Mesenchymal stem cell-secreted prostaglandin E2 ameliorates acute liver failure via attenuation of cell death and regulation of macrophage polarization

Background Acute liver failure (ALF) is an acute inflammatory liver disease with high mortality. Previous preclinical and clinical trials have confirmed that mesenchymal stem cell (MSC) is a promising therapeutic approach; however, the effect is not satisfied as the underlying molecular mechanisms of MSC in treating ALF remain unclear. Methods MSC isolated from 4- to 6-week-old C57BL/6 mice were used to treat ALF. Histological and serological parameters were analyzed to evaluate the efficacy of MSC. We explored the molecular mechanism of MSC in the treatment of ALF by detecting liver inflammatory response and hepatocyte death. Results In this study, we found that the therapeutic potential of MSC on ALF is dependent on the secretion of prostaglandin E2 (PGE2), a bioactive lipid. MSC-derived PGE2 inhibited TGF-β-activated kinase 1 (TAK1) signaling and NLRP3 inflammasome activation in liver macrophages to decrease the production of inflammatory cytokines. Meanwhile, macrophages in the liver could be induced to anti-inflammatory (M2) macrophages by MSC-derived PGE2 via STAT6 and mechanistic target of rapamycin (mTOR) signaling, which then promote inflammatory resolution and limit liver injury. Finally, administrating EP4 antagonist significantly ameliorated the therapeutic ability of MSC, which promoted liver inflammation and decreased M2 macrophages. Conclusions Our results indicate that PGE2 might be a novel important mediator of MSC in treating ALF, which is through inhibiting the liver inflammatory response and hepatocyte death.


Introduction
Acute liver failure (ALF) is a clinical syndrome with high mortality rate, and liver transplantation is the only effective way to cure ALF now [1].Due to the shortage of organ donor resources, alternative treatments are needed urgently [2].It has been shown that mesenchymal stem cell (MSC) not only rescued several animal models of liver injury, but that it showed an effective therapeutic potential in clinical trials, which is based on their ability to secrete various trophic factors [3].Our previous study confirmed that MSC-derived PGE 2 could promote hepatocyte proliferation in ALF; however, the role of MSCderived PGE 2 on liver inflammation still remains unclear.
Several studies confirmed the cell death and hepatic inflammation in the progression of ALF [4].Inflammatory mediators of toxic liver injury include cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1β, and reactive oxygen species (ROS) which accelerate liver injury [5].Most of these inflammatory mediators were secreted by innate immune cells in the liver, including monocytes, macrophages, and dendritic cells (DC), which are activated by damage-associated molecular patterns (DAMPs) released by damaged hepatocytes.Liver macrophages hold central functions in initiating, perpetuating, and restricting inflammation.Macrophages can be classified into pro-inflammatory (M1) or antiinflammatory (M2) macrophages [6].M1 cells secrete large amounts of pro-inflammatory cytokines, such as TNF and IL-1β.M2 cells secrete immunosuppressive cytokines, like IL-10, which plays a vital role in the resolution of inflammation.Recent studies have confirmed the role of MSC on macrophage polarization.For example, the therapeutic ability of MSC on cartilage repair and sepsis was dependent on the induction of M2 macrophages [7,8].In various liver disease, the therapeutic ability of MSC also relies on macrophage polarization [9,10].However, the mechanism of MSC on macrophage polarization remains unclear.
Sterile inflammation, which is defined as an inflammatory response triggered by damage mediated by noninfectious sources, plays a major role in ALF [4].The nucleotide-binding and oligomerization domain-like receptor 3 (NLRP3) inflammasome is the main kind of sterile inflammation, which contains three parts: NLRP3, apoptosis-associated speck-like protein (ASC), and procaspase 1.The inflammasome is activated by DAMPs or pathogen-associated molecular patterns (PAMPs), which contribute to the maturation and secretion of IL-1β and IL-18 [11].Recent studies confirmed that inflammasome activation contributes to the progression of various liver disorders, like viral fulminant hepatitis and nonalcoholic fatty liver disease [12].Therefore, targeting the NLRP3 inflammasome might be an effective strategy for reducing liver inflammation.
In this study, we focused on the inflammatory response in ALF.We confirmed that MSC could attenuate liver inflammation and NLRP3 inflammasome activation; meanwhile, it promoted M2 macrophages to resolve inflammation, thus to cure ALF.

Isolation of mouse bone marrow MSC
MSCs were isolated from 4-to 6-week-old C57BL/6 mice as we previously described [13].We used flow cytometry (BD FACSAria II; BD Biosciences, San Jose, CA, USA) to identify the MSCs; the antibodies used were against mice antigens CD29, CD34, CD44, CD45, and CD90 (BD Biosciences).Positive cells were counted and compared to the signals of corresponding immunoglobulin isotypes.Mediums were supplemented with 10% fetal calf serum (Sciencell, San Diego, CA, USA), 100 U/ml penicillin, and 100 g/ml streptomycin.To deplete or overexpress COX2 in MSC to inhibit or increase the synthesis and secretion of PGE 2 , the short hairpin RNA against COX2 or COX2 gene lentivirus (GeneChem, Shanghai, China) was transfected to MSCs following the manufacturer's instructions [named MSC-COX2(−) and MSC-COX2(+)].

Bone marrow-derived macrophage (BMDM) isolation and culture
BMDMs were prepared and cultured as described [14].In brief, BM cells were flushed from femurs and tibias of mice and dispersed mechanically.Cell suspensions were filtered through a 200-mesh filter, and the remaining cells were collected by centrifugation at 300×g for 4 min.After centrifugation, the cells were resuspended and cultured in RIP 1640 supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF, 20 ng/ ml, eBioscience) for 7 days.
EP4 antagonist was added 30 min prior to treatment with PGE 2 or MSC.For macrophage polarization, BMDMs were stimulated with PGE 2 or MSC for 24 h.

Real-time quantitative PCR and western blotting
Total RNA or protein for liver tissues and cells were extracted as previously described [13].The primer sequences and primary antibodies are listed in Supplementary Table S1 and S2.
Small interfering RNA and transfection siRNA-specific EP4 as well as control siRNAs were obtained from RiboBio (GuangZhou, China).The detailed sequences of siRNAs are listed in Supplementary Table S3.Transfection with siRNAs was completed using ribo-FECT™ CP (RiboBio) according to the manufacturer's instruction.

Measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
Serum ALT and AST were determined using an automatic biochemical analyzer (Olympus, Tokyo, Japan) in the Affiliated Drum Tower Hospital of Nanjing University Medical School.

TUNEL and PI assay
Cell apoptosis and death in the liver were measured using the one-step TUNEL Apoptosis Assay kit and propidium iodide (PI) staining (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions.

IL-1β and IL-10 analysis
IL-1β levels in serum or cell supernatant and IL-10 levels in serum were detected by an enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Thermo Scientific, Vienna, Australia).

Caspase 1 activity
Caspase-1 activity in liver tissues was measured by Caspase 1 Activity Assay Kit (Beyotime Biotechnology) according to the manufacturer's instructions.

Statistical analysis
Data were expressed as means ± SD.Differences between groups were assessed by a two-tailed t test assuming unequal variance.Five to 8 mice/group were analyzed using Prism software (GraphPad).p values less than 0.05 were considered as statistically significant.

MSC protects against ALF via PGE 2
Paralyzed with our previous study [13], MSC protected against ALF through PGE 2 , as revealed by plasma levels of ALT and AST (Fig. 1a) and liver damage in HE staining (Fig. 1b).TUNEL and PI staining confirmed the hepatocyte necrosis and apoptosis were decreased when infused with MSC, especially in the MSC-COX2(+) group; however, MSC-COX2(−) failed to decrease hepatocyte necrosis and apoptosis (Fig. 1c, d).Taken together, MSC protected hepatocyte death against ALF via PGE 2 .

MSC ameliorates inflammation in ALF
Liver inflammation is associated with hepatocyte damage and death; therefore, we examined the inflammatory response in the liver after MSC infusion.Notably, mRNA expression levels of inflammatory cytokines and chemokines, including CCL2, IL-1β, iNOS, and TNF-α were significantly inhibited in the MSC group, and in the MSC-COX2(+) group, the levels of these cytokines decreased more obviously.By contrast, the MSC-COX2(−) group, which inhibited secretion of PGE 2 from MSC, failed to decrease these inflammatory cytokines (Fig. 2a).NF-κB signaling plays a great role in inflammation [15].We demonstrated that LPS/D-Gal-induced ALF significantly activated NF-κB signaling in the liver, as the increased expression of the phosphorylation of IKK-β and P65 (p-IKK-β and p-P65) (Fig. 2b).The MSC or MSC-COX2(+) group exhibited an ameliorated phenotype, while MSC-COX2(−) did not show this phenotype (Fig. 2b).Next, we aimed to explore the mechanism of MSC on NF-κB signaling inhibition.TGF-β-activated kinase 1 (TAK1) is an intracellular hub molecule that regulates NF-κB signaling pathways which plays a key role in cell survival and death [16].Our results demonstrated that TAK1 signaling was inhibited after MSC infusion, as the expression of the phosphorylation of TAK1 (p-TAK1), JNK (p-JNK), P38 (p-P38), and c-Jun (p-c-Jun) were significantly reduced in the MSC or MSC-COX2(+) group, while these TAK1 substrates still expressed high in the MSC-COX2(−) group (Fig. 2c).
To confirm the role of TAK1 in ALF, we further used 5Z-7-ox, a specific TAK1 inhibitor, to inhibit TAK1 before LPS/D-Gal administration.Notably, TAK1 inhibition protected against liver damage, shown by normalized serum ALT and AST and reduced necrosis on the histological analysis (Fig. 2d, e).Treatment of 5Z-7-ox significantly suppressed TAK1 activation and reduced activation of its downstream signaling pathways, including p-JNK, p-P38, p-c-Jun, and NF-κB signaling (Fig. 2e).Collectively, these observations suggested that MSC-derived PGE 2 protected ALF through suppressing liver inflammation in a TAK1-NF-κB pathway.

MSC inhibits inflammasome activation of macrophages in ALF
Previous studies have confirmed the role of inflammasome activation in LPS/D-Gal-induced liver injury, which accelerated liver inflammation [17].Next, we explored whether MSC infusion could inhibit NLRP3 inflammasome activation in the liver.The protein levels of NLRP3, cas-pase1 p20, and mature-IL-1β were decreased in the MSC or MSC-COX2(+) group (Fig. 3a).In parallel, serum concentrations of IL-1β and the activity of caspase 1 enzyme in liver tissues confirmed the inhibition of NLRP3 inflammasome by MSC-derived PGE 2 (Fig. 3b, c).However, MSC-COX2(−) failed to inhibit NLRP3 inflammasome activation in liver tissues.In order to identify the cell population responsible for increased production of NLRP3 inflammasome, the double immunohistochemistry staining was performed.The results demonstrated that NLRP3 was mostly activated in macrophages in the liver and MSC or MSC-COX2(+) could inhibit NLRP3 activation in liver macrophages (Fig. 3d).To confirm this result, we treated mouse hepatocyte cell line AML12 or BMDM with 1 μg/ml LPS for 24 h in vitro.As showed in Fig. 3e and f, the activation of NLRP3 and levels of IL-1β secretion was much higher in BMDM, which indicated that macrophages were more sensitive to DAMPs than hepatocytes.Collectively, our results indicated that MSC-derived PGE 2 inhibited liver macrophage inflammasome activation to protect against liver injury.

MSC induces M2 macrophage polarization in ALF
Mouse models of acute liver injury have revealed the role of macrophages in accelerating damage through cytokines and chemokines releasing.However, liver macrophages are plastic and adapt their phenotype to promote tissue repair according to signals derived from the hepatic microenvironment [6].Several studies confirmed the role of MSC on macrophage polarization in different models [19,20].Thus, we tended to explore whether MSC could induce M2 macrophages in ALF to promote inflammation resolution.We found that gene expression associated with M2-like (Arg1, Mgl1, Mgl2, and Ym1) macrophages were increased in the MSC and MSC-COX2(+) groups (Fig. 5a); meanwhile, the serum levels of IL-10, which is produced by M2 macrophages, were also increased in the MSC and MSC-COX2(+) groups (Fig. 5b).However, MSC-COX2(−) failed to induce M2 macrophages.In vitro studies confirmed the MSCderived PGE 2 on macrophage polarization, as the expression of M2 markers and numbers of CD206-positive cells of BMDM treated with MSC-conditioned medium or PGE 2 were increased (Fig. 5c, d).Taken together, our results demonstrated that MSC induced M2 macrophages to resolute inflammation in ALF through PGE 2 .
Next, we investigated the mechanism of MSC-derived PGE 2 on macrophage polarization.Western blotting results showed that not only the classical M2 pathway, STAT6, was activated when treated with MSC or PGE 2 , but also resulted in mTOR activation, as increased phosphorylation of the mTOR substrates mTOR, S6K, AKT, and GSK-3β (Fig. 6a), which correlated with previous studies [21].In contrast, MSC-COX2(−)-conditioned medium failed to activate mTOR and STAT6 pathway (Fig. 6a).Treatment of BMDM with mTOR inhibitor, rapamycin (Rap), or AKT inhibitor, MK-2206, confirmed the role of mTOR signaling in MSC-derived PGE 2 on M2 macrophage polarization, as the mRNA levels of M2 markers were significantly increased after administration of Rap or MK-2206 (Fig. 6b-d).Taken together, our

MSC protects against liver inflammation via PGE 2 receptor (EP) 4
Our previous studies showed that MSC-derived PGE 2 promoted hepatocyte proliferation through EP4 [13]; we explored whether EP4 also played a role in liver inflammation resolution.We treated mice with EP4 inhibitor (EP4i), GW627368X.The mRNA levels of inflammatory cytokines increased when treated with EP4i (Fig. 7a).Western blot results confirmed that inhibition of TAK1 and NF-κB signaling by MSC was also mediated by EP4, as the expression of TAK1 and NF-κB signaling substrates was higher when EP4i was administered (Fig. 7b, c).Meanwhile, the expression of NLRP3 and the activity of caspase 1 enzyme were also higher when EP4i was administered, even in the MSC-COX2(+) group (Fig. 7d, e).In vitro studies using EP4i or siRNA to inhibit or deplete EP4 in BMDM, the western blot and IL-1β secretion confirmed the role of EP4 on NLRP3 inflammasome inhibition (Fig. 7f, g).Taken together, these results showed that MSC-derived PGE 2 ameliorated liver inflammation through EP4.

EP4 mediates MSC-derived PGE 2 on macrophage polarization
Next, we explored whether M2 macrophage polarization was also mediated by EP4.The mRNA levels of M2 macrophage markers and serum levels of IL-10 were also inhibited when treated with EP4i (Fig. 8a, b).And mRNA levels of PGE 2 receptors (EP1-EP4) in BMDM confirmed the role of EP4 on M2 macrophage polarization (Fig. 8c).EP4i or siRNA treatment demonstrated the role of EP4 in macrophage polarization, as the mRNA levels of M2 markers were decreased when treated with EP4i or siRNA (Fig. 8e, g).Meanwhile, the protein levels of STAT6 and mTOR signaling substrates confirmed the role of EP4 on STAT6 and mTOR signaling (Fig. 8d, f).Overall, these results demonstrated that EP4 played a major role in MSC-derived PGE 2 on macrophage polarization.

Discussion
Cell death and inflammation play great roles in the progression of ALF [4].Accordingly, we speculated that reduced cell death and hepatic inflammation would be beneficial for the prevention and treatment of ALF.As described previously, MSC could regulate these important pro-inflammatory cytokines via a variety of effector mechanisms [22].In our study, we clarified that MSCderived PGE 2 reduced hepatocyte death and liver inflammation to attenuate ALF.Meanwhile, MSC-derived PGE 2 also modulated macrophage polarization to promote inflammation resolution in ALF.
PGE 2 is a bioactive lipid which is catalyzed by the COX2 from arachidonic acid.PGE 2 exerts a wild range of biological effects associated with inflammation and cancer [23].Several studies confirmed that PGE 2 promoted the resolution of inflammation and tissue repair.In a concanavalin A (ConA)-induced liver injury model, depleted COX2 in mice accelerated liver injury [24].Hepatocyte COX2 expression protected against liver ischemia-reperfusion injury (IRI); meanwhile, in patients who underwent liver transplantation, there was a significant positive correlation of plasma PGE 2 levels and graft function [25].These results indicated that PGE 2 might have great therapeutic potentials in treating inflammatory liver diseases.In this study, we demonstrated the therapeutic role of MSC secreted PGE 2 on LPS/D-Galinduced ALF based on its anti-apoptosis and antiinflammation ability.
We confirmed that MSC-derived PGE 2 reduced proinflammatory cytokine production in the liver through inhibiting TAK1-NF-κB signaling, which is important in regulating the expression of pro-inflammatory cytokines.TAK1 is a central regulator of cell death and inflammation through activating downstream effectors such as NF-κB and mitogen-activated protein kinases (MAPKs) [16].Recent studies have demonstrated the role of TAK1 in the progression of various liver diseases, like liver IRI, nonalcoholic steatohepatitis (NASH), and hepatocellular carcinoma [26][27][28].In our study, we found that MSC infusion could decrease the expression of TAK1 downstream substrates and NF-κB, thus alleviating liver inflammation.In parallel, administration of the TAK1-specific inhibitor, 5Z-7-ox, could also protect against ALF in vivo.
Inflammasome activation has been identified as a major contributor to hepatocyte damage in ALF [29].Our results showed that administration of MSC, especially MSC-COX2(+), significantly suppressed LPS/ D-Gal-induced inflammasome activation, while MSC-COX2(−) failed to show this phenomenon, which demonstrated the role of PGE 2 on NLRP3 inflammasome inhibition.Next, we confirmed that macrophages in the liver are the main source of NLRP3, which then accelerated liver inflammatory response.A previous study has showed that TAK1 restricted NLRP3 activation and NF-κB was important for NLRP3 activation [18,30].We found that mice pretreated with 5Z-7-ox significantly inhibited NLRP3 inflammasome activation, which correlated with the in vitro model results, showing the role of TAK1 on NLRP3 inflammation activation.These results indicated that MSC-derived PGE 2 restricted macrophage NLRP3 activation through TAK1.
Liver macrophages hold a central position in maintaining homeostasis in the liver as well as in the pathogenesis of acute or chronic liver injury.We showed that MSC-derived PGE 2 ameliorated macrophage activation-induced inflammatory response.Recent studies have revealed macrophages to be heterogeneous; M2 macrophages protected against exacerbated inflammation and thus limited tissue injury [6].Several studies have showed the role of MSC on macrophage polarization [19,20].Our studies demonstrated the role of MSC-derived PGE 2 on M2 macrophage induction in ALF thus limiting liver injury, as the increased expression of M2 markers and plasma levels of IL-10.Next, we found that MSC-derived PGE 2 not only activated STAT6 signaling in BMDM, the classical pathway on M2 macrophage polarization [31], but also enhanced the expression of mTOR signaling substrates, which has shown to control macrophage metabolism and thus to shape their properties [21,32].Administration of the mTOR inhibitor or AKT-specific inhibitor significantly inhibited the ability of MSC-derived PGE 2 on M2 macrophage polarization.PGE 2 elicits a wide range of biological effects through its receptors, EP1-EP4 [23].Our previous study found that MSC-derived PGE 2 promoted hepatocyte proliferation via EP4.In this study, we administed EP4 antagonist to mice, which significantly inhibited the effects of MSC on attenuating liver inflammation and M2 macrophage polarization.And the administration of an agonist for EP4 has been found to confer protection against IRI in the liver, ischemic heart diseases, and intestinal injury [33][34][35].Collectively, MSC-derived PGE 2 protected against ALF through EP4.
In conclusion, our data confirmed that MSC attenuated ALF dependent on the secretion of PGE 2 .MSCderived PGE 2 inhibited TAK1 signaling and NLRP3 inflammasome activation in liver macrophages; meanwhile, MSC-derived PGE 2 could also induce M2 macrophages to secret anti-inflammatory cytokines, like IL-10 to promote inflammation resolution and limit liver injury through activating STAT6 and mTOR signaling in macrophages, finally to inhibit the liver inflammatory response and hepatocyte apoptosis induced by LPS/D-Gal.All these phenomena were relied on the PGE 2 receptor, EP4.Inhibiting EP4 significantly alleviated the therapeutic potential of MSC on ALF.This study provided a new therapeutic mechanism of MSC on ALF, which might provide a new direction to enhance the functionality of MSC.

Fig. 1
Fig. 1 MSC protects LPS/D-Gal-induced liver injury via PGE 2 .a Serum levels of ALT and AST in each group (n = 6).b Representative HE-stained liver sections from each group and quantitation of the necrosis area in each group (n = 4).c Representative images of TUNEL-stained liver sections from each group and the number of TUNEL-positive cells in each group (n = 4).d Representative images of PI staining to indicate necrosis cells and the number of PI-positive cells in each group (n = 4) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 2
Fig. 2 MSC-derived PGE 2 restrains inflammatory responses in the liver during LPS/D-Gal-induced ALF. a The mRNA levels of pro-inflammatory cytokines (IL-1β, CCL2, iNOS, TNF-α) in the liver in each group (n = 4).b The protein expression levels of NF-κB signaling in each group.c The protein expression levels of TAK1 signaling in each group.d Serum levels of ALT and AST in mice pretreated with TAK1 inhibitor, 5Z-7-ox (n = 4).e Representative HE-stained liver sections and quantitation of necrosis area in each group (n = 4).f Protein levels of TAK1 and NF-κB signaling in each group (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7
Fig. 7 MSC-derived PGE 2 protects against liver inflammation via EP4. a The mRNA levels of inflammatory cytokines (IL-1β, CCL2, iNOS, TNF-α) in the liver from each group pretreated with EP4 inhibitor (EP4i) (n = 4).b The protein expression levels of NF-κB signaling in each group pretreated with EP4i.c The protein expression levels of TAK1 signaling in each group pretreated with EP4i.d The protein expression levels of NLRP3 inflammasome signaling in each group pretreated with EP4i.e Measurement of caspase-1 enzymatic activity in the liver of each group pretreated with EP4i (n = 4).f Protein levels of NLRP3 inflammasome and TAK1 signaling in BMDM pretreated with EP4i or knocking down of EP4 via siRNA.g The levels of IL-1β in supernatants of BMDM pretreated with EP4i or knocking down of EP4 via siRNA (*p < 0.05, **p < 0.01)

Science
Foundation Grant (2018 M642222), Jiangsu Province Natural Science Foundation (BK20190114), the Nanjing Medical Science and Technique Development Foundation (QRX17129), the Nanjing health science and technology development project for Distinguished Young Scholars (JQX19002), the Nanjing health science and technology development project for Medical and health research (YKK19070), the Nanjing Science and technology project (201911039), and Project of Nanjing University Hospital Management Institute, Aid project of Nanjing Drum Tower Hospital Health, Education & Research Foundation (NDYG2020047).