Recent experimental and clinical studies have shown that liver nonparenchymal cells (including dendritic cells [DCs], liver sinusoidal endothelial cells, and Kupffer cells [KCs]) play important roles in tolerance induction (1–3). KCs, the most important resident macrophages of the liver, comprise a major proportion (20%) of hepatic nonparenchymal cells, and the tolerogenic properties of the liver are generally accepted (4, 5). Over the last decade, we have found that KCs play a dual role in the pathologic changes that occur after liver transplantation through a mechanism that may be closely associated with the modulation of antigen-specific T-cell responses and the T helper 1 and 2 (Th1/Th2) balance (6–11). The question of how to regulate the function of KCs toward immunosuppression is the next step.
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate the expression of target genes involved in numerous physiologic processes. MiRNAs exert profound effects on the development and differentiation of immune cells and on the regulation of innate and acquired immune responses (12–14). Among these miRNAs, miRNA-155 (miR-155) is a key regulator of immune homeostasis, or the balance between immune response and immune tolerance (15, 16). Although several previous reports have shown that miR-155 is involved in the development of DCs (17, 18), no report has detailed its role in regulating the maturation, function, or maintenance of KCs, another antigen-presenting cell subset, or in immune tolerance induction.
Many of the current research works on miRNAs regarding immunity and inflammation are focused on inflammatory diseases, and allograft studies have also demonstrated that miR-155 expression is associated with inflammation and upregulation of proinflammatory mediators in transplant patients (19–21). The aim of the present study is to assess the expression of miR-155 in KCs in relation to the balance of Th1/Th2 cytokines, degree of antigen-specific T-cell response, number of apoptotic T cells, and survival of liver allografts.
miR-155 Regulates Phenotype Alteration on the KC Surface
Real-time polymerase chain reaction (PCR) results showed that the expression of miR-155 in the miR-155 mimic group led to a 210-fold increase compared to the control mimic group (Fig. 1A), whereas the expression of miR-155 in the miR-155 inhibitor group led to a 360-fold decrease compared to the control inhibitor group (Fig. 1B). These results indicated that miR-155 mimic and inhibitor can effectively enhance or inhibit miR-155 expression, respectively, in KCs. To determine the role of upregulation or inhibition of miR-155 expression in the antigen-presenting function of KCs, we performed flow cytometric (FCM) analysis to evaluate the expression of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC-II), CD40, and CD86 on the surface of transfected KCs. The results demonstrated that overexpression of miR-155 (mimic) resulted in relatively high levels of expression of these molecules, whereas knockdown of miR-155 expression (inhibitor) lowered their expression (Fig. 1C, D).
miR-155 Regulates Th1/Th2-Associated Cytokine Secretion By Modulation of the SOCS1/JAK/STAT Signaling Pathways in KCs
Interferon-gamma (IFN-γ), a representative Th1 cytokine, participates in graft rejection, whereas interleukin (IL)-10, a representative Th2 cytokine, induces graft acceptance. We thus assessed the effect of miR-155 on Th1/Th2-associated cytokine secretion in KCs. As shown in Figure 2, the level of IFN-γ was significantly suppressed (Fig. 2A), whereas that of IL-10 was significantly enhanced in the miR-155 inhibitor group compared with those in the control and miR-155 mimic groups (Fig. 2B). These data suggested that miR-155 suppression in KCs induced a predominantly Th2 cytokine profile, whereas miR-155 enhancement produced a predominantly Th1 cytokine profile.
We next set to determine the major target of miR-155 that shifts the Th1/Th2 cytokine profile. Earlier studies indicated that miR-155 and suppressor of cytokine signaling 1 (SOCS1) interact in regulatory T cells, macrophages, and human breast cancer cells (22–24). SOCS1 has been shown to negatively regulate various immune responses (25). Therefore, we examined whether the translation level of SOCS1 in KCs is regulated by miR-155. As expected, western blot results indicated that SOCS1 protein level was increased by miR-155 inhibition and decreased by miR-155 overexpression (Fig. 2C), whereas the SOCS1 mRNA level was not obviously altered (Fig. 2D).
SOCS1 is reportedly recruited to Janus kinase (JAK), in turn blocking JAK tyrosine kinase activity and downstream signal transducer and activator of transcription (STAT) phosphorylation. Therefore, we assessed miR-155-regulated immune response through SOCS1 by examining JAK2 and STAT3 phosphorylation. As shown in Figure 2E and F, miR-155 inhibition suppressed JAK2 and STAT3 phosphorylation, whereas miR-155 overexpression enhanced JAK2 and STAT3 phosphorylation, consistent with the regulatory mechanism of SOCS1. Taken together, these data showed that miR-155 posttranscriptionally regulated SOCS1 expression and activated JAK/STAT signaling in KCs, resulting in Th1/Th2 cytokine profile deviation.
miR-155 Indirectly Regulates T-Cell Proliferation and Apoptosis Induced by KCs in Mixed Lymphocyte Reactions
The purity of the T cells was 75.42% after Nylon Fiber Column T purification; the proportion of CD4-positive T cells was 52.63%, and the proportion of CD8-positive T cells was 24.71% (CD4+/CD8+=2.13; Fig. 3A).
The allostimulatory activities of transfected KCs were investigated in allogeneic mixed lymphocyte reactions. As shown in Figure 3B, miR-155 knockdown in KCs induced weaker proliferation of allogeneic T cells compared to the control inhibitor group, but enhancement of miR-155 in KCs led to stronger proliferation of T cells when comparing to the control mimic group. We also assessed apoptosis of allogeneic T cells in mixed lymphocyte reactions by fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) FCM analysis and demonstrated that apoptosis of T cells in the miR-155 inhibitor group (35.62%) was much higher than that in the control inhibitor (20.81%) or miR-155 mimic (14.36%) group (P<0.05; Fig. 3C). To address the mechanism underlying the different degrees of allogeneic T-cell apoptosis in transfected KCs, the expression of Fas ligand (FasL) protein in KCs was investigated. As shown in Figure 3D, western blot results indicated that FasL protein level was increased by miR-155 inhibition and decreased by miR-155 overexpression.
miR-155 Short Hairpin RNA (shRNA) Lentivirus Treatment Prolongs Liver Allograft Survival and Improves Liver Function
We then determined the effect of miR-155 shRNA lentivirus on allograft survival in vivo. The results showed that miR-155 shRNA lentivirus administration effectively downregulated miR-155 expression in KCs and liver tissues (Fig. 4A). In addition, statistically significant improvement in survival occurred in mice administered miR-155 shRNA lentivirus; 58.3% of the mice survived at least 30 days. Mice administered normal saline or negative control lentivirus survived for shorter periods (median, 12–13 days; Fig. 4B).
Next, we examined the pathology and function of liver allografts. As shown in Figure 4C, obvious acute rejection was present in the three groups at day 7 postoperatively; however, milder vacuolation of hepatic cells and less inflammatory cell infiltration were observed in mice treated with miR-155 shRNA lentivirus compared to mice treated with normal saline or negative control lentivirus. The serum concentrations of liver enzymes (alanine aminotransferase and aspartate aminotransferase) were significantly decreased after miR-155 shRNA lentivirus treatment compared with those after treatment with normal saline or negative control lentivirus (Fig. 4D).
miR-155 shRNA Lentivirus Treatment Shifts Th1/Th2 Balance and Enhances T-Cell Apoptosis in Liver Allografts
To address the molecular mechanism of miR-155 knockdown on improved liver allograft function, we assessed serum IFN-γ and IL-10 concentrations and T-cell apoptosis in allografts on postoperative day 7. As indicated by real-time PCR, miR-155 shRNA lentivirus treatment decreased production of the proinflammatory cytokine IFN-γ while enhancing production of the anti-inflammatory cytokine IL-10 (Fig. 5A). At the protein level, as analyzed by western blot (Fig. 5B) and enzyme-linked immunosorbent assay (ELISA; Fig. 5C), miR-155 shRNA lentivirus administration inhibited IFN-γ expression and increased IL-10 expression. In addition, these changes were associated with intrahepatic levels of SOCS1 protein after miR-155 shRNA lentivirus treatment (Fig. 5B). Further, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of hepatic tissues showed substantially more apoptotic T cells in the transfection group than in other groups, and more apoptotic T cells were present in the portal area (Fig. 5D). Altogether, these results were consistent with our in vitro data.
Our present results showed that an increased level of miR-155 in KCs was implicated in proinflammatory cytokine secretion and acute rejection after liver transplantation. MiR-155 knockdown exhibited regulatory effects, causing immune deviation from the Th1 to the Th2 cytokine profile, provoking antigen-specific T-cell hyporesponsiveness and prolonging liver allograft survival.
Currently, several target genes for miR-155 have been identified including PU.1, AID, SOCS1, SHIP1, and others (15, 16). Of these, SOCS1 is considered to be a central regulator of immune cell function and inflammation response. SOCS1 regulates macrophage and DC activation, development, and differentiation (25–27). SOCS1 also regulates the intensity, duration, and quality of cytokines secretion, such as interleukins, IFN-γ, and most of these cytokines use the so-called JAK/STAT signaling pathway (28, 29). In our in vitro experiments, we found that the translation level of SOCS1 was also regulated by miR-155 in KCs. In addition, we found that silencing SOCS1 with miR-155 mimic in KCs enhanced antigen presentation, with greater expression of MHC-II and costimulatory molecules, and induced a stronger Th1 cytokine profile through the JAK/STAT signaling pathway. In contrast, SOCS1 overexpression with miR-155 inhibitor maintained KCs in a steady, immature state, with less surface expression of costimulatory and MHC-II molecules, and reduced the proliferation of allogeneic T cells. Acute rejection is closely associated with activated T lymphocyte–mediated immune response (30, 31). T-cell activation requires two signals: those of the MHC and antigenic peptide complex, and costimulatory molecules. Activation of T cells with lower levels of MHC and costimulation molecules may lead to antigen-specific T-cell hyporesponsiveness or the development of immune tolerance. Taken together, these findings support the immunosuppressive function of miR-155 downregulation in KCs.
Fas is a prominent member of the tumor necrosis factor receptor superfamily. Its binding with its ligand induces apoptosis, resulting in immune tolerance modulation (32) and cancer progression (33). Our previous studies have indicated that Fas/FasL-mediated apoptosis plays a crucial role in KC-induced T-cell depletion, and nuclear factor κB (NF-κB) activation is intimately involved in FasL gene transcription and protein synthesis in KCs (6). These results are consistent with other findings (34, 35). A few more recent publications support miR-155 as an NF-κB transactivational target involved in a negative feedback loop through downregulation of IκB kinases and other genes (36). Given these findings, we speculate that miR-155 repression in KCs may induce NF-κB activation and consequently increase apoptosis of T cells by promoting the expression of FasL on KCs. Interestingly, our present data showed that miR-155 suppression in KCs enhanced FasL expression and increased the number of apoptotic T cells. We also found a greater quantity of TUNEL-positive, apoptotic T cells in grafted liver after miR-155 shRNA lentivirus treatment.
To further investigate the effects of miR-155 shRNA lentivirus on tolerance induction in vivo, we performed orthotopic liver transplantation in mice after caudal vein injection and portal vein perfusion of the donor liver with miR-155 shRNA lentivirus specific for mice. We evaluated liver function, histology, and postoperative survival. The results showed that miR-155 shRNA lentivirus treatment prolonged liver allograft survival and improved liver function. The underlying molecular mechanism may be correlated with Th1/Th2 cytokine profile deviation through SOCS1 targeting, and apoptotic T cell increase.
Th1 activity is generally accepted to be associated with rejection, whereas a Th2 response may favor graft acceptance and tolerance. Our present study demonstrated that miR-155 suppression in KCs or graft livers efficiently decreased Th1-associated cytokine expression while it enhanced anti-inflammatory cytokine expression. Whether other miRNAs are involved in the expression of these cytokines is not well understood and requires further investigation. In addition to KCs, miR-155 is expressed in and regulates the functions of T cells, B cells, and DCs, among others (37, 38). Thus, the possibility remains that miR-155 downregulation may work through its effect on additional cell types. Therefore, we need to look for a better KC-targeted approach in future studies.
In conclusion, we have shown that miR-155 can regulate the balance of Th1/Th2 cytokines and the maturation and function of KCs. Inhibiting the expression of miR-155 in KCs positively induced immune suppression through downregulation of T-cell response and pro-inflammatory cytokine secretion and increases in FasL expression to induce T-cell apoptosis. Moreover, silencing miR-155 prolonged liver allografts survival after transplantation in mice. Hence, miR-155 might be a key component in liver transplantation immunology and a promising target for the development of novel drugs or gene therapies for inducing tolerance.
MATERIALS AND METHODS
Inbred male BALB/c (H-2d) mice and C3H (H-2k) mice (25–30 g) were obtained from Chongqing Medical University (Chongqing, China) and Vital River Laboratory Animal Technology Co. Ltd (Beijing, China) and housed in a temperature- and humidity-controlled environment with free access to tap water and rat chow. All experimental procedures were approved by the institutional animal care and use committee.
Isolation and Treatment of KCs
KCs were isolated from BALB/c mice and recipients using collagenase digestion and differential centrifugation with Percoll according to a previously published protocol (6). Cells isolated in this manner were 85% KCs and 95% viable cells. For in vitro experiments, 100 nM/L miR-155 mimic (double-stranded RNA oligonucleotides) and miR-155 inhibitor (single-stranded chemically modified oligonucleotides) (GenePharma Co., Ltd., Shanghai, China) were used for the overexpression and inhibition of miR-155 in KCs, respectively. Transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Scrambled mimic or inhibitor (GenePharma) was transfected as matched control. After transfection for 24 hr, cells were harvested to measure the levels of SOCS1/JAK/STAT proteins and surface molecules.
Orthotopic Liver Transplantation in Mice
A total of 81 orthotopic BALB/c to C3H mouse liver transplantations were performed under a surgical microscope according to the method of Kamada and Calne (39) with slight modification. This model is an acute rejection model reported by Qian and colleagues (40). A volume of 0.2 mL normal saline, negative control (scrambled-miR-155 shRNA) lentivirus, and miR-155 shRNA lentivirus (1 × 109 TU/mL) were injected to the donor through the caudal vein (0.1 mL/min) before surgery. They were also perfused through the portal vein (0.1 mL/min) for 20 min during the cold storage period in the normal saline group, negative control group, and transfection group (each group, n=27). Three recipients in each group were sacrificed for miR-155 analysis in KCs and transplanted livers at days 1, 4, 7, and 14 posttransplantation. Three recipients in each group were also killed for histologic inspection, protein and mRNA analysis, liver function test, and ELISA at day 7 after transplantation. Other cases of orthotopic liver transplantation were carried out to assess survival.
The expression of FasL (Santa Cruz Biotech, CA), SOCS1, JAK2, p-JAK2, STAT3, and p-STAT3 protein (Cell Signaling Technology, Danvers, MA) in transfected KCs, and expression of SOCS1, IFN-γ, and IL-10 protein (Santa Cruz Biotech, CA) in liver allografts was assessed by western blot following a method described previously (10, 11).
Real-Time Polymerase Chain Reaction
Levels of miR-155 and SOCS1 mRNA in transfected KCs, and levels of IFN-γ and IL-10 mRNA in liver allografts were examined by real-time PCR. Total RNA from KCs or liver allografts was extracted with TRIzol reagent (Invitrogen). Real-time PCR was performed according to a standard protocol with sense and antisense primers for miR-155, SOCS1, IFN-γ, IL-10, U6, and β-actin. Relative gene expression was quantified using U6 as an internal control for miR-155 and β-actin as an internal control for other RNAs.
FCM Analysis of KC Surface Phenotypes
KC surface phenotypes were detected by FCM analysis. In brief, cells were washed and stained with FITC-, phycoerythrin-, or allophycocyanin-conjugated anti-mouse CD40, CD86, and MHC-II antibodies (all from eBioscience, San Diego, CA), diluted to optimal concentrations. Cells were then washed twice and FCM analysis was performed with appropriate isotype controls.
Isolation of Splenic CD4+ T Cells and Mixed Lymphocyte Reaction Assay
For the mixed lymphocyte reaction assay, CD4+ T cells were first harvested from C3H mouse spleen and purified over a Nylon Fiber Column T (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were then cocultured with KCs from BALB/c mice that had been transfected with miR-155 mimic, control mimic, miR-155 inhibitor, or control inhibitor at a 10:1 ratio. After coculture for 72 hr, supernatants were collected and added to 96-well plates. Next, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (10 mg/mL; Sigma, St Louis, MO) was added to each well and the plates were incubated for an additional 4 hr at 37°C. After centrifugation, the MTT solution was removed, and 200 μL dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO) was added to each well to solubilize the formazan crystals.
In Vitro Apoptosis of T cells
Apoptosis of T cells in the mixed lymphocyte reaction was measured by FITC-annexin V and PI FCM analysis (Becton Dickinson, San Jose, CA), according to the manufacturer’s instructions. Cells were harvested, washed twice with cold phosphate-buffered saline, resuspended in binding buffer, stained with FITC-annexin V, and then stained with PI. FCM was performed on a FACS Calibur system (Becton Dickinson, San Jose, CA). For each analysis, a minimum of 10,000 cells was counted.
Histology and Liver Function Post-Orthotopic Liver Transplantation
Liver tissues were immersion-fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. The histological findings of hematoxylin-eosin staining were graded according to the Banff scheme (41). The rejection activity index (RAI) was calculated from the three individual scores (venous endothelial inflammation, bile duct damage, and portal inflammation) (42). Functional markers of hepatocyte injury (alanine aminotransferase and aspartate aminotransferase) were quantified with an autobiochemical analyzer (CX7; Beckman Coulter, Inc., Brea, CA).
In Vivo Apoptosis of T Cells
Apoptosis of T cells in liver grafts was assessed by TUNEL assay, as described previously (7).
For cytokine measurements, conditioned medium from KCs and serum samples were kept at –70°C until assay. IFN-γ and IL-10 levels were quantified with commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.
Data are shown as mean±standard deviation (SD). Statistical analyses were performed using analysis of variance and Student t test. The Kaplan-Meier method was used for survival analysis. Differences were considered to be statistically significant at P<0.05.
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