Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation
Stem Cell Research & Therapy volume 14, Article number: 48 (2023)
Mesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs.
This work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models.
Our results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization.
PGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.
Acute lung injury (ALI) and its subsequent form acute respiratory distress syndrome (ARDS) are important causes of morbidity and mortality worldwide. They have become one of the most global burdens in the twenty-first century due to coronavirus disease 2019 (COVID-19) [1, 2]. ALI occurs due to massive inflammatory processes that cause epithelial and endothelial lung injury, leading to increased vascular permeability. Furthermore, several reports show that macrophages, neutrophils, and their related factors play crucial roles in lung inflammation [3, 4]. Therefore, there is an urgent need to find an effective therapeutic to target these abnormalities, which could be an effective strategy to prevent and treat ALI.
Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into numerous cell types and are present in several tissues [5,6,7]. Increasing evidence of MSC migration to lung injury and their contribution to lung regeneration attracts attention for the treatment of ALI/ARDS . For these reasons, the use of MSCs in preclinical and clinical settings has been extensively investigated. In animal and human lung perfusion models, both intravenous and intratracheal administration of MSCs for ALI significantly improved alveolar permeability and inflammation [9, 10]. Various paracrine factors produced by MSCs play a crucial role in influencing the microenvironment of injured tissues and modulating the immune response. These factors are transforming growth factor (TGF)-β1, hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin (IL-10), and nitric oxide (NO) [11, 12]. Consequently, therapeutic applications of MSCs remain promising for ALI/ARDS.
Prostaglandin E2 (PGE2) is a lipid signaling molecule that plays an important role in the modulation of inflammatory and fibrotic diseases [13, 14]. It can be synthesized by many tissue cells, such as epithelial cells, fibroblasts, and inflammatory cells, that infiltrate tissues after partial injury [15,16,17,18]. By interacting with the E-type prostaglandin receptor (EP) family, it plays a role in a wide range of physiological processes and, as a result, facilitates the regeneration of multiple organ systems following injury. The production of PGE2 in damaged tissues is significantly increased, and many studies have reported PGE2-regulated roles in activating endogenous stem cells, the immune response, angiogenesis, and other processes [19, 20]. Due to these functions, PGE2 was hypothesized and selected to strengthen the protective effects of MSCs against LPS-induced ALI models.
Macrophages are considered a key component of the innate immune system. Due to their plasticity, macrophages play a very important role in tissue regeneration. They have been divided into two categories, classically activated and proinflammatory M1 macrophages or alternatively activated and anti-inflammatory M2 macrophages [21,22,23]. The role of MSCs and their secretomes in macrophage immunomodulation has been studied in a wide range of models. Previous studies reported that MSCs play a critical role in mediating macrophage polarization from the proinflammatory (M1) form to the anti-inflammatory (M2) form by regulating the production of cytokines such as IL-10, IL-1β, IL-6 and TNF-α [18, 24,25,26]. Hence, regulating macrophage polarization is critical for lung repair and regeneration.
In recent years, the priming strategy has been considered to stimulate and improve the therapeutic effects of MSCs. Different stimuli have previously been used, including cytokines, hypoxia, biochemical factors, and biomaterials [27, 28]. The lipid signaling molecule prostaglandin E2 (PGE2), an inflammatory mediator, can enhance tissue regeneration and repair after injury in various organ systems [19, 29]. In this study, we used PGE2 to prime MSCs and investigated the therapeutic potential of PGE2-MSCs for acute lung injury. Furthermore, we highlighted the antifibrotic effects and possible mechanisms of PGE2-MSCs in ALI. We found that PGE2-MSCs significantly alleviated ALI and mediated lung regeneration. We also explored whether macrophage polarization plays an essential role in the anti-inflammatory effect of PGE2-MSCs in ALI mice.
Materials and methods
ALI models were established as previously reported [30, 31]. In summary, 2.5% avertin was administered intraperitoneally to anesthetize C57BL/6 mice (8–10 weeks old, weighing 22–25 g), and lipopolysaccharide (LPS, O55:B5; 5 mg/kg; Sigma-Aldrich, dissolved in PBS) was administered intratracheally to cause lung injury. Then, intravenously administered MSCs and PGE2-MSCs were applied 6 h later. The International Guiding Principles for Biomedical Research Involving Animals, which the Council published for the International Organizations of Medical Sciences, were followed by all experiments, which were approved by the Nankai University Animal Care and Institutional Animal Care Committees (approval no. 20170022).
C57BL/6 mice (female, 8–10 weeks old, weighing 22–25 g) were randomly divided into five groups: sham group, ALI group (LPS only, 5 mg/kg), PBS group, MSC group (1 \(\times\) 106) and PGE2-MSCs group (1 \(\times\) 106). The mice were anesthetized with inhaled isoflurane (2% to 3%), and then LPS (5 mg/kg) was administered intratracheally to induce lung injury. After 6 h of LPS administration, the ALI mice were intravenously injected with 250 µl of PBS, which was used as a solvent control as previously described . At different time points, survival rates and body weight ratios were calculated. Then, bronchoalveolar lavage fluid (BALF) and lung tissue samples were obtained and used for hematoxylin and eosin (H&E) staining, quantitative real-time polymerase chain reaction (qRT‒PCR), Western blot assay (WB), etc.
As previously reported [33, 34], human placental MSCs (hP-MSCs) were cultured in DMEM/F12 media with 10% fetal bovine serum (FBS) (Australia), 1% NEAA (Gibco), 1% L-glutamine (Gibco), and 1% penicillin and streptomycin (Gibco). MSCs were transduced with a self-inactivating lentiviral vector containing a ubiquitin promoter driving firefly luciferase and an enhanced green fluorescence protein (Fluc-eGFP) double fusion (DF) reporter gene to monitor transplanted cells in vivo [6, 32]. MSCs were incubated at 37 °C in a humidified incubator containing 5% CO2. At the beginning of the experiment, PGE2 (CAS 363–24-6; Santa Cruz Biotechnology) was added to the culture medium of MSCs. The final concentration was 2 µmol/L. After 12 h of culture, cells were washed and resuspended with PBS prepared for mice injection. The total collected cell was 1 × 106 cells, and the route of administration was intravenous.
Bioluminescence imaging of Fluc-eGFP-labeled MSCs
For bioluminescence imaging (BLI), firefly luciferase (Fluc) was used for MSCs as previously described [35, 36]. For intravital imaging, ALI mice were established by intratracheal injection of LPS. After that, ALI models received intravenous injections of Fluc-labeled 1 × 106 total MSC cells in a volume of 250 μL. After 24 h, ALI mice were imaged using the IVIS Lumina Imaging System (Xenogen Corporation, Hopkinto, MA) after intraperitoneal injection of the substrate of D-luciferin (150 mg/kg; Biosynth International, USA).
Cell counting and protein concentration assay of BALF
To collect BALF, all mice were euthanized after LPS challenge and treatment with PGE2-MSCs and MSCs. The BALF samples were centrifuged to pellet the cells, washed twice with ice-cold PBS and collected before being centrifuged for 5 min at 4 °C. Next, the sedimented cells were resuspended in PBS to obtain total cell counts using a hemocytometer. Neutrophils and macrophages were counted using the Wright-Giemsa staining method .
The left and right upper lobes (RUL) were collected from all groups that were not subjected to BALF collection. The lung tissue samples were fixed for 48 h in 4% PFA, dehydrated in a series of graded ethanol, embedded in paraffin wax, and cut into 5-μm-thick sections. The paraffin-embedded sections were stained with hematoxylin and eosin (H&E) for pathological analysis.
Tissue samples from the lungs were fixed in 4% PFA and dehydrated in 30% sucrose solution before being embedded in (OCT) (Sakura Finetek, 4583, Japan). All samples were cut into 5 µm sections transversely. Anti-rabbit F4/80 antibodies (28,463–1-AP, Proteintech) and anti-rabbit CD206 antibodies (Abcam) were used to stain the samples (18,704–1-AP, Proteintech). The nuclei of each sample were stained with 4′,6-diamidino-2-phenylindole (DAPI, C0065, Solarbio), and all samples on glass slides were mounted with mounting medium and antifade (Solarbio, S2100). Fluorescence images were captured using an Olympus fluorescence microscope. Complete details on antibodies can be found in Additional file 1: Table S1.
Quantitative real-time PCR
Total RNA was isolated from the cells and tissues using TRIzol reagent (Takara, Japan) according to the manufacturer’s instructions. cDNA was generated based on TransScript Fly First-Strand cDNA Synthesis SuperMix (YEASEN, China). qRT‒PCR was performed using SYBR Green PCR Master Mix (YEASEN, China). A CFX96 TM Real-Time PCR System was used for the real-time PCR analysis (Bio-Rad, USA). The estimate of gene expression was normalized to actin expression and calculated using the 2(−ΔΔCt) method. The primer sequences can be found in Additional file 1: Table S2.
Western blot analysis
Tissues prepared for western blotting were lysed in radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (GenStrar, China). A total of 30 µg of protein from each sample was run on 10% SDS‒PAGE gels in electrophoresis buffer and transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Darmstadt, Germany). Then, skim milk (5%) was used as a blocking buffer for 2 h, and the membrane was incubated with primary antibodies overnight at 4 °C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Signals were visualized with a Pierce-enhanced chemiluminescence Western blotting substrate (Millipore). GAPDH was used as the loading control. Complete details on antibodies can be found in Additional file 1: Table S1.
Total RNA samples from MSCs and PGE2-MSCs were isolated with Trizol (Invitrogen) for RNA sequencing (RNA-seq). Three samples from each group were harvested for RNA isolation. RNA quantity and quality were determined using a Nanodrop (Thermo Scientific, Waltham, MA). RNA-seq was performed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA). Gene ontology (GO) classification and distribution analysis of gene function were done with the Gene Ontology Consortium (Gene Ontology http://geneontology.org/). Gene set enrichment analysis (GSEA, www.broadinstitute.org/gsea) was performed. The Kyoto Encyclopedia of Genes and Genomes (http://www.kegg.jp/) database was used for the genome information and system functions analysis.
All data are shown as the mean ± SEM. of at least three independent replicates. GraphPad Prism version 5.01 was used to analyze all statistical comparisons one-way for multigroup comparisons (GraphPad Software, San Diego, CA, USA). Asterisks denote statistical significance in each figure and the reference group. The basis for comparison was displayed in plus sign if needed.
Characterization and biodistribution of PGE2-MSCs and MSCs
To monitor PGE2-MSCs in vivo in real time, DFMSCs were transduced into MSCs for labeling and injected intravenously (Fig. 1A–D). BLI analysis revealed a significant linear correlation between the amount of PGE2-MSCs and Fluc activity. The determination of the concentration of LPS to establish ALI models has been achieved using different doses of LPS for various animal models. Based on preliminary data, 5 mg/kg LPS was used for LPS-ALI models, and 2 µmol/L PGE2 was used as a priming suitable dose for MSCs culture (Additional file 1: Fig. S1A-B). We intraperitoneally injected Fluc substrate (D-luciferin; 150 mg/kg; Biosynth International, USA) at different time points. Average radiance quantified the BLI signal from the region of interest (ROI) in the lung after LPS-induced ALI (Fig. 1E, F). The results revealed that the labeling and priming by PGE2 were specific for the targeting of the lung of LPS-induced ALI models and revealed therapeutic potential, encouraging further investigation.
Treatment with PGE2-MSCs protects LPS-induced ALI mice
ALI models and BALF samples were collected to investigate the therapeutic effects of (PGE2-MSCs and MSCs) on lung radiance, total cells, total protein concentration, survival rate, and weight rate as designed in Fig. 2A. First, as previously described, we used BLI to track PGE2-MSC and MSC survival in LPS-induced ALI models longitudinally. In summary, we observed the retention and stability of PGE2-MSCs and MSCs in vivo. A total of 1 \(\times\) 106 Fluc-labeled PGE2-MSCs suspended in PBS were intravenously injected into ALI mice at a total volume of 250 μL. At different times, mice were imaged immediately after intraperitoneal injection of D-luciferin (150 mg/kg) using the IVIS Lumina Imaging System. The BLI findings indicated that the robust Fluc signals in all groups indicated successful injection of PGE2-MSCs and MSCs, and the survival of PGE2-MSCs were better than those of MSCs, as shown in Fig. 2B, C. Our results also found that, based on Kaplan‒Meier survival curves, the highest overall survival rates were observed in the PGE2-MSC group compared with the control groups (Fig. 2 D). Furthermore, the PGE2-MSC-treated models improved the weight reduction of the LPS-ALI models compared to the control groups (Fig. 2E). Cell counting and protein levels were measured in BALF to analyze lung damage and inflammation. The total protein level and cell number increased in the ALI and PBS groups but were reduced in the PGE2-MSC and MSC groups (Fig. 2F, G). These results indicate that PGE2-MSCs may effectively reduce cellular infiltration as well as lung injury in ALI models.
PGE2-MSCs affect lung tissue repair and the immune response
The protective effects of PGE2-MSCs against LPS-ALI have been explored in histopathological and immune cell profile evaluations. We assessed the therapeutic effects of PGE2-MSCs (Fig. 3A-F). The histopathological evaluation of lung tissues was examined with a light microscope. Our results showed that the normal structure of alveolar and interstitial tissues was destroyed in LPS-ALI mice, and inflammatory infiltration was prominent, while the PGE2-MSC-treated groups evidently improved the histopathological changes in the LPS-ALI models by reducing inflammatory cells and alveolar hemorrhage compared with the control groups. PGE2-MSCs also improved the lung injury score in LPS-ALI mice compared to the control groups (Fig. 3A, B). Furthermore, PGE2-MSC and MSC treatments remarkably reduced the total number of cells, macrophages, and neutrophils per field compared to the control treatments (Fig. 3C-F). Collectively, our findings found that PGE2-MSCs alleviated LPS-induced ALI.
Identification of the potential mechanisms of PGE2-MSCs in LPS-ALI Mice by RNA-Seq
To further investigate the molecular mechanisms by which PGE2-MSCs attenuate LPS-ALI models, comprehensive RNA-seq analyses were performed for both PGE2-MSCs and MSCs. Cluster analysis was used to evaluate the expression patterns of differentially expressed genes under different experimental conditions; genes with high expression levels between samples were classified into different categories. These genes are involved in certain biological processes or in certain metabolic processes. There is a real connection in the signaling pathways. Therefore, we can discover unknown biological connections between genes by clustering expressions. We used a heatmap package to perform a two-way cluster analysis on the union and samples of different genes in all comparison groups, clustering according to the expression level of the same gene in different samples and the expression pattern of different genes in the same samples. Our results showed that the top 40 expressed genes were related to immune response processes such as macrophages and neutrophils and cytokine signaling (Fig. 4A).
To count the set of significantly differentially expressed genes and make a histogram of differentially expressed genes between other comparison groups, Venn diagram analyses were used to provide a comprehensive profile of PGE2-MSCs and MSCs. It also counts the number of differentially expressed genes that were upregulated and downregulated in each comparison group. In total, 1163 genes were significantly upregulated in PGE2-MSCs compared with MSCs, and most of them were related to functional biological processes, including regulation of cell proliferation, cytokine signaling, immune responses, and metabolic processes (Fig. 4B).
A volcano map was drawn using the ggplot2 package for mapping differentially expressed genes. The volcano map shows the distribution of genes, the difference in gene expression multiples, and the significant results. Under normal conditions, the distribution of differential genes on the left and right of the figure should be roughly symmetric, with the left side being Case compared to Control. The dot plot also represents an overview of pathway enrichment analysis for up-regulated genes and down regulated genes (Fig. 4C, D).
To understand the biological functions of the proteins contained in PGE2-MSCs and MSCs, we used gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of coexpressed proteins. Most of the factors identified were related to the cytokine signaling pathway, metabolic proteins and catabolic proteins (Fig. 5A, B & Additional file 1: Fig. S2A-B). The enrichment analyses of the gene set (GSEA) of PGE2-MSCs-RNA-seq analysis indicated that transcription of genes associated with the acute inflammatory response and macrophage activation was significantly downregulated (Fig. 5C, D). Taken together, these findings suggest that PGE2-MSCs positively regulate several signaling pathways with significant effects on macrophages and other immune cells. The general impact of this positive regulation could be responsible for lung amelioration and modulation of the immune response in LPS-ALI mice.
Treatment with PGE2-MSCs attenuates LPS-induced ALI in mice through regulating macrophage polarization and cytokine production
To assess the potential mechanism of PGE2-MSCs and MSCs to protect LPS-induced ALI mice, we tested M1 and M2 polarization experimentally and supported these findings by complete analysis of RNA-Seq and immunofluorescence staining for F4/80 and CD 206, as illustrated in Fig. 6A-G & Additional file 1: Fig. S3. The same immunohistochemical results were also obtained for F4/80 (Fig. 6A, B) and CD 206 (Fig. 6C, D). By qRT‒PCR, the relative levels of the expression of the M2 polarization markers Arg-1 and CD 206 in the PGE2-MSC group were the highest compared to those in the other groups (Fig. 6E-G). In contrast, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1 β), and inducible nitric oxide synthase (iNOS) were reduced after administration (Fig. 7E-H). TNF-α, IL-6 and iNOS are markers of the inflammatory state of M1 polarization, while interleukin-10 (IL-10), CD206, and Arginase-1 (Arg-1) represent the anti-inflammatory state of M2 polarization [38,39,40]. Collectively, M2 polarization is critical for ALI-lung attenuation of PGE2-MSC administration.
Therapeutic mechanisms of PGE2-MSCs in LPS-ALI mice via the SMAD3, α-SMA and MMP2 pathways
The accumulation of immune cells and immune mediators such as cytokines increases the fibrotic risk of lung tissue. Therefore, we investigated the expression of SMAD3, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-2 (MMP2). Our data showed that in agreement with RNA-seq analysis, PGE2-MSC treatment with PGE2-MSCs significantly downregulated SMAD3, α-SMA and MMP2 (Fig. 7A-D, Additional file 1: Fig. S4), and cytokine profiles have been previously described (Fig. 7 E–H). Experimental evidence in specific organisms can be generalized to other organisms through genomic information. To investigate the potential mechanisms of PGE2-MSCs, we applied bioinformatics tools based on RNA sequencing analysis (Fig. 5A, B, Additional file 1: Fig. S5A-D, Fig. S6). We used the GO and KEGG pathways to detect genes related to inflammation. Therefore, several pathways have been represented as potential mechanisms, such as the MAPK-NIK/NF-kappaB-TLR/JAK-STAT pathways.
The summary of our present study is as follows: intravenous administration of PGE2-MSCs to mice successfully delivered to the lung significantly improved survival and weight ratio and obviously reduced lung inflammation, total cell number and protein permeability in LPS-induced ALI mice. The therapeutic effects of PGE2-primed MSCs were better than those of MSCs as a single treatment. Histopathological changes and immune cell findings of PGE2-MSCs support our hypothesis of the therapeutic potential of PGE2-priming MSCs, which could improve lung regeneration and mediate the balance of immune response cells in LPS-induced ALI models. The comprehensive genomic analysis provides other evidence of the therapeutic potential and molecular mechanisms of PGE2 priming MSCs for LPS-induced ALI models, and macrophage polarization plays an essential role in anti-inflammation and regeneration of the injured alveolus with clear antifibrotic efficacy in ALI mice (Fig. 8).
Macrophage polarization is thought to be a dynamic, developing, and heterogeneous phenomenon. The microenvironment affects macrophage phenotypes and functions. Regulating the production of cytokines and transcription factors may control cellular function during polarization [41, 42]. Macrophages play a critical role in the inflammatory response following ALI by releasing inflammatory mediators. Macrophage-polarized M1 macrophages are proinflammatory macrophages that can be found in the early stage of tissue injury, while M2 macrophages contribute significantly to tissue regeneration [43, 44]. In a pathological state, the expression level of proinflammatory cytokines such as IFN-γ, TNF-α IL-6 and IL-1-β increases with a reduction in macrophage-associated mediators such as IL-10 [30, 45]. Here, we found that the administration of PGE2-MSCs induced macrophages to shift toward M2 macrophages, suggesting that the critical regulator of PGE2-MSCs in tissue regeneration could be controlled by mediating macrophage polarization.
Both primary and preclinical applications of MSC-based therapy have powerfully attracted attention for the development of ARDS treatments and other lung disorders [11, 46]. By transferring several bioactive molecules, MSCs play a crucial role in physiological and pathological conditions [32, 47]. The interaction between MSCs and target cells is critical for their MSC functions [38, 48, 49]. Increasing evidence shows that the therapeutic efficacy and paracrine factors of MSCs could be influenced by biological, biochemical, and/or biophysical factors. In fact, priming of MSCs is required to improve their roles in the microenvironment of injured tissues [27, 50,51,52].
PGE2, a major prostaglandin generated by COX-1 and COX-2 enzymes, mediates several physiological and pathological roles [19, 53]. Several cell types can produce PGE2, such as fibroblasts and inflammatory cells, and the distinct secretion of PGE2 occurs during the immune response [54, 55]. It has been reported that PGE2 may play proinflammatory and anti-inflammatory roles by binding to EP1-EP4 receptors [35, 56, 57]. Via PGE2 secretion, MSCs mediate the activation of macrophage (M2) polarization and inhibit the proliferation of activated T, NK and NKT cells [49, 58]. Recent work using the bioactive molecule PGE2 showed promise for angiogenesis functions and regenerative therapy .
Different strategies have been introduced as suggestions for approaches to enhance the therapeutic potential of stem cells. Priming MSCs with biofactors and chemical factors improved the therapeutic efficacy of MSCs by regulating their secretion [27, 28]. Previous works have extensively studied the potential roles of MSC priming by a wide range of biofactors, such as IFN-γ [59,60,61], TNF-α [62, 63], IL-1α-β , FGF-2 , LPS , IL-17A , TLR3  and IGF-1 [69, 70], which represent promising findings in improving MSC treatment profiles for different diseases. This work is the first study to use PGE2 to prepare MSCs with therapeutic potential for LPS-induced ALI models. Our previous work reported the therapeutic potential of MSC-EVs in radiation-induced lung injury and their roles in endothelial cell damage, vascular permeability, inflammation, and fibrosis . At present, we reveal the protective effects of soothing and regenerating. Related work reported the therapeutic efficacy of MSCs and their extracellular vesicles for lung inflammation, including COVID-19, by secreting a wide range of paracrine factors and balancing immune response processes [71,72,73,74].
In addition to our preliminary investigation, several studies reported histopathological changes in ALI models with increasing adaptive immune cell infiltration (macrophages and neutrophils) and capillary permeability [31, 75]. Our findings showed that PGE2-primed MSCs markedly reduced the numbers of macrophages and neutrophils in BALF. As in previous reports, LPS-induced ALI leads to edema with increasing protein concentration and total cell numbers in BALF [76, 77], and our treatment with PGE2 priming MSCs evidently reduced both in BALF. Inflammatory regulators, especially macrophage-associated cytokines, upregulate inflammatory reactions and are known as the etiology of the cytokine storm, which is a considerable obstacle for lung treatments. They also represent the hallmark of acute lung injury [31, 78, 79]. Using genomic, proteomic and experimental analyses, we observed that PGE2-primed MSCs show a marked ability to balance proinflammatory and anti-inflammatory cytokines.
Our strategy has some limitations. First, this work achieved acute lung injury, but ALI can progress to ARDS in many cases and cause substantial respiratory system failure. Delaying the transition from ALI to ARDS should therefore be considered as well. In addition, we employed a single dose of MSCs and PGE2-MSCs that was set arbitrarily and with the help of prior experience. Dose‒response studies, which compare the effects of varying doses of MSCs and PGE2-MSCs, should be a standard part of future research to find optimal and minimally effective doses. Finally, we described the genomic and molecular mechanisms contributing to the protective effects of MSCs and PGE2-MSCs. Nevertheless, it would be clinically and pharmacologically useful to investigate which small molecules and RNAs are the functional components mediating the protective effects of PGE2-MSCs.
In summary, this work was designed to use PGE2 priming to enhance the therapeutic potential of MSCs for ALI models. Our findings revealed that PGE2-primed MSCs conclusively protected and regenerated lung injury after LPS challenge. PGE2 priming of MSCs elevates the polarization of macrophages from the proinflammatory subset (M2) to the anti-inflammatory subset (M2) by regulating cytokine production and blocking polymorphonuclear neutrophil influx into the injured tissue and preventing further damage. Therefore, M2 macrophage polarization is a critical mediator of PGE2 priming of MSCs in ALI models. This study provides a candidate for ALI treatment and deserves attention in future clinical settings.
Availability of data and materials
The transcriptome sequence data have been deposited in the NCBI Sequence Read Archive under the primary accession code PRJNA938188 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA938188). All other data are included in the article and its Supplementary Information files or available from the corresponding authors upon reasonable request.
Acute lung injury
Acute respiratory distress syndrome
Green fluorescent protein
Enhanced green fluorescence protein
Human placental Mesenchymal stem cell
Mesenchymal stem cells
Coronavirus disease 2019
Fetal bovine serum
Hematoxylin and eosin
Polymerase chain reaction
Metwaly SM, Winston BW. Systems biology ARDS research with a focus on metabolomics. Metabolites. 2020;10(5):207.
Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med. 2005;353(16):1685–93.
Dushianthan A, Grocott MP, Postle AD, Cusack R. Acute respiratory distress syndrome and acute lung injury. Postgrad Med J. 2011;87(1031):612–22.
Saguil A, Fargo M. Acute respiratory distress syndrome: diagnosis and management. Am Fam Physician. 2012;85(4):352–8.
Li R, Wang C, Zhou M, Liu Y, Chen S, Chai Z, et al. Heparan sulfate proteoglycan-mediated internalization of extracellular vesicles ameliorates liver fibrosis by targeting hepatic stellate cells. Extracellular Vesicle. 2022;1: 100018.
Jia PP, Zhao XT, Liu Y, Liu MA, Zhang QA, Chen S, et al. The RGD-modified self-assembling D-form peptide hydrogel enhances the therapeutic effects of mesenchymal stem cells (MSC) for hindlimb ischemia by promoting angiogenesis. Chem Eng J. 2022;450: 138004.
Ren J, Liu Y, Yao Y, Feng L, Zhao X, Li Z, et al. Intranasal delivery of MSC-derived exosomes attenuates allergic asthma via expanding IL-10 producing lung interstitial macrophages in mice. Int Immunopharmacol. 2021;91: 107288.
Behnke J, Kremer S, Shahzad T, Chao CM, Bottcher-Friebertshauser E, Morty RE, et al. MSC based therapies-new perspectives for the injured lung. J Clin Med. 2020;9(3):682.
Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol. 2007;179(3):1855–63.
Martens A, Ordies S, Vanaudenaerde BM, Verleden SE, Vos R, Van Raemdonck DE, et al. Immunoregulatory effects of multipotent adult progenitor cells in a porcine ex vivo lung perfusion model. Stem Cell Res Ther. 2017;8(1):159.
Hezam K, Mo R, Wang C, Liu Y, Li Z. Anti-inflammatory effects of mesenchymal stem cells and their secretomes in pneumonia. Curr Pharm Biotechnol. 2022;23(9):1153–67.
Najar M, Raicevic G, Fayyad-Kazan H, Bron D, Toungouz M, Lagneaux L. Mesenchymal stromal cells and immunomodulation: a gathering of regulatory immune cells. Cytotherapy. 2016;18(2):160–71.
Cheng H, Liu F, Zhou M, Chen S, Huang H, Liu Y, et al. Enhancement of hair growth through stimulation of hair follicle stem cells by prostaglandin E2 collagen matrix. Exp Cell Res. 2022;421(2): 113411.
Chen S, Huang H, Liu Y, Wang C, Chen X, Chang Y, et al. Renal subcapsular delivery of PGE2 promotes kidney repair by activating endogenous Sox9(+) stem cells. iScience. 2021;24(11):103243.
Park JY, Pillinger MH, Abramson SB. Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin Immunol. 2006;119(3):229–40.
Kondeti V, Al-Azzam N, Duah E, Thodeti CK, Boyce JA, Paruchuri S. Leukotriene D4 and prostaglandin E2 signals synergize and potentiate vascular inflammation in a mast cell-dependent manner through cysteinyl leukotriene receptor 1 and E-prostanoid receptor 3. J Allergy Clin Immunol. 2016;137(1):289–98.
Sander WJ, O’Neill HG, Pohl CH. Prostaglandin E as a modulator of viral infections. Front Physiol. 2017;8:89.
Friedman EA, Ogletree ML, Haddad EV, Boutaud O. Understanding the role of prostaglandin E2 in regulating human platelet activity in health and disease. Thromb Res. 2015;136(3):493–503.
Cheng H, Huang H, Guo Z, Chang Y, Li Z. Role of prostaglandin E2 in tissue repair and regeneration. Theranostics. 2021;11(18):8836–54.
Cao X, Duan L, Hou H, Liu Y, Chen S, Zhang S, et al. IGF-1C hydrogel improves the therapeutic effects of MSCs on colitis in mice through PGE-mediated M2 macrophage polarization. Theranostics. 2020;10(17):7697–709.
Chen X, Tang J, Shuai W, Meng J, Feng J, Han Z. Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome. Inflamm Res. 2020;69(9):883–95.
Kim J, Hematti P. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol. 2009;37(12):1445–53.
Martinez FO, Helming L, Gordon S. Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol. 2009;27:451–83.
Lo Sicco C, Reverberi D, Balbi C, Ulivi V, Principi E, Pascucci L, et al. Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: endorsement of macrophage polarization. Stem Cells Transl Med. 2017;6(3):1018–28.
Morrison TJ, Jackson MV, Cunningham EK, Kissenpfennig A, McAuley DF, O’Kane CM, et al. Mesenchymal stromal cells modulate macrophages in clinically relevant lung injury models by extracellular vesicle mitochondrial transfer. Am J Respir Crit Care Med. 2017;196(10):1275–86.
Zhu Y, Xu L, Collins JJP, Vadivel A, Cyr-Depauw C, Zhong S, et al. Human umbilical cord mesenchymal stromal cells improve survival and bacterial clearance in neonatal sepsis in rats. Stem Cells Dev. 2017;26(14):1054–64.
Noronha NC, Mizukami A, Caliari-Oliveira C, Cominal JG, Rocha JLM, Covas DT, et al. Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies. Stem Cell Res Ther. 2019;10(1):131.
Zhang SY, Ren JY, Yang B. Priming strategies for controlling stem cell fate: applications and challenges in dental tissue regeneration. World J Stem Cells. 2021;13(11):1625–46.
Zhang S, Liu Y, Zhang X, Zhu D, Qi X, Cao X, et al. Prostaglandin E hydrogel improves cutaneous wound healing via M2 macrophages polarization. Theranostics. 2018;8(19):5348–61.
Lv H, Liu Q, Sun Y, Yi X, Wei X, Liu W, et al. Mesenchymal stromal cells ameliorate acute lung injury induced by LPS mainly through stanniocalcin-2 mediating macrophage polarization. Ann Transl Med. 2020;8(6):334.
Lv H, Liu Q, Wen Z, Feng H, Deng X, Ci X. Xanthohumol ameliorates lipopolysaccharide (LPS)-induced acute lung injury via induction of AMPK/GSK3β-Nrf2 signal axis. Redox Biol. 2017;12:311–24.
Huang A, Liu Y, Qi X, Chen S, Huang H, Zhang J, et al. Intravenously transplanted mesenchymal stromal cells: a new endocrine reservoir for cardioprotection. Stem Cell Res Ther. 2022;13(1):253.
Zhang K, Chen X, Li H, Feng G, Nie Y, Wei Y, et al. A nitric oxide-releasing hydrogel for enhancing the therapeutic effects of mesenchymal stem cell therapy for hindlimb ischemia. Acta Biomater. 2020;113:289–304.
Liu Y, Cui J, Wang H, Hezam K, Zhao X, Huang H, et al. Enhanced therapeutic effects of MSC-derived extracellular vesicles with an injectable collagen matrix for experimental acute kidney injury treatment. Stem Cell Res Ther. 2020;11(1):161.
Huang H, Chen S, Cheng H, Cao J, Du W, Zhang J, et al. The sustained PGE2 release matrix improves neovascularization and skeletal muscle regeneration in a hindlimb ischemia model. J Nanobiotechnology. 2022;20(1):95.
Leng L, Wang Y, He N, Wang D, Zhao Q, Feng G, et al. Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy. Biomaterials. 2014;35(19):5162–70.
Lei X, He N, Zhu L, Zhou M, Zhang K, Wang C, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate radiation-induced lung injury via miRNA-214-3p. Antioxid Redox Signal. 2021;35(11):849–62.
Shi Y, Wang Y, Li Q, Liu K, Hou J, Shao C, et al. Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nat Rev Nephrol. 2018;14(8):493–507.
Cheng P, Li S, Chen H. Macrophages in lung injury, repair, and fibrosis. Cells. 2021;10(2):436.
Lee J-W, Chun W, Lee HJ, Min J-H, Kim S-M, Seo J-Y, et al. The role of macrophages in the development of acute and chronic inflammatory lung diseases. Cells. 2021;10(4):897.
Cao J, Dong R, Jiang L, Gong Y, Yuan M, You J, et al. LncRNA-MM2P identified as a modulator of macrophage M2 polarization. Cancer Immunol Res. 2019;7(2):292–305.
Saleh LS, Vanderheyden C, Frederickson A, Bryant SJ. Prostaglandin E2 and its receptor EP2 modulate macrophage activation and fusion. ACS Biomater Sci Eng. 2020;6(5):2668–81.
Herold S, Mayer K, Lohmeyer J. Acute lung injury: how macrophages orchestrate resolution of inflammation and tissue repair. Front Immunol. 2011;2:65.
Johnston LK, Rims CR, Gill SE, McGuire JK, Manicone AM. Pulmonary macrophage subpopulations in the induction and resolution of acute lung injury. Am J Respir Cell Mol Biol. 2012;47(4):417–26.
Haddad JJ, Fahlman CS. Redox- and oxidant-mediated regulation of interleukin-10: an anti-inflammatory, antioxidant cytokine? Biochem Biophys Res Commun. 2002;297(2):163–76.
Islam D, Huang Y, Fanelli V, Delsedime L, Wu S, Khang J, et al. Identification and modulation of microenvironment is crucial for effective mesenchymal stromal cell therapy in acute lung injury. Am J Respir Crit Care Med. 2019;199(10):1214–24.
Zhang K, Li R, Chen X, Yan H, Li H, Zhao X, et al. Renal endothelial cell-targeted extracellular vesicles protect the kidney from ischemic injury. Adv Sci (Weinh). 2023;10:e2204626.
Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles. 2014;3:3.
Volarevic V, Gazdic M, Simovic Markovic B, Jovicic N, Djonov V, Arsenijevic N. Mesenchymal stem cell-derived factors: Immuno-modulatory effects and therapeutic potential. BioFactors. 2017;43(5):633–44.
Baldari S, Di Rocco G, Piccoli M, Pozzobon M, Muraca M, Toietta G. Challenges and strategies for improving the regenerative effects of mesenchymal stromal cell-based therapies. Int J Mol Sci. 2017;18(10):2087.
Hu C, Li L. Preconditioning influences mesenchymal stem cell properties in vitro and in vivo. J Cell Mol Med. 2018;22(3):1428–42.
Noone C, Kihm A, English K, O’Dea S, Mahon BP. IFN-gamma stimulated human umbilical-tissue-derived cells potently suppress NK activation and resist NK-mediated cytotoxicity in vitro. Stem Cells Dev. 2013;22(22):3003–14.
Chu CH, Chen SH, Wang Q, Langenbach R, Li H, Zeldin D, et al. PGE2 inhibits IL-10 production via EP2-mediated beta-arrestin signaling in neuroinflammatory condition. Mol Neurobiol. 2015;52(1):587–600.
Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther. 2004;103(2):147–66.
Jiang W, Xu J. Immune modulation by mesenchymal stem cells. Cell Prolif. 2020;53(1): e12712.
Jiang J, Dingledine R. Role of prostaglandin receptor EP2 in the regulations of cancer cell proliferation, invasion, and inflammation. J Pharmacol Exp Ther. 2013;344(2):360–7.
Lee BC, Kim HS, Shin TH, Kang I, Lee JY, Kim JJ, et al. PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact. Sci Rep. 2016;6:26298.
Gazdic M, Volarevic V, Arsenijevic N, Stojkovic M. Mesenchymal stem cells: a friend or foe in immune-mediated diseases. Stem Cell Rev Rep. 2015;11(2):280–7.
Sonoda S, Yamaza H, Ma L, Tanaka Y, Tomoda E, Aijima R, et al. Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells. Sci Rep. 2016;6:19286.
He X, Jiang W, Luo Z, Qu T, Wang Z, Liu N, et al. IFN-gamma regulates human dental pulp stem cells behavior via NF-kappaB and MAPK signaling. Sci Rep. 2017;7:40681.
Strojny C, Boyle M, Bartholomew A, Sundivakkam P, Alapati S. Interferon gamma-treated dental pulp stem cells promote human mesenchymal stem cell migration in vitro. J Endod. 2015;41(8):1259–64.
Shi L, Fu S, Fahim S, Pan S, Lina H, Mu X, et al. TNF-alpha stimulation increases dental pulp stem cell migration in vitro through integrin alpha-6 subunit upregulation. Arch Oral Biol. 2017;75:48–54.
Qin Z, Fang Z, Zhao L, Chen J, Li Y, Liu G. High dose of TNF-alpha suppressed osteogenic differentiation of human dental pulp stem cells by activating the Wnt/beta-catenin signaling. J Mol Histol. 2015;46(4–5):409–20.
Redondo-Castro E, Cunningham C, Miller J, Martuscelli L, Aoulad-Ali S, Rothwell NJ, et al. Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro. Stem Cell Res Ther. 2017;8(1):79.
Gorin C, Rochefort GY, Bascetin R, Ying H, Lesieur J, Sadoine J, et al. Priming dental pulp stem cells with fibroblast growth factor-2 increases angiogenesis of implanted tissue-engineered constructs through hepatocyte growth factor and vascular endothelial growth factor secretion. Stem Cells Transl Med. 2016;5(3):392–404.
Croes M, Oner FC, Kruyt MC, Blokhuis TJ, Bastian O, Dhert WJA, et al. Proinflammatory mediators enhance the osteogenesis of human mesenchymal stem cells after lineage commitment. PLoS ONE. 2015;10(7): e0132781.
Sivanathan KN, Rojas-Canales DM, Hope CM, Krishnan R, Carroll RP, Gronthos S, et al. Interleukin-17A-induced human mesenchymal stem cells are superior modulators of immunological function. Stem Cells. 2015;33(9):2850–63.
Qiu Y, Guo J, Mao R, Chao K, Chen BL, He Y, et al. TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in TNBS-induced colitis via the TLR3-Jagged-1-Notch-1 pathway. Mucosal Immunol. 2017;10(3):727–42.
Ma S, Liu G, Jin L, Pang X, Wang Y, Wang Z, et al. IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla. Sci Rep. 2016;6:36922.
Feng X, Huang D, Lu X, Feng G, Xing J, Lu J, et al. Insulin-like growth factor 1 can promote proliferation and osteogenic differentiation of human dental pulp stem cells via mTOR pathway. Dev Growth Differ. 2014;56(9):615–24.
Monsel A, Zhu YG, Gennai S, Hao Q, Hu S, Rouby JJ, et al. Therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice. Am J Respir Crit Care Med. 2015;192(3):324–36.
Park J, Kim S, Lim H, Liu A, Hu S, Lee J, et al. Therapeutic effects of human mesenchymal stem cell microvesicles in an ex vivo perfused human lung injured with severe pneumonia. Thorax. 2019;74(1):43–50.
Zhu YG, Feng XM, Abbott J, Fang XH, Hao Q, Monsel A, et al. Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin-induced acute lung injury in mice. Stem Cells. 2014;32(1):116–25.
Mei SH, Haitsma JJ, Dos Santos CC, Deng Y, Lai PF, Slutsky AS, et al. Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med. 2010;182(8):1047–57.
Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;342(18):1334–49.
Grommes J, Soehnlein O. Contribution of neutrophils to acute lung injury. Mol Med. 2011;17(3–4):293–307.
Tsushima K, King LS, Aggarwal NR, De Gorordo A, D’Alessio FR, Kubo K. Acute lung injury review. Intern Med. 2009;48(9):621–30.
Cross LJ, Matthay MA. Biomarkers in acute lung injury: insights into the pathogenesis of acute lung injury. Crit Care Clin. 2011;27(2):355–77.
Gomez-Escobar LG, Hoffman KL, Choi JJ, Borczuk A, Salvatore S, Alvarez-Mulett SL, et al. Cytokine signatures of end organ injury in COVID-19. Sci Rep. 2021;11(1):12606.
This study was financially supported by the National Natural Science Foundation of China (U2004126), the Tianjin Natural Science Foundation (22JCZXJC00170, 21JCZDJC00070), Research Project on Skin Injury & Repair (BKJ21J016), and Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-043A).
Ethics approval and consent to participate
The Ethics Committee for the Use of Animals of Nankai University, approved the experimental protocols for studies of mesenchymal stem cells (MSCs) for acute lung injury therapy (project title: The therapeutic effects and mechanisms of mesenchymal stem cells (MSCs) for acute lung injury; Approval no. 20170022; Date of approval: Nov 20, 2017).
Consent for publication
The authors have declared that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
. Complete details of antibodies. Table S2. Primers Used for Real-Time PCR. Fig. S1. Dose and time point effects of LPS-induced ALI in mouse models. Fig. S2. RNA-Seq analysis to assess the expression patterns of differentially expressed genes under different experimental conditions. Fig. S3. PGE2-MSCs regulate macrophage polarization and cytokine production. Fig. S4. Images of the uncropped immunoblots are shown in Fig. 7A.
About this article
Cite this article
Hezam, K., Wang, C., Fu, E. et al. Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation. Stem Cell Res Ther 14, 48 (2023). https://doi.org/10.1186/s13287-023-03277-9