Liver transplantation is the only effective treatment for end-stage liver diseases and acute liver failure.1,2 Although major advances in prevention and treatment of acute rejection (AR) have been made, AR is still a major cause of graft dysfunction after liver transplantation. Currently, immunosuppressants are the main treatment for AR, but their therapeutic effects are still poor. More importantly, the use of immunosuppressant has serious complications, such as susceptibility to malignancy and serious infections.3-5 Therefore, new therapeutic strategies are essential to improve the therapeutic effect and reduce the incidence of complications.
Macrophages, a vital component of innate and adaptive immunity, play an important role in regulating the immune response to organ transplantation. Macrophages, including monocyte-derived macrophages and resident macrophages, have been classified into two types: M1 macrophages (classically activated phenotype) and M2 macrophages (alternatively activated phenotype).6,7 M1 macrophages secrete high levels of proinflammatory cytokines and participate in the type 1 helper T cell response, whereas M2 macrophages secrete high levels of anti-inflammatory cytokines and participate in the Th2 response.8,9 In response to different microenvironment cues, macrophages can flexibly switch their phenotypes between M1 and M2, generating different cell populations with distinct functions.10,11 The role of Kupffer cells (KCs), the resident macrophages in liver derived from fetal liver and embryonic yolk, in liver transplantation and other hepatic diseases has been emphasized in previous research.12-15 Moreover, multiple studies reported that the M2 phenotype KCs contributed to the induction and maintenance of liver transplantation tolerance.13,14,16 Krenkel and Tacke17 also demonstrated that the M2 phenotype KCs suppressed the proinflammatory response of bone marrow-derived macrophages infiltrated into the liver via secretion of anti-inflammatory cytokines. Therefore, inducing KC M2 polarization may offer a novel treatment option for the AR of liver transplantation.
IL-34, a functional ligand for colony stimulating factor receptor (CSF-1R), is widely expressed in the liver, brain, thymus, heart, kidney, lung, and spleen.18 Although both IL-34 and CSF-1, another functional ligand for CSF-1R, play important roles in the survival, differentiation, proliferation, and chemotaxis of macrophages,19 their bioactivities and signal activations are not identical due to their different spatial and temporal expression.20,21 Multiple studies have shown that IL-34 is implicated in several immune diseases. IL-34 expression is upregulated in salivary glands from patients with Sjogren syndrome.22 IL-34 can be used as a biomarker to assess the progression and therapeutic effectiveness in rheumatoid arthritis,23-25 and there is a positive correlation between IL-34 serum level and the severity of insulin-resistant type II diabetes chronic inflammation.26,27 In malignant diseases, IL-34 promotes tumor progression, accelerates the metastatic process, induces chemo-resistance and shortens the time of recurrence via recruiting and inducing immunosuppressive tumor-associated macrophages.28-30 In addition, the inhibitor of CSF-1R inhibits the malignant progression and prolongs the survival of tumor-bearing mice by altering the phenotype of tumor-associated macrophages.31 More significantly, Bézie et al32 have demonstrated that IL-34 is a regulatory T cells (Tregs)-specific cytokine with immunosuppressive function. They also found that IL-34 could maintain and expand the immunosuppressive capacity of Tregs.32 However, the role and mechanism of IL-34 in AR of liver transplantation are still unreported. Here, we found that IL-34 could switch the phenotype of KCs from M1 to M2 by activating the PI3K/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. In addition, we found that IL-34 could inhibit the AR of liver transplantation. More importantly, the inhibitory effect of IL-34 on AR of liver transplantation was M2 KC-dependent. Taken together, our study indicates that IL-34 has promise as a novel therapeutic strategy for AR of liver transplantation.
MATERIALS AND METHODS
Animals and Orthotopic Liver Transplantation Models
Brown Norway (BN) and Lewis (LEW) rats (male, 220–250 g) were purchased from Chongqing Medical University experimental animal center (Chongqing, China). The allotransplantation model (LEW rat as donor, BN rat as recipient, LEW to BN) and syngeneic transplantation model (LEW rat as donor, LEW rat as recipient, LEW to LEW) of rat orthotopic liver transplantation were performed by modified two-cuff technique as described by Kamada.33,34 Rats in the pseudo-operation group had only the laparotomy performed without liver transplantation. All animal experiments were approved by the institutional animal care and use committee.
Isolation, Culture, and Treatment of KCs
KCs were isolated from LEW rat liver according to the method described previously,35 and cultured in Roswell Park Memorial Institute 1640 containing 10% fetal bovine serum (FBS) as well as 1% streptomycin and penicillin. KCs were divided into the following groups: Control group (KCs cultured in normal media), lipopolysaccharide (LPS) group (KCs were treated with LPS (Cell Signaling Technology, USA) for 12 h), IL-34 group (KCs were treated with rat IL-34 recombinant protein (Cloud Clone corporation, USA) for 12 h after treatment with LPS), LY294002 group (KCs were pretreated with LY294002 (Cell Signaling Technology, USA) 2 h before treatment with rat IL-34 recombinant protein and LPS), Rapamycin group (KCs were pretreated with rapamycin (Sangon Biotech, China) 8 h before treatment with rat IL-34 recombinant protein and LPS).
Enzyme-Linked Immunosorbent Assay
The supernatants were collected 12 h after treatment with rat IL-34 recombinant protein. The serum samples were collected 7 days after liver transplantation through the caudal vein. The concentrations of IL-34 (Elabscience, USA), IL-10 (4A Biotech, China), IL-12p70 (4A Biotech, China), TGF-β1 (NeoBioscience, China), and IFN-γ (NeoBioscience, China) in serum and supernatant were detected by enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s instructions.
Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated by using TRIzol reagent (Takara, Japan) and reverse-transcribed by using All-in-one cDNA Synthesis SuperMix (Bio-tool, USA) according to the manufacturer’s instructions. Target gene fragments were amplified by using special primers (summarized in Table 1, Thermo Fisher Scientific, USA).
The expression levels of T-Akt1/2, T-Akt1, T-Akt2, T-mTOR, T-eukaryotic translation initiation factor 4E binding protein (4eBP), T-ribosomal protein s6 kinase (S6K) T-p65, T-p38, IκBα, arginine enzyme 1 (Arg-1) (Santa Cruz Biotechnology, USA), P-Akt1/2, P-Akt1, P-Akt2, P-mTOR, P-4E-BP, P-p38, P-p65, and P-S6K (Abcam, U.K.) were detected by western blot following the manufacturer’s instructions.
Flow Cytometry Analysis
KCs isolated from liver tissue were stained with anti-F4/80-FITC (BD Bioscience, USA) to assess the purity. The ratio of M2 KCs was assessed by staining with anti-F4/80-FITC and anti-CD206-PE (BD Bioscience, USA). KCs were suspended as single cells with PBS, stained with the antibody for 2 h in a dark room, and then washed twice with PBS. The staining was assessed by Amnis Flowsight (Millipore, USA) and analyzed by FlowJo7.6.5 software.
Adeno-associated Virus Construction and Use in Vivo
The adeno-associated Viral Vectors Helper-Free System was purchased from HanBio (Shanghai, China). The coding sequence (CDS, Gene ID: 498951) for IL-34 protein was cloned into pHBAAV-CMV-MCS-EGFP vectors to construct the pHBAAV-CMV-IL-34-EGFP stably expressing IL-34. The pHBAAV-CMV-IL-34-EGFP was packaged with pAAV-RC and pHelper to produce serotype 9 adeno-associated virus (AAV) vectors. The AAV vectors were used to transfect AAV-293 cells and the supernatant containing virus particles was harvested and purified. The recipient rats were injected with 100 μL/rat of AAV (1.4 × 1013 μg/mL) or normal saline through the caudal vein 30 days before liver transplantation. The concentration of IL-34 in serum was analyzed by ELISA to assess the in vivo expression of IL-34.
Histology and Liver Function
The grafts were fixed in 10% paraformaldehyde and then embedded in paraffin. The sections were stained with hematoxylin-eosin. The rejection activity index (RAI) was graded based on the Banff pattern according to histological changes.36,37 The serum samples for assessing liver function were obtained from the caudal vein of recipients on day 0, 1, 3, 5, and 7 after liver transplantation. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) (Jiancheng Bioengineering Institute, China) were detected according to the manufacturers’ instructions.
Liposome-encapsulated Clodronate Treatment and Adoptive Transfer of KCs
Liposome-encapsulated clodronate (78-BI, Shanghai, China) was injected into the donors before liver transplantation to deplete KCs.38 KCs isolated from LEW rats were injected into the recipient rats through the portal vein during transplantation.39,40
One-way analysis of variance was used to test the statistical significance of differences. The Kaplan-Meier method with the log-rank test was used to analyze survival. A P value less than 0.05 was considered statistically significant.
Inhibitory Effect of IL-34 on AR of Rat Liver Transplantation
To determine IL-34 expression in recipient rats after liver transplantation, ELISA and reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze the recipient rat serum and graft samples, respectively. We found that serum IL-34 in the syngeneic transplantation group was higher than that in the allotransplantation group on day 7 after liver transplantation, and IL-34 expression at mRNA level in the allotransplantation group was significantly lower than that in the syngeneic transplantation group (Figure 1A). These results suggested that IL-34 might be involved in AR and exerted an immunoregulatory effect on liver transplantation. To test this hypothesis, recipient rats were treated with either AAV-IL-34 or AAV-green-fluorescent protein (GFP) before liver transplantation. According to the kinetics of AAV expression in vivo, AAV-GFP and AAV-IL-34 were injected into recipient rats 30 days before transplantation to acquire optimal in vivo expression.41,42 The ELISA results showed that the serum IL-34 of AAV-IL-34-treated rats was higher than that of normal saline (NS)-treated rats and AAV-GFP-treated rats (Figure 1B), suggesting that IL-34 was expressing efficiently in vivo. The results of histological examination showed that the mixed inflammatory cells infiltrated into the portal area were lessened, and the injuries to hepatic cells and vessels were milder in the AAV-IL-34 group compared with the control and AAV-GFP groups (Figure 1C). The RAI of the AAV-IL-34 group was significantly lower than that of the control and AAV-GFP groups (Figure 1D). More importantly, AAV-IL-34 treatment prolonged the survival of recipient rats after liver transplantation (Figure 1E). In addition, the analyses of representative inflammatory cytokines in serum from recipient rats showed that AAV-IL-34 treatment decreased IL-12 expression and increased IL-10 expression at the serum level after liver transplantation (Figure 1F). Furthermore, the analyses of serum ALT, AST, and TBIL on days 0, 1, 3, 5, and 7 after transplantation showed that AAV-IL-34 treatment significantly improved the liver function of recipients (Figure 1G).
IL-34 Induces KCs M2 Polarization In Vivo and In Vitro
It has been established that the M2 phonotype KCs contributed to the induction and maintenance of liver transplantation tolerance.13,14,16 To investigate the phenotypes of KCs in grafts after liver transplantation, KCs were isolated on day 7 after liver transplantation. Compared with the control and AAV-GFP groups, the expression levels of Arg-1, IL-10, and TGF-β1 in KCs were higher, whereas IL-12 expression was lower in the AAV-IL-34 group (Figures 2A-B). These results suggested that IL-34 could induce KC M2 polarization in vivo after liver transplantation. To determine whether IL-34 induces the KC M2 polarization directly in vitro, KCs were isolated from rat liver tissues and treated with LPS and rat IL-34 recombinant protein12,43,44 (Figures 2C-E). We found that IL-12 expression was higher in LPS-treated KCs at both the mRNA and supernatant levels compared with nontreated KCs (Figure 2D-E). Next, KCs were treated with IL-34. The results showed that although IL-34 attenuated the increase of IL-12 expression at the supernatant and mRNA levels, it also significantly increased the expression levels of IL-10 and TGF-β1 in a concentration-dependent manner (Figure 2D-E). To analyze the effects of IL-34 on polarization of KCs in more detail, we also measured the expression of Arg-1. As expected, Arg-1 expression in LPS-treated KCs was also increased after treatment with IL-34 (Figure 2F). In addition, we treated KCs with IL-34 alone, but we did not observe M2 polarization of KCs (Figure 2G). These results showed that IL-34 could switch the phenotype of KCs from M1 to M2 in vitro.
IL-34 Induces KC M2 Polarization Through the PI3K/Akt Pathway
The PI3K/Akt pathway, downstream of CSF-1R, plays a central role in CSF-1R signaling.19 To determine whether the PI3K/Akt pathway was associated with KC M2 polarization induced by IL-34, the phosphorylation status of Akt1/2 in KCs was analyzed. The phosphorylation status of Akt1/2 in LPS-treated KCs was enhanced compared with nontreated KCs. The result showed that IL-34 treatment enhanced the phosphorylation status of Akt1/2. Furthermore, these IL-34–mediated effects were partly abolished by the PI3K inhibitor LY294002 (Figure 3A). Moreover, LY294002 attenuated the increase of Arg-1, IL-10, and TGF-β1 expression as well as the decrease of IL-12 expression at both the mRNA and protein levels (Figures 3A-C). The result of flow cytometry (FCM) showed that the ratio of M2 KCs in LPS-treated KCs was increased after treatment with IL-34, whereas LY294002 pretreatment partly abolished the IL-34-mediated effect on KC polarization (Figures 3D-E). Previous research demonstrated that Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.45 To further address the roles of Akt1 and Akt2 in M2 polarization of KCs induced by IL-34, the phosphorylation status of Akt1 and Akt2 in KCs was examined. We found that the phosphorylation status of both Akt1 and Akt2 in LPS-treated KCs was higher than that in the nontreated KCs. The phosphorylation status of Akt1 was significantly enhanced in response to IL-34 treatment, whereas that of Akt2 was not increased. Moreover, LY294002 pretreatment attenuated the enhanced phosphorylation status of Akt1 induced by IL-34 treatment (Figure 3F). These results suggested that IL-34–induced KC M2 polarization partially through the PI3K/Akt1 pathway.
To learn more about the mechanisms of the PI3K/Akt signal pathway augmentation in KC M2 polarization induced by IL-34, mediators, such as mTOR, NF-κB, and p-38 mitogen-activated protein kinase (p38 MAPK), were investigated. We found that IL-34 treatment enhanced the phosphorylation status of mTOR, S6K, and 4E-BP (Figure 4A). It is worth noting that the IL-34–mediated effect on KC M2 polarization was partially blocked by the mTOR inhibitor rapamycin (Figure 4B-F). Furthermore, we also found that IL-34 increased the IκB expression and decreased the phosphorylation status of p65 and p38 MAPK; these effects of IL-34 were also partially abolished by LY294002 (Figure 4G).
The AR Inhibition Induced by IL-34 Is Due to the M2 Polarization of KCs
M2 KCs play a crucial role in AR inhibition of liver transplantation, and our study has demonstrated that IL-34 could inhibit AR of liver transplantation and polarize KCs to the M2 phenotype. Therefore, we hypothesized that the AR inhibition induced by IL-34 was due to the M2 polarization of KCs. To determine that the protection against AR of liver transplantation observed in AAV-IL-34-treated rats was due to KC M2 polarization, the donor KCs were depleted by clodronate treatment and the nontreated KCs or LPS-treated KCs were transferred into AAV-IL-34-treated rats. Histological analyses of the grafts (Figures 5A-B), monitoring survival (Figure 5C), and detecting the serum ALT, AST, and TBIL levels (Figure 5D) showed that the transfer of nontreated KCs into AAV-IL-34–treated rats reduced the severity of AR. More significantly, the transfer of LPS-treated KCs into AAV-IL-34–treated rats also reduced the severity of AR after liver transplantation (Figures 5A-D). These results suggested that the AR inhibition induced by IL-34 was due to the M2 polarization of KCs.
Liver transplantation is a lifesaving procedure for patients with chronic end-stage liver diseases and acute liver failure. Although immunosuppressants are widely used after organ transplantation, AR is still a life-threatening complication. Thus, effective treatments for AR of liver transplantation are needed. Here, we provide evidence that IL-34 could inhibit AR of liver transplantation by inducing M2 polarization of KCs, suggesting that IL-34 is a viable treatment strategy for AR of liver transplantation.
In the present study, we found that IL-34 expression was decreased in grafts and serums from the allotransplantation group compared with the syngeneic transplantation group. It was consistent with the study of Bézie et al32 in heart transplantation. Based on the well-demonstrated evidence that IL-34 serves as a Tregs–specific cytokine,32 it is not hard to speculate that the Tregs accumulation in graft increased IL-34 expression, and IL-34 could be upregulated in serum as an inhibitory cytokine. Thus, we speculated that IL-34 might exert an immunoregulatory function in rat liver transplantation. As expected, we observed that IL-34 overexpression in recipients significantly prolonged survival, improved liver function, alleviated hepatic tissue injury of recipient rats, and decreased the concentration of serum proinflammatory cytokine. In addition, we found that IL-34 could induce KC M2 polarization in vivo after liver transplantation and switch the phenotype of KCs from M1 to M2 in vitro.
Recent studies have demonstrated that the PI3K/Akt pathway played a crucial role in CSF-1R signaling,19 and activation of the PI3K/Akt pathway is a pivotal step in macrophage M2 polarization.46,47 Previous studies also demonstrated that the production of Arg-1 and IL-10 was controlled by the PI3K/Akt pathway. Moreover, many transcriptional factors that regulate the induction of inflammatory cytokines were also controlled by PI3K/Akt pathway.48,49 The activation of mTOR is a crucial step in M2 polarization of macrophages.50 Mercalli et al51 and Chen et al52 have demonstrated that rapamycin could enhance the M1 polarization of macrophages. The PI3K/Akt/mTOR signaling pathway is involved in the M2 polarization of macrophages induced by IL-10 and BMP-7.46,53 In our study, inhibitors of PI3K and mTOR were used to determine the role of the PI3K/Akt/mTOR pathway in KC M2 polarization induced by IL-34. The results showed that IL-34 significantly enhanced the phosphorylation status of mTOR, Akt1/2, S6K, and 4E-BP. The pretreatments with LY294002 and rapamycin partially blocked KC M2 polarization induced by IL-34, indicating that the activation of PI3K/Akt/mTOR signaling was associated with KC M2 polarization induced by IL-34. In addition, previous research has demonstrated that Akt1 and Akt2 protein kinases played opposite roles in macrophage polarization.45 Androulidaki et al54 demonstrated that Akt1, activated by LPS through the TLR4 pathway, could regulate the polarization of macrophages via increasing the expression of let-7e and miR-181c while decreasing that of miR-155 and miR-125b. Moreover, deletion of Akt1 and induction of miR-155–induced macrophages M1 polarization.55 In contrast to Akt1, Akt2 ablation suppressed the TLR4 signaling pathway and induced M2 polarization.56,57 Our results showed that the phosphorylation status of both Akt1 and Akt2 was increased in LPS-treated KCs compared with nontreated KCs, whereas only the phosphorylation status of Akt1 was increased by the IL-34 treatment. Thus, we speculated that the phosphorylation of Akt1, but not Akt2, was acquired for KC M2 polarization induced by IL-34. NF-κB and MAPK play crucial roles in macrophage M1 polarization, and IL-12 induction was regulated by the p38 MAPK-activating pathway in dendritic cells.48,58,59 Previous research has also demonstrated that activation of the PI3K/Akt pathway plays an important role in feedback inhibition of LPS signals by suppressing MAPK and NF-κB in monocytes and dendritic cells.60,61 In the present study, we observed that the activation of p65 and p38 MAPK in LPS-treated KCs was significantly impaired by IL-34 treatment, and the effects of IL-34 were partially abolished by LY294002 pretreatment. These results indicated that suppressions of both the NF-κB pathway and p38 MAPK pathway, downstream of the PI3K/Akt pathway, were also involved in the M2 polarization of KCs induced by IL-34.
The M1/M2 polarization status of KCs was associated with the severity of AR.13,14,16 The results also showed that treating AAV-IL-34–treated rats with clodronate aggravates the severity of AR of liver transplantation. In addition, transfer of nontreated KCs into rats treated by AAV-IL-34 and clodronate reduced the severity of AR. These results suggested that the inhibitory effect of IL-34 on AR was at least partially mediated by KCs. More significantly, transfer of LPS-treated KCs into rats treated with AAV-IL-34 and clodronate also reduced the severity of AR. This suggested that IL-34 switched the phenotype of KCs from M1 to M2 in vivo and inhibited the AR of liver transplantation. Our study has demonstrated that rapamycin could block the IL-34–mediated effect on KC M2 polarization through inhibiting mTOR signaling pathway, which seemed to indicate that rapamycin could block the inhibitory effect of IL-34 on AR of liver transplantation by blocking the effect of IL-34 on KC polarization. However, rapamycin is widely used in the clinical setting to suppress the AR of liver transplantation through inhibiting T-cell proliferation and inducing Tregs differentiation. We speculated that the inhibitory effect of rapamycin on AR by inhibiting T-cell proliferation and inducing Tregs differentiation was more powerful than the hastened effect on AR by blocking KC M2 polarization. However, it is required for further study that tests our hypothesis.
In summary, data in our study showed that IL-34 could inhibit the AR of liver transplantation through inducing KC M2 polarization, indicating that delivering IL-34 into human recipients is a promising therapeutic strategy for the AR of liver transplantation. However, further studies are needed to elucidate the detailed molecular mechanism of IL-34 in AR of liver transplantation. In addition, more research is needed to assess the safety and efficacy of IL-34 use in the human body.
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