Pharmacological preconditioning with adenosine A1 receptor agonist induces immunosuppression and improves graft survival in novel allogeneic transplantation models

Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.

In contrast, we have shown that early desensitization of A 1 R can alter the balance towards an immunosuppressive A 2A R environment 11,12 . In previous studies, we characterized the immunosuppressive effects of A 1 R desensitization by pharmacological preconditioning (PPC) 11 . We found that 24 h pre-activation of the A 1 R by a selective adenosine A 1 R agonist, 2-Chloro-n(6)-cyclopentyladenosine (CCPA), led to downregulation of A 1 R and upregulated A 2A R, mitigated the inflammatory response against invading bacteria, decreasing the number of blood lymphocytes and their reactivity to mitogen and MLR 13,14 .
In a recent work, we showed that A 1 R elimination by genetic manipulation or by desensitization with PPC, is associated with cAMP elevation and lymphopenia 14 . T-cell dysfunction and lymphocyte apoptosis are known to be linked with cAMP elevation [15][16][17] . Moreover, Tregs induce suppression in effector T cells either by direct transfer of cAMP via gap junctions 18 , or by PGE 2 19 and adenosine secretion 5 . Organ transplantation is the general treatment for end failure of heart, kidneys, lungs and other essential organs. Suppressing the patient's natural defense mechanism from rejection of the graft by immunosuppressive drugs is paramount in this procedure. A 2A R activation was shown to improve transplantation outcome. For example; Lappas et al. showed that, during early reperfusion in a lung transplantation model, treatment with an A 2A R agonist reduced lung inflammation and preserved the pulmonary function 20 . Similarly, Sevigny et al. demonstrated enhanced skin allograft transplant survival by activating A 2A R with specific agonists 21 . We hypothesize that pre-and post-operative modulation of the immune system with adenosine A 1 receptor agonist, will upregulate the immunosuppressive A 2A R and improve outcome in transplant recipients.
The aim of the current study was to challenge the immunosuppressive effects of A 1 R-PPC in models of allogeneic grafts.

Results
A 1 R receptor reduction affects blood cell lineage. We previously established that adenosine activation downregulates lymphocyte activation both in vitro in MLR assays and in vivo in sepsis models. We have showed that this downregulation is affiliated to A 1 R pre-activation and its desensitization.
In this study, our aim was to challenge the immunosuppressive effects of A 1 R-PPC in models of allogeneic grafts.
We conducted blood counts for Vehicle-treated, PPC and A 1 R-KO mice. The latter is used to illustrate the total absence of A 1 R. As shown in Fig. 1, we found a significant reduction in WBC (Fig. 1A) and lymphocyte ( Fig. 1B) counts in both PPC-treated and A 1 R-KO mice. The lymphocyte cells were the main cell population affected, with only 3.05 ± 0.4 lymphocytes in A 1 R-KO mice (p = 0.012) compared to 6.46 ± 1.52 cells × 10 3 /μl in WT mice -less than 50%. 24 h PPC and 72 h PPC was 4.45 ± 1.29 cells x10 3 /μl (p = 0.0202) and 4.14 ± 0.69 cells x10 3 /μl (p = 0.083), respectively. Another decrease was also found in basophils and neutrophils at 24 h (Fig. 1C,D respectively). In addition, there were no changes in hematocrit, hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) at any time course (data not shown).
A 1 R ppc upregulates A 2A R. We determined mRNA levels in the A 2A R of mice following PPC with the A 1 R agonist (CCPA, 0.1 mg/kg) and found elevation of A 2A R mRNA levels compared to that of untreated mice (Fig. 2).

Desensitization of A 1 R restrains leukocyte infiltration and muscle decay in vivo. Our previous
in-vitro results showed that leukocytes from preconditioned mice reduced both proliferation and reactivity.
To evaluate both the leukocytes immunological and ischemic effect in vivo, we created an innovative, simple allogeneic graft model. We grafted the Pectoralis Major muscle from Balb/c donor mice in an artificial pocket in nape of control C57BL/6 mice. This procedure allows us to evaluate both the infiltrate of leukocytes in the graft and the duration of graft necrosis. The implants were removed on day 10 for further analysis, such as for histological evaluation and scoring. Grafts were graded blindly by a pathologist in an adapted score of ISHLT 22 . Syngeneic muscle grafts shown a transient mild inflammation in the course of 21 days (data not shown). Whether, allogeneic Vehicle-treated muscle histology revealed massive infiltration of medium-to-large atypical leukocytes with both round and irregular nuclei infiltrating T cells, accompanied by infiltrating eosinophils, plasma cells, and neutrophils (Fig. 3A). We also observed blood vessel injury (vasculitis). In addition, there was wide-scale damage to the muscle tissue that matched the state of acute allograft rejection, scored by ISHLT (Fig. 3B). In contrast, PPC muscle histology exposed profoundly less infiltration and less muscle necrosis compared to allografts vehicle-treated mice. Muscles from A 1 R-KO mice, used as a positive control to the A 1 R desensitization by PPC, were also showed moderate signs of inflammation and rejection. The blind grade score confirmed our observation, showing significant differences in favor of PPC and A 1 R-KO muscles (p = 0.0093) and (p = 0.0490), respectively. A 1 R desensitization attenuates MLR proliferation of mice challenged with allogeneic grafts. To associate our findings to the above reduction of circulating lymphocytes and alloreactivity, we removed the spleens from the grafted mice. We cultured C57BL/6 responder's splenocytes from the three groups: Vehicle-treated allogeneic, PPC-treated allogeneic and A 1 R-KO allogenic with stimulators Balb/c splenocytes, in a Two-way MLR. We found that PPC splenocytes that re-encountered allogeneic splenocytes were significantly (p = 0.0442) depressed and showed decreased proliferation. A 1 R-KO showed an even greater decrease in proliferation, approximately 50% compared to proliferation in Vehicle-treated allogeneic mice group (Fig. 4).

PPC attenuates skin allograft rejection.
To support our findings on muscle grafts, we also tested the effect of PPC in a skin graft model, in which rejection could be followed continuously without further intervention. In this model, ear skin grafts from Balb/c donor mice were grafted on the dorsal area of vehicle CCPA (0.1 mg/kg)-treated C57BL/6 mice. Graft survival was followed daily by visual inspection and photography from day 5 (removal of bandage) till rejection (loss of all viable skin). Figure 5 shows representative pictures taken at day 6 which illustrate the marked differences between grafts in the vehicle-treated (Fig. 5A) 5B). In the vehicle-treated group, we observed clear rejection signs and inflammation such as redness, swelling, and loss of viable skin -effects that were markedly reduced in the pre-conditioned mice. The most significant finding was when PPC was administrated both on the donor and recipient (Fig. 5C).
As shown in Fig. 6, at day five, upon removal of the bandage, significant differences (p = 0.0016) were observed between the single PPC group to the vehicle-treated groups. In the vehicle-treated group, only 70% of the allografts remained implanted, while all grafts in the single and double PPC groups were intact. Initial rejection in the PPC group began at day 7 until day 13, when the entire allogeneic population of recipients rejected their grafts with a shift in favor of PPC mice.

Discussion
Adenosine is a potent modulator of lymphocyte development, proliferation, and activity, and its effect depends both on its bioavailability and on cell surface receptor expression. The role of adenosine signaling in regulating tumor immunity has been widely described for its potential therapeutic role in cancer 23 .
In this study we have shown that modulation of adenosine receptors by A 1 R PPC prolongs allogeneic graft survival, exhibiting lymphocyte blood count reduction and T-cell response moderation. www.nature.com/scientificreports www.nature.com/scientificreports/ The PPC protocol was well established in our previous studies 2,13,14 , where we found that stimulation of the pro-inflammatory Gi-coupled receptor A 1 R causes it to become dysfunctional by desensitization, and in parallel subsequentially upregulate the Gs-coupled A 2A R as the dominant receptor for extracellular adenosine. In a SIRS model, this phenomenon induced leukopenia and reduced T-cell reactivity 14 and, therefore, led to immunosuppression 2 . These studies led us to test whether PPC could moderate allograft reactions.
It is a common finding that immunosuppressive drugs induce lymphopenia [24][25][26] . We have shown that PPC significantly reduced lymphocytes in complete blood counts (CBC) in grafted mice. CBC revealed that the white blood cell linage, mainly lymphocyte counts, remained significantly low up to 72 h post-PPC. Lymphocytes are accountable for a majority role in graft rejection elevation, pro-inflammatory cytokines, and stimulation of the immune response 27 . On the other hand, other leukocyte lineages and the red cell linage were not significantly    www.nature.com/scientificreports www.nature.com/scientificreports/ we believe that dysfunction of A 1 R is, by itself, immunosuppressive, as indicated by our observation that A 1 R-KO mice had a reduced MLR and a moderate reaction towards allogeneic challenge 13 .
For that reason, we established a unique and novel model that allows us to evaluate the immunological and ischemic reaction towards allogeneic grafts. We grafted a thin flap muscle in the nape of allogenic matched strain, in a way that we can excised the flap and examined its viability and alloreactivity. This model can be easily applied to various pharmacological or anti-inflammatory treatments.
We have blindly and significantly showed that the treatment groups were either PPC 24 h before instillation or were A 1 R-KO mice were better preserved and exhibited fewer infiltrating cells compared to vehicle-treated mice. Furthermore, splenocytes from the grafted mice that were exposed for 10 days to allogeneic implants, were challenged for MLR. It is known that low MLR response is predictive to successful transplantation [34][35][36] . We have shown that a single PPC treatment effectively and significantly reduced lymphocyte response. This anergic effect in accordance with our previous findings indicating that the suppression in MLR is associated with an elevation of A 2A R following PPC. Similar results are also shown when A 2A R agonist decreased proliferation in allogeneic MLR assays 10,21 .
This PPC imprinting process, by which a brief stimulation period of the lymphocytes in vivo establishes a long-lasting depressed response is a new strategy and can be easily applied. This phenomenon of a continued response was previously shown by Koshiba et al. and named the "memory" of T cells, which suggests that brief exposure of T cells to adenosine in vitro is sufficient to observe the inhibition of TCR-triggered effector functions 37 .
Finally, we challenged the PPC method in the skin allograft model. For this evaluation, we managed to establish a skin grafting model that had the advantages of being relatively easy to reproduce and not requiring sutures. When we conducted syngeneic transplantation, all five mice remained alive with the graft up to 8 months post transplantation. We showed that PPC treatment significantly attenuated skin graft rejection compared to vehicle-treated grafts. In addition we noticed that when PPC was administered to both the donor and recipient, we were able to propone the course of rejection in the early days after bandage removal.
The first hours post-graft implantation are critical for survival. The stressed ischemic tissue induces the release of ROS, cytokines, chemokines, and adenosine 38,39 . Activation of adenosine A 2A R has already been shown to have a protective effect during liver transplantation 40 . Therefore, it is possible that this effect could be intensified with PPC, allowing endogenous extracellular adenosine to act mainly on A 2A R, without the contradictory effects of A 1 R.
In conclusion, we have demonstrated that PPC moderates graft rejection. We believe that A 1 R activation, followed by its desensitization and induction of A 2A R, shifts the pro-inflammatory danger signal of extracellular adenosine in the graft milieu to an anti-inflammatory response.
Considering the minimal side effects of this treatment, this approach is relevant to the recipient, as well as to healthy or brain-dead live donors. We believe that PPC can be integrated as a pre-transplantation preparation in the future, along with the concept of treating both the donor and the recipient, thereby improving the treatment.

Materials and Methods
Mice. All  pharmacological preconditioning (ppc). We treated mice as previously described 11,13 In brief, for PPC, mice were treated by intraperitoneal injection (i.p.) using 2-chloro-N6-cyclopentyladenosine (CCPA 0.1 mg/kg), a specific adenosine A 1 R agonist, 24 h and 12 h before conducting any of the listed below procedures. Muscle allogeneic challenge model. In order to evaluate the immunological and ischemic reaction of PPC in allografts we established a simple, easy to reproduce, novel model of allogeneic graft. We performed a small, sterile incision in the nape of recipient mice. To this artificial pocked we grafted the Pectoralis Major muscle from donor mice. Due to his thin structure the diffusion of nutrients and oxygen is effective and the ischemic stress is minimal. This procedure allows us to remove the graft in any time point, easily without damage to the recipient mice. The grafts were then sent for histological and immunological evaluation as described below.
Recovery of organs. Grafts were removed, and biopsies of the parietal muscle were fixed and stained with hematoxylin and eosin (H&E). Grafts were graded blindly by our pathologist in an adapted ISHLT score 22 . In brief, the revised categories of cellular rejection were as follows: Grade 0 -no rejection, Grade 1 -mild rejection, Grade 2 -moderate rejection, and Grade 3 -severe rejection.
Isolation of mononuclear cells from spleens. As we described earlier 14 , spleens were removed from mice and disrupted under sterile conditions in phosphate buffer saline (PBS) through 40-μm BD Falcon cell strainers (Fisher Scientific; Pittsburgh, PA, USA). Mononuclear cells were then isolated via density gradient centrifugation using Histopaque 1083 (Sigma-Aldrich). Cells were washed twice, and total leukocytes were counted after trypan blue staining using an improved Neubaur hemocytometer. Cells were grown in RPMI 1640 medium and supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 μg/ ml streptomycin (Biological Industries; Bet Haemek, Israel).
Activation of leukocytes. Leukocyte activation was performed using 96-well flat-bottom culture plates (Greiner Bio-One; Germany) for 72 h at 37 °C in the presence of 5% CO2. For standard two-way MLR assays, responder cells (total splenocytes, 2 × 10 5 ) were co-cultured with an equal number of stimulator splenocytes in 200 μl medium 14,16 . Thymidine (1 μ Ci/well; PerkinElmer Life and Analytical Sciences) was added 18-24 h before recovering (Inotech Biosystems International Inc.) using Type A filter mats (PerkinElmer Life and Analytical Sciences) and a beta-plate scintillation mixture (PerkinElmer). CPM were determined using a liquid scintillation analyzer (Packard 1900CA, Packard Instrument Co.). Data were expressed as the mean CPM of triplicate determination, and converted into proliferation percentages. 100% proliferation refers to vehicle treated allogeneic group. Splenocytes background readout values (medium alone) were deducted from the results. Skin transplant procedure. For skin grafts we used half-thickness ear skin (~0.7 cm 2 ) that were from donor mice, and were grafted on the dorsal area of the recipient mice. After the procedure, the grafts were wrapped in a sterile bandage (with the non-adhesive gauze segment placed over the skin graft), and tied loosely enough to allow for breathing and free arm mobility. Recipient mice were monitored daily for any signs of distress, and an analgesic was administered if needed for pain relief. Mice were anesthetized using the above procedure, and the bandages were cut and removed using blunt-end scissors. Grafts were checked in the first hours for signs of scabbing or contraction. If present, then grafts did not vascularize and were considered to be failures. Grafts were monitored daily for signs of rejection (usually defined as ~80% necrosis of the donor tissue).
To asses graft survival we performed daily recordings from day 6 to day 9. Later on we analyzed it by visual inspection in a masked fashion. Statistical analysis. The comparisons was carried out using one of the following: a Mann-Whitney nonparametric t-test or by a one-way ANOVA followed by a Tukey post-test. All comparison were preform using Graphpad Prism 5 software (GraphPad; San Diego, CA). Survival grafts were analyzed by Mantel-Cox test. P values below 0.05 were considered significant. Values are presented as mean ± SEM.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.