Artemisinin Attenuates Transplant Rejection by Inhibiting Multiple Lymphocytes and Prolongs Cardiac Allograft Survival

Immunological rejection is an important factor resulting in allograft dysfunction, and more valid therapeutic methods need to be explored to improve allograft outcomes. Many researches have indicated that artemisinin and its derivative exhibits immunosuppressive functions, apart from serving as a traditional anti-malarial drug. In this assay, we further explored the therapeutic effects of artemisinin for transplant rejection in a rat cardiac transplantation model. We found that it markedly attenuated allograft rejection and histological injury and significantly prolonged the survival of allograft. Upon further exploring the mechanism, we demonstrated that artemisinin not only attenuated T cell-mediated rejection (TCMR) by reducing effector T cell infiltration and inflammatory cytokine secretion and increasing regulatory T cell infiltration and immunoregulatory cytokine levels, but also attenuated antibody-mediated rejection (ABMR) through inhibition of B cells activation and antibody production. Furthermore, artemisinin also reduced macrophage infiltration in allografts, which was determined to be important for TCMR and ABMR. Moreover, we demonstrated that artemisinin significantly inhibited the function of pure T cells, B cells, and macrophages in vitro. All in all, this study provide evidence that artemisinin significantly attenuates TCMR and ABMR by targeting multiple effectors. Therefore, this agent might have potential for use in clinical settings to protect against transplant rejection.


INTRODUCTION
Cardiac transplantation has become the gold-standard long-term medical treatment for end-stage heart failure and has achieved remarkable success (1). However, its biggest challenge is concomitant rejection, resulting from interactions between the recipient immune system and allograft. Immune responses could result in the rejection to cardiac allografts, which including chronic rejection (CR) and acute rejection (AR). And AR is a leading cause to the development of CR (2). Furthermore, transplant rejection mainly consists of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) (3). Although immunosuppression has achieved good results for TCMR therapy, side effects such as infection and graft toxicities are likely to be a significant impact on patient prognosis (4). Moreover, ABMR, caused by donorspecific antibodies (DSAs), has emerged as an important immunological barrier to successful transplantation (5,6). Current therapeutic strategies for ABMR focus on removing the generated antibodies in the peripheral blood, or eliminating B cells to inhibit the generation of antibodies, but these strategies are not as effective as expected (7). Therefore, more effective and safe strategies must be explored to improve transplant outcomes.
Artemisinin (C 15 H 22 O 5 , ART), derived from sweet wormwood (Artemisia annua L), has been used to treat malaria in China for a long time. Taking the high safety without noticeable side effects and adverse reactions into consideration, ART has been used to treat millions patients in the world (8). Recently, ART and its derivatives were found to have other properties, including immunosuppressive and antiinflammatory effects (9).
ART and its derivatives exhibit immunosuppressive and against inflammation via regulation of the immune system, inhibiting T lymphocyte proliferation, enhancing Treg differentiation, and increasing the proliferation of Treg (10)(11)(12). Dihydroartemisinin (derivative of ART) treatment also decreases autoantibodies in lupus model (13). However, as ART could obviously inhibit the activation and proliferation of T cells, the decrease of antibody level was considered as a secondary effect on the inhibition of T cells. and the direct effects of these drugs on B cells (especially for DSAs) are unclear. Moreover, ART can to regulate innate immune cells. Studies have indicated that ART significantly reduces peritoneal macrophage phagocytosis and the phagocytic index in vivo and can also inhibit monocyte/macrophage adhesion to HUVECs to protect against the development of early atherosclerotic lesions (14,15). To date, the anti-inflammatory and immunosuppressive properties of ART have been discussed, but their effect on immunological rejection has not been reported.
Therefore, here, we investigated the therapeutic effects of ART on rejection, including TCMR and ABMR, using a rat cardiac transplantation model, providing a novel therapy choice for the patients undergoing transplant rejection.

Experimental Protocol and Groups
The model of cardiac allograft transplant rejection was established by transplanting Brown Norway (BN) hearts into Lewis recipient rats. Recipients were random assigned to two groups as follows: (a) ART (n=6), ART was diluted in peanut oil (100 g/L) and administered intragastrically daily at 22 mg/kg/day from day 0 until cardiac graft rejection; (b) control (n=6), an equal volume of peanut oil was applied to vehicle-treated recipients. Cardiac Lewis-to-Lewis rat transplantation served as the isograft control (n=5), and these animals all received a daily equal volume of peanut oil administered intragastrically. For rats only undergoing skin transplantation, animals were divided into two groups as follows: (1) ART, ART was fed intragastrically (22 mg/kg/day) from day 0 to day 36 post skin transplantation; (2) control, an equal volume of peanut oil was applied to control group animals every day.

Subjects and Animals
Peripheral blood was obtained from healthy volunteers between the ages of 18 and 26 years who were acquired from Sun Yat-sen University. All subjects signed informed consent forms and our study was approved by the Ethical Committee of Sun Yatsen University.
Male rats weighing 200-250 g were obtained from Vital River Laboratory Animal Technology Co., Ltd. Animals used in this assay were proved by the Sun Yat-sen University Institutional Ethical Guidelines and consistent with the Guide for the Care and Use of Laboratory Animals.

Rat Skin Transplantation
For skin transplantation, the donor rat was anesthetized with isoflurane, the tail was excised, and full-thickness tail skin was obtained. The recipient was anesthetized, and full-thickness skin grafts (1-2.0 × 2 cm 2 ) were acquired from BN donors. The grafts were then transplanted onto the dorsal area of Lewis rats with an 8-0 nylon suture.

Rat Heterotopic Cardiac Transplantation
Detailed processes of cardiac transplantation were described previously (16,17). Briefly, the pulmonary artery and ascending aorta of the heart were anastomosed end-to-end to the vena cava and abdominal aorta, respectively. Survival of cardiac graft function was judged everyday by abdominal palpation. Rejection was defined as total cessation of contractions.

Detection of Circulating Donor-Specific Antibodies
Graft recipient sera were obtained at the indicated time points. Circulating DSA (IgM and IgG) was evaluated using flow cytometry. Briefly, donor splenocytes were incubated with recipient sera for 30 min at 37°C, washed, and then incubated with anti-rat IgM and IgG (Bio Legend) for 1 h at 4°C. The mean fluorescence intensity was used to reflect individual DSAs.

Histology and Immunohistochemistry
Five days post cardiac transplantation, cardiac grafts were fixed and embedded. Hematoxylin and eosin (H&E), anti-CD3, anti-CD4, anti-CD8, anti-CD68, and anti-Foxp3 were used for further assessment. Sections were examined by light microscopy to evaluate the pathologic features of cardiac allografts. For TUNEL staining, the one-step TUNEL apoptosis assay kit (Roche TMR-RED; 12156792910) was used and strict limited to the protocol. Sections were counterstained with DAPI to label nuclei. Fields containing both the rejection area and the normal zone were scanned.
Quantitative Real-Time Polymerase Chain Reaction mRNA levels were determined using RT-qPCR. RNA was acquired from fresh tissues or cells using TRIzol reagent (Invitrogen, USA). PrimeScript RT master mix (TAKARA, Japan) was used for reversing transcribed. SYBR Green I master mix (Roche, Switzerland) was used for RT-qPCR in a LightCycler480 system (Roche), with GAPDH as an internal control. Detailed Primers used could be found in Table 1.

Isolation and In Vitro Culture of T Cells, B Cells, and Macrophages
Corresponding microbeads (Miltenyi Biotec, Germany) were used for cells isolation from peripheral blood mononuclear cells (PBMCs). Flow cytometry was used for determining purity of the cells, and the purity was consistently >95%.

Assessment of Apoptosis
An annexin V apoptosis detection kit (R&D Systems) was used for apoptosis detection according to the manufacturer's instructions using flow cytometry (BD, San Diego, CA).

Statistical Analysis
The KS normality test was first used to test if the data were normally distributed; all data met this criterion. The Student's t tests were used for analysis of significant differences. The logrank test was used for graft survival. Data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using Prism (GraphPad). *P < 0.05 was considered significant.

ART Reduces Rejection and Prolongs Cardiac Allograft Survival
Recipients treated with ART had longer graft survival than controls (17.33 ± 4.89 vs 6.83 ± 0.75 days); approximately 66% of grafts in the ART group survived over 2 weeks, while survival time of grafts were less than 8 day in the control group ( Figure  1A). Subsequently, allografts were harvested on day 5 after cardiac transplantation. As shown by representative H&E photomicrographs, the rejection area of the ART group was noticeably thinner than that of the control group. Interstitial vasculitis, hemorrhage, and edema were evident in the control group, which were significantly diminished by ART treatment ( Figure 1B). Moreover, TUNEL staining revealed apoptotic cells (red) in the ART group were significantly less than that in the control group, especially in the rejection area. DAPI staining (blue) indicated that nuclei in the control group were deformed and broken, which indicated that more cells were in a state of apoptosis. Together, these data indicate that ART reduced rejection and protected cardiomyocytes ( Figure 1C).

ART Attenuates T Cell-Mediated Rejection, and Increasing Regulatory T Cell Infiltration in Allografts
Five days after transplantation, inflammatory cell (CD45 + ) frequencies and numbers were significantly lower in the cardiac grafts obtained from the ART group compared with the control group (Figures 2A-C). As shown in Figures 2D-K, infiltrative CD3 + , CD3 + CD4 + , and CD3 + CD8 + cell numbers were significantly less in cardiac grafts of the ART group than in

Gene
Forward primer Reverse primer those of the control group, although the percentages of these cells did not change significantly. As shown by representative IHC stains, ART treatment significantly decreased the infiltration of T cells ( Figures 2L-N), and the results showed the same trend as flow cytometry results. Intriguingly, ART treatment also dramatically enhanced Treg cell (Foxp3 + ) expansion based on IHC results ( Figure 2O). Additionally, mRNA levels of the T cell-associated genes IL-2, IFN-g, CD3, CD8, CD4, and IL-17 were significantly reduced following ART treatment ( Figures 3A-F). Furthermore, those of Treg cell-associated genes, Foxp3, IL-10, and TGF-b, were significantly elevated ( Figures 3G-I).
ART Inhibits B Cells Activation and Donor-Specific Antibodies Production and Attenuates Allograft Antibody-Mediated Rejection ABMR, caused by B cell activation and DSA production, is an important factor in allograft dysfunction. By flow cytometry, we examined B cell (B220 + ) populations in spleens and allografts of recipients obtained 5 days after transplantation and quantitatively analyzed frequencies and cell numbers. Although there was no effect on B cell frequencies, ART treatment decreased the number of B cells in the spleens and cardiac grafts compared with the control group ( Figures 4A-F). Furthermore, changes in DSA levels (IgG and IgM) at 0 and 5 days after cardiac transplantation were detected; ART treatment conspicuously inhibited this upward trend ( Figure 4G). No significantly difference was found regarding the levels of IgM between the ART -treated and control groups ( Figure 4H).  Figure 4I). On the contrary, limited response was found regrading IgM levels post skin allograft transplantation ( Figure 4J). Moreover, ART treatment significantly reduces diffuse C4d and IgG deposition in capillaries ( Figure 4K).

ART Reduces Macrophage Infiltration Into Allografts
According to the diagnosis of heart rejection, interstitial mononuclear and macrophage cell infiltration is the typical feature of AR, and macrophage is of vital importance in the development of TCMR and ABMR (18,19). Therefore, macrophage infiltration in the allografts were detected. Less frequencies of macrophages (CD11b + ) were found in the ART -treated group than the control group (49.34 ± 5.77% vs. 60.16 ± 7.76%, P < 0.05), and macrophage numbers were reduced more clearly ( Figures 5A-C). Representative histological analysis also revealed ART treatment inhibited infiltration of CD68 cells ( Figures 5D, E). Moreover, levels of pro-inflammatory cytokines (IL-6 andIL-1b) were decreased in the ART group when compared with the control group, indicating that macrophage function was inhibited by ART treatment ( Figures  5F, G).

Artemisinin Significantly Suppresses T Cell, B Cell, and Macrophage Function In Vitro
To further explore mechanisms underlying the suppression of lymphocyte function by ART, we performed in vitro assays using PBMCs. Because ART can induce apoptosis, to exclude the potential confounding effects of reduced cytokine secretion, which might result from apoptosis, we independently assessed the proapoptotic effect of ART on total lymphocytes. Specifically, PBMCs were cultured with ART at concentrations ranging from  2 to 500 µM for 24 h, and PI-Annexin V staining was performed to evaluate cell survival. ART promoted apoptosis in a dosedependent manner ( Figure 6A). Finally, our results showed that at 20 µM, the pro-apoptotic effect of ART was mild, while apoptosis occurred when cells were cultured over 20 µM ART ( Figure 6B). Moreover, measurements of ART (20 µM)-induced cell death overtime revealed the rapid accumulation of annexin V + PI − cells at the early stage of culture, which transitioned to annexin V + PI + cells over time ( Figure 6C). Therefore, subsequent in vitro experiments were performed with a range of ART centered around 20 µM. Next, we purified specify cells, including B cells (CD19 + ), T cells (CD3 + ), and monocytes (CD14 + ) from human peripheral mononuclear leukocytes and cultured them with different ART concentrations to evaluate cytokine secretion in vitro. As shown in Figure 6D, purified CD3 + T cells from PBMCs produced high levels of IFN-g following stimulation of anti-CD3 plus anti-CD28. Further, cytokine productions were inhibited by ART in a dose-dependent manner. A significant inhibitory effect was observed at 10 µM, and a decrease in cytokines was induced at higher concentrations (~100 µM). B cells (CD19 + ) purified from PBMCs were stimulated with CD40L and anti-IgM for 9 days with or without ART at different concentrations and supernatant IgG and IgM levels were evaluated by ELISA. ART also inhibited IgG production in a dose-dependent manner at 10 µM. Except for with 100 µM ART, IgM levels were not significantly changed ( Figures 6E, F). As mentioned, purified CD14 + cells produced high levels of IL-1b following LPS stimulation. However, ART, from 10 to 100 µM, significantly inhibited this in a dosedependent manner ( Figure 6G).

DISCUSSION
ART and its derivatives are associated with potent immunosuppressive function and have been involved in some clinical trials dealing with autoimmune diseases (20,21). Here, we provide the first evidence demonstrating the effect of ART on transplant immunity using a rat cardiac transplantation model. Specifically, it significantly inhibited rejection by targeting multiple effectors, attenuated allograft injury, and thus prolonged cardiac allograft survival. Currently, transplant rejection is recognized as a dangerous factor for allograft loss (22). Thus, sustainable treatment of rejection remains critical for long-term graft function. Although immunosuppressive therapy has dramatically prolonged the lives of patients, the notable problems associated with these drugs remain. First of all, significant toxicities which affect allograft survival should be taken into consideration. Second, their longterm use inevitably increases infection risks and it might lead to death, especially in the first year post-transplantation (23). Therefore, safe and effective immunosuppression in clinical practice is expected.
Fully MHC-mismatched cardiac transplantation was used to establish a rat transplant rejection model. Rejection diagnosis and classification were based on histological changes, which occur with allograft injury, and inflammatory cell infiltration, and experiments have indicated that rejection is a mix of humoral and cellular rejection (24). In this assay, macrophages and T cells were the major subsets of immune cells infiltrating into the grafts, in accordance with previous results (25). We also observed B cell infiltration and C4d and IgG deposition in the allografts, suggesting that ABMR was involved in rejection in this model. Further, ART significantly attenuated TCMR and ABMR by inhibiting T cell, B cell, and macrophage activation and infiltration. More importantly, approximately 66% of recipients administered ART survived beyond 14 days without combinations with any other drugs or strategies, this is unprecedented.
T cell is of vital importance in rejection post allogeneic transplantation. Recognize allo-antigens by T cell is the primary basis for allograft rejection (23). ART and its derivative were shown to inhibit T lymphocytes. Wang et al. demonstrated that artemether (an ART derivative) has direct inhibitory effects on T cells, suppressing T cell proliferation and activation via the Ras-Raf1-ERK1/2 (11). Another study found that SM934 (an ART derivative) inhibits the accumulation and differentiation of Th1 and Th17 cells and induces the differentiation and expansion of Treg cells (26). In our study, all cardiac grafts were rejected within 8 days post transplantation when no other interventions were applied. Flow cytometric and histological analysis showed that ART treatment significantly inhibited infiltration of lymphocytes, especially effector T cells, in allografts and reduced inflammatory cytokine secretion. Moreover, functions of pure T cells were inhibited by ART in vitro in a dose-dependent manner. These results indicated that ART could significantly inhibit the occurrence of TCMR. Intriguingly, ART treatment also dramatically enhanced the expansion of Treg cells, which play a central role in transplant tolerance. This is consistent with other assays and our previously published data showing that Treg cell therapy could significantly alleviate renal allograft injury and induce immune tolerance in animal and clinical trials (27,28).
Our data thus showed that ART could effectively inhibit TCMR and induce Treg cell expansion. ABMR occurs when recipients are presensitized to donor antigens before surgery or due to de novo DSA production postoperatively. B cells are the main effectors of ABMR. To explore the changes in B cells, we first observed that ART decreased these cell numbers in recipient spleens and allografts. Although function of infiltration B cell in grafts is a mystery, Hippen et al. demonstrated that B cells might be involved in the deterioration of allograft function and anti-donor antibody production (29). These data provided evidence that infiltration of B cell as a critical parameter of ABMR. Therefore, ART treatment could reduce B cell infiltration in the recipient spleen and allografts.
Although some studies have demonstrated that ART family drugs reduce antibodies in a model of systemic lupus erythematosus, it was not known if ART could decrease DSAs with organ transplantation (13,30). We generated a new animal model of BN skin transplantation into Lewis recipients to detect changes in DSAs in the circulation of different treatment groups. This study, for the first time, demonstrated that ART dramatically reduces circulating DSA-IgG levels in this model. Moreover, the changes in DSA-IgG levels at 0 and 5 days after cardiac transplantation were similar. Histological changes, including IgG and C4d were decreased in ART-treated group. Current therapeutic strategies to control DSAs are focused on antibody removal with plasma exchange, intravenous immunoglobulin, and monoclonal antibodies (31,32). However, these strategies also have some limitations, although a combination of multiple strategies could contribute to a synergistic effect (33,34). Owing to its excellent suppressive effects on production of DSAs, ART might be an economical and effective choice for therapy of AMR in clinical settings. Apart from adaptive immune cells, other innate immune cells including macrophages, monocytes, and NK cells infiltrate allografts during rejection and were proven to aggravate allograft injuries (18,35,36). Moreover, macrophages are important innate immune cells; in human cardiac transplants, the infiltrates associated with rejection are predominantly composed of mononuclear cells and macrophages with macrophages outnumbering T cells, especially in the prophase of rejection (37,38). Moreover, recent studies have found that innate myeloid immune cells, such as macrophages and monocytes, can form antigen-specific immune memory and thus serve as an central component in immune rejection (39). All of these results indicate that macrophage is of vital importance during transplantation immune response. Accordingly, our results showed that  macrophages were outnumbered T cells, and ART treatment could significantly reduce the frequency and number of macrophages compared to those in the control group. Pro-inflammatory cytokine IL-1b were also decreased in the ART group. These results also proved that ART treatment can not only inhibit the number of infiltrating macrophages but also reduce their function. Although our results indicated that ART could significantly attenuate rejection by inhibiting multiple pathways, several limitations should be noted. First, effects of ART are wide, and we did not conduct studies on such mechanisms in depth. Second, the research on immune cell function was not thorough, and especially, the subtype analysis of cytokines was not sufficient. Finally, the concentration of ART in vivo was determined based on our experience and references, and thus, the side effects of ART should be further explored.
In summary, we established a transplant rejection model and demonstrated that ART can suppress TCMR, ABMR, and macrophage infiltration in vivo and provided evidence that ART attenuates allograft injury and rejection after rat cardiac transplantation. Subsequently, we demonstrated the inhibitory effect of ART on the function of various purified lymphocytes in vitro. Although this was a pilot study of the suppressive effects of ART on rejection, this study provides novel evidence for the therapy of rejection in patients who suffer transplantation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Sun Yat-sen University Institutional Ethical Guidelines for animal experiments.