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Original Basic Science—General

Improvement of Islet Allograft Function Using Cibinetide, an Innate Repair Receptor Ligand

Yao, Ming MD1,2; Watanabe, Masaaki MD, PhD1,2; Sun, Sune MD1,2; Tokodai, Kazuaki MD, PhD1,2; Cerami, Anthony MD, PhD3; Brines, Michael MD, PhD3; Östenson, Claes-Göran MD, PhD4; Ericzon, Bo-Göran MD, PhD1,2; Lundgren, Torbjörn MD, PhD1,2; Kumagai-Braesch, Makiko MD, PhD1,2

Author Information
doi: 10.1097/TP.0000000000003284
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Abstract

INTRODUCTION

Pancreatic islet transplantation (PITx) offers a treatment option for type 1 diabetes mellitus patients with hypoglycemic unawareness, despite carefully monitored exogenous insulin therapy.1 PITx has been proven to improve metabolic control by lowering HbA1C and reduce hypoglycemic unawareness events, which dramatically increases the quality of life.2 However, a gradual reduction of insulin independence following PITx indicates that improvements can be made to increase the efficacy of this treatment option. The current standard procedure for clinical PITx is to transplant islet-grafts into the recipient’s liver via a percutaneous route into the portal vein under steroid-free immunosuppression (IS).3 When islets are embolized into the liver, tissue factor expression on islets activates the coagulation and complement cascade,4,5 resulting in severe inflammation at the transplantation site, causing tissue injury and production of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-12.6,7 Macrophages secrete macrophage inflammatory protein (MIP)1-β and monocyte chemoattractant protein (MCP)-I, which potentiate a devastating inflammatory reaction to the islet grafts.8,9 As a consequence, >50% of the transplanted islets are immediately destroyed after PITx.10,11 In the allogeneic setting, initial inflammatory reactions activate macrophages and neutrophils to secrete cytokines that recruit and potentiate immune-stimulatory antigen-presenting cells, such as dendritic cells (DCs), which play a key role in the transition from innate to subsequent adaptive immune responses, leading to allograft rejection.12-14

To achieve an insulin-independent state, and long-term graft function, recipients need to receive large numbers of viable islets, which in turn may require multiple PITx’s from several donors.15,16 Considering the limited number of donors, and the risk immunizing the recipient, an efficient strategy for preventing islet graft loss after PITx is of great importance for a further improvement in clinical outcomes, as well as leading to a possible decrease in the islet graft-mass needed for PITx. As the understanding that innate immune reactions immediately after PITx cause the subsequent adaptive immunological progression, the importance of reducing cytokine-induced damage and preventing early inflammatory reactions induced by PITx is increasingly recognized and not yet fully countered by the current standard PITx specific immunosuppressive arsenal.17

Erythropoietin (EPO) exerts antiinflammatory, antiapoptotic, and cytoprotective effects through its binding to a heterodimeric receptor consisting of 1 isomer of the EPO receptor and the β-common subunit of type 1 cytokine receptor CD131.18,19 The heterodimeric receptor expression is upregulated in damaged tissues under inflammation and is therefore called the innate repair receptor (IRR). IRR can be expressed by a wide variety of cells, including monocytes, T-cells,20 and pancreatic islets.21 EPO downregulates proinflammatory immune effector pathways in response to lipopolysaccharide (LPS) stimulation22 and chemical tissue damage.23,24 However, clinical use of EPO can cause side effects, such as increased hematocrit levels, and thrombocyte reactivity and aggregation, with the risk of thromboembolism.25,26

Cibinetide (previously ARA 290), the pyroglutamate helix B surface peptide, is engineered from the structure of EPO that displays a high specificity for the IRR, thus having the beneficial cell-protective effects, and lacking the hematopoietic, and thromboembolic properties.27-29 We have previously reported that cibinetide treatment ameliorated inflammatory status in the liver, protected rodent pancreatic islets from cytokine-induced damage, and improved marginal-mass-graft survival in a syngeneic mouse PITx model.30 In the clinical PITx setting, islets that survive the early innate insults also risk later impact of the adaptive immunity leading to rejection. As a stepwise approach toward clinical adaptation, we here investigated cibinetide’s antiinflammatory and islet-protective efficacy early after allogeneic PITx, as well as its effect on the activation of adaptive immune responses and its impact on long-term graft function in combination with conventional IS following allogeneic PITx.

MATERIALS AND METHODS

Animals

Male C57BL/6N (H-2b) and BALB/c (H-2d) were purchased from Charles River, Inc. (Sulzfeld, Germany). The mice were maintained in a pathogen-free facility at Karolinska Institute, Stockholm, Sweden, and were used for experiments aged 12–15 wk. All experiments were approved by the local ethics committee (S30-15 and 78-15). The study was conducted according to the Guidelines for the Use of Laboratory Animals of Karolinska Institute.

Reagents and Antibodies

Cibinetide was provided by Araim Pharmaceuticals, Inc. (Tarrytown, NY). Recombinant mouse interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and IL-12 were purchased from PeproTech Inc. (Rocky Hill, NJ), LPS (E. Coli:055: B5) from Sigma-Aldrich (St. Louis, MO), antimouse CD11c antibody-allophycocyanin or fluorescein isothiocyanate (FITC) (N418), and antimouse major histocompatibility complex (MHC) Class II antibody-phycoerythrin (PE) (M5/114.15.2) from Miltenyi Biotech (Bergisch Gladbach, Germany), antimouse CD8A-PE (54-67), antimouse CD3E-PE-CYN7 (145-2C11), and antimouse CD4FITC (GK1.5) from Invitrogen (Waltham, MA).

Culture Medium

Mixed lymphocyte culture (MLC)-medium and islet culture medium consisted of RPMI-1640, supplemented with 10% fetal bovine serum, 2-mercaptoethanol (50 μmol/L), penicillin (100 U/mL), and streptomycin (100 µg/mL), all from Invitrogen. The medium used for differentiation of mouse DCs (DC-medium) consisted of mouse-MLC medium-plus GM-CSF and IL-4 (10 ng/mL each).

Diabetes Mellitus Induction in C57BL/6N Mice

C57BL/6N mice were rendered diabetic using a single IP injection of 150 mg/kg streptozotocin (Sigma-Aldrich). The blood glucose levels were monitored as previously described.30 The mice with blood glucose levels exceeding 25 mmol/L (450 mg/dL) during 2 consecutive measurements were considered diabetic and used as recipients.

Mouse Pancreatic Islet Isolation and Allogeneic PITx and Treatment Protocol

Pancreatic islets from BALB/c mice were isolated, as previously described.17,31 Islets of ≤200 μm in diameter were used in the transplantation. The islets were cultured overnight and transplanted into the recipient livers of diabetic C57BL/6N mice via the portal vein. To investigate the short-term islet protective efficacy of cibinetide in an allogeneic setting, a marginal mass of 320 islets were transplanted. In order to evaluate cibinetide effect on long-term islet graft survival, 450 allogeneic islets were transplanted as previously described.30,31 In the cibinetide monotherapy group, cibinetide (120 µg/kg) was administered perioperative via IP injections, that is, just before transplantation, directly after the infusion of islets, and 6 h after the transplantation procedure followed by daily injections during 14 consecutive days. In the control group, the recipients received injections with the same volume of phosphate buffered saline. In the tacrolimus treatment group, tacrolimus (0.4 mg/kg) (Astellas, Tokyo, Japan) was administered in daily IP injections days 4–14. In the combination treatment group, both cibinetide and tacrolimus were administered as described above. Nonfasting blood samples taken from the tail vein were used for monitoring the blood glucose levels. Normoglycemia was defined when the glucose level was below 11.1 mmol/L (200 mg/dL). Graft rejection was defined when the glucose levels had exceeded 19.5 mmol/L (351 mg/dL) for 2 consecutive days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on the recipients that maintained euglycemia at 65 d after PITx as previously described.30

At the time of euthanasia, recipient livers and pancreases were procured. The relative mRNA expression of proinsulin in the liver was examined by quantitative polymerase chain reaction. The insulin from the pancreas was extracted with acid ethanol, and insulin amount was measured by mouse insulin ELISA (Mercodia, Uppsala, Sweden). Some animals were euthanized at 16 h or day 5 after PITx. The graft-bearing liver, the spleen, regional lymph nodes, and peripheral blood mononuclear cells (PBMCs) were procured.

mRNA Preparation and Quantification

Total RNA was extracted from the quick frozen-recipient liver, and 1 µg RNA was converted to cDNA, as described previously.30 Quantification of mRNA was performed using TaqMan real-time PCR on an Applied Biosystems 7500 Fast Real-Time PCR System purchased from ThermoFisher (Waltham, MA). All samples were analyzed as described before.30 The primers targeting specific mRNAs shown in Table 1 were purchased from Applied Biosystems (Waltham, MA).

TABLE 1. - The TaqMan primers/probes targeting specific mRNAs used for mRNA quantification
Gene ID name
PPIA Mm00620857_s1
Mouse TNF-α Mm00443258_m1
Mouse IL-1β Mm00434228_m1
Mouse MCP-1 Mm00441242_m1
Mouse IL-6 Mm00446190_m1
Mouse MIP-1β Mm00443111_m1
Mouse IFN-γ Mm01168134_m1
Mouse proinsulin Mm00731595_gH
Mouse CD11c Mm00498701_m1
Mouse IL-12 Mm01288989_m1
IFN-γ, interferon-gamma; IL-12, interleukin 12; IL-1β, interleukin 1β; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein 1; MIP-1β, macrophage inflammatory protein 1β; PPIA, peptidyl-prolyl cis-trans isomerase A; TNF-α, tumor necrosis factor α.

Flow Cytometry Analysis

Mononuclear cells from spleen, lymph nodes, and peripheral blood were stained with direct-conjugated antibodies and examined by flow cytometry (FACS-Canto, BD Bioscience, San Jose, CA, USA), and analyzed by FlowJo version 10 (Ashland, OR, USA).

Enzyme-linked Immunospot Assay

C57BL/6N splenocytes (2.0 × 105 cells/well) were cocultured with irradiated (20Gy,137Cs) syngeneic or allogeneic donor (BALB/c) splenocytes (2.0 × 105 cells/well) for 24 h in an antimouse IFN-γ mAb-precoated enzyme-linked immunospot (ELISpot) plate at 37°C with 5% CO2 in humidified air. The cytokine-producing cells were detected by using a mouse IFN-γ ELISpot kit (Mabtech AB, Nacka, Sweden). The spots were enumerated using ELISpot software (AID, Strasburg, Germany).

Mouse Mixed Leukocyte Reaction

The responder cells (C57BL/6N mouse; 2.0 × 105 cells/well) were cocultured with the irradiated syngeneic or allogeneic BALB/c mice in 96-well round-bottom plates, at 37°C and in humidified air with 5% CO2, for 48–96 h, in the MLC-medium. The cells were pulsed with3H-thymidine (1 μCi/well) (PerkinElmer, Waltham, MA) 18 h before estimating the thymidine incorporation with a β-counter (MicroBeta TrilLux Wallac Sverige AB, Upplands Väsby, Sweden).

Immunohistological Examination

After euthanasia, the recipient livers were mounted with optimal cutting temperature compounds (Sigma-Aldrich) and immediately frozen in liquid nitrogen. Each liver was sliced into approximately 200 different sections (5 µm thick/slice). Every 10th section was stained with hematoxylin and eosin (Sigma-Aldrich) and screened using an optical microscope (Olympus, Solna, Sweden). If an islet-like structure was observed, the consecutive section was stained for insulin using rhodamine-conjugated antiinsulin antibodies (clone 2D11-H5) (Mabtech AB). The procedure was followed, as described previously.32

Mouse Dendritic Cells Preparation, Culture, and Stimulation

C57BL/6N murine bone marrow-derived DCs (BMDCs) were generated as previously described.31,33 The cells were cultured in 24-well plates at a density of 106 cells/mL/well in DC-medium for a total of 7 d at 37°C with 5% CO2 in a humidified atmosphere. On days 2 and 4, the medium was replaced with fresh DC-medium, with or without cibinetide (100 nmol/L). On day 6, briefly, adherent immature DCs were collected and seeded to new 24-well plates at the concentration of 5.0 × 105 cells/500 µl/well and stimulated with LPS (100 ng/mL) for final maturation, for 20 h. In the cibinetide treatment group, the cells were incubated with cibinetide 60 min before LPS stimulation. The maturation of the DCs was assessed using MHC class II expression of CD11c+ cells by flow cytometry. The IL-12 concentration in culture supernatants was assessed using ELISA (Mabtech AB).

Dendritic Cell-mouse Mixed Leukocyte Reaction

LPS matured BMDCs were washed at least 3 times to remove unbound LPS and cytokines. T-cells were purified from splenocytes of C57BL/6N or BALB/c by using a pan T-cell isolation kit II (MiltenyiBiotec). The isolated T-cells were used as responders or syngeneic/allogeneic antigens. Antigens and DCs were irradiated at 20G. Antigens (irradiated T-cells 105 cells/well), and DCs (104 cells/well) were cocultured with the responder T-cells (105 cells/well) in 96-well round-bottom plates in 200-µl MLC medium, at 37°C in humidified air with 5% CO2, for 96 h. The cells were pulsed with3H-thymidine (1 μCi/well) 18 h before estimating the thymidine incorporation by using β-counter. Culture supernatants were collected at 72 h after incubation, and IFN-γ concentration was measured by ELISA (Mabtech AB).

Statistical Analysis

Statistical analysis was performed using the Mann-Whitney U-test or Student t test for comparing 2 groups, or ANOVA with multiple comparison tests for >3 variables. Differences in the graft survival rate between groups were evaluated using a log-rank test using the Kaplan-Meier method. A p-value <0.05 was considered statistically significant, and all calculations were performed using GraphPad Prism software version 8 (GraphPad Software Inc., San Diego, CA).

RESULTS

Cibinetide Improved Glycemic Control After Marginal Mass Allogeneic PITx

To evaluate if cibinetide treatment could reduce the immediate loss of islet grafts after PITx, thus lower the number of islets needed to improve glycemic control, we used a marginal allogeneic PITx model of diabetic C57BL/6N mice. In the marginal model (320 islets), none of the control group mice showed lowered blood glucose levels after PITx. In contrast, cibinetide-monotreated mice showed significantly improved glycemic control during the 15 d of follow-up time (P < 0.0001, Figure 1).

FIGURE 1.
FIGURE 1.:
As a marginal allogeneic PITx, 320 islets of BALB/c mice were transplanted into the diabetic C57BL/6N mice liver via the portal vein. Mean ± SD of nonfasting blood glucose levels of the vehicle-treated control group and the cibinetide-monotreated groups are shown. The marginal islet graft volume was not sufficient to normalize the blood glucose levels of control group animals. While cibinetide-treated mice showed significantly improved glycemic control during the 15 d of follow-up time (P < 0.0001, 2-way ANOVA, n = 3 in each group). PITx, pancreatic islet transplantation.

Cibinetide Reduced the Inflammatory Reactions at the Transplantation Site After Allogeneic PITx

In order to examine cibinetide effect on the inflammation at the transplantation site, the mRNA expression levels of IL-1β, IL-6, MIP-1β, MCP-1, and TNF-α in the recipient livers at 16 h, after allogeneic PITx were compared.

The upregulation of IL-1β and IL-6 mRNA expressions were significantly suppressed (Figure 2A and B), and the mRNA expressions of TNF-α, MIP-1β, and MCP-1 were lowered in the livers of the cibinetide-treated group (Figure 2C–E), compared to the control group.

FIGURE 2.
FIGURE 2.:
The relative gene expression of proinflammatory cytokines was examined using PCR from livers extirpated 16 h after PITx from the cibinetide-treated mice and vehicle-treated control group mice. Boxes represent the relative gene expression compared to housekeeping genes (mRNA/PPIA). Box floor and roof represent 25th and 75th percentile. Whiskers represent the maximum and minimum value. The horizontal line within box represents median. Cibinetide treatment significantly reduced IL-1β (A) and IL-6 (B) and had lowering tendency of TNF-α (C), MIP-1β (D), and MCP-1 (E) mRNA expression levels, compared to the vehicle-treated control group (Mann-Whitney U-test, n = 5 in each group). IL-1β, interleukin 1β; IL-6, interleukin 6; MCP-1, monocyte chemoattractant protein 1; MIP-1β, macrophage inflammatory protein 1β; PCR, polymerase chain reaction; PITx, pancreatic islet transplantation; PPIA, peptidyl-prolyl cis-trans isomerase A; TNF-α, tumor necrosis factor α.

Cibinetide Treatment, in Combination With Delayed Onset Tacrolimus, Prolonged Islet Graft Survival in Allogeneic PITx

When 450 BALB/c islets were transplanted to diabetic C57BL/6N mice, all of the mice could achieve euglycemia. Cibinetide monotherapy could significantly prolong graft survival time compared to the control group (median survival time 20 and 11 d, respectively, P = 0.0283) (Figure 3A). However, when no IS (tacrolimus) was used, all of the recipients rejected their grafts within 25 d. In the groups administered with tacrolimus, treatment with cibinetide perioperatively and during the 14 d after PITx (combination group) significantly improved islets graft survival time compared to the group treated with tacrolimus monotherapy from day 4 to 14 (n = 9 in each group, P = 0.0381, Figure 3A). At the end of the follow-up time, 70 d after PITx, 67% of the recipients in the combination group maintained euglycemia, compared to 22% in the tacrolimus monotherapy group (Figure 3A, B3, and B4).

FIGURE 3.
FIGURE 3.:
A, Islet graft survival of C57BL/6N mice after PITx with 450 allogeneic islets from BALB/c mice are depicted in Kaplan-Meier. Nonfasting blood samples taken from the tail vein were used for monitoring the blood glucose levels. Normoglycemia was defined as being when the blood glucose level was below 11.1 mmol/L (200 mg/dL). Graft rejection was defined as when the blood glucose levels had exceeded 19.5 mmol/L (351 mg/dL) for 2 consecutive d. Vehicle-treated control group mice obtained a short period of improved glycemic control after PITx; all these subjects lost the function of islet grafts within 20 d. Cibinetide monotreatment could significantly prolong graft survival time (MST control; 11 d n = 5 and cibinetide mono treatment; 20 d n = 7) (P = 0.0283; Log-rank Mantel-Cox test). Treatment with cibinetide and delayed onset tacrolimus administration (combination) significantly prolonged graft survival time compared to the group that were only administered with tacrolimus (P = 0.0381, Log-rank Mantel-Cox test). B, blood glucose levels of each mouse from the study groups are depicted in individual glucose plot values (B1 control; B2, cibinetide monotreatment; B3, tacrolimus; B4, combination treatment). PITx, pancreatic islet transplantation.

IPGTT was performed on remaining euglycemic mice at 65 d after PITx in order to assess the graft function. The blood glucose recovery of the transplanted groups was significantly slower compared to the nondiabetic naïve mice (Figure 4A). Intraportally transplanted islet recipients can perform worse in glucose provocations compared to healthy subjects.34 Impaired IPGTT could also be due to low amount of surviving beta cells after 50 d without any of the studied drugs.

FIGURE 4.
FIGURE 4.:
A, At 65 d after PITx, IPGTT was performed on mice that maintained normoglycemic levels of nonfasting blood glucose sampling. Transplanted mice showed a slower recovery of blood glucose levels compared with nondiabetic control (small dotted line n = 9). The blood glucose recovery of tacrolimus monotreated mice (bigger dotted line n = 2) and combination-treated mice (solid line n = 6) that had acceptable graft function at the end of the follow-up showed similar recovery rates. Each time point of glucose sampling is depicted in mean ± SD. B, When sacrificed at the end of the follow-up, in normoglycemic mice at d 70 (combination n = 5 [1 liver used for histology], tacrolimus n = 2, black line) and on the d of rejection (combination n = 3, tacrolimus n = 6, grey line). The extirpated livers of normoglycemic mice showed a tendency of higher proinsulin mRNA expression in the combination treatment group compared to the tacrolimus-monotreated group. The proinsulin mRNA levels in the extirpated livers of the rejected mice were diminished in both combination and tacrolimus groups. C, Liver sections from a euglycemic mouse in the combination group at 70 d after allogeneic PITx: hematoxylin and eosin staining ×10 magnification show engrafted islets adjacent to a portal branch. Immunofluorescent staining of insulin. Indicates engrafted islets containing insulin. IPGTT, intraperitoneal glucose tolerance test; PITx, pancreatic islet transplantation.

After euthanasia, the livers of all mice were retrieved, and mRNA was extracted. Proinsulin mRNA was detected in all of the livers from the euglycemic recipients and diminished in the livers of graft-rejected recipients, confirming graft loss (Figure 4B). Histological sections of extirpated liver from euglycemic mice from the combination group showed engrafted islets positive for insulin (Figure 4C).

The amount of pancreatic insulin was diminished in all of the streptozotocin-induced mice at euthanasia, confirming there was no recovery of the innate pancreatic insulin production (data not shown).

Cibinetide Ameliorated the Inflammation at the Transplant Site and Reduced DC Activity 5 d After PITx

To investigate the inflammatory status and the activity of DCs at the transplant site at the time point of the activation of the adaptive immunity, the recipient livers were retrieved at 5 days after allogeneic PITx, and inflammatory cytokines/chemokines and DC activity markers were evaluated. The relative mRNA gene expressions of MCP-1, MIP-1β were suppressed in the cibinetide-treated group compared to the control group (P = 0.0143 and P = 0.0286, respectively, Figure 5A and B). The genes-related DC activities, that is, CD11c and IL-12, were also significantly reduced in the cibinetide-treated group (P = 0.0286 and P = 0.0286) (Figure 5C and D). At the same time point, the cibinetide-treated recipient spleens showed a tendency of lower amount of MHC class II within the CD11c+ cells compared to the control group (P = 0.0975 Student t test) (data not shown).

FIGURE 5.
FIGURE 5.:
The relative gene expression of proinflammatory cytokines (MCP-1, MIP-1β) and DC expression markers (CD11c and IL-12) were examined using PCR from livers extirpated 5 d after PITx from the cibinetide-treated and PBS-treated control group mice. The roof and floor of boxes represent 75th and 25th percentile, whiskers represent maximum and minimum value, and horizontal lines represent median. Livers from cibinetide-treated mice had significantly lower expression of MCP-1 (A) and MIP-1β (B) compared to the livers from the vehicle treated control group mice. (Mann-Whitney U-test) n = 4 in each group. The expressions of DC-specific CD11c (C) and IL-12 (D) were also significantly reduced in the livers of cibinetide-treated recipients. DC, dendritic cell; IL-12, interleukin 12; MCP-1, monocyte chemoattractant protein 1; MIP-1β, macrophage inflammatory protein 1β; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PPIA, peptidyl-prolyl cis-trans isomerase A.

Cibinetide-reduced Dendritic Cell Maturation and the Subsequent T-cell Alloactivity In Vitro

The effect of cibinetide on the maturation of BMDCs was examined in vitro. The addition of LPS to immature mouse BMDCs enhanced the maturation and activation of these cells, manifested by an increased expression of MHC class II, and IL-12 production. In this setting, the addition of cibinetide in the DC culture significantly reduced the upregulation of MHC class II (Figure 6A) and IL-12 production (Figure 6B) from the LPS-stimulated DCs.

FIGURE 6.
FIGURE 6.:
The in vitro maturation of the bone marrow-derived DC stimulated with LPS was evaluated by measuring the MFI of MHC class II of cells and concentration of IL-12 in supernatant samples (ELISA). (A) The cibinetide-treated DCs showed reduced MHC class II expression (P = 0.0272), compared to without cibinetide treatment. (B) The concentration of IL-12 in the culture supernatant after 20 h LPS stimulation was reduced in the supernatant in the cibinetide-pretreated DCs, compared to the nonpretreated DCs (P = 0.0243). (Mann-Whitney U-test, n = 10 in each group.) To investigate cibinetide-treated DCs T-cell activation capabilities, a DC-MLR was designed (C, D). The gray bars represent a C56BL/6 T cell activity in culture with immature DCs (no LPS maturation). White bars represent T-cell activity in culture with mature DCs by LPS stimulation. The black bars represent T-cell activity in culture with cibinetide-pretreated-LPS matured DCs. DCs matured with LPS effectively activated the recipient T-cells during stimulation with allogeneic antigens. DCs that matured with LPS in the presence of 100 nmol/L cibinetide-reduced T-cell activating capabilities, which was indicated by lower allogeneic proliferation (C) and production of IFN-γ (D), compared to DCs matured in LPS without cibinetide (P = 0.05, Mann-Whitney U-test, n = 3 in all groups). DC, dendritic cell; IFN-γ, interferon-gamma; IL-12, interleukin 12; LPS, lipopolysaccharide; MFI, medium fluorescent intensity; MLR, mixed leukocyte reaction.

During the DC-mixed leukocyte reaction (MLR) (described in Materials and Methods section), the DCs that underwent the maturation process with LPS and cibinetide subsequently lead to a reduced allogeneic T-cell activity, which is shown by significantly reduced T-cell proliferation, and secretion of IFN-γ, when stimulated with allogeneic (BALB/c) antigens, compared to DCs matured without cibinetide (Figure 6C and D).

Cibinetide-treated Recipients Splenocytes Showed Delayed Allogeneic Reactivity During Ex Vivo Antigen Rechallenge

By limiting the innate responses, including DC maturation, the activation of adaptive immune responses might subsequently be reduced. To evaluate this potential effect, we investigated the immunological aspect with splenocytes procured at 5 d after allogeneic PITx. When encountering donor (BALB/c) antigens, the recipient splenocytes from the control group animals had higher numbers of IFN-γ producing cells and proliferated quicker compared to splenocytes from the cibinetide-treated recipients (Figure 7A and 7B1). Stimulation index of the proliferation tests at days 2 and 3 with splenocytes from the cibinetide-treated recipients was significantly lower compared to the splenocytes from the control group (Figure 7B2).

FIGURE 7.
FIGURE 7.:
Splenocytes extirpated from mice at 5 d after PITx was assessed against syngeneic and donor-specific antigens in MLR. (A) The IFN-γ secreting cell number was assessed by ELISpot after 24 h incubation. The spleens from the cibinetide-treated recipients showed lower amount of alloreactive IFN-γ producing cells against donor antigens, compared to the spleens from the control group (P = 0.0075, Mann-Whitney U-test, n = 5 in each group). (B1) The proliferation was assessed by thymidine incorporation at d 2–4. Splenocytes from cibinetide-treated recipients showed lower proliferative responses against donor-specific antigen until d 3 in the MLR assays as compared to spleens from the vehicle treated control group. (B2) Stimulation Index calculated from the proliferation tests was significantly lower in the cibinetide group compared to the control group at d 2 and 3 (Student t test, n = 4). ELISpot, enzyme-linked immunospot; IFN-γ, interferon-gamma; MLR, mixed leukocyte reaction; PITx, pancreatic islet transplantation.

Concordantly, the cibinetide-treated recipient splenocytes at 5 d after PITx showed a lower percentage of CD8+ T-cells compared to the control group (P = 0.0311 Student t test) (data not shown).

DISCUSSION

This study demonstrates cibinetide’s beneficial effects in allogeneic PITx. First, we showed that cibinetide could reduce intrahepatic inflammatory reactions directly after allogeneic PITx and reduced the marginal limit of islets needed to improve glycemic control in diabetic mice, confirming previous findings from the syngeneic model.30 Second, our studies suggested that cibinetide-reduced BMDC maturation, which subsequently lead to reduced T-cell alloactivity. Concordantly, we showed that cibinetide could delay the onset of adaptive immune reactivity after PITx. Finally, we demonstrated that cibinetide, in combination with low-dose tacrolimus, could improve long-term islet-graft function compared to either drug alone.

Islets are subjected to mechanical and chemical injury during the isolation process, and thereafter ischemia-reperfusion injury during the transplantation.35,36 When the islets are transplanted into the bloodstream, the already injured islets are also subjected to the instant blood-mediated inflammatory reaction.37 Injured islets release inflammatory cytokines,38 that is, MCP1, IL-8, and activated endothelial cells secrete IL-6 and IL-8 to attract inflammatory cells such as monocytes and macrophages, which in turn secrete various proinflammatory cytokines/chemokines that enhance the inflammation at the transplant site. These innate reactions are believed to cause the immediate loss of more than half of the transplanted islets.10,11 Recently, several substances that counteract the initial innate/inflammatory reactions have been suggested.2 The inclusion of TNF-α blocker has been clinically adopted by many transplant centers.39 Similarly, using IL-1 receptor antagonist has been suggested. However, the evidence in favor of these antiinflammatory agents is limited to single-center trials and syngeneic mouse PITx model,40,41 or trials have not shown any improvement.42,43 In nonhematopoietic tissues during stress and inflammation, an externalization and upregulation of CD131 occurs in the cell surface, forming a receptor complex of EPO receptor/CD131 (IRR). This alternative pathway of EPO signaling leads to a switch of the cell’s energy expenditure to cytoprotective functions.27 Mechanistically, agonizing the IRR using nonhematopoietic EPO-analog reduces the binding affinity of NF-κB to DNA, which leads to a reduced secretion of downstream mediators, such as MCP-1 and MIP1β, leading to reduced macrophage infiltration,44,45 and diminished macrophage secretion of IL-6, IL-1β, and TNF-α in an inflammatory milieu.30 By administering cibinetide, we could attenuate the mRNA expression of proinflammatory cytokines IL-6, IL-1β, MCP-1, and MIP1β, in the transplant site directly after allogeneic PITx, which indicates a reduction of the intrahepatic innate immune response. This mechanism protected the islet grafts and reduced the number of islets needed to achieve glycemic control in allogeneic PITx (Figure 1). The effect is due to cibinetide ability to lower the inflammation at the transplant site and to increase robustness of the islet grafts in a proinflammatory milieu, as discussed in the previous reported syngeneic PITx model.30

In the allogeneic setting, the surviving grafts from early damage caused by innate immunity will still be subjected to the risk of allograft rejection by adaptive immune responses. A high risk of rejection and most of the neovascularization process is occurring within the first 2 wk,46,47 which is the most vulnerable period for the transplanted islet grafts, hence in this present study focusing on allogenic PITx, we extended the cibinetide treatment period in the allogeneic setting to 14 d.

The physiology of rejection starts with inflammation. Innate reactions in turn signal DCs to become inflammatory and increases the expression of costimulatory molecules that promote activation of T-cells, enhancing the risk of allograft rejection.48 Maintaining DCs in an immature state inhibits their expression of costimulatory and antigen-presenting molecules, thus reducing their capabilities of activating T-cells.49 Here, we demonstrate that cibinetide can reduce the maturation of BMDC and the subsequent T-cell activity during allogeneic stimulation (Figure 6). Concordantly, splenocytes from cibinetide-treated recipients showed delayed reactivity in ex vivo antigen restimulation (Figure 7). In addition, it was previously demonstrated that cibinetide could reduce macrophage activation following LPS stimulation.30 These data suggest that cibinetide’s effect might be limited to the maturation stages of DCs and macrophages.

There are studies reporting that EPO has proinflammatory properties on DCs and macrophages.50,51 These studies were, however, performed using recombinant human EPO, and not selective IRR agonists, as in our case. The homodimer EPO receptor activation leads to the activation of JAK2/STAT5, and in parallel, mitogen-activated kinases and NF-κB pathways.52,53 Nearly all studied tissues only express the heterodimeric IRR receptor following metabolic stress and injury.54 Tissue under stress locally produces a higher concentration of EPO, which is required to activate IRR, that mediates a switch of the intracellular signaling to cytoprotective and antiinflammatory properties.55 Studies performed using a selective IRR agonist show predominantly antiinflammatory and cytoprotective reactions.27,28,30,45

This study shows that initial treatment with cibinetide in allogeneic PITx is beneficial by reduced early inflammation and increased robustness of the grafts in the inflammatory milieu. However, although a higher proportion of surviving islets early after transplantation can improve glycemic control initially, it cannot maintain a euglycemia state during a constant loss due to rejection for a longer period. In the combination treatment group, 66% of mice had functioning grafts at the end of the long-term follow-up (70 d), compared to 22% in the tacrolimus monotherapy group. This indicates that cibinetide’s effect is not limited to its initial graft protective mechanism. Its suggested effect on DC and macrophages may also have an added effect to tacrolimus to reduce the risk of rejection.

The safety of cibinetide treatment has already been demonstrated in clinical trials with no thromboembolic complications.56,57 These studies also showed reduction of clinical symptoms in both diabetes- and sarcoidosis-associated neuropathy. Interestingly, glucometabolic control was also improved in these patients with diabetes, which has also been shown in a rodent model.58

This study indicates that treatment with cibinetide could reduce early inflammation, subsequent alloresponses, and significantly prolong the long-term allogeneic graft function. These encouraging results, together with a favorable safety profile, make us propose cibinetide as a promising candidate drug to add to the clinical PITx treatment arsenal.

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