Abstract
Background
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.
Methods
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.
Results
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.
Conclusion
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.
Supplementary Information
The online version contains supplementary material available at
10.1186/s13287-023-03277-9.
Keywords: Acute lung injury (ALI), Acute respiratory distress syndrome
(ARDS), Prostaglandin E2 (PGE2), Mesenchymal stem cells (MSCs), Priming
Introduction
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)
[[45]1, [46]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 [[47]3, [48]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 [[49]5–[50]7]. Increasing evidence of MSC migration to lung
injury and their contribution to lung regeneration attracts attention
for the treatment of ALI/ARDS [[51]8]. 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 [[52]9,
[53]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)
[[54]11, [55]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
[[56]13, [57]14]. It can be synthesized by many tissue cells, such as
epithelial cells, fibroblasts, and inflammatory cells, that infiltrate
tissues after partial injury [[58]15–[59]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 [[60]19,
[61]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
[[62]21–[63]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 [[64]18, [65]24–[66]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 [[67]27, [68]28]. The lipid signaling
molecule prostaglandin E2 (PGE2), an inflammatory mediator, can enhance
tissue regeneration and repair after injury in various organ systems
[[69]19, [70]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
Animals
ALI models were established as previously reported [[71]30, [72]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).
Experimental protocol
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
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10^6) and PGE2-MSCs group (1
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10^6). 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 [[73]32]. 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.
Cell culture
As previously reported [[74]33, [75]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
[[76]6, [77]32]. MSCs were incubated at 37 °C in a humidified incubator
containing 5% CO[2]. 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 × 10^6 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 [[78]35, [79]36]. For intravital
imaging, ALI mice were established by intratracheal injection of LPS.
After that, ALI models received intravenous injections of Fluc-labeled
1 × 10^6 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 [[80]37].
Histopathological evaluation
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.
Immunofluorescence staining
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 [81]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 [82]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
[83]1: Table S1.
RNA sequencing
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
[84]http://geneontology.org/). Gene set enrichment analysis (GSEA,
[85]www.broadinstitute.org/gsea) was performed. The Kyoto Encyclopedia
of Genes and Genomes ([86]http://www.kegg.jp/) database was used for
the genome information and system functions analysis.
Statistical 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.
Results
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. [87]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 [88]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. [89]1E, F). The results
revealed that the labeling and priming by PGE[2] were specific for the
targeting of the lung of LPS-induced ALI models and revealed
therapeutic potential, encouraging further investigation.
Fig. 1.
[90]Fig. 1
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Characterization and biodistribution of PGE2-MSCs and MSCs. A Schematic
representation of the double-fusion (DF) firefly luciferase (Fluc) and
green fluorescent protein reporter gene containing Fluc and GFP driven
by ubiquitin promotion. B Brightfield and fluorescence microscopy
showing GFP expression in MSCs and PGE2-MSCs. C, D The quantification
BLI of MSCs and PGE2-MSCs shows a robust correlation between cell
number and Fluc activity. The signal activity was represented by
photons/s/cm^2/steradian. E, F Fluc signals were tracked by BLI
analysis to monitor the retention of transplanted PGE2-MSCs and MSCs in
vivo. The signal activity was represented by photons/s/cm^2/steradian
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. [92]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
[MATH: × :MATH]
10^6 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. [93]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. [94]2 D). Furthermore, the
PGE2-MSC-treated models improved the weight reduction of the LPS-ALI
models compared to the control groups (Fig. [95]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. [96]2F, G). These results indicate that PGE2-MSCs may effectively
reduce cellular infiltration as well as lung injury in ALI models.
Fig. 2.
[97]Fig. 2
[98]Open in a new tab
Protective effects of PGE2-MSC treatment against LPS-ALI in mice. A
Schematic of the priming of PGE2-MSCs and study design. B
Representative BLI of mice transplanted with PGE2-MSCs and MSCs for in
vivo monitoring. C Quantitative analysis of BLI signals showed that the
survival of PGE2-MSCs were better than those of MSCs. Data are
expressed as the mean ± SEM. *P < 0.05. D Kaplan–Meier survival curves
of LPS-induced ALI mice after administration of PGE2-MSCs, MSCs and PBS
for 5 days. E Body weight was measured daily to observe the severity of
ALI. F, G Total cell count and total protein concentration in BALF of
LPS-induced ALI mice with or without MSC treatment at 24 h 48 h and
72 h. Data are presented as the mean ± SEM. *P < 0.05 vs ALI;^#P < 0.05
vs PBS; ^&P < 0.05 vs MSCs
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. [99]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. [100]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. [101]3C-F). Collectively, our findings found
that PGE2-MSCs alleviated LPS-induced ALI.
Fig. 3.
[102]Fig. 3
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Effects of PGE2-MSC treatment on lung tissue repair and the immune
response. A, B HE staining of the lungs of the mice (n = 5) of each
experimental group was processed for histological evaluation 24 h after
the LPS challenge, and the lung injury score was determined as a
percentage. C-F Total cell counts per field, macrophages, and
neutrophils from the BALF were counted using a hemocytometer, and the
Wright-Giemsa staining method was used for cytosine staining (HE
magnification is 100 × and 200x, the Wright-Giemsa is 40 × and 100x).
Data are represented as the mean ± SEM. *P < 0.05 vs ALI;^#P < 0.05 vs
PBS; ^&P < 0.05 vs MSCs
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. [104]4A).
Fig. 4.
[105]Fig. 4
[106]Open in a new tab
RNA analysis of PGE2-MSCs. A Heatmap of the RNA-seq analysis showing
differences in the expression of the phagocytosis, anti-inflammation
and fibrogenic components of MSCs primed with PGE2. N represents MSCs
(without treatments), and P represents PGE2-MSCs. B Venn diagrams
showing the overlap between genes from MSCs primed with PGE2 and
untreated MSCs. C The volcano plot of differentially expressed genes
was drawn by the ggplots2 software package. The volcano plot shows the
distribution of genes, the fold difference of gene expression, and the
significance results. D Dotplot pathway enrichment map showing the
significantly overrepresented pathways
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. [107]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. [108]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. [109]5A, B & Additional file [110]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. [111]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.
Fig. 5.
[112]Fig. 5
[113]Open in a new tab
PGE2-primed MSCs revealed a unique protein expression profile. A Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway analysis of PGE2-MSC
protein expression. B GO analysis of proteins in PGE2-MSCs. C, D Gene
set enrichment analysis (GSEA) of PGE2-MSCs, revealing the regulation
of the acute inflammatory response and macrophage activation. The p
value was calculated by GSEA
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. [114]6A-G & Additional file [115]1: Fig. S3. The same
immunohistochemical results were also obtained for F4/80 (Fig. [116]6A,
B) and CD 206 (Fig. [117]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. [118]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. [119]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 [[120]38–[121]40]. Collectively, M2 polarization is
critical for ALI-lung attenuation of PGE2-MSC administration.
Fig. 6.
[122]Fig. 6
[123]Open in a new tab
PGE2-MSCs regulate macrophage polarization and cytokine production. A
Representative immunofluorescence images of F4/80 expression (red) in
LPS-induced ALI mice. Scale bar, 200 µm. B Quantification of
F4/80 + cells in each group. C Immunofluorescence images of CD206
expression (green) in LPS-induced ALI mice. Scale bar, 100 µm. D
Quantification of CD206 + cells in each group. E–G qRT‒PCR analysis of
macrophage-related gene expression (Arg1, CD206, IL-6 and iNOS). Data
are represented as the mean ± SEM. *P < 0.05 vs ALI;^#P < 0.05 vs PBS;
^&P < 0.05 vs MSC
Fig. 7.
[124]Fig. 7
[125]Open in a new tab
Therapeutic mechanisms of PGE2-MSCs in LPS-ALI mice A-D Representative
immunoblot and expression analysis of SMAD3, MMP2, and α-SMA in
LPS-induced ALI mice. E qRT‒PCR analysis of IL-6 mRNA in LPS-ALI mice
monitored for 24 h, 48 h, and 72 h. F qRT‒PCR analysis of IL-1β mRNA in
LPS-ALI mice monitored for 24 h, 48 h, and 72 h. G qRT‒PCR analysis of
TNF-α mRNA in LPS-ALI mice monitored for 24 h, 48 h, and 72 h. H
qRT‒PCR analysis of IL-10 mRNA in LPS-ALI mice monitored for 24 h,
48 h, and 72 h. Data are represented as the mean ± SEM. *P < 0.05 vs
ALI;^#P < 0.05 vs PBS; ^&P < 0.05 vs MSC
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. [126]7A-D, Additional file
[127]1: Fig. S4), and cytokine profiles have been previously described
(Fig. [128]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. [129]5A, B,
Additional file [130]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.
Discussion
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. [131]8).
Fig. 8.
[132]Fig. 8
[133]Open in a new tab
Schematic diagram of the PGE2-MSC treatment mechanisms for LPS-ALI by
regulating macrophage polarization. Both RNA-seq analysis and
experimental results prove that treatment with PGE2-MSCs effectively
protected LPS-induced ALI induced by LPS by several mechanisms that
mediate the regulation of immune cells, anti-inflammation, fluid
clearance, and tissue regeneration. This scheme was created by authors
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
[[134]41, [135]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
[[136]43, [137]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 [[138]30, [139]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 [[140]11, [141]46]. By transferring several
bioactive molecules, MSCs play a crucial role in physiological and
pathological conditions [[142]32, [143]47]. The interaction between
MSCs and target cells is critical for their MSC functions [[144]38,
[145]48, [146]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 [[147]27, [148]50–[149]52].
PGE2, a major prostaglandin generated by COX-1 and COX-2 enzymes,
mediates several physiological and pathological roles [[150]19,
[151]53]. Several cell types can produce PGE2, such as fibroblasts and
inflammatory cells, and the distinct secretion of PGE2 occurs during
the immune response [[152]54, [153]55]. It has been reported that PGE2
may play proinflammatory and anti-inflammatory roles by binding to
EP1-EP4 receptors [[154]35, [155]56, [156]57]. Via PGE2 secretion, MSCs
mediate the activation of macrophage (M2) polarization and inhibit the
proliferation of activated T, NK and NKT cells [[157]49, [158]58].
Recent work using the bioactive molecule PGE2 showed promise for
angiogenesis functions and regenerative therapy [[159]35].
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 [[160]27, [161]28]. Previous works
have extensively studied the potential roles of MSC priming by a wide
range of biofactors, such as IFN-γ [[162]59–[163]61], TNF-α [[164]62,
[165]63], IL-1α-β [[166]64], FGF-2 [[167]65], LPS [[168]66], IL-17A
[[169]67], TLR3 [[170]68] and IGF-1 [[171]69, [172]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 [[173]37]. 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
[[174]71–[175]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 [[176]31, [177]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
[[178]76, [179]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 [[180]31, [181]78, [182]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.
Conclusion
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.
Supplementary Information
[183]13287_2023_3277_MOESM1_ESM.docx^ (5.8MB, docx)
Additional file 1. Table S1. 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. [184]7A.
Acknowledgements