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reperfusion injury
Biomedicine & Pharmacotherapy 123 (2020) 109793
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CD47 blockade alleviates acute rejection of allogeneic mouse liver transplantation by reducing ischemia/reperfusion injury
T
Ding-yang Lia, Shu-li Xieb, Guang-yi Wangb, Xiao-wei Danga,* a b
Department of Hepatobiliary & Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, Henan Province, China Department of Hepatobiliary& Pancreatic Surgery, The First Norman Bethune Hospital Affiliated to Jilin University, Changchun 130021, Jilin Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute rejection CD47 ischemia/reperfusion injury Liver transplantation thrombospondin-1
Despite advances in immunosuppressive therapies, acute rejection response is still a serious concern especially in the early phase after liver transplantation. This study aimed to evaluate whether blocking the TSP1-CD47 signaling pathway could attenuate the acute rejection after liver transplantation. An allogeneic mouse orthotopic liver transplantation model (Balb/c→C3H) with prolonged cold ischemic phase was used to induce severe IRI and lethal acute rejection. CD47mAb or isotype matched-control IgG2a was administered to donor liver during graft perfusion. Recipients were sacrificed at 1d, 3d, 5d and 7d after reperfusion. Blood samples were collected to evaluate serum alanine aminotransferase, total bilirubin, HMGB-1,TNF-α, IL-2 and INF-γ level. Flow cytometric analysis was used to detect the strength of innate and adaptive immune response. Liver tissue was obtained for HE, TUNEL staining and F4/80 immumohistochemical staining. Moreover, we conducted a mixed lymphocyte reaction treated with IgG2a or CD47mAb. Mice in CD47mAb-treated group demonstrated improved survival and significantly lower increase in Suzuki score, apoptosis index, acute rejection index, serum alanine aminotransferase, total bilirubin, HMGB-1, TNF-α, IL-2, INF-γ level and the degree of Kupffer cells' activation especially in the early phase of acute rejection. In addition, Pearson's correlation analysis confirmed significant correlation between Suzuki score/ALT and acute rejection index. The in vitro inhibition assay showed that CD47 blockade couldn’t directly inhibit recipient lymphocyte proliferation. Based on the evidence that TSP1-CD47 signaling blockade with CD47mAb could alleviate acute rejection by reducing the extent of IRI after liver transplantation indirectly, this study provided a basis for new interventions and management methods to support better transplant outcomes.
1. Introduction Orthotopic liver transplantation is currently the only effective treatment option for end-stage liver disease and acute liver failure. Despite advances in immunosuppressive therapies that have improved the outcome of liver transplantation, acute graft rejection is still a serious complication. Concerns include the increased possibility of primary graft non-function or dysfunction, considerable medical cost, a decreased quality of life for the recipient, and a reduced survival rate of the graft or the recipient [1]. Therefore, it is essential that protective strategies are sought to alleviate the extent of acute rejection, especially during the early phase after transplantation.
Ischemia/reperfusion injury (IRI), which inevitably arises after liver transplantation, is associated with induction and activation of the innate immune response, endothelial cell damage, and ultimately, hepatocyte necrosis [2].There is an active but controversial debate on the role of IRI in the induction and exacerbation of acute liver graft rejection based on a body of clinical and experimental data [3–6]. Supporters argue that IRI could result in a varying extent of liver inflammation and injury, which could subsequently lead to enhanced allogenicity of grafts through stimulation of innate immunity-related inflammatory pathways. CD47, a member of the immunoglobulin (Ig) superfamily that is ubiquitously expressed in all tissues, combines with signal regulatory
Abbreviations: ALT, alanine aminotransferase; CD47mAb, monoclonal antibody specific to CD47; ELISA, enzyme-linked immunosorbent assay; FACS, flow cytometric analysis; HMGB-1, high mobility group box -1; IRI, ischemia/reperfusion injury; MLT, mouse orthotopic liver transplantation; RAI, rejection activity index; ROS, reactive oxygen species; SEM, standard error of the mean; SIRPα, signal regulatory protein-α; TBIL, total bilirubin; TSP1, hrombospondin-1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling ⁎ Corresponding author at: Department of Hepatobiliary & Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, 1 Jianshe road, Zhengzhou 450000, Henan Province, China. E-mail address: [email protected] (X.-w. Dang). https://doi.org/10.1016/j.biopha.2019.109793 Received 18 August 2019; Received in revised form 6 December 2019; Accepted 10 December 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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procedure, the liver was exposed to cold ischemia for about 4 h. Recipient blood and hepatic tissue were sampled under anesthesia at 1, 3, 5 and 7 days after reperfusion. Blood was used for serum assays and flow cytometric analysis. Liver samples were fixed in 10% formaldehyde and embedded in paraffin. Twenty recipients were followed for 10 days after surgery. Mice that died or were euthanized (because of conjectural surgical complications) within 24 h after transplantation were excluded from analysis.
protein-α (SIRPα), and it is a receptor for the secreted matricellular protein thrombospondin-1 (TSP1) [7,8]. TSP1-CD47 signaling has been shown to correlate with various important cellular pathways, including nitric oxide, VEGF, cGMP, cAMP, and reactive oxygen species (ROS) [9,10]. A large body of evidence suggests that TSP1 signaling through CD47 exerts a considerable influence on animal models of IRI [11,12]. Additionally, recent studies have reported that liver survival and perfusion after IRI was limited through up-regulating CD47 expression in liver tissue, and targeting CD47 inhibition before IRI could reduce the extent of IRI and enhance the survival of steatotic liver allografts [13]. These relationships among TSP1-CD47 signaling, IRI, and acute graft rejection led us to hypothesize that CD47 blockade could indirectly alleviate the severity of acute liver rejection. To test this hypothesis, a well-established allogeneic mouse orthotopic liver transplantation (MLT) model with prolonged cold ischemia time was used to investigate the correlation between IRI and acute rejection, and to investigate the effect of TSP1–CD47 signaling blockade on tissue injury and the immune response.
2.3. Histological examination Fixed livers were sectioned at a thickness of 4 μm and stained with HE for general histopathologic evaluation of IRI, in accordance with Suzuki’s criteria [15]. In addition, the Banff rejection activity index (RAI) was used to assess the severity of acute rejection [16]. Hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using a TransDetectIn Situ Fluorescein TUNEL Cell Apoptosis Detection Kit (Transgen Biotech, China). Only hepatocytes were included in this analysis. The apoptotic index of the TUNEL assay was determined as the percentage of the apoptotic events per hepatic cell population in five random fields at ×400 magnification (Olympus IX51, Japan). Formalin-fixed, paraffin-embedded tissue slices (4 μm) were immunostained with rat anti-mouse F4/80 antibody diluted at 1:200 (eBioscience, USA) to evaluate the degree of Kupffer cells' maturation and activation. The proportion of F4/80 positive cells in liver was calculated in five randomly selected high power field(×400) for each slice using a microscope (Olympus IX51, Japan).
2. Material and methods 2.1. Animals Male 8- to 12-week-old Balb/c and C3H mice (Vital River Laboratories, Beijing, China) weighing 20–25 g were used as donors and recipients, respectively, for the experiments. Mice were housed under standard specific pathogen free (SPF)-level conditions on a 12-h dark/ light cycle, and they had free access to water and food. Donor and recipient mice were fasted without water deprivation for 12 h before liver transplantation. One hundred pairs of mice were randomly assigned to two groups of equal size, and these groups received pretreatment with either rat anti-CD47 monoclonal antibody (CD47mAb) MIAP301 (Santa Cruz Biotechnology, USA) or an isotype matched-control rat IgG2a (Santa Cruz Biotechnology, USA) before graft retrieval. Ten C3H mice were used as the sham control, which underwent opening and closing of the venter that lasted for 30 min. Animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
2.4. Serum assays After centrifugation of blood samples at 4000 rpm for 15 min at 4 °C, the serum supernatant was collected for serological testing. Serum alanine aminotransferase (ALT) and total bilirubin (TBIL) were assayed with a standard automatic biochemistry analyzer (Vitros 350, Johnson & Johnson, USA). The serum HMGB-1 level was measured using an HMGB-1 ELISA kit II (Shino-Test, Japan). Cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-2, and interferon (IFN)-γ secretion were measured by enzyme-linked immunosorbent assay (ELISA), in accordance with the kit manufacturer’s protocol (Biolegend, USA).
2.2. Surgical procedures
2.5. Flow cytometric analysis
All surgical procedures were performed under clean, but not sterile conditions using inhalation anesthesia with isoflurane. A surgical microscope (Olympus SZX 7.0, Japan) was used, and the procedure was a slight modification of techniques described by Qian et al. [14]. Briefly, the ligaments of the liver, right adrenal vessels, right renal arteries and veins, the portal vein, hepatic artery, and common bile duct were separated. After a polyethylene tube was inserted into the lumen of the common bile duct, the gallbladder was flushed initially with 0.4 mL Ringer’s solution followed by 0.2 mL Ringer’s solution containing 0.2 μg CD47mAb or IgG2a via the fundus of the gallbladder. Following removal of the gallbladder, the pyloric vein was ligated, the right adrenal vessels were dissected by electrocoagulation and the right renal vein and artery were ligated and divided. After heparinization, the liver was perfused through the portal vein with 3 mL cold Ringer’s Lactate solution, followed by 1 mL Ringer’s solution containing 1.3 μg CD47mAb or IgG2a. The resected liver was stored at 4 °C in Ringer’s solution for further preparation. The suprahepatic vena cava (SHVC) anastomosis was constructed with a continuous microsuture using 10-0 prolene (Ethicon, USA). Reconstruction of the portal vein and infrahepatic vena cava was performed using the cuff technique. Biliary’s continuity was restored by inserting a stent into the recipient’s common bile duct. After reconstruction, cefoperazone sodium, sulbactam sodium (Pfizer Inc., USA), and tramadol (Grunenthal Co Ltd., Germany) were administered to the recipient without immunosuppression. During each
White blood cells were separated from peripheral blood following treatment with red blood cell lysis buffer (Beyotime, China). The following antibodies were used in the flow cytometric analysis (FACS) of innate and adaptive immune response: anti-mouse CD11b-PerCP/Cy5.5 (eBioscience, USA); anti-mouse CD16/32-APC (Biolegend, USA); antimouse CD4-FITC (BD Biosciences, USA); and anti-mouse CD45RB-PE (BD Biosciences, USA). Rat IgG2a (BD Biosciences, USA) was used as an isotype control. Labeled cells were analyzed using a FACSCalibur (BD LSRFortessa, USA). 2.6. Mixed lymphocyte reaction (MLR) The T cells that were purified from spleen of normal C3H mice by the magnetic cell separation method using a MACSxpressPan T cell isolation kit (Miltenyi Biotec, Germany) were prepared as responders. There were 2 × 105 cells placed into each well in the 96-well roundbottom plates, and an equal number of gamma-irradiated (20 Gy) C3H (syngeneic), BALB/c (donor), or C57BL/10 (third party) spleen cells were added as stimulators, and then treated with 0.2 μg control Ig or CD47 mAb. The cultures were incubated in 200 μL of 10% FCS RPMI 1640 medium for 72 h at 37 °C. [3H]-TdR (1 μCi) was added to each well 16 h before the cells were harvested, and [3H]-TdR incorporation 2
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coagulation necrosis were observed in the IgG2a-treated group. However, significantly decreased areas of hepatocyte ballooning were seen in recipients that had CD47mAb pretreatment compared with the IgG2a-treated group (Fig. 2A, B). Although the apoptotic index of all the recipients continually increased, significantly fewer apoptotic cells were observed in liver tissue from the CD47mAb-treated group compared with the IgG2a-treated group at all time-points (Fig. 2D). Serum ALT and TBIL levels were used as markers of hepatocyte and bile duct injuries. Compared with the IgG2a -treated group, the ALT level remained consistently low in the group treated with CD47mAb (Fig. 3A). For TBIL level, the same tendency was observed at 5 and 7 days after MLT (Fig. 3B).
was measured using a liquid scintillation counter. Results were expressed as the mean count per minute (cpm) ± standard error of the mean (SEM) from triplicate cultures. 2.7. Statistical analysis Results are shown as the mean ± SEM. Animal survival was assessed using a Kaplan–Meier analysis and the log-rank test. Direct comparison of two study groups was performed using the Student’s ttest for means. Pearson’s correlation analysis was performed to detect any correlation between Suzuki scores/ALT values and the acute rejection index. All analyses were conducted using SPSS® version 17.0 (SPSS, Chicago, IL, USA). All P-values were two-tailed, and P < 0.05 was considered to be statistically significant. GraphPad Prism 6.0 (La Jolla, CA, USA) was used to generate the graphs.
3.3. CD47 blockade alleviated the expansion and activation of recipient myeloid cells in the early phase after liver transplantation The innate immune system plays the role of a bridge between IRI and acute rejection after liver transplantation. To further investigate the mechanisms leading to improved outcomes, we evaluated the effect of CD47 blockade on regulation of innate immune cell expansion and activation. Significant increases in the percentage of CD11b+, CD11b+CD16/32+, and CD11b+CD16/32high in white blood cells were detected in treated groups compared with the sham group at 1 day and 3 days after MLT, while the CD47mAb-treated group showed this increase to a relatively lower extent (Fig. 4A–C). The percentage of both CD16/32+ and CD16/32high in CD11b+ cells was also decreased compared with IgG2a-treated group (P < 0.05, data not shown). The proportion of F4/80 positive cells demonstrated similar trend as the above flow cytometry analysis (Fig. 4D). A lower HMGB-1 and TNF-α releasing level was observed in the CD47mAb-treated group compared with the IgG2a-treated group in the early phase after reperfusion (Fig. 3C,D).
3. Results 3.1. Pretreatment with CD47mAb improved the survival after mouse liver transplantation Three mice were excluded from the study because of death from SHVC bleeding of unknown cause within 24 h post-transplantation, which was considered to indicate an unsuccessful surgical operation. The anhepatic time and total cold ischemia time were 16.8 ± 1.7 min and 225.4 ± 24.6 min, respectively, and there was no significant difference between the two groups (P > 0.05). The 7-day and 10-day survival rates in the CD47mAb-treated group were 80% and 30%, respectively, and these survival rates were significantly higher compared with the IgG2a-treated group (30% and 10%). The longest survival time was 12 days for recipients that were treated with IgG2a, and the survival time was 15 days for mice treated with CD47mAb. Comparison of the two survival curves showed that CD47mAb treatment contributed to improved post-transplantation survival (P < 0.05, Fig. 1).
3.4. CD47 blockade decreased the severity of acute rejection of liver transplants in mice
3.2. CD47 blockade protected liver allografts from ischemia/reperfusion injury
The extent of acute rejection in the transplanted liver tissues gradually increased in the treated groups. The RAI of the transplanted liver tissues in the IgG2a-treated group significantly increased compared with the CD47mAb-treated group at 3 days and 5 days after MLT (Fig. 2C). Similar to the IgG2a-treated group, infiltration by mononuclear cells was observed in the portal area of CD47mAb-blocked liver tissue, but at a decreased level. Little infiltration was observed in the CD47mAbtreated hepatic lobule. However, in the IgG2a-treated group, there was evident infiltration and even perivascular hepatic necrosis at 5 days after reperfusion (Fig. 2A). The statistical data showed a high correlation (r = 0.805/0.733, each P < 0.01) between the graft Suzuki score/ ALT value and the RAI. Subpopulations of CD4+ T cells, which express high or low levels of D45RB, have different cytokine secretion profiles and mediate distinct immunological functions. The proportion of the different subsets was used to evaluate the severity of acute rejection in our study. FACS analysis showed the accumulation of both CD4+CD45RBhigh and CD4+CD45RBlow cells in all recipients. Consistent with the rejection scores, the CD4+CD45RBhigh-to-CD4+CD45RBlow ratio increased gradually. Twice as much proliferation was observed in CD4+CD45RBhigh T cells compared with CD4+CD45RBlow T cells at 7 days after reperfusion. The proportion in the CD47mAb-treated group was lower compared with the IgG2a-treated group at 3, 5, and 7 days after reperfusion (Fig. 4E). Serum IL-2 and IFN-γ are cytokines that are associated with transplant rejection, and their levels were assessed to determine the severity of acute rejection. Following activation of T lymphocytes, the concentrations of these two kinds of cytokines increased. In the CD47mAbtreated group, serum IL-2 was significantly lower at 5 days and 7 days post-transplantation, as was IFN-γ at 7 days after reperfusion, compared
Data of ALT and TBIL levels and HE and TUNEL staining were assessed to determine the extent of liver injury. CD47mAb conferred protection against preservation–reperfusion injury in liver grafts undergoing 4 h of cold ischemia, as evidenced by the significantly lower increase in ALT, TBIL, Suzuki score, and apoptosis index compared with pretreatment with IgG2a. Compared with the sham group, histological alterations were observed in treated groups, including marked sinusoidal dilatation, hepatic necrosis, and hepatocyte ballooning degeneration. Extensive areas of hepatocyte ballooning around the central vein and scattered areas of
Fig. 1. Recipient survival outcome when CD47mAb or isotype matched- control rat IgG2a was administered to the donor liver during graft perfusion. Compared with IgG2a control group, the survival curves showed a significantly improved outcome in CD47mAb-treated group (p = 0.045) (n = 10 for each group). 3
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Fig. 2. Histological evaluation of liver tissue at specific time point. (A) HE staining of liver tissue from sham, IgG2a, and CD47mAb-treated groups at 5 days after MLT (×200) (n = 10 for each group). Infiltratioyablen by mononuclear cells was found limited in the portal area of the CD47-blocked liver tissue, however, in IgG2a-treated group, the infiltration could reach the hepatic lobule and even resulted in perivascular hepatic necrosis. (B) Significantly lower Suzuki score, (C) Banff rejection activity index and (D) apoptotic index were shown in the CD47mAbtreated group versus the IgG2a control group. Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005.
Fig. 3. The results of serum analysis. Significantly lower increase in ALT (A) and total bilirubin (B) was found in CD47mAb treated group, compared with treatment of IgG2a. Concentrations of the serum HMGB-1(C), TNF-α(D), IL-2 (E), and INF-γ (F) were significantly reduced in the CD47mAb-treated group versus the IgG2a group(n = 10 for each group). Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005.
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Fig. 4. Cytometric analysis of peripheral blood about innate and adaptive immune response. (A) Representative FACS plot of WBCs stained with anti-CD11b and anti-CD16/32 in sham, IgG2a and CD47mAb-treated groups at 24 h after liver transplantation. The percentage of CD11b+CD16/32+ (B) and CD11b+ CD16/32high (C) cells in WBCs were decreased in the CD47mAb-treated group versus the IgG2a group at 1d and 3d after liver transplantation. CD47mAb could reduce the proportion of F4/80 positive cells in liver tissue (D) and the CD4+CD45RBhigh-to-CD4+CD45RBlow ratio in the peripheral blood (E), compared with IgG2a control group (n = 10 for each group). Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.
lymphocyte proliferation, no matter if the stimulator was from a third party or from the donor mice.
4. Discussion Although the incidence of allograft rejection is reduced significantly with immunosuppressive therapy, acute rejection episodes still occur in 15–45% of recipients within the first several months after liver transplantation. In the current study, we found that blocking the TSP1–CD47 signaling pathway using an anti-CD47 monoclonal antibody markedly reduced liver IRI, alleviated the extent of early acute rejection, and prolonged survival time. We identified an important association between the extent of IRI and the severity of histopathology that proved acute rejection. The pathophysiology of IRI includes direct cellular injury that is caused by ischemia and delayed damage that results from activation of the inflammatory response. Although the mechanisms of IRI have long been investigated, little progress has been made in reducing its impact on liver graft function. There continues to be great interest in the role of CD47 in liver IRI. Blockade of TSP1–CD47 signaling has been shown to reduce IRI by preventing the inhibition of eNOS activity and VEGF signaling, decreasing the production of ROS, and limiting the recruitment of inflammatory leukocytes [10,11,13]. Our results confirmed the protective effect of CD47 blockade on mouse liver IRI, which was consistent with a report that CD47mAb400 was administered to both the donor and the recipient using a rat model of liver transplantation [12]. However, CD47mAb could provide a survival benefit to the
Fig. 5. Effect of CD47 blockade on mixed lymphocyte reaction. The T cells isolated from spleen of C3H mice, were stimulated in vitro by an equal number of gamma-irradiated C3H (syngeneic), BALB/c (donor) or C57BL/10 (third party) spleen cells, with the treatment of 0.2ug CD47mAb or control Ig. No significant difference in recipient T cells' proliferation between IgG2a and CD47mAb treatment was seen for any stimulator.
with the IgG2a-treated group (Fig. 3E, F). To exclude the possibility that CD47 blockade directly ameliorates acute rejection rather than an indirect effect through alleviating IRI, we conducted an in vitro inhibition assay. The proliferative activity in recipient spleen T cells that were stimulated by BALB/c or C58BL/10 spleen cells showed a stronger response compared with C3H spleen cells. However, there was no significant difference in proliferation between IgG2a and CD47mAb treatment for any stimulator (Fig. 5). These results indicated that CD47 blockade could not directly inhibit C3H
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prolonged cold ischemia did not induce lethal liver graft rejection or accelerate the progress of acute rejection [6]. Thus, we sought to further confirm this issue. We performed Balb/c→C3H liver transplantation, which was associated with lethal acute rejection and a survival time of about 2 weeks, and the results corroborated the significant correlation of liver IRI and acute rejection. In our opinion, there are mainly four reasons accounting for this phenomenon. First, liver IRI could enhance the immunogenicity and the immune recognition through increasing antigen exposure [27,28]. Second, IRI could improve the expression and secretion of adhesion molecules and cytokines, such as TNF-α, IL-2, and IFN-γ, which could trigger and accelerate the graft rejection [29]. Third, injured hepatic sinusoidal endothelial cells could upregulate the expression of the B7 molecule and then activate the B7-1/B7-2/B7-3–CD28/CTLA-4 costimulatory signal pathway [30]. Finally, the apoptosis-Fas/FasL–cytotoxic lymphocyte–rejection axis provides the link between IRI and rejection [31]. Because the possibility that CD47 blockade could directly inhibit recipient lymphocyte proliferation was ruled out through the in vitro inhibition assay, all of the above evidence suggests that CD47mAb treatment could alleviate acute rejection of allogeneic mouse liver transplantation through indirectly reducing IRI.
recipient even when the antibody was administered only to the donor graft in our study. This contradiction is most likely explained by the change of perfusion fluid and the effect of CD47mAb treatment on biliary system. Ringer’s solution containing CD47mAb we used did not need to be flushed out before liver reconstruction, which is different from UW solution. Additionally, we considered that the effect of abundant CD47mAb on the recipient immune system via CD47-SIRPα signaling cannot be ruled out. It is well known that lymphocyte infiltration and activation plays an important role in the acute rejection response, and the severity of graft rejection could be determined by the ratio of the specific lymphocyte subpopulations [17]. CD45RB, a particular CD45 isoform, is expressed on leukocytes and involved in T lymphocyte activation. Several groups have shown that T cells expressing high levels of CD45RB in mice were potent effector cells that are capable of promoting transplant rejection and systematic inflammation. However, T cells expressing low levels showed abilities to inhibit graft rejection [18,19]. In the present study, the shift in balance between the CD4+CD45RBhigh subset and CD4+CD45RBlow subset was consistent with the extent of histopathology-proven acute rejection. In addition, we chose IL-2 and IFN-γ, which are known as key cytokines in the alloresponse, as biomarkers for the characterization of acute rejection. These results suggest that CD47 blockade alleviates the extent of acute rejection in the early phase following liver transplantation. There is increasing evidence that cells in the innate immune system, such as monocytes, macrophages, and neutrophils, participate in liver IRI especially in the early phase. Accumulation and adhesion of neutrophils and the release of pro-inflammatory cytokines and ROS derived from activated macrophages are involved in the pathological process [20]. Although, acute rejection has been considered as response of adaptive immune system for decades, it is gradually becoming more accepted that the innate immune response could function as a pivotal trigger in acute rejection and regulate the strength of the adaptive immune response through antigen presentation and the release of cytokines [21]. Therefore, improved understanding of the mechanisms involved in the innate immune responses may help to develop more effective approaches to attenuate IRI and acute rejection. CD11b, expressed on myeloid-derived cells, is implicated in various adhesive interactions of monocytes, macrophages and granulocytes. CD16/32 acts synergistically with CD11b to promote phagocyte adherence and phagocytosis, and it is rapidly up-regulated following activation of myeloid cells [22,23]. F4/80 antigen, as part of the EGF-TM7 family, is expressed on a wide range of mature tissue macrophages including intragraft Kupffer cells. Overall, the FACS results, serum TNF-α levels, the degree of Kupffer cells' maturation and activation that were observed in the current study provided strong evidence that the CD47 blockade inhibited the recipient myeloid cell expansion and activation. In our opinion, the initial vigorous cell injury, apoptosis, or death that is initiated by IRI causes the release of massive amounts of damage-associated molecular patterns, like HMGB-1, and then induces a heavy influx of inflammatory cells, such as infiltrating neutrophils and macrophages. This, in turn, results in endothelial injury and produces further cellular components that are readily presented by MHC molecules on dendritic cells and macrophages, augmenting graft immunogenicity, accelerating antigen processing and presentation, and facilitating T-cell recognition. CD47 blockade could sever the bond between IRI and rejection to some extent. A large-scale clinical retrospective review demonstrated that prolonged cold ischemia time was associated with a higher extent of graft injury, and a subsequent increase in the frequency of acute rejection, regardless of whether a living or deceased donor liver graft was transplanted [24]. Association between IRI and acute rejection was also confirmed using a rat model of allogeneic liver transplantation [25]. However, Killackey et al. reported that the association between the degree of IRI and the incidence of acute rejection was not statistically significant [26]. In BN-Lew and ACI-Lew liver transplantation models,
5. Conclusions Taken together, TSP1-CD47 signaling blockade with CD47mAb can attenuate lethal acute rejection indirectly through reducing the extent of IRI after liver transplantation. This finding is based on the confirmed significant correlation between the extent of IRI and the severity of acute rejection. However, because of the species specificity and the limited number of mice that were used, additional studies in large animals or clinical trials are required to verify this phenomenon. This study provides evidence that may be used as a basis for new interventions and management methods to support better transplant outcomes. Funding This work was supported by the youth innovation fund project from the first affiliated hospital of Zhengzhou University. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments We thank Wei Li and Guodong Wang for the help to build the mouse liver transplantation model. References [1] M. Charlton, J. Levitsky, B. Aqel, J. O’Grady, J. Hemibach, M. Rinella, J. Fung, M. Ghabril, R. Thomason, P. Burra, E.C. Little, M. Berenguer, A. Shaked, J. Trotter, J. Roberts, M. Rodriguez-Davalos, M. Rela, E. Pomfret, C. Heyrend, J. GallegosOrozco, F. Saliba, International liver transplantation society consensus statement on immunosuppression in liver transplant recipients, Transplantation 102 (5) (2018) 727–743. [2] K. Nakamura, S. Kageyama, B. Ke, T. Fujii, R.A. Sosa, E.F. Reed, N. Datta, A. Zarrinpar, R.W. Busuttil, J.W. Kupiec-Weglinski, Sirtuin 1 attenuates inflammation and hepatocellular damage in liver transplant ischemia/Reperfusion: from mouse to human, Liver Transpl. 23 (10) (2017) 1282–1293. [3] T.H.C. Oliveira, P.E. Marques, P. Proost, M.M.M. Teixeira, Neutrophils: a cornerstone of liver ischemia and reperfusion injury, Lab. Invest. 98 (1) (2018) 51–62. [4] Y. Zhai, H. Petrowsky, J.C. Hong, R.W. Busuttil, J.W. Kupiec-Weglinski, Ischaemiareperfusion injury in liver transplantation–from bench to bedside, Nat. Rev. Gastroenterol. Hepatol. 10 (2) (2013) 79–89. [5] K. Nakamura, M. Zhang, S. Kageyama, B. Ke, T. Fujii, R.A. Sosa, E.F. Reed, N. Datta, A. Zarrinpar, R.W. Busuttil, J.A. Araujo, J.W. Kupiec-Weglinski, Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury, J. Hepatol. 67 (6) (2017) 1232–1242.
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[6] H. Jin, U. Dahmen, A. Liu, H. Huang, Y. Gu, O. Dirsch, Prolonged cold ischemia does not trigger lethal rejection or accelerate the acute rejection in two allogeneic rat liver transplantation models, J. Surg. Res. 175 (2) (2012) 322–332. [7] H.D. Yang, H.S. Kim, S.Y. Kim, M.J. Na, G. Yang, J.W. Eun, H.J. Wang, J.Y. Cheong, W.S. Park, S.W. Nam, HDAC6 suppresses Let-7i-5p to elicit TSP1/CD47-mediated anti-tumorigenesis and phagocytosis of hepatocellular carcinoma, Hepatology 70 (4) (2019) 1262–1279. [8] P. Burger, P. Hilarius-Stokman, D. de Korte, T.K. van den Berg, R. van Bruggen, CD47 functions as a molecular switch for erythrocyte phagocytosis, Blood 119 (23) (2012) 5512–5521. [9] N.M. Rogers, M. Sharifi-Sanjani, G. Csanyi, P.J. Pagano, J.S. Isenberg, Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease, Matrix Biol. 37 (2014) 92–101. [10] S. Kaur, T. Chang, S.P. Singh, L. Lim, P. Mannan, S.H. Garfield, M.L. Pendrak, D.R. Soto-Pantoja, A.Z. Rosenberg, S. Jin, D.D. Roberts, CD47 signaling regulates the immunosuppressive activity of VEGF in T cells, J. Immunol. 193 (8) (2014) 3914–3924. [11] M. Xu, X. Wang, B. Banan, D.L. Chirumbole, S. Garcia-Aroz, A. Balakrishnan, D.K. Nayak, Z. Zhang, J. Jia, G.A. Upadhya, J.P. Gaut, R. Hiebsch, P.T. Manning, N. Wu, Y. Lin, W.C. Chapman, Anti-CD47 monoclonal antibody therapy reduces ischemia-reperfusion injury of renal allografts in a porcine model of donation after cardiac death, Am. J. Transplant. 18 (4) (2018) 855–867. [12] Z.Y. Xiao, B. Banan, J. Jia, P.T. Manning, R.R. Hiebsch, M. Gunasekaran, G.A. Upadhya, W.A. Frazier, T. Mohanakumar, Y. Lin, W.C. Chapman, CD47 blockade reduces ischemia/reperfusion injury and improves survival in a rat liver transplantation model, Liver Transpl. 21 (4) (2015) 468–477. [13] Z. Xiao, B. Banan, M. Xu, J. Jia, P.T. Manning, R.R. Hiebsch, M. Gunasekaran, G.A. Upadhya, W.A. Frazier, T. Mohanakumar, Y. Lin, W.C. Chapman, Attenuation of ischemia-reperfusion injury and improvement of survival in recipients of steatotic rat livers using CD47 monoclonal antibody, Transplantation 100 (7) (2016) 1480–1489. [14] S.G. Qian, J.J. Fung, A.V. Demetris, S.T. Ildstad, T.E. Starzl, Orthotopic liver transplantation in the mouse, Transplantation 52 (3) (1991) 562–564. [15] S. Suzuki, L.H. Toledo-Pereyra, F.J. Rodriguez, D. Cejalvo, Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine, Transplantation 55 (6) (1993) 1265–1272. [16] Banff schema for grading liver allograft rejection: an international consensus document, Hepatology 25 (3) (1997) 658–663. [17] H. Kim, H. Kim, S.K. Lee, X.L. Jin, T.J. Kim, C. Park, J.I. Lee, H.S. Kim, S.K. Hong, K.C. Yoon, S.W. Ahn, K.B. Lee, N.J. Yi, J. Yang, K.W. Lee, W.J. Hawthorne, K.S. Suh, Memory T cells are significantly increased in rejected liver allografts of rhesus monkeys, Liver Transpl. 24 (2) (2018) 256–268. [18] G. Chen, P.P. Luke, H. Yang, L. Visser, H. Sun, B. Garcia, H. Qian, Y. Xiang, X. Huang, W. Liu, G. Senaldi, A. Schneider, S. Poppema, H. Wang, A.M. Jevnikar,
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CD47 blockade alleviates acute rejection of allogeneic mouse liver transplantation by reducing ischemia/reperfusion injury
T
Ding-yang Lia, Shu-li Xieb, Guang-yi Wangb, Xiao-wei Danga,* a b
Department of Hepatobiliary & Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, Henan Province, China Department of Hepatobiliary& Pancreatic Surgery, The First Norman Bethune Hospital Affiliated to Jilin University, Changchun 130021, Jilin Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute rejection CD47 ischemia/reperfusion injury Liver transplantation thrombospondin-1
Despite advances in immunosuppressive therapies, acute rejection response is still a serious concern especially in the early phase after liver transplantation. This study aimed to evaluate whether blocking the TSP1-CD47 signaling pathway could attenuate the acute rejection after liver transplantation. An allogeneic mouse orthotopic liver transplantation model (Balb/c→C3H) with prolonged cold ischemic phase was used to induce severe IRI and lethal acute rejection. CD47mAb or isotype matched-control IgG2a was administered to donor liver during graft perfusion. Recipients were sacrificed at 1d, 3d, 5d and 7d after reperfusion. Blood samples were collected to evaluate serum alanine aminotransferase, total bilirubin, HMGB-1,TNF-α, IL-2 and INF-γ level. Flow cytometric analysis was used to detect the strength of innate and adaptive immune response. Liver tissue was obtained for HE, TUNEL staining and F4/80 immumohistochemical staining. Moreover, we conducted a mixed lymphocyte reaction treated with IgG2a or CD47mAb. Mice in CD47mAb-treated group demonstrated improved survival and significantly lower increase in Suzuki score, apoptosis index, acute rejection index, serum alanine aminotransferase, total bilirubin, HMGB-1, TNF-α, IL-2, INF-γ level and the degree of Kupffer cells' activation especially in the early phase of acute rejection. In addition, Pearson's correlation analysis confirmed significant correlation between Suzuki score/ALT and acute rejection index. The in vitro inhibition assay showed that CD47 blockade couldn’t directly inhibit recipient lymphocyte proliferation. Based on the evidence that TSP1-CD47 signaling blockade with CD47mAb could alleviate acute rejection by reducing the extent of IRI after liver transplantation indirectly, this study provided a basis for new interventions and management methods to support better transplant outcomes.
1. Introduction Orthotopic liver transplantation is currently the only effective treatment option for end-stage liver disease and acute liver failure. Despite advances in immunosuppressive therapies that have improved the outcome of liver transplantation, acute graft rejection is still a serious complication. Concerns include the increased possibility of primary graft non-function or dysfunction, considerable medical cost, a decreased quality of life for the recipient, and a reduced survival rate of the graft or the recipient [1]. Therefore, it is essential that protective strategies are sought to alleviate the extent of acute rejection, especially during the early phase after transplantation.
Ischemia/reperfusion injury (IRI), which inevitably arises after liver transplantation, is associated with induction and activation of the innate immune response, endothelial cell damage, and ultimately, hepatocyte necrosis [2].There is an active but controversial debate on the role of IRI in the induction and exacerbation of acute liver graft rejection based on a body of clinical and experimental data [3–6]. Supporters argue that IRI could result in a varying extent of liver inflammation and injury, which could subsequently lead to enhanced allogenicity of grafts through stimulation of innate immunity-related inflammatory pathways. CD47, a member of the immunoglobulin (Ig) superfamily that is ubiquitously expressed in all tissues, combines with signal regulatory
Abbreviations: ALT, alanine aminotransferase; CD47mAb, monoclonal antibody specific to CD47; ELISA, enzyme-linked immunosorbent assay; FACS, flow cytometric analysis; HMGB-1, high mobility group box -1; IRI, ischemia/reperfusion injury; MLT, mouse orthotopic liver transplantation; RAI, rejection activity index; ROS, reactive oxygen species; SEM, standard error of the mean; SIRPα, signal regulatory protein-α; TBIL, total bilirubin; TSP1, hrombospondin-1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling ⁎ Corresponding author at: Department of Hepatobiliary & Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, 1 Jianshe road, Zhengzhou 450000, Henan Province, China. E-mail address: [email protected] (X.-w. Dang). https://doi.org/10.1016/j.biopha.2019.109793 Received 18 August 2019; Received in revised form 6 December 2019; Accepted 10 December 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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procedure, the liver was exposed to cold ischemia for about 4 h. Recipient blood and hepatic tissue were sampled under anesthesia at 1, 3, 5 and 7 days after reperfusion. Blood was used for serum assays and flow cytometric analysis. Liver samples were fixed in 10% formaldehyde and embedded in paraffin. Twenty recipients were followed for 10 days after surgery. Mice that died or were euthanized (because of conjectural surgical complications) within 24 h after transplantation were excluded from analysis.
protein-α (SIRPα), and it is a receptor for the secreted matricellular protein thrombospondin-1 (TSP1) [7,8]. TSP1-CD47 signaling has been shown to correlate with various important cellular pathways, including nitric oxide, VEGF, cGMP, cAMP, and reactive oxygen species (ROS) [9,10]. A large body of evidence suggests that TSP1 signaling through CD47 exerts a considerable influence on animal models of IRI [11,12]. Additionally, recent studies have reported that liver survival and perfusion after IRI was limited through up-regulating CD47 expression in liver tissue, and targeting CD47 inhibition before IRI could reduce the extent of IRI and enhance the survival of steatotic liver allografts [13]. These relationships among TSP1-CD47 signaling, IRI, and acute graft rejection led us to hypothesize that CD47 blockade could indirectly alleviate the severity of acute liver rejection. To test this hypothesis, a well-established allogeneic mouse orthotopic liver transplantation (MLT) model with prolonged cold ischemia time was used to investigate the correlation between IRI and acute rejection, and to investigate the effect of TSP1–CD47 signaling blockade on tissue injury and the immune response.
2.3. Histological examination Fixed livers were sectioned at a thickness of 4 μm and stained with HE for general histopathologic evaluation of IRI, in accordance with Suzuki’s criteria [15]. In addition, the Banff rejection activity index (RAI) was used to assess the severity of acute rejection [16]. Hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using a TransDetectIn Situ Fluorescein TUNEL Cell Apoptosis Detection Kit (Transgen Biotech, China). Only hepatocytes were included in this analysis. The apoptotic index of the TUNEL assay was determined as the percentage of the apoptotic events per hepatic cell population in five random fields at ×400 magnification (Olympus IX51, Japan). Formalin-fixed, paraffin-embedded tissue slices (4 μm) were immunostained with rat anti-mouse F4/80 antibody diluted at 1:200 (eBioscience, USA) to evaluate the degree of Kupffer cells' maturation and activation. The proportion of F4/80 positive cells in liver was calculated in five randomly selected high power field(×400) for each slice using a microscope (Olympus IX51, Japan).
2. Material and methods 2.1. Animals Male 8- to 12-week-old Balb/c and C3H mice (Vital River Laboratories, Beijing, China) weighing 20–25 g were used as donors and recipients, respectively, for the experiments. Mice were housed under standard specific pathogen free (SPF)-level conditions on a 12-h dark/ light cycle, and they had free access to water and food. Donor and recipient mice were fasted without water deprivation for 12 h before liver transplantation. One hundred pairs of mice were randomly assigned to two groups of equal size, and these groups received pretreatment with either rat anti-CD47 monoclonal antibody (CD47mAb) MIAP301 (Santa Cruz Biotechnology, USA) or an isotype matched-control rat IgG2a (Santa Cruz Biotechnology, USA) before graft retrieval. Ten C3H mice were used as the sham control, which underwent opening and closing of the venter that lasted for 30 min. Animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
2.4. Serum assays After centrifugation of blood samples at 4000 rpm for 15 min at 4 °C, the serum supernatant was collected for serological testing. Serum alanine aminotransferase (ALT) and total bilirubin (TBIL) were assayed with a standard automatic biochemistry analyzer (Vitros 350, Johnson & Johnson, USA). The serum HMGB-1 level was measured using an HMGB-1 ELISA kit II (Shino-Test, Japan). Cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-2, and interferon (IFN)-γ secretion were measured by enzyme-linked immunosorbent assay (ELISA), in accordance with the kit manufacturer’s protocol (Biolegend, USA).
2.2. Surgical procedures
2.5. Flow cytometric analysis
All surgical procedures were performed under clean, but not sterile conditions using inhalation anesthesia with isoflurane. A surgical microscope (Olympus SZX 7.0, Japan) was used, and the procedure was a slight modification of techniques described by Qian et al. [14]. Briefly, the ligaments of the liver, right adrenal vessels, right renal arteries and veins, the portal vein, hepatic artery, and common bile duct were separated. After a polyethylene tube was inserted into the lumen of the common bile duct, the gallbladder was flushed initially with 0.4 mL Ringer’s solution followed by 0.2 mL Ringer’s solution containing 0.2 μg CD47mAb or IgG2a via the fundus of the gallbladder. Following removal of the gallbladder, the pyloric vein was ligated, the right adrenal vessels were dissected by electrocoagulation and the right renal vein and artery were ligated and divided. After heparinization, the liver was perfused through the portal vein with 3 mL cold Ringer’s Lactate solution, followed by 1 mL Ringer’s solution containing 1.3 μg CD47mAb or IgG2a. The resected liver was stored at 4 °C in Ringer’s solution for further preparation. The suprahepatic vena cava (SHVC) anastomosis was constructed with a continuous microsuture using 10-0 prolene (Ethicon, USA). Reconstruction of the portal vein and infrahepatic vena cava was performed using the cuff technique. Biliary’s continuity was restored by inserting a stent into the recipient’s common bile duct. After reconstruction, cefoperazone sodium, sulbactam sodium (Pfizer Inc., USA), and tramadol (Grunenthal Co Ltd., Germany) were administered to the recipient without immunosuppression. During each
White blood cells were separated from peripheral blood following treatment with red blood cell lysis buffer (Beyotime, China). The following antibodies were used in the flow cytometric analysis (FACS) of innate and adaptive immune response: anti-mouse CD11b-PerCP/Cy5.5 (eBioscience, USA); anti-mouse CD16/32-APC (Biolegend, USA); antimouse CD4-FITC (BD Biosciences, USA); and anti-mouse CD45RB-PE (BD Biosciences, USA). Rat IgG2a (BD Biosciences, USA) was used as an isotype control. Labeled cells were analyzed using a FACSCalibur (BD LSRFortessa, USA). 2.6. Mixed lymphocyte reaction (MLR) The T cells that were purified from spleen of normal C3H mice by the magnetic cell separation method using a MACSxpressPan T cell isolation kit (Miltenyi Biotec, Germany) were prepared as responders. There were 2 × 105 cells placed into each well in the 96-well roundbottom plates, and an equal number of gamma-irradiated (20 Gy) C3H (syngeneic), BALB/c (donor), or C57BL/10 (third party) spleen cells were added as stimulators, and then treated with 0.2 μg control Ig or CD47 mAb. The cultures were incubated in 200 μL of 10% FCS RPMI 1640 medium for 72 h at 37 °C. [3H]-TdR (1 μCi) was added to each well 16 h before the cells were harvested, and [3H]-TdR incorporation 2
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coagulation necrosis were observed in the IgG2a-treated group. However, significantly decreased areas of hepatocyte ballooning were seen in recipients that had CD47mAb pretreatment compared with the IgG2a-treated group (Fig. 2A, B). Although the apoptotic index of all the recipients continually increased, significantly fewer apoptotic cells were observed in liver tissue from the CD47mAb-treated group compared with the IgG2a-treated group at all time-points (Fig. 2D). Serum ALT and TBIL levels were used as markers of hepatocyte and bile duct injuries. Compared with the IgG2a -treated group, the ALT level remained consistently low in the group treated with CD47mAb (Fig. 3A). For TBIL level, the same tendency was observed at 5 and 7 days after MLT (Fig. 3B).
was measured using a liquid scintillation counter. Results were expressed as the mean count per minute (cpm) ± standard error of the mean (SEM) from triplicate cultures. 2.7. Statistical analysis Results are shown as the mean ± SEM. Animal survival was assessed using a Kaplan–Meier analysis and the log-rank test. Direct comparison of two study groups was performed using the Student’s ttest for means. Pearson’s correlation analysis was performed to detect any correlation between Suzuki scores/ALT values and the acute rejection index. All analyses were conducted using SPSS® version 17.0 (SPSS, Chicago, IL, USA). All P-values were two-tailed, and P < 0.05 was considered to be statistically significant. GraphPad Prism 6.0 (La Jolla, CA, USA) was used to generate the graphs.
3.3. CD47 blockade alleviated the expansion and activation of recipient myeloid cells in the early phase after liver transplantation The innate immune system plays the role of a bridge between IRI and acute rejection after liver transplantation. To further investigate the mechanisms leading to improved outcomes, we evaluated the effect of CD47 blockade on regulation of innate immune cell expansion and activation. Significant increases in the percentage of CD11b+, CD11b+CD16/32+, and CD11b+CD16/32high in white blood cells were detected in treated groups compared with the sham group at 1 day and 3 days after MLT, while the CD47mAb-treated group showed this increase to a relatively lower extent (Fig. 4A–C). The percentage of both CD16/32+ and CD16/32high in CD11b+ cells was also decreased compared with IgG2a-treated group (P < 0.05, data not shown). The proportion of F4/80 positive cells demonstrated similar trend as the above flow cytometry analysis (Fig. 4D). A lower HMGB-1 and TNF-α releasing level was observed in the CD47mAb-treated group compared with the IgG2a-treated group in the early phase after reperfusion (Fig. 3C,D).
3. Results 3.1. Pretreatment with CD47mAb improved the survival after mouse liver transplantation Three mice were excluded from the study because of death from SHVC bleeding of unknown cause within 24 h post-transplantation, which was considered to indicate an unsuccessful surgical operation. The anhepatic time and total cold ischemia time were 16.8 ± 1.7 min and 225.4 ± 24.6 min, respectively, and there was no significant difference between the two groups (P > 0.05). The 7-day and 10-day survival rates in the CD47mAb-treated group were 80% and 30%, respectively, and these survival rates were significantly higher compared with the IgG2a-treated group (30% and 10%). The longest survival time was 12 days for recipients that were treated with IgG2a, and the survival time was 15 days for mice treated with CD47mAb. Comparison of the two survival curves showed that CD47mAb treatment contributed to improved post-transplantation survival (P < 0.05, Fig. 1).
3.4. CD47 blockade decreased the severity of acute rejection of liver transplants in mice
3.2. CD47 blockade protected liver allografts from ischemia/reperfusion injury
The extent of acute rejection in the transplanted liver tissues gradually increased in the treated groups. The RAI of the transplanted liver tissues in the IgG2a-treated group significantly increased compared with the CD47mAb-treated group at 3 days and 5 days after MLT (Fig. 2C). Similar to the IgG2a-treated group, infiltration by mononuclear cells was observed in the portal area of CD47mAb-blocked liver tissue, but at a decreased level. Little infiltration was observed in the CD47mAbtreated hepatic lobule. However, in the IgG2a-treated group, there was evident infiltration and even perivascular hepatic necrosis at 5 days after reperfusion (Fig. 2A). The statistical data showed a high correlation (r = 0.805/0.733, each P < 0.01) between the graft Suzuki score/ ALT value and the RAI. Subpopulations of CD4+ T cells, which express high or low levels of D45RB, have different cytokine secretion profiles and mediate distinct immunological functions. The proportion of the different subsets was used to evaluate the severity of acute rejection in our study. FACS analysis showed the accumulation of both CD4+CD45RBhigh and CD4+CD45RBlow cells in all recipients. Consistent with the rejection scores, the CD4+CD45RBhigh-to-CD4+CD45RBlow ratio increased gradually. Twice as much proliferation was observed in CD4+CD45RBhigh T cells compared with CD4+CD45RBlow T cells at 7 days after reperfusion. The proportion in the CD47mAb-treated group was lower compared with the IgG2a-treated group at 3, 5, and 7 days after reperfusion (Fig. 4E). Serum IL-2 and IFN-γ are cytokines that are associated with transplant rejection, and their levels were assessed to determine the severity of acute rejection. Following activation of T lymphocytes, the concentrations of these two kinds of cytokines increased. In the CD47mAbtreated group, serum IL-2 was significantly lower at 5 days and 7 days post-transplantation, as was IFN-γ at 7 days after reperfusion, compared
Data of ALT and TBIL levels and HE and TUNEL staining were assessed to determine the extent of liver injury. CD47mAb conferred protection against preservation–reperfusion injury in liver grafts undergoing 4 h of cold ischemia, as evidenced by the significantly lower increase in ALT, TBIL, Suzuki score, and apoptosis index compared with pretreatment with IgG2a. Compared with the sham group, histological alterations were observed in treated groups, including marked sinusoidal dilatation, hepatic necrosis, and hepatocyte ballooning degeneration. Extensive areas of hepatocyte ballooning around the central vein and scattered areas of
Fig. 1. Recipient survival outcome when CD47mAb or isotype matched- control rat IgG2a was administered to the donor liver during graft perfusion. Compared with IgG2a control group, the survival curves showed a significantly improved outcome in CD47mAb-treated group (p = 0.045) (n = 10 for each group). 3
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Fig. 2. Histological evaluation of liver tissue at specific time point. (A) HE staining of liver tissue from sham, IgG2a, and CD47mAb-treated groups at 5 days after MLT (×200) (n = 10 for each group). Infiltratioyablen by mononuclear cells was found limited in the portal area of the CD47-blocked liver tissue, however, in IgG2a-treated group, the infiltration could reach the hepatic lobule and even resulted in perivascular hepatic necrosis. (B) Significantly lower Suzuki score, (C) Banff rejection activity index and (D) apoptotic index were shown in the CD47mAbtreated group versus the IgG2a control group. Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005.
Fig. 3. The results of serum analysis. Significantly lower increase in ALT (A) and total bilirubin (B) was found in CD47mAb treated group, compared with treatment of IgG2a. Concentrations of the serum HMGB-1(C), TNF-α(D), IL-2 (E), and INF-γ (F) were significantly reduced in the CD47mAb-treated group versus the IgG2a group(n = 10 for each group). Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005.
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Fig. 4. Cytometric analysis of peripheral blood about innate and adaptive immune response. (A) Representative FACS plot of WBCs stained with anti-CD11b and anti-CD16/32 in sham, IgG2a and CD47mAb-treated groups at 24 h after liver transplantation. The percentage of CD11b+CD16/32+ (B) and CD11b+ CD16/32high (C) cells in WBCs were decreased in the CD47mAb-treated group versus the IgG2a group at 1d and 3d after liver transplantation. CD47mAb could reduce the proportion of F4/80 positive cells in liver tissue (D) and the CD4+CD45RBhigh-to-CD4+CD45RBlow ratio in the peripheral blood (E), compared with IgG2a control group (n = 10 for each group). Values were presented as mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.
lymphocyte proliferation, no matter if the stimulator was from a third party or from the donor mice.
4. Discussion Although the incidence of allograft rejection is reduced significantly with immunosuppressive therapy, acute rejection episodes still occur in 15–45% of recipients within the first several months after liver transplantation. In the current study, we found that blocking the TSP1–CD47 signaling pathway using an anti-CD47 monoclonal antibody markedly reduced liver IRI, alleviated the extent of early acute rejection, and prolonged survival time. We identified an important association between the extent of IRI and the severity of histopathology that proved acute rejection. The pathophysiology of IRI includes direct cellular injury that is caused by ischemia and delayed damage that results from activation of the inflammatory response. Although the mechanisms of IRI have long been investigated, little progress has been made in reducing its impact on liver graft function. There continues to be great interest in the role of CD47 in liver IRI. Blockade of TSP1–CD47 signaling has been shown to reduce IRI by preventing the inhibition of eNOS activity and VEGF signaling, decreasing the production of ROS, and limiting the recruitment of inflammatory leukocytes [10,11,13]. Our results confirmed the protective effect of CD47 blockade on mouse liver IRI, which was consistent with a report that CD47mAb400 was administered to both the donor and the recipient using a rat model of liver transplantation [12]. However, CD47mAb could provide a survival benefit to the
Fig. 5. Effect of CD47 blockade on mixed lymphocyte reaction. The T cells isolated from spleen of C3H mice, were stimulated in vitro by an equal number of gamma-irradiated C3H (syngeneic), BALB/c (donor) or C57BL/10 (third party) spleen cells, with the treatment of 0.2ug CD47mAb or control Ig. No significant difference in recipient T cells' proliferation between IgG2a and CD47mAb treatment was seen for any stimulator.
with the IgG2a-treated group (Fig. 3E, F). To exclude the possibility that CD47 blockade directly ameliorates acute rejection rather than an indirect effect through alleviating IRI, we conducted an in vitro inhibition assay. The proliferative activity in recipient spleen T cells that were stimulated by BALB/c or C58BL/10 spleen cells showed a stronger response compared with C3H spleen cells. However, there was no significant difference in proliferation between IgG2a and CD47mAb treatment for any stimulator (Fig. 5). These results indicated that CD47 blockade could not directly inhibit C3H
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prolonged cold ischemia did not induce lethal liver graft rejection or accelerate the progress of acute rejection [6]. Thus, we sought to further confirm this issue. We performed Balb/c→C3H liver transplantation, which was associated with lethal acute rejection and a survival time of about 2 weeks, and the results corroborated the significant correlation of liver IRI and acute rejection. In our opinion, there are mainly four reasons accounting for this phenomenon. First, liver IRI could enhance the immunogenicity and the immune recognition through increasing antigen exposure [27,28]. Second, IRI could improve the expression and secretion of adhesion molecules and cytokines, such as TNF-α, IL-2, and IFN-γ, which could trigger and accelerate the graft rejection [29]. Third, injured hepatic sinusoidal endothelial cells could upregulate the expression of the B7 molecule and then activate the B7-1/B7-2/B7-3–CD28/CTLA-4 costimulatory signal pathway [30]. Finally, the apoptosis-Fas/FasL–cytotoxic lymphocyte–rejection axis provides the link between IRI and rejection [31]. Because the possibility that CD47 blockade could directly inhibit recipient lymphocyte proliferation was ruled out through the in vitro inhibition assay, all of the above evidence suggests that CD47mAb treatment could alleviate acute rejection of allogeneic mouse liver transplantation through indirectly reducing IRI.
recipient even when the antibody was administered only to the donor graft in our study. This contradiction is most likely explained by the change of perfusion fluid and the effect of CD47mAb treatment on biliary system. Ringer’s solution containing CD47mAb we used did not need to be flushed out before liver reconstruction, which is different from UW solution. Additionally, we considered that the effect of abundant CD47mAb on the recipient immune system via CD47-SIRPα signaling cannot be ruled out. It is well known that lymphocyte infiltration and activation plays an important role in the acute rejection response, and the severity of graft rejection could be determined by the ratio of the specific lymphocyte subpopulations [17]. CD45RB, a particular CD45 isoform, is expressed on leukocytes and involved in T lymphocyte activation. Several groups have shown that T cells expressing high levels of CD45RB in mice were potent effector cells that are capable of promoting transplant rejection and systematic inflammation. However, T cells expressing low levels showed abilities to inhibit graft rejection [18,19]. In the present study, the shift in balance between the CD4+CD45RBhigh subset and CD4+CD45RBlow subset was consistent with the extent of histopathology-proven acute rejection. In addition, we chose IL-2 and IFN-γ, which are known as key cytokines in the alloresponse, as biomarkers for the characterization of acute rejection. These results suggest that CD47 blockade alleviates the extent of acute rejection in the early phase following liver transplantation. There is increasing evidence that cells in the innate immune system, such as monocytes, macrophages, and neutrophils, participate in liver IRI especially in the early phase. Accumulation and adhesion of neutrophils and the release of pro-inflammatory cytokines and ROS derived from activated macrophages are involved in the pathological process [20]. Although, acute rejection has been considered as response of adaptive immune system for decades, it is gradually becoming more accepted that the innate immune response could function as a pivotal trigger in acute rejection and regulate the strength of the adaptive immune response through antigen presentation and the release of cytokines [21]. Therefore, improved understanding of the mechanisms involved in the innate immune responses may help to develop more effective approaches to attenuate IRI and acute rejection. CD11b, expressed on myeloid-derived cells, is implicated in various adhesive interactions of monocytes, macrophages and granulocytes. CD16/32 acts synergistically with CD11b to promote phagocyte adherence and phagocytosis, and it is rapidly up-regulated following activation of myeloid cells [22,23]. F4/80 antigen, as part of the EGF-TM7 family, is expressed on a wide range of mature tissue macrophages including intragraft Kupffer cells. Overall, the FACS results, serum TNF-α levels, the degree of Kupffer cells' maturation and activation that were observed in the current study provided strong evidence that the CD47 blockade inhibited the recipient myeloid cell expansion and activation. In our opinion, the initial vigorous cell injury, apoptosis, or death that is initiated by IRI causes the release of massive amounts of damage-associated molecular patterns, like HMGB-1, and then induces a heavy influx of inflammatory cells, such as infiltrating neutrophils and macrophages. This, in turn, results in endothelial injury and produces further cellular components that are readily presented by MHC molecules on dendritic cells and macrophages, augmenting graft immunogenicity, accelerating antigen processing and presentation, and facilitating T-cell recognition. CD47 blockade could sever the bond between IRI and rejection to some extent. A large-scale clinical retrospective review demonstrated that prolonged cold ischemia time was associated with a higher extent of graft injury, and a subsequent increase in the frequency of acute rejection, regardless of whether a living or deceased donor liver graft was transplanted [24]. Association between IRI and acute rejection was also confirmed using a rat model of allogeneic liver transplantation [25]. However, Killackey et al. reported that the association between the degree of IRI and the incidence of acute rejection was not statistically significant [26]. In BN-Lew and ACI-Lew liver transplantation models,
5. Conclusions Taken together, TSP1-CD47 signaling blockade with CD47mAb can attenuate lethal acute rejection indirectly through reducing the extent of IRI after liver transplantation. This finding is based on the confirmed significant correlation between the extent of IRI and the severity of acute rejection. However, because of the species specificity and the limited number of mice that were used, additional studies in large animals or clinical trials are required to verify this phenomenon. This study provides evidence that may be used as a basis for new interventions and management methods to support better transplant outcomes. Funding This work was supported by the youth innovation fund project from the first affiliated hospital of Zhengzhou University. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgments We thank Wei Li and Guodong Wang for the help to build the mouse liver transplantation model. References [1] M. Charlton, J. Levitsky, B. Aqel, J. O’Grady, J. Hemibach, M. Rinella, J. Fung, M. Ghabril, R. Thomason, P. Burra, E.C. Little, M. Berenguer, A. Shaked, J. Trotter, J. Roberts, M. Rodriguez-Davalos, M. Rela, E. Pomfret, C. Heyrend, J. GallegosOrozco, F. Saliba, International liver transplantation society consensus statement on immunosuppression in liver transplant recipients, Transplantation 102 (5) (2018) 727–743. [2] K. Nakamura, S. Kageyama, B. Ke, T. Fujii, R.A. Sosa, E.F. Reed, N. Datta, A. Zarrinpar, R.W. Busuttil, J.W. Kupiec-Weglinski, Sirtuin 1 attenuates inflammation and hepatocellular damage in liver transplant ischemia/Reperfusion: from mouse to human, Liver Transpl. 23 (10) (2017) 1282–1293. [3] T.H.C. Oliveira, P.E. Marques, P. Proost, M.M.M. Teixeira, Neutrophils: a cornerstone of liver ischemia and reperfusion injury, Lab. Invest. 98 (1) (2018) 51–62. [4] Y. Zhai, H. Petrowsky, J.C. Hong, R.W. Busuttil, J.W. Kupiec-Weglinski, Ischaemiareperfusion injury in liver transplantation–from bench to bedside, Nat. Rev. Gastroenterol. Hepatol. 10 (2) (2013) 79–89. [5] K. Nakamura, M. Zhang, S. Kageyama, B. Ke, T. Fujii, R.A. Sosa, E.F. Reed, N. Datta, A. Zarrinpar, R.W. Busuttil, J.A. Araujo, J.W. Kupiec-Weglinski, Macrophage heme oxygenase-1-SIRT1-p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury, J. Hepatol. 67 (6) (2017) 1232–1242.
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