PPARγ-dependent hepatic macrophage switching  acts as a central hub for hUCMSCs-mediated alleviation of decompensated liver cirrhosis in rats

doi:10.21203/rs.3.rs-1999570/v1

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Research Article

PPARγ-dependent hepatic macrophage switching acts as a central hub for hUCMSCs-mediated alleviation of decompensated liver cirrhosis in rats

https://doi.org/10.21203/rs.3.rs-1999570/v1

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Journal Publication

published 27 Jul, 2023

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Background: Decompensated liver cirrhosis (DLC), a terminal-stage complication of liver disease, is a major cause of morbidity and mortality in patients with hepatopathie. Human umbilical cord mesenchymal stem cell (hUCMSCs) therapy has emerged as a treatment novel alternative for the treatment of DLC. However, optimized therapy protocols and the associated mechanisms are not completely understood.

Methods: We constructed a DLC rat model consistent with the typical clinical characteristics combined use of PB and CCL₄. By performing dynamic detection of liver morphology and function in rats for 11 weeks, the various disease characteristics of DLC and the therapeutic effect of hUCMSCs on DLC in experimental rats were fully investigated, according to ascites examination, histopathological and related blood biochemical analyses. Flow-cytometry analysis of rat liver, immunofluorescence and RT-qPCR were performed to examine the changes of liver immune microenvironment after hucMSCs treatment. RNA-seq analysis of liver and primary macrophages and hUCMSCs co-culture system in vitro were performed to explore possible signaling pathways. PPARγ antagonist, GW9662, and clodronate liposomes were used to inhibit PPAR activation and pre-exhaustion of macrophages in DLC rats’ liver respectively.

Results: We found that changing of the two key issues, the frequency and initial phase of hUCMSCs infusion can affect the efficacy of hUCMSCs and the optimal hUCMSCs treatment schedule is once every week for three weeks at the early-stage of DLC progression, providing the best therapeutic effect in reducing mortality and ascites, and improving liver function in DLC rats. hUCMSCs treatment skewed the macrophage phenotype from M1-type to M2-type through activating PPARγ signaling pathway in liver, which was approved by primary macrophages and hUCMSCs co-culture system in vitro. Both inhibition of PPARγ activation with GW9662 and pre-exhaustion of macrophages in DLC rats’ liver abolished the regulation of hUCMSCs on macrophage polarization, thus attenuating the beneficial effect of hUCMSCs treatment in DLC rats.

Conclusions: HUCMSCs treatment on DLC were attributed to the activation of the PPARγ signaling pathway in liver macrophages of DLC rats, which polarizes M1-type macrophages to M2-type, thus inhibiting inflammation and promoting the repair of damaged liver tissue. Our results about different infusion regimens comparison and mechanisms exploration provide a robust theoretical foundation for the future study design of Mesenchymal Stem Cells therapy on DLC.

decompensated liver cirrhosis

hUCMSCs

stem cell therapy

macrophages

PPARγ

Decompensated liver cirrhosis (DLC) is the end stage of chronic liver disease, that is induced by various etiologies, such as viral hepatitis and steatohepatitis.¹ The prognosis of patients with DLC is quite poor, and the estimated 5-year survival rate is 14%-35%.² Currently, liver transplantation is the definitive treatment for DLC, however, this strategy is limited by a shortage of available donors and several adverse effects. Therefore, alternative effective strategies for DLC therapy are urgently needed.

Cell therapy is a promising treatment for end-stage liver diseases. Hepatocyte-based therapy has been demonstrated to improve the survival of animals with experimentally induced acute-chronic liver damage, however, limited cell sources and low proliferation have restricted their large-scale application.^3–5 Mesenchymal stem cells (MSCs) have attracted increasing attentions for the treatment of hepatic diseases owing to their abundance, high proliferative activity, and low immunogenicity.^6–8 According to several clinical studies, MSCs therapy improves liver function and alleviates related complications in patients with liver cirrhosis.^9–14 However, only one study found a long-term survival benefit at the 75-month follow-up after a combination of UC-MSC infusion and conventional drug therapy.¹⁴ Moreover, other studies revealed the limited effects of MSCs and even a lack of effect on liver cirrhosis.^15–16 To some extent, MSCs therapy has different therapeutic effects owing to variations in key factors vary, including cell quality, administration route, optimal dose and the starting time of infusion. Among them, administration route and optimal dose have been widely studied and reported. However, only few studies revealed the starting time of MSCs injection at which stage of DLC progress, the corresponding injection frequency, and the interval of cell infusion. This is because it is difficult to directly conduct clinical studies on the initiation time, treatment frequency, and interval of MSCs infusion. Addressing these issues in DLC animal models is thus of great significance for clinical application.

Compared with other MSCs (such as adipose and bone marrow derived-MSCs), human umbilical cord-derived MSCs (hUCMSCs) have more advantages for many reasons, including potential noninvasive isolation, with a higher proliferative activity, and a stronger anti-inflammatory effect.^17–19 Most importantly, hUCMSCs secrete large amounts of hepatocyte growth factor, which has been demonstrated to promote the growth of hepatocytes and inhibit fibrosis of hepatic stellate cells.^20–21 As a result, hUCMSCs are the most favorable seed cells for the treatment of liver disease. Based on previous studies, hUCMSCs exsert therapeutic effects on experimental hepatic fibrosis and cirrhosis.^22–24 Clinical studies revealed that hUCMSCs infusion can improve cirrhosis with ascites in patients with decompensated cirrhosis, and improve liver function in patients with acute and chronic liver failure.^{7, 14} Currently, highly formalized strategies are employed in most MSCs-based studies for the treatment of chronic liver disease; however, the optimal treatment regimens and molecular mechanisms of hUCMSCs on DLC remain obscure. An in-depth exploration of these issues is thus of positive significance for promoting basic research on hUCMSCs for clinical transformation.

To date, no animal model has completely demonstrated the disease progression of decompensated cirrhosis through continuous weekly monitoring of DLC model. Although the combination of phenobarbital and CCl₄ has become the standard method to construct decompensated cirrhosis animal models, as reported previously, only few indexes are used to carry out an evaluation after the completion of modeling.^25–28 Consequently, comprehensive evaluation and dynamic monitoring of disease occurrence and development process are lacking. More importantly, lack of preclinical animal models to closely simulate the clinical and pathologic features of human DLC has limited the execution of studies on the therapeutic mechanism of MSCs and the improvement of clinical treatment regimens.

In this study, we constructed a DLC rat model consistent with the typical clinical characteristics. By performing dynamic detection of liver morphology and function in rats for 11 weeks, the various stages and corresponding disease characteristics of DLC in experimental rats were fully understood. Using DLC rat models, we evaluated the influences of key therapeutic options and the therapeutic effects of hUCMSCs on DLC, which indicated that the optimal hUCMSCs treatment schedule is once every week for three weeks in the early-stage of DLC progression; this schedule could significantly improve various typical characteristics of DLC rats. Mechanistically, hUCMSCs polarize M1-type macrophages to M2-type macrophages by activating the PPARγ signaling pathway in the liver macrophages of DLC rats. Collectively, this study provides a theoretical basis and treatment regimen selection for the clinical application of hUCMSCs in DLC patients, thereby serving as innovative research of significant valuable in clinical application.

Isolation and identification of human umbilical cord mesenchymal stem cells

Human umbilical cord tissue was obtained from three healthy donors at the Sichuan Maternal and Child Health Hospital, following their consent according to procedures approved by the Medical Ethics Committee of Sichuan University (K2018109-1). Human umbilical cord mesenchymal stem cells(hUCMSCs) were isolated and purified, and their the immunophenotype and differentiation potential determined according to reported procedures ^29–30; the results are shown in Figure S1. HUCMSCs were cultured in mesenchymal stem cell basal medium (DAKEWE, Beijing, China) supplemented with 5% UltraGROTM (HPCFDCRL50, Helios). Cells between passage 5 and 6 were used for all experiments.

Tracking of hUCMSCs distribution in vivo

To monitor the biodistribution of hUCMSCs in DLC rats, hUCMSCs labeled with the fluorescent lipophilic tracer, DiR, were intravenously injected into DLC rats after four weeks of modeling. HUCMSCs were labeled according to the manufacturer’s instructions. Fluorescence imaging distribution was observed using a Small Animal Optical Imaging System (IVIS Spectrum,Perkin Elmer). The results are shown in Figure S2.

Isolation and identification of Kupffer and Peritoneal macrophages

Adult male Wistar rats (approximately 200g) were used to obtain rat kupffer cells and peritoneal macrophages; the detailed isolation methods according to the reported protocols.^31–32 The primary cells were cultured in MaM medium (basal medium + 5% FBS + 1% MaGS + 1% P/S). All cells were cultured in a 37°C, 5% CO₂ incubator.

DLC rat model and hUCMSCs treatment

Male Wistar rats were purchased from Beijing Huafu-kang Biotechnology Co., Ltd. (China). All experimental procedures involving animal were approved by the Sichuan University Medical Ethics Committee (K2018109-2).To induce decompensated liver cirrhosis model, 8 weeks old male Wistar rats (220 ± 30g) were intraperitoneally injected with CCl₄ (0.5mL/kg body weight, dissolved in olive oil, 1:1; Sigma-Aldrich) twice per week for 11 weeks, and at the same time, with 0.35 g/L phenobarbital (Sigma-Aldrich) in the rats’ drinking water. Rats were anesthetized by intramuscular injection of Zoletil®50 (zolazepam–tiletamine) 100–200µL according to the body weight at the 11th week of modeling and then sacrificed at a dose of 1-2ml. The typical features of decompensated cirrhosis, including ascites, impaired liver function, and high mortality were observed at the end of week 11; the results are shown in Figure S3. According to our experimental design, hucMSCs(6×10⁶ cells/kg) were administered intravenously once per week or three times per week for three consecutive weeks at the 5th and 8th of modeling, respectively. Samples were harvested at the end of 11 weeks modeling to evaluate the treatment efficacy of hucMSCs.

Abdominal ultrasound and imaging acquisition

All rats underwent abdominal ultrasonography once per week during DLC modeling using a PHILIPS real-time ultrasonography with a low-frequency convex array probe at 42Hz.

Examination of rats blood biochemical index

The venous blood of rats was placed in procoagulant tubes or anticoagulant tubes and then centrifuged at 1800rpm for 15 min at 4℃ to obtain serum and plasma, respectively. The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), ALkaline phosphatase (ALP), albumin (ALB), total bilirubin (TBIL), gamma-glutamyl transpeptidase (GGT), and creatinine (CREA) were determined at GLP workshop of the National New Drug Safety Evaluation Center (WestChina-Frontier PharmaTech).

Cytokine detection using the Luminex Assay

Rat serum cytokines were detected using multifactor Luminex Assay according to the manufacturer’s instructions. First, the antibody arrays were incubated with the blocking buffer at room temperature for 30 minutes. The serum sample diluted in blocking buffer was added to the corresponding well at 4℃ overnight. The wells were rinsed 3 times with the wash buffer I (5 minutes each time), and then 2 times with wash buffer II at room temperature (10 minutes each time). The antibody arrays were incubated with the biotinylated antibody cocktail diluted in the blocking buffer at room temperature for 2 hours, and washed as mentioned above; The HRP-antiprotein-streptomycin was diluted with blocking buffer, added to the wells, incubated at room temperature for 2 h, and then washed as mentioned above; After mixing the detection buffer C and detection buffer D at a ratio of 1:1, the mixture was incubated with the antibody arrays for 2 min. Finally, the signal was read using the Luminex instrument.

Flow cytometry

Animals were euthanized at the end of week 11. The livers were harvested, minced, and digested in RPMI-1640 medium containing collagenase IV (0.1%; Gibco), nuclease, and 1% fetal bovine serum (FBS) at 37℃ for 40–60 min, and the cell suspensions were filtered. Fixable viability stain 620 (BD Biosciences) was used to discriminate between live and dead cells. Finally, the cells were blocked with Fc-Block (BD Biosciences) and stained with antibodies. The data were acquired using a NovoCyte flow cytometer.

Histological analysis and immunohistochemistry

Liver tissues were harvested, immediately fixed with 4% paraformaldehyde, and embedded in paraffin for subsequent use. For liver histology, deparaffinized liver sections were sectioned at 4-µm thickness and stained with hematoxylin and eosin (H&E) red. For immunohistochemistry, deparaffinized liver sections were subjected to citric acid buffer (PH6.0) microwave antigen retrieval and then treated with 0.3% H₂O₂ solution to block endogenous peroxidase. After washing, the sections were blocked with non-immun serum and incubated overnight with primary antibodies at 4℃. These sections were then incubated with a chromogenic reagent until the liver sections tuned brown. Four to eight independent liver sections were randomly collected and the number of positive cells was quantified using ImageJ software.

Immunofluorescence

Liver tissues were harvested, fixed with 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose, and embedded in OCT compound. Briefly, 4-µm thick frozen liver sections were incubated with primary antibody at 4°C overnight after antigen recovery. The secondary antibody was subsequently added for 1 h at room temperature. Nuclei were stained with Hoechst 33258 at room temperature for 10min. Frozen sections were observed and photographed using a fluorescent microscope (Leica, Germany). Four to eight independent liver sections were randomly collected and the number of positive cells was quantified using ImageJ software.

Real-time PCR

Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). After the concentration was measured, RNA was reverse-transcribed, and mRNA expression analysis was performed using the PrimeScript RT Reagent Kit (TaKaRa, Japan) on a LightCycler 96 System (Roche, Basel, Swizerland). Gene expression was normalized to that of the housekeeping gene, β-actin. The antibodies used are listed in Supplementary Table 1.

RNA-seq

Total RNA from the liver tissues was isolated as described above. RNA purity was assessed using the AMPure XP system (Beckman Coulter, Beverly, USA). Sequencing libraries were prepared using the NEBNext® UltraTM RNA Library Prep Kit (Illumina, NEB, USA) according to the manufacturer’s protocol. Samples were ligated to unique adaptors and subjected to PCR amplification. Libraries were validated using an Agilent Bioanalyzer 2100, normalized, and pooled for sequencing. RNA-seq libraries prepared from at least three biological replicates for each group were sequenced on an Illumina Novaseq using barcoded multiplexing and a 150-bp read length. The raw data were normalized using DESeq2. The RNA-seq raw data are deposited in the NCBI GEO under accession nos. SRR12807514, SRR12807514, SRR12807515, SRR12807516, SRR12807517, SRR12807518, SRR12807519, SRR12807520, SRR12807521, SRR12807522

Western blotting

Proteins were extracted from the treated cells or tissues, as indicated using RIPA lysis buffer (Beyotime, Nanjing, China) containing 1% protease inhibitor cocktail (Merck Millipore, Birrika, USA), and prepared for SDS-PAGE loading buffer (Abclonal, Wuhan, China).The antibodies used are listed in Supplementary Table 2. GAPDH was used as the loading control for all experiments.

Statistical analysis

Data were analyzed using Prism software (GraphPad Prism version 5). Statistical significance was analyzed using Two-tailed Student’s Test. Animal survival was presented using Kaplan–Meier survival curves and analyzed using the log-rank test. Differences were considered statistically significant at P < 0.05. The symbols used to denote significance are as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns (no statistical significance).

Establishment and evaluation of the DLC rat model

To evaluate the effect of hUCMSCs-infusion on DLC, a rat model that largely conforms to the typical clinical features of DLC was established, according to published research. Based on histopathological examination, the combined use of PB and CCL₄ could induce necrosis and the infiltration of inflammatory cells in rat liver. At week 2, the fibroid tissue in the hepatic portal area was found to gradually increase, and liver fibrosis was observed (Figure S4A and B). After four weeks of drug treatment, pseudo lobular-like structures were found in the liver of most DLC rats, and were accompanied by ascites formation (Figure S4D), which is one of the most typical features of decompensated cirrhosis, indicating that 4-week cumulative drug treatment could effectively promote the formation of DLC in rats. After continued treatment for 7 weeks, the liver structure of DLC rats was destroyed. Consistently, extensive hepatocyte necrosis, numerous inflammatory cell infiltrations, and the formation of massive ascites were observed (Figure S4A-D). Additionally, the hydroxyproline content in the liver of model rats increased linearly during the modeling period and reached a high value at 8–11 weeks (Fig S4E). Based on the examination of plasma prothrombin time (PT)and serum albumin (ALB), total bilirubin (TBIL), and the content of creatinine (CREA), the results suggest that the liver function of the model rats was most severely damaged at 8–11 weeks after modeling (Figure S4F). According to the changes in liver structure, the degree of liver fibrosis, liver function, and ascites in rats, 5–7 weeks of rat modeling can be considered to correspond to the early formation stage of DLC in patients, while 8–11 weeks of rat modeling can be considered to correspond to the end-stage of DLC in patients.

Optimizing the infusion regimen is essential to the hUCMSCs-based therapy for DLC

Adjusting key factors, such as the frequency of MSCs infusion and the time selection for MSCs infusion, and optimizing the treatment regimen will further improve the therapeutic effect of MSCs. As shown in the schematic diagram (Fig. 1A), the hUCMSCs infusion was performed in the early (at week 5 of modeling) and end stages (at week 8 of modeling) in DLC rats, and each group was administered two treatment regimens: single and triple infusions (once a week). The four treatment regimens were labeled T-A, T-B, T-C, and T-D, respectively.

After 11 weeks of modeling, only half of rats in the DLC group survived, whereas the survivability of rats in all hUCMSCs-treated groups was improved, especially the T-B group with a survival rate of 100% (Fig. 1B). Furthermore, the level of ascites, the most distinctive feature of decompensated cirrhosis, was significantly reduced in all hUCMSCs treatment groups. In particular, ascites development was completely prevented in the T-B group (Fig. 1C). Evidently, sclerosis of the liver was significantly reduced in the T-B and T-C groups according to the liver organ coefficients (Fig. 1D). Based on the HE staining results, hUCMSC treatments reduced the inflammatory infiltration in the liver and restored the damaged liver structure (Fig. 1F). The level of hydroxyproline, the main component in collagen tissue, was also reduced in all hUCMSC treatment groups (Fig. 1E), aligning with the Sirius red staining results (Fig. 1G). Notably, the most significant decrease was observed in the T-B group. The four hUCMSC treatment groups did not display a uniform response to liver function compared to the DLC group; however, the T-B group showed significant improvements in all indexes, including ALT, AST, ALP, PT and TBIL levels, and meanwhile the ALB and CREA levels increased (Fig. 1H). To further explore the effect of the infusion interval on hUCMSCs therapy, weekly and biweekly infusions of hUCMSCs were compared, as shown in Figure S5A. Surprisingly, changes in hUCMSCs infusion intervals had no significant effects on DLC rat therapy (Figure S5B-L). Altogether, these results confirm the potential therapeutic effect of hUCMSCs on DLC. More interestingly, performing hUCMSCs-based treatment at the early stage of DLC, with triple hUCMSCs infusion, could produce the best therapeutic effects, thereby providing a great reference for future basic research and the formulation of clinical treatment regimens.

hUCMSCs improve DLC by modulating the immune microenvironment in rats, especially by shifting intrahepatic macrophages from M1 to the M2 type

Based on the above findings, different hUCMSCs infusion regimens are extremely important for hUCMSCs therapy in DLC rats. To determine the cause of this difference, flow cytometry analysis of the liver immune cells of DLC rats treated with hUCMSCs was performed. The proportion of total T cells among CD45 + cells was significantly increased by hUCMSCs treatment, whereas the proportions of B cells among CD45 + cells remained unchanged (Figure S6A). Further analysis of the ratio of CD4+/CD8 + T cells demonstrated that there was significantly decreased only in the T-B group, but not changed in the other treatment groups (Figure S6A). The percentage of neutrophils was significantly decreased in all hUCMSCs treatment groups compared with the DLC group, however, there was no significant change in the proportion of monocytes and their subpopulations among CD45 + cells (Figure S6B). Additionally, considering the leading role of macrophages in the immune regulation of liver diseases, we determined the proportion of total macrophages and their subtypes, M1 and M2 macrophages. Flow cytometric analysis revealed no difference in the percentage of total macrophages among all groups, whereas the proportion of M1 macrophages in total macrophages decreased and the proportion of M2 macrophages increased significantly in the T-B and T-C hUCMSCs treatment groups compared with the DLC group (Fig. 2A and B). To determine the effect of hUCMSCs treatment on the changes in M1 macrophages and M2 macrophages, the T-B group with the most obvious changes in M1/M2 macrophages was selected for subsequent studies. RNA expression analysis of the liver tissue from the T-B group showed that the expression levels of M1-related genes, such as IL-6, MCP-1, and IL-1β, were up-regulated in DLC rats compared with normal rats, however, the levels of these genes were significantly downregulated after hUCMSCs treatment (Fig. 2C). In contrast, the expression levels of CD163, Arg1, IL10, and other M2-related genes were significantly downregulated in the DLC group compared with the NC group, and significantly up-regulated in the hUCMSCs group (Fig. 2D). To further determine whether M2 macrophages polarization can improve systemic inflammatory levels, the expression of immune-related factors in the serum of rats was examined using an Inflammation Antibody Array. The serum levels of various pro-inflammatory factors, such as IL-1β, IL-7, M-CSF, GM-CSF, and IFN-γ, were significantly decreased after hUCMSCs treatment, while the expression level of the anti-inflammatory factor, IL-10, was significantly up-regulated (Fig. 2E), which was further confirmed by examining the mRNA expression levels of inflammatory factors in the liver tissue (Fig. 2F). Although the exact molecular mechanism between different infusion regimens remains unclear, our results revealed that the change in M1/M2 macrophages proportion plays a decisive role in different infusion regimens and further research also proved that optimal hUCMSCs infusion treatment promotes the expression of M2-related genes while inhibits the expression of M1-related genes.

hUCMSCs significantly increase PPARγ in the liver of DLC rats

To further investigate the genomic changes in the liver of DLC rats treated with hUCMSCs, transcriptome sequencing (RNA-SEQ) of tissue samples from the normal control group (NC), decompensated cirrhosis group (DLC), and hUCMSCs treated group (hUCMSCs) was performed at the end of 11 weeks. Principal component analysis (PCA) suggested that samples in the same group had good uniformity, and samples in the hUCMSCs group were closer to those in the NC group than the DLC group (Figure S7). By performing a Venn analysis, 1871 differentially expressed genes in the DLC group compared with the NC group and 784 differentially expressed genes were identified in the MSC group compared with the DLC group. By comparing the NC group with the MSC group, 510 differentially expressed genes were identified; such finding aligns with the conclusion that the liver gene expression profiles of DLC rats treated with hUCMSCs were closer to those of normal rats (Fig. 3A). For the selected differentially expressed mRNAs(545 + 85), KEGG pathways enrichment analysis revealed that the gene set was mainly involved in processes related to the PPAR signaling pathway and arachidonic acid metabolism (Fig. 3B). Furthermore, heat map analysis of the immune-related genes and immune-process-related genes revealed that Cxcl1, Cxcl12, Ccr1, IL1α, IL23a, PPARγ, Ln2, and other immune-related genes in the liver of DLC rats treated with hUCMSCs were significantly different from those in the DLC group and tended to be reversed into the normal group (Fig. 3C), which illustrates that these immune factors play an important role in the development of inflammation. To further validate the RNA-seq results, RT-qPCR was performed to detect the expression of PPARγ in liver tissues. The expression of PPARγ in the DLC group was found to be significantly lower than that in the NC group. In contrast, the expression of PPARγ in the hUCMSCs group was significantly up-regulated compared with that in the DLC group, aligning with the results of RNA-seq (Fig. 3D). Additionally, the western blot results showed that hUCMSCs treatment reversed the low protein level of PPARγ in liver tissues of rats in the DLC group (Fig. 3E). As a result, RNA-seq analysis of the liver tissues combined with further validation experiments indicated that PPARγ played an important role in the hUCMSCs treatment of DLC rats.

hUCMSCs skewed the macrophage phenotype from M1-like to M2-like through the activation of PPARγ

Based on the animal hUCMSCs treatment experiment of DLC rats that the proportion of total macrophages remained unchanged while that of the M1 type significantly decreased and that of the M2 type increased after hUCMSCs treatment. Thus, we speculated whether hUCMSCs directly affect the macrophage phenotype. To determine whether hUCMSCs would directly affect proinflammatory macrophages phenotype in vitro, primary peritoneal macrophages (marked M0) were isolated and stimulated for transformation into M1-type macrophages by LPS and IFN-γ. Thereafter, hUCMSCs were co-cultured with M1 macrophages to observe the polarization of M1-type macrophages. Simultaneously, M2 phenotype macrophages were generated, with factors, such as IL4, IL13, and IL10 as positive control. Flow cytometry revealed that the proportion of M1-type macrophages decreased from 10.97–2.6% after hUCMSCs treatment, whereas that of M2-type macrophages increased from 26.35–58.69% (Fig. 4A and B). The co-culture of hUCMSCs with macrophages decreased the expression levels of iNOS, TNF-α, CD86, and other M1-type macrophage-related genes in macrophages while increased the expression levels of M2-type macrophage-related genes such as Ym1, Arg1, IL10 and CD206 (Fig. 4C). These results indicate that hUCMSCs can directly promote macrophage polarization from the M1-phenotype to the M2-phenotype, however, the underlying mechanism needs to be further elucidated.

Based on the results of differential gene enrichment analysis mentioned above, we focused on the PPARγ signaling pathway, which is also enriched in immune-related differential gene clusters. PPARγ, a subtype of the peroxisome proliferator-activated receptor family, has been demonstrated to be an important nuclear transcription factor with anti-inflammatory function. Both PPARγ and its ligands have been reported to be involved in the cellular regulation of monocytes and macrophages, and play an important role in the deinflammatory phase³³. Therefore, we hypothesized that PPARγ is a potential target of hUCMSCs therapy. To validate this hypothesis, we detected the expression of PPARγ and the macrophage surface marker, CD68, in liver tissues by immunofluorescence staining of CD68 and PPARγ positive cells. Results showed that double-positive CD68 + PPARγ + cells were significantly higher in the hUCMSCs group than the DLC group (Fig. 4D). In addition, the M1-type macrophages were treated with Rosiglitazone (Rosi), a PPARγ agonist, to verify the effect of PPARγ activation on phenotypic changes in macrophages. Flow cytometry showed that PPARγ activation could reduce the proportion of M1-type macrophages while increase the proportion of M2-type macrophages (Figure S8A). RT-qPCR analysis confirmed these results owing to the increased mRNA expression of M2-related genes (Arg1, CD206, and CD163) and decreased expression of M1-related genes (iNOS, TNF-α, and IL-1β) after rosiglitazone treatment (FigureS8B). Finally, macrophages and hUCMSCs were co-cultured to examine the directly effect of hUCMSCs on PPARγ in macrophages. The results demonstrated that the expression level of PPARγ in M1 macrophages was significantly up-regulated after co-culture with hUCMSCs (Fig. 4E). Moreover, the expression levels of downstream PPARγ genes (CD36, SCD1, FABP4, LXRa, Arg1, and STAT6) were significantly increased in the hUCMSCs-treated group compared to the control group (Fig. 4F). Taken together, hUCMSCs activated PPARγ and its downstream genes in macrophages, thereby promoting the polarization of macrophages from the M1 to M2 type.

PPARγ antagonist, GW9662, abolishes the regulation of hUCMSCs on macrophage polarization in vitro

To further investigate whether hUCMSCs affect the macrophage phenotype through the PPARγ pathway, the effect of PPARγ on the inflammatory phenotype of macrophages was determined by treating the co-cultured cells described above with the PPARγ antagonist, GW9662. The immunofluorescence assay revealed that the number of CD68 + and PPARγ + double positive macrophages were significantly increased after co-culture with hUCMSCs, whereas the numbers of double-positive cells and fluorescence intensity were significantly decreased after the addition of the PPARγ antagonist GW9662 (Fig. 5A). RT-qPCR further proved that the up-regulated expression of macrophage PPARγ was abolished in the presence of GW9662(Fig. 5B), which aligned with the result that the up-regulation of PPARγ protein levels in macrophages co-cultured with hUCMSCs was inhibited once treated with GW9662 (Fig. 5C and D). Additionally, the expression of M1-type and M2-type macrophage-related genes showed no significant changes in the co-culture system with the PPARγ antagonist GW9662 (Fig. 5E and F). These results confirm that hUCMSCs promoted the polarization of macrophages from pro-inflammatory M1-type to anti-inflammatory M2-type, relying on the activation of the PPARγ signaling pathway in macrophages and the anti-inflammatory treatment effects of hUCMSCs disappeared once PPARγ activation was inhibited.

The hUCMSCs-PPARγ-macrophage axis plays a key role in DLC treatment

Although several studies have reported the therapeutic effects of hUCMSCs on cirrhosis involving in macrophages, the mechanisms underlying the progression of cirrhosis are not completely understood. In this study, we confirmed the macrophages phenotype switches under hUCMSCs treatment in vivo and in vitro; however, whether this effect plays a key role in DLC disease progression and hUCMSCs treatment needs to be further explored. Therefore, macrophages were depleted using clodronate liposomes during hUCMSCs treatment to investigate whether the repair effect of DLC treatment on the liver was partially affected by macrophages. The acquisition and treatment regimen of macrophage-depleted DLC rats are shown in Fig. 6A; macrophage depletion was the only difference between the DLC + hUCMSCs and DLC-Liposome + hUCMSCs groups. We observed that macrophage depletion using liposomes significantly decreased the proportion of macrophages in the liver and blood of DLC rats, suggesting the successful establishment of the DLC rats with hepatic macrophage depletion (Figure S9A and B). Further investigation revealved that hUCMSCs treatment could significantly promote the body weight increase of DLC rats, but could not improve the rapidly decreasing body weight of macrophage-depleted DLC rats (Figure S9C). The livers of rats in the macrophage-depleted group were swollen, rough in texture, and stiff, with small nodules. Furthermore, there was no significant difference in the appearance of the liver after hUCMSCs treatment and the liver organ coefficient also showed no differences (Figure S9D and Fig. 6B). What’s more, HE staining results showed that hUCMSCs treatment could not restore the damaged liver structure, improve the fatty degeneration of the liver, and reduce the inflammatory infiltration in the liver, and hepatonecrosis in the DLC-Lipsome group (Fig. 6C). Evidently, the administration of hUCMSCs in the macrophage-depleted DLC-Lipsome group failed to reduce the serum biochemical indexes, including ALT, AST, ALP, PT, and TBIL; increase the liver function indexes, such as ALB and CREA levels; and improve PT coagulation function (Fig. 6D and E). All these results indicate that the depletion of intrahepatic macrophages aggravates disease progression in DLC rats, and hUCMSCs treatment cannot improve the disease characteristics in macrophage depleted DLC rats.

As mentioned above, activation of the PPARγ pathway has been proved to promote the polarization of M2 macrophages in vitro. However, whether the activation of PPARγ plays an important role in DLC rats with hUCMSCs treatment needs to be further elucidated. Consequently, the PPARγ antagonist, GW9662, was employed during hUCMSCs treatment to induce the systemic inhibition of PPARγ in DLC rats. HE staining revealed that the damaged liver structure and hepatonecrosis did not significantly improved, with worse inflammatory infiltration in DLC rats treated with hUCMSCs and GW9662 (Figure S10A). Moreover, Sirian red staining revealed no decreased collagen fibers in DLC rats treated with hUCMSCs and GW9662 compared with DLC rats only treated with hUCMSCs (Figure S10B). In addition, the level of serum ALB, which can reflect the protein synthetic function of the liver, was significantly up-regulated to 38.2 ± 4.2 g/L in the hUCMSCs group, while difference was not found between the hUCMSCs group treated with GW9662 (34.8 ± 3g/L) and the DLC group(33.6 ± 1.6g/L) (Fig. 6F). Based on these results, the inhibition of PPARγ attenuated the beneficial effect of hUCMSCs treatment in DLC rats, suggesting that the activation of the PPARγ pathway plays an indispensable role in hUCMSCs treatment in DLC rats.

In summary, a schematic diagram of the mechanisms of hUCMSCs treatment in DLC rats is shown in Fig. 7. Briefly, hUCMSCs treatment can reconstruct liver structure, reduce ascites, hepatocyte necrosis, neutrophil infiltration and collagen deposition, inhibit the activation of hepatic stellate cells, and decrease the expression level of inflammatory factors in DLC rats. Mechanistically, hUCMSCs treatment induces the polarization of proinflammatory macrophages into repair macrophages by activating the nuclear transcription factor, PPARγ, thereby eliminating the inflammatory response and promoting tissue repair.

Owing to the growing burden of liver diseases worldwide, the prevention and treatment of patients with decompensated liver cirrhosis will become a substantial health care challenge. Among the different types of therapies, hUCMSCs transplantation has emerged as the best alternative to liver transplantation for the treatment of DLC. However, the optimal treatment regimens and molecular mechanisms of hUCMSCs on DLC remain unclear.^34–36 The findings of this study not only confirmed the excellent effects found in previous studies, but also revealed that the optimal hUCMSCs infusion regimen is weekly infusion for three consecutive weeks at the early stage of DLC rats and the mechanism of hUCMSCs treatment is the polarization of pro-inflammatory M1-type macrophages to anti-inflammatory M2-type through PPARγ activation.

In this study, our results demonstrated that hUCMSCs infusion at week 8 of modeling, corresponding to the end-stage of DLC, had a certain therapeutic effect on DLC. Notably, repeat hUCMSCs infusion in the T-B group (once per week for 3 consecutive weeks) at the early stage of DLC (week 5 of modeling) can significantly improve the efficacy of hUCMSCs treatment in DLC, and especially prevent ascites production and the death of DLC rats. The multifaceted efficacies of hUCMSCs are as follows 1. reduce the necrosis of liver cells and the release of cytoplasmic enzymes ALT, AST, and ALP; 2. improve liver function, increase albumin, decrease total bilirubin and prothrombin time; 3. improve the liver microenvironment, reduce the level of inflammatory factors, and increase the expression of the anti-inflammatory factor, IL10; 4. reduce fiber hyperplasia and restore the structural disorder caused by fiber septum; Based on our treatment regimen results, the infusion starting time and infusion times should be considered in clinical hUCMSCs therapy on DLC. Of note, the infusion interval might not be important.

A clinical observational study demonstrated that extending the treatment course (more than four times) may be an option to improve the efficacy of hUCMSCs, aligning with our results that three injections of hUCMSCs were superior to one ingection at the early-stage of DLC.³⁷ Surprisingly, three injections of hUCMSCs was less efficacious than one infusion at the end-stage of DLC, indicating that the disease stage must be considered in the formulation of the frequency schedule for hUCMSCs treatment. Most clinical trials focus on the dosage and duration of MSCs treatment while ignoring the initial infusion time points corresponding to clinical characteristics.^{9, 38–40} Our results strongly support that hUCMSCs infusion combined with the optimized initial infusion time and frequency could be a very promising treatment approach for DLC. Importantly, hUCMSCs-based therapy should not only be regarded as the last option for the treatment of advanced diseases, but should also be combined with other conventional treatments as early as possible to improve its therapeutic effect in patients with DLC. Although a certain gap exists between the treatment benefits in animal models and clinical applications, our findings enrich the preclinical study of MSCs treatment for DLC, providing an important reference for future clinical treatment.

To evaluate the effects of hUCMSCs-infusion on DLC, a rat model that largely conforms to the typical clinical features of DLC was established in this study. In contrast to previous reports that defined experimental DLC using a single or few indicators,^41–44 we performed real-time dynamic detection of the liver structure, degree of liver fibrosis, liver function, and the amount of ascites in rats for 11 weeks, and fully understood the stage of disease development and the corresponding disease characteristics of DLC in DLC rats. Based on our results, 5–7 weeks of rat modeling could correspond to the early formative stage of DLC in patients, and 8–11 weeks of rat modeling could be considered to correspond to the end stage of DLC in patients; Thereby laying a solid foundation for further studies on the treatment and mechanism of decompensated cirrhosis. In addition, to observe the homing effect of hUCMSCs in DLC model rats, intravenous injection of hUCMSCs at week 5 of modeling revealed that hUCMSCs mainly homed in the damaged liver, as shown in Figure S2, which consistent with other studies on MSCs homing.^45–47

MSCs exert immunomodulatory and immunosuppressive effects in vivo and in vitro^48–49. In several mouse models of acute liver failure, MSCs significantly reduce inflammatory cell infiltration, reduce apoptosis, and increase hepatocyte proliferation, thus promoting liver regeneration and improving survival^50–52. MSCs have been reported to modulate adaptive immune responses, induce DC to up-regulate the anti-inflammatory cytokine, IL-10, and reduce the secretion of the pro-inflammatory cytokines, TNF-α and IL-12⁵³. In this study, hUCMSCs treatment changed the immune microenvironment of the liver based on a significant decrease in neutrophils compared with that in the DLC model group, a decrease in the proportion of M1 macrophages, and an increase in the proportion of M2 macrophages. Accordingly, we will investigate the dynamic effects on liver macrophages after hUCMSCs infusion to further clarify the therapeutic mechanism of hUCMSCs on macrophages, as the activity of immune cells was found to vary at different stages of disease.

Hepatic macrophages, including resident Kupffer cells (KCs) and recruited monocyte-derived macrophages, are highly plastic and can adjust their phenotypes according to signals from the microenvironment.⁵⁴ MSCs treatment restores liver macrophage homeostasis to ameliorates the progression of liver diseases including acute liver injury, liver fibrosis, and cirrhosis.^55–57. Consistently, different hUCMSCs infusion regimens were found to exhibit differential effects on macrophage homeostasis regulation ability, among which the T-B group could significantly down-regulate the M1/M2 macrophages ratio. Apparently, our findings suggest that macrophage polarization plays a central role in hUCMSCs therapy for decompensated cirrhosis and the therapeutic effect of hUCMSCs affected by infusion time point was highly correlated with M1/M2 polarization.

According to previous studies, PPARγ, which is essential for macrophage polarization, is involved in the regulation of M1-type macrophage polarization to M2-type in inflammation and injury-related diseases, such as Parkinson’s desease, sepsis, and atherosclerosis.^58–60 Besides, the activation of PPARγ promotes the polarization of M2-type macrophages to prevent the development of non-alcoholic fatty liver disease and liver injury.^{31, 61–62} In this study, hUCMSCs were found to induce the polarization of M1-type macrophages into M2-type macrophages by activating PPARγ in vivo and in vitro. Previous studies on PPARγ -deficient macrophages revealed their resistance to M2 polarization⁶³. Consistently, our findings revealed that hUCMSCs infusion had no therapeutic effect in DLC rats after intrahepatic macrophages depletion with clodronate liposomes, which was validated by the significantly reduced therapeutic effect of hUCMSCs after injection of GW9662 (PPARγ antagonist) during the DLC treatment period, proving that the existence and activation of macrophages PPARγ are indispensable for DLC therapy. This study had some limitations as we only verified that the effect of hUCMSCs on macrophage polarization disappeared when PPARγ antagonists were used in vitro, however, further verification was not carried out in macrophage-specific PPARγ knockout rats.

In conclusion, we examined the DLC rat model for 11 consecutive weeks and assessed the typical clinical features corresponding to each disease stage, according to that, formulated the different hUCMSCs infusion regimens, and found that the optimal hUCMSCs infusion regimen was once per week for three consecutive weeks at the early stage of DLC. This regimen can effectively inhibit the occurrence of ascites and significantly improve liver structure and function in DLC rats. The therapeutic mechanism of hUCMSCs is mainly attributed to the polarization of M1-type macrophages to M2-type macrophages through the activation of the PPARγ signaling pathway in DLC rat liver macrophages, thereby inhibiting inflammation and promoting the repair of damaged liver tissue. This study laid a solid experimental foundation for elucidating the functions and mechanisms of hUCMSCs treatment in DLC, and at the same time, our studies on hUCMSCs-based therapy with optimal regiments reveal that this therapy might serve as an effective alternative to fulfill the needs for the treatment of DLC, ultimately providing a new approach for promoting and advancing the clinical application of cell-based therapy.

DLC

decompensated liver cirrhosis

MSCs

Mesenchymal stem cells

hUCMSCs

human Umbilical Cord Mesenchymal Stem Cells

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

ALP

Alkaline phosphatase

ALB

Albumin

α-SMA

alpha smooth muscle Actin

CCl₄

Carbon tetrachloride

Col1a1

Collagen Type I Alpha 1

CREA

Creatinine

DLC

Decompensated Liver Cirrhosis

GGT

gamma-glutamyl transpeptidase

Gene ontology

HRP

Horseradish peroxidase

H&E

Hematoxylin and Eeosin staining

IL-6

Interleukin-6

IL-10

Interleukin-10

IFN-γ

Interferon-γ

KEGG

Kyoto Encyclopedia of Genes and Genomes

Phenobarbital

PPARγ

Peroxisome proliferator-activated receptorγ

Prothrombin time

TBIL

Total bilirubin

TGF-β1

Transforming growth factor β1

TNF-α

Tumor necrosis factor-α

Western Blot.

Supplemental Information

Supplemental information can be found online at

Ethics approval and consent to participate

Human umbilical cord tissue was obtained from three healthy donors at the Sichuan Maternal and Child Health Hospital, following their consent according to procedures approved by the Medical Ethics Committee of Sichuan University (K2018109-1). Experimental procedures involving animals were approved by the Sichuan University Medical Ethics Committee (K2018109-2).

Consent for publication

(Not applicable)

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the

corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This study was supported by a National Key R&D Program of China grant (2017YFA0105702) and the technology Major Project in Sichuan Province of China (2022YFS0153, 2022ZDZX0024) and 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University（ZYGD20003）.

Authors' contributions

Y.Y. and L.Z. carried out the experiments and analysis the data. Q.J provided human Umbilical Cord tissues. Y.Y., Y.R., Y.H., and D.S. helped to complete experiments including flow cytometry and mIHC assay. F.C, L.C., G.S and L.D helped to interpret data and revised the manuscript. H.D was involved in obtaining funding and study supervision.

Acknowledgements

We acknowledge National Key R&D Program of China, the technology Major Project in Sichuan Province of China and 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University.

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PPARγ-dependent hepatic macrophage switching acts as a central hub for hUCMSCs-mediated alleviation of decompensated liver cirrhosis in rats

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Isolation and identification of human umbilical cord mesenchymal stem cells

Tracking of hUCMSCs distribution in vivo

Isolation and identification of Kupffer and Peritoneal macrophages

DLC rat model and hUCMSCs treatment

Abdominal ultrasound and imaging acquisition

Examination of rats blood biochemical index

Cytokine detection using the Luminex Assay

Flow cytometry

Histological analysis and immunohistochemistry

Immunofluorescence

Real-time PCR

RNA-seq

Western blotting

Statistical analysis

Results

Establishment and evaluation of the DLC rat model

Optimizing the infusion regimen is essential to the hUCMSCs-based therapy for DLC

hUCMSCs significantly increase PPARγ in the liver of DLC rats

hUCMSCs skewed the macrophage phenotype from M1-like to M2-like through the activation of PPARγ

PPARγ antagonist, GW9662, abolishes the regulation of hUCMSCs on macrophage polarization in vitro

The hUCMSCs-PPARγ-macrophage axis plays a key role in DLC treatment

Disscussion

Conclusions

Abbreviations

Declarations

Supplemental Information

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

References

Supplementary Files

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