small protein changes related to inflammation. The protein network analysis showed that the levels of proteins involved in metabolism were increased and that these proteins interacted with each other. ACLY catalyzes acetyl-CoA synthesis from citrate leading to lipid synthesis. NAMPT is the rate-determining enzyme in the mammalian NAD+ biosynthesis salvage pathway that is also essential for maintaining de novo lipogenesis [23]. PYGB, AGL, and PYGL degrade glycogen, activate the pentose phosphate pathway, and thereby mediate the production of NADPH, which can also aid de novo lipid synthesis [24]. C3 is known to trigger inflammation and can degrade triglycerides [25]. The changes in the miRNA levels were small, which made it difficult to conclude their involvement in inflammation. However, the expression of several miRNAs showed low p-values; therefore, their possible influence on the pathology cannot be ruled out. We found marginal upregulation of hsa-miR-21-5p expression. Previous studies have shown that this miRNA is one of the lipid metabolism-related miRNAs, and its upregulation correlates with hyperglycemia, hyperlipidemia, and inflammation markers, such as CRP and IL-1β [26]. hsa-miR-21-5p can also regulate lipid metabolism and mitochondrial metabolism [27]. NF-κB increases the expression of hsa-miR-21-5p, which induces the proliferation of smooth muscle cells in the human pulmonary artery [28, 29]. hsa-miR-21-5p can also induce monocyte differentiation in dendritic cells [30]. These results suggest that hsa-miR-21-5p is a potential therapeutic target for regulating inflammation after ischemic recanalization. In addition, a small increase in miR-23a expression and a decrease in the expression of miR-124-3p were observed. The expression of miR-23a has been reported to decrease in response to NF-κB activation and to enable negative feedback [31], suggesting that an increase in its expression may disrupt this self-regulatory mechanism. Similarly, miR-124-3p exerts anti-inflammatory effects by inducing and maintaining the M2 macrophage phenotype [32, 33]. Therefore, reducing this miRNA level may lead to the dominance of the M1 phenotype in macrophages, causing inflammatory outcomes [33, 34]. Due to the nature of this study, causality cannot be determined but our results suggest that miRNAs and proteins may cooperatively and adversely affect lipid metabolism, leading to induction of the subsequent inflammation. In fact, despite the decrease in free fatty acid levels (arachidonic, docosahexaenoic, and stearic acid), an increase in levels of unsaturated fatty acid containing LPCs (LPC16:1, LPC18:1, LPC20:1) was seen. Increased levels of unsaturated LPCs have been demonstrated to trigger systemic inflammatory response via the NF-κB pathway [12]. As mentioned above, NF-κB increases the expression of hsa-miR-21-5p, which can induce monocyte differentiation in dendritic cells [30–32]. In summary, hypoxic stimuli may be inducing abnormal lipid metabolism by proteins and miRNAs, which triggers NF-kB-mediated inflammation.

There are several limitations to this study. First, this study was conducted in vitro, and an experimental system was adopted to simplify the cause of intestinal ischemia. Thus, an in vivo study is required to validate our results for clinical translation. Second, although we used hypoxia stimuli in IECs to mimic the in vivo intestinal ischemia-reperfusion injury, this simplified protocol needs to be validated in more detail. Furthermore, as these cells are derived from human adenocarcinoma, their response to stimuli may vary from that of IECs in a physiological state. Third, exosomal RNA concentration measurements were performed using Nanodrop and Qubit, which are less reliable than a bioanalyzer.

Conclusion

We performed a comprehensive analysis of IEC exosomal contents in hypoxic conditions, which shed light on the pathogenesis of systemic inflammation after intestinal injury. The results of this study have partially clarified the mechanism of multiple organ failure following ischemic recanalization, thereby providing a target for treatment.

Supporting information

S1 Table. Annotation of proteins related to inflammation in Gene Ontology terms.

The annotation was obtained from the UniProt database (https://www.uniprot.org/).

(DOCX)

Click here for additional data file. (21.5KB, docx)
S1 Fig. Schematic of the experiment.

(PDF)

Click here for additional data file. (181KB, pdf)
S2 Fig. Heat map showing the changes in protein expression after ischemic stimulation.

(PDF)

Click here for additional data file. (242.3KB, pdf)
S1 Methods. List of primers used in the miRNA PCR array.

(DOCX)

Click here for additional data file. (18.8KB, docx)

Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing and Dr. Kana Ariga for creating a schema.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

MK, Scientific Research from the Japan Society for the Promotion of Science (18K16509) AS, Scientific Research from the Japan Society for the Promotion of Science (21K16585).

References

  • 1.Dewar D, Moore FA, Moore EE, Balogh Z. Postinjury multiple organ failure. Injury. 2009;40: 912–918. doi: 10.1016/j.injury.2009.05.024 [DOI] [PubMed] [Google Scholar]
  • 2.Faist E, Baue AE, Dittmer H, Heberer G. Multiple organ failure in polytrauma patients. J Trauma. 1983;23: 775–787. doi: 10.1097/00005373-198309000-00002 [DOI] [PubMed] [Google Scholar]
  • 3.Fry DE, Pearlstein L, Fulton RL, Polk HC. Multiple system organ failure. The role of uncontrolled infection. Arch Surg. 1980;115: 136–140. doi: 10.1001/archsurg.1980.01380020006003 [DOI] [PubMed] [Google Scholar]
  • 4.Deitch EA, Adams C, Lu Q, Xu DZ. A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability. Surgery. 2001;129: 39–47. doi: 10.1067/msy.2001.109119 [DOI] [PubMed] [Google Scholar]
  • 5.Adams CA, Hauser CJ, Adams JM, Fekete Z, Xu DZ, Sambol JT, et al. Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation. Shock. 2002;18: 513–517. doi: 10.1097/00024382-200212000-00005 [DOI] [PubMed] [Google Scholar]
  • 6.Deitch EA, Adams CA, Lu Q, Xu DZ. Mesenteric lymph from rats subjected to trauma-hemorrhagic shock are injurious to rat pulmonary microvascular endothelial cells as well as human umbilical vein endothelial cells. Shock. 2001;16: 290–293. doi: 10.1097/00024382-200116040-00010 [DOI] [PubMed] [Google Scholar]
  • 7.Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg. 1998;228: 518–527. doi: 10.1097/00000658-199810000-00008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200: 373–383. doi: 10.1083/jcb.201211138 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Essandoh K, Yang L, Wang X, Huang W, Qin D, Hao J, et al. Blockade of exosome generation with GW4869 dampens the sepsis-induced inflammation and cardiac dysfunction. Biochim Biophys Acta. 2015;1852: 2362–2371. doi: 10.1016/j.bbadis.2015.08.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Kojima M, Gimenes-Junior JA, Langness S, Morishita K, Lavoie-Gagne O, Eliceiri B, et al. Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: unlocking the mystery of post-shock multiple organ failure. J Trauma Acute Care Surg. 2017;82: 42–50. doi: 10.1097/TA.0000000000001296 [DOI] [PubMed] [Google Scholar]
  • 11.Kojima M, Gimenes-Junior JA, Chan TW, Eliceiri BP, Baird A, Costantini TW, et al. Exosomes in postshock mesenteric lymph are key mediators of acute lung injury triggering the macrophage activation via toll-like receptor 4. FASEB J. 2018;32: 97–110. doi: 10.1096/fj.201700488R [DOI] [PubMed] [Google Scholar]
  • 12.Senda A, Morishita K, Kojima M, Doki S, Taylor B, Yagi M, et al. The role of mesenteric lymph exosomal lipid mediators following intestinal ischemia-reperfusion injury on activation of inflammation. J Trauma Acute Care Surg. 2020;89: 1099–1106. doi: 10.1097/TA.0000000000002897 [DOI] [PubMed] [Google Scholar]
  • 13.Williams EC, Coimbra R, Chan TW, Baird A, Eliceiri BP, Costantini TW. Precious cargo: modulation of the mesenteric lymph exosome payload after hemorrhagic shock. J Trauma Acute Care Surg. 2019;86: 52–61. doi: 10.1097/TA.0000000000002093 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Dong ZW, Liu H, Su FF, Fan XZ, Zhang Y, Liu P. Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway. World journal of gastroenterology. 2022;28: 918–932. doi: 10.3748/wjg.v28.i9.918 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kip AM, Soons Z, Mohren R, Duivenvoorden AAM, Röth AAJ, Cillero-Pastor B, et al. Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis. 2021;12: 95. doi: 10.1038/s41419-020-03379-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Stierum R, Gaspari M, Dommels Y, Ouatas T, Pluk H, Jespersen S, et al. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2. Biochim Biophys Acta. 2003;1650: 73–91. doi: 10.1016/s1570-9639(03)00204-8 [DOI] [PubMed] [Google Scholar]
  • 17.Willms E, Johansson HJ, Mäger I, Lee Y, Blomberg KE, Sadik M, et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep. 2016;6: 22519. doi: 10.1038/srep22519 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, et al. Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Sci Rep. 2017;7: 15297. doi: 10.1038/s41598-017-15717-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999;20: 3551–3567. doi: [DOI] [PubMed] [Google Scholar]
  • 20.Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37: 911–917. doi: 10.1139/o59-099 [DOI] [PubMed] [Google Scholar]
  • 21.Li W. Volcano plots in analyzing differential expressions with mRNA microarrays. J Bioinform Comput Biol. 2012;10: 1231003. doi: 10.1142/S0219720012310038 [DOI] [PubMed] [Google Scholar]
  • 22.Record M, Carayon K, Poirot M, Silvente-Poirot S. Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies. Biochim Biophys Acta. 2014;1841: 108–120. doi: 10.1016/j.bbalip.2013.10.004 [DOI] [PubMed] [Google Scholar]
  • 23.Monica D, Bernhard B, Dmitry N. Exploring the role of ATP-citrate lyase in the immune system. Front Immunol. 2021;12: 632526. http://www.ncbi.nlm.nih.gov/pubmed/632526 doi: 10.3389/fimmu.2021.632526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ritterson Lew C, Guin S, Theodorescu D. Targeting glycogen metabolism in bladder cancer. Nat Rev Urol. 2015;12: 383–391. doi: 10.1038/nrurol.2015.111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Barbu A, Hamad OA, Lind L, Ekdahl KN, Nilsson B. The role of complement factor C3 in lipid metabolism. Mol Immunol. 2015;67: 101–107. doi: 10.1016/j.molimm.2015.02.027 [DOI] [PubMed] [Google Scholar]
  • 26.Simionescu N, Niculescu LS, Sanda GM, Margina D, Sima AV. Analysis of circulating microRNAs that are specifically increased in hyperlipidemic and/or hyperglycemic sera. Mol Biol Rep. 2014;41: 5765–5773. doi: 10.1007/s11033-014-3449-2 [DOI] [PubMed] [Google Scholar]
  • 27.Nasci VL, Chuppa S, Griswold L, Goodreau KA, Dash RK, Kriegel AJ. miR-21-5p regulates mitochondrial respiration and lipid content in H9C2 cells. Am J Physiol Heart Circ Physiol. 2019;316: H710–H721. doi: 10.1152/ajpheart.00538.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kang BY, Kleinhenz JM, Murphy TC, Hart CM. The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol. 2011;301: L881–L891. doi: 10.1152/ajplung.00195.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yang S, Banerjee S, de Freitas A, Cui H, Xie N, Abraham E, et al. miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling. Am J Physiol Lung Cell Mol Physiol. 2012;302: L521–L529. doi: 10.1152/ajplung.00316.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hashimi ST, Fulcher JA, Chang MH, Gov L, Wang S, Lee B. MicroRNA profiling identifies miR-34a and miR-21 and their target genes JAG1 and WNT1 in the coordinate regulation of dendritic cell differentiation. Blood. 2009;114: 404–414. doi: 10.1182/blood-2008-09-179150 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Peng P, Li Z, Liu X. Reduced expression of miR-23a suppresses A20 in TLR-stimulated macrophages. Inflammation. 2015;38: 1787–1793. doi: 10.1007/s10753-015-0156-7 [DOI] [PubMed] [Google Scholar]
  • 32.Liang Y, Xie J, Che D, Zhang C, Lin Y, Feng L, et al. MiR-124-3p helps to protect against acute respiratory distress syndrome by targeting p65. Biosci Rep. 2020;40. doi: 10.1042/BSR20192132 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Essandoh K, Li Y, Huo J, Fan GC. MiRNA-mediated macrophage polarization and its potential role in the regulation of inflammatory response. Shock. 2016;46: 122–131. doi: 10.1097/SHK.0000000000000604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Qin Z, Wang PY, Su DF, Liu X. miRNA-124 in immune system and immune disorders. Front Immunol. 2016;7: 406. doi: 10.3389/fimmu.2016.00406 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Jon M Jacobs

24 Nov 2022

PONE-D-22-23365Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injuryPLOS ONE

Dear Dr. Kojima,

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injury

Title

A major focus of the recently updated guidelines of the International Society for Extracellular Vesicles (ISEV) on minimal information for studies of extracellular vesicles (MISEV) is the need for appropriate nomenclature in reporting extracellular vesicle (EV) research. So I advise the authors to use a more precise term

Abstract

Many unimportant technical details are mentioned in the abstract. I advise the authors get rid of them.

Introduction

The introduction did not sufficiently shed light on the studies related to hypoxia and its effect on the components of exosomes.

Methods:

The mentioned method is not well explained. I believe that hypoxia tests should be conducted in a standard atmosphere such as the hypoxia chamber.

The authors mentioned that the cell culture was free of serum, what is the reason?

ExoQuick-TC Exosome Precipitation Solution Kit is not an accurate method for isolating exosomes. So, the authors should use a more precise phrase. For example, the concentration of extracellular vesicles.

The authors did not clarify the amount of media used to obtain a sufficient amount of exosomes.

In the Exo-Check Antibody Array, its method is not well explained. As well as the positive sample, which appears to have not worked, shows a clear result.

Nanodrop are not a valid method for measuring exosomal RNA concentration and quality. It often shows inaccurate results and residual salts from the precipitate. The best way is by bioanalyzer

How the authors ensured that their exosome content was not contaminated with some of the components present in the media. Especially since the isolation was done here by precipitation.

Results:

Isolation and characterization of exosomes is a major and important experiment. So it is supposed to be in the main body of the manuscript and not in the appendices.

Discussion

The discussion sheds light on the possible role and impact of exosomes after hypoxia. This discussion was not deep enough.

Reviewer #2: Atsushi Senda et al. investigated whether the isolated intestinal epithelial cell-derived exosomes ameliorated intestinal epithelia cells hypoxia reoxygenation injury. I have some concerns.

The data without in vivo experiment cannot support any valuable conclusion for clinical translation.

The study is too simple and too observational. It needs more mechanism investigation.

**********

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Reviewer #1: Yes: Faisal A A

Reviewer #2: No

**********

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PLoS One. 2023 Mar 29;18(3):e0283702. doi: 10.1371/journal.pone.0283702.r002

Author response to Decision Letter 0

17 Feb 2023

RESPONSE TO REVIEWERS’ COMMENTS

We would like to thank the reviewers for their insightful comments and suggestions. Please see our point-by-point responses to reviewers’ comments below. Our responses are all labelled as “Response.”

Reviewer #1: Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injury

Title

A major focus of the recently updated guidelines of the International Society for Extracellular Vesicles (ISEV) on minimal information for studies of extracellular vesicles (MISEV) is the need for appropriate nomenclature in reporting extracellular vesicle (EV) research. So I advise the authors to use a more precise term

Response: We thank the reviewer for their suggestion. We have changed the title to “Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model.”

Abstract

Many unimportant technical details are mentioned in the abstract. I advise the authors get rid of them.

Response: We thank the reviewer for their comment. We agree and have reduced the description of technical details in the abstract as suggested.

Introduction

The introduction did not sufficiently shed light on the studies related to hypoxia and its effect on the components of exosomes.

Response: We thank the reviewer for their constructive comments. We have added the following sentence to the third paragraph of the Introduction in line with their suggestion to show the findings between hypoxia and its effects on exosome components:

------------------------------------------------------------------------------------------------------------------

Corresponding to the ischemic state, cultured IECs were first placed under hypoxic conditions. Subsequently, they were returned to an oxygenated environment corresponding to the reperfusion state. Accordingly, this hypoxia reoxygenation model has been used to simulate ischemia-reperfusion injury in an in vitro setting in previous studies [14,15].

14. Zw D, H L, Ff S, Xz F, Y Z, P L. Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway. World journal of gastroenterology. 2022;28. doi:10.3748/wjg.v28.i9.918

15. Kip AM, Soons Z, Mohren R, Duivenvoorden AAM, Röth AAJ, Cillero-Pastor B, et al. Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis. 2021;12: 95. doi:10.1038/s41419-020-03379-9

Methods:

The mentioned method is not well explained. I believe that hypoxia tests should be conducted in a standard atmosphere such as the hypoxia chamber.

Response: To generate an environment analogous to that of the reperfusion period in hemorrhagic shock, cells in the hypoxia and normoxia groups were subsequently incubated in the normoxic environment for 24 h. All tests, including biological activity tests, proteome analyses, lipid analyses, and miRNA quantification, were performed in a normoxic environment. We have made appropriate changes to the revised manuscript to clarify this point.

The authors mentioned that the cell culture was free of serum, what is the reason?

Response: We thank the reviewer for their question. We used serum-free cell culture to avoid the potential contamination of the exosomes that could derive from the serum. We have changed our description in the revised manuscript to ensure that readers understand this point.

ExoQuick-TC Exosome Precipitation Solution Kit is not an accurate method for isolating exosomes. So, the authors should use a more precise phrase. For example, the concentration of extracellular vesicles.

The authors did not clarify the amount of media used to obtain a sufficient amount of exosomes.

Response: We would like to thank the reviewer for raising this point. We used 80 mL of media to obtain sufficient amount of exosomes. We have added this clarification to our revised manuscript.

In the Exo-Check Antibody Array, its method is not well explained. As well as the positive sample, which appears to have not worked, shows a clear result.

Response: We have included a more detailed explanation in the section kindly pointed out. Our results were positive for ICAM, ALIX, CD81, ANXA5, TSG101, and positive control; intermediate for FLOT1 and EpCAM; and negative for GM130, CD63, and Blank. The difference in concentration between the two positive controls may be caused by a concentration irregularity in the sample or in the blocking agent.

As for CD63 expression, the amount and type of surface antigens depends on the cell type from which the exosomes are extracted. In addition, CD63 may not be detected using the kit due to the reactivity between the antibody and cell type. The figure below (manufacturer's manual) also shows that the surface markers on exosomes in human serum and HEK cells are very different. We believe that the relatively low distribution of CD63 on the cell surface of exosomes extracted from the Caco-2 cells used in this study could not be detected, unlike other markers such as CD81. Since this is a simple kit test, if most of the exosome markers are detected, it is considered evidence for the presence of exosomes.

Figures from Exo-Check Antibody Array User-manual P.5

http://www.systembio.com/wp/wp-content/uploads/Exo-Check_User-Manual.pdf

Nanodrop are not a valid method for measuring exosomal RNA concentration and quality. It often shows inaccurate results and residual salts from the precipitate. The best way is by bioanalyzer

Response: We appreciate the reviewer’s highly appropriate remarks. To avoid the complexity of the description, we originally referred to this as Nanodrop, but in the actual measurement, we used both Qubit and Nanodrop. As pointed out, there were samples that showed unexpected values with Nanodrop; thus, we did not perform further measurements on them. In the revised manuscript, we have amended the Methods section regarding this point and added text that we did not use a bioanalyzer as a study limitation.

How the authors ensured that their exosome content was not contaminated with some of the components present in the media. Especially since the isolation was done here by precipitation.

Response: We are deeply concerned about the points raised by the reviewer. We used serum-free media to avoid contamination. However, as the reviewer pointed out, the existence of contamination cannot be completely ruled out. Therefore, we decided to present a relative evaluation between the hypoxia group and normoxia group in our experimental system:

In a preliminary experiment, the protein concentration and exosome particle size distribution were compared between exosome samples subjected to centrifugation at 10,000 × g and those subjected to the ExoQuick-TC followed by 0.2-µm filtration. As pointed out by the reviewer, the amount of 30–150-nm particles was lower in the exosomes obtained using ExoQuick followed by filtration than in those obtained with ultracentrifugation (82% in ultracentrifuge versus 78% ExoQuick, please refer to the figure below).

To determine whether the above difference is significant, we compared the protein amount in exosomes obtained by ultracentrifuge with that in the exosomes obtained by ExoQuick.

Additionally, as shown in the figure below, the correlation coefficients between the protein amounts between the Ultracentrifuge and ExoQuick groups were over 99.5% (99.3% between ultracentrifuge-isolated exosome compartment and ExoQuick-isolated exosome compartment in the hypoxia group, and 99.6% in the normoxia group).Therefore, we concluded that there was no significant difference between the ExoQuick and ultracentrifugation results and chose to perform the experiment using the Centrifugal Filter Unit for better experimental efficiency.

Results:

Isolation and characterization of exosomes is a major and important experiment. So it is supposed to be in the main body of the manuscript and not in the appendices.

Response: We agree with the comment and have revised our manuscript as suggested. Please see Figure 1.

Discussion

The discussion sheds light on the possible role and impact of exosomes after hypoxia. This discussion was not deep enough.

Response: We thank the reviewer for this useful suggestion. Since this study is only an observation of qualitative relationships, we have added some more to the Discussion section, but being mindful not to exceed the extent to which conclusions can be stated.

Reviewer #2: Atsushi Senda et al. investigated whether the isolated intestinal epithelial cell-derived exosomes ameliorated intestinal epithelia cells hypoxia reoxygenation injury. I have some concerns.

The data without in vivo experiment cannot support any valuable conclusion for clinical translation. The study is too simple and too observational. It needs more mechanism investigation.

Response: We appreciate the valuable comments made by the reviewer. To better understand the significance of this study, please allow us to explain the background of our previous studies.

We have focused on in vivo studies using animal models. As described in the background, we focused on in vitro experiments to prove the following assumptions.

1. We first confirmed the presence of exosomes in the lymph fluid of rats in 2017[1].

2. We have shown that exosomes in mesenteric lymph fluid have biological activity after hemorrhagic shock in an animal model and that these exosomes induce lung injury when administered to the naive animal [2].

3. Exosomes extracted from rat lymph fluid in a rat intestinal ischemia-reperfusion model have been shown to induce immune cell inflammation, as in the trauma hemorrhagic shock model [3].

4. The lipid concentration in exosomes was altered after ischemia-reperfusion injury, and some lipids, such as polyunsaturated fatty acids and lysophosphatidylcholines, revealed significant changes in exosomes [3].

The above in vivo studies suggest that inflammatory exosomes extracted from the intestinal tract travel through the lymphatic system to cause inflammation in the lungs and other organs. However, lymphatic fluid contains not only exosomes derived from intestinal cells, but also exosomes secreted from vascular endothelial cells and blood cell components, and not only exosomes derived from intestinal epithelial cells. Therefore, in this study we decided to focus only on intestinal epithelial cells to clarify how their composition is altered by hypoxic stimuli and how they are involved in inflammation.

We fully agree with the reviewer's point. However, we have now presented an in vitro focused study with the aim of selecting targets for future animal studies by analyzing lipids, proteins, and miRNAs in detail.

We appreciate the valuable comments made by the reviewer. To better contextualize the significance of this study, we would like to provide additional information about our previous research. Our previous work has mainly focused on in vivo studies using animal models. As described in the background, we conducted in vitro experiments to confirm the following hypotheses:

1. We confirmed the presence of exosomes in the lymph fluid of rats in 2017 [1].

2. We demonstrated that exosomes in mesenteric lymph fluid have biological activity after hemorrhagic shock in an animal model, and that these exosomes induce lung injury when administered to naive animals [2].

3. Exosomes extracted from rat lymph fluid in a rat intestinal ischemia-reperfusion model induced immune cell inflammation, similar to the trauma hemorrhagic shock model [3].

4. Lipid concentration in exosomes was found to be altered after ischemia-reperfusion injury, with significant changes in polyunsaturated fatty acids and lysophosphatidylcholines [3].

The above in vivo studies suggest that inflammatory exosomes extracted from the intestinal tract may cause inflammation in the lungs and other organs. However, since lymphatic fluid contains exosomes from various sources, including vascular endothelial cells and blood cell components, we decided to focus on intestinal epithelial cell-derived exosomes in this study to investigate how their composition is altered by hypoxic stimuli and their potential role in inflammation.

We agree with the reviewer's point that our study is limited in scope and lacks in vivo experiments. However, we present an in vitro study to provide insights into the mechanisms underlying the protective effects of intestinal epithelial cell-derived exosomes against hypoxia reoxygenation injury, which could serve as a basis for future animal studies. We analyzed the lipids, proteins, and miRNAs in detail to identify potential targets for future studies.

References

1. Kojima M, Gimenes-Junior JA, Langness S, Morishita K, Lavoie-Gagne O, Eliceiri B, et al. Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure. J Trauma Acute Care Surg. 2017;82: 42–50. doi:10.1097/TA.0000000000001296

2. Kojima M, Gimenes-Junior JA, Chan TW, Eliceiri BP, Baird A, Costantini TW, et al. Exosomes in postshock mesenteric lymph are key mediators of acute lung injury triggering the macrophage activation via Toll-like receptor 4. FASEB J. 2018;32: 97–110. doi:10.1096/fj.201700488R

3. Senda A, Morishita K, Kojima M, Doki S, Taylor B, Yagi M, et al. The role of mesenteric lymph exosomal lipid mediators following intestinal ischemia-reperfusion injury on activation of inflammation. J Trauma Acute Care Surg. 2020;89: 1099–1106. doi:10.1097/TA.0000000000002897

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Decision Letter 1

Jon M Jacobs

14 Mar 2023

Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model

PONE-D-22-23365R1

Dear Dr. Kojima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jon M. Jacobs, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: I have no more comment. My concerns have been addressed. I suggest language editing before fully acceptance.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

**********

Acceptance letter

Jon M Jacobs

20 Mar 2023

PONE-D-22-23365R1

Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model

Dear Dr. Kojima:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

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    Supplementary Materials

    S1 Table. Annotation of proteins related to inflammation in Gene Ontology terms.

    The annotation was obtained from the UniProt database (https://www.uniprot.org/).

    (DOCX)

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    S1 Fig. Schematic of the experiment.

    (PDF)

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    S2 Fig. Heat map showing the changes in protein expression after ischemic stimulation.

    (PDF)

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    S1 Methods. List of primers used in the miRNA PCR array.

    (DOCX)

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    Submitted filename: Reviewer_0217.docx

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    Data Availability Statement

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