DsbA-L deletion attenuates LPS-induced acute kidney injury by modulating macrophage polarization.

Disulfide bond A oxidoreductase-like protein (DsbA-L) drives acute kidney injury (AKI) by directly upregulating the expression of voltage-dependent anion-selective channels in proximal tubular cells. However, the role of DsbA-L in immune cells remains unclear. In this study, we used an LPS-induced AKI mouse model to assess the hypothesis that DsbA-L deletion attenuates LPS-induced AKI and explore the potential mechanism of DsbA-L action. After 24 hours of LPS exposure, the DsbA-L knockout group exhibited lower serum creatinine levels compared to the WT group. Furthermore, peripheral levels of the inflammatory cytokine IL-6 were decreased. Transcriptomic data analysis revealed a significant down-regulation in the IL-17 and tumor necrosis factor pathways in DsbA-L knockout mice following LPS induction. Metabolomic analysis suggested that arginine metabolism was significantly different between the WT and DsbA-L knockout groups after LPS treatment. Notably, the M1 polarization of macrophages in the kidneys of DsbA-L knockout AKI mice was significantly reduced. Expression of the transcription factors NF-κB and AP-1 was downregulated after DsbA-L knockout. Our results suggest that DsbA-L regulates LPS-mediated oxidative stress, promotes M1 polarization of macrophages, and induces expression of inflammatory factors via the NF-κB/AP-1 pathway.

Quantitative proteomic analysis of exosomes from umbilical cord mesenchymal stem cells and rat bone marrow stem cells.

Exosomes derived from mesenchymal stem cells (MSCs) have been used for cancer treatment, however, an in-depth analysis of the exosomal proteomes is lacking. In this manuscript, we use the diaPASEF (parallel accumulation serial fragmentation combined with the data-independent acquisition) method to quantify exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) and rat bone marrow stem cells (BMSCs), resulting in identification of 4200 human proteins and 5362 rat proteins. Comparison of human exosomal proteins and total cellular proteins reveals that some proteins exist in the exosomes exclusively that can be served as potential markers for exosomes. Quantitative proteomic analysis of exosomes from different passages of BMSCs shows that the proteins involved in TGF-ß signaling pathway are regulated in abundance, which could be markers for the therapeutic ability of BMSC exosomes. Collectively, the data presented by this study can be a resource for further study of exosome research.

TIPE2 deficiency prolongs mouse heart allograft survival by facilitating immature DCs-induced Treg generation.

It has been reported that deletion of tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2, TIPE2) facilitates the activation of T-cell receptors. However, the role of TIPE2 in T-cell-mediated acute transplant rejection remains unclear. To illustrate the underlying cellular mechanisms, we transplanted BALB/c hearts into C57BL/6 wild-type (WT) or C57BL/6 mice deficient for TIPE2 (TIPE2-/-) and found that TIPE2-/- recipient mice showed significantly prolonged survival of heart allografts and suppressed maturation of CD11c+ dendritic cells (DCs), which largely abolished the activation and proliferation of alloreactive T cells and their cytotoxic activity. TIPE2-/- DCs increased CD4+CD25+Foxp3+CD127- regulatory T cells (Tregs)generation, likely by inhibiting DCs maturation and CD80 and CD86 expression. Administration of anti-CD25 abolished the allograft survival induced by TIPE2 deficiency. Moreover, TIPE2 deficiency increased IL-10 production in T cells and in recipient serum and allografts. Mechanistic studies revealed that TIPE2-/- restrained the maturation of DCs via inhibition of PI3K/AKT phosphorylation during alloantigen stimulation. Taken together, TIPE2 deficiency in recipient mice inhibited acute rejection by increasing Tregs generated by immature DCs. Thus, TIPE2 could be a therapeutic target for suppressing rejection in organ transplantation.

Divergent and Stereoselective Synthesis of Ustusal A, (-)-Albrassitriol, and Elegansin D.

The first synthesis of ustusal A as well as expeditious access to (-)-albrassitriol is described as featuring a singlet oxygen [4 + 2] cycloaddition, achieving the desired stereoselectivity for the 1,4-cis-hydroxyl groups. Transformation of (+)-sclareolide to III followed by a key Horner-Wadsworth-Emmons (HWE) reaction and stereospecific allylic oxidation facilitated the first synthesis of elegansin D. The biological evaluation of these natural products together with seven elegansin D analogues was performed, among which several elegansin D analogues exhibited potential anticancer activity against liver cancer HepG2 cells (IC50 = 11.99-25.58 µM) with low cytotoxicity on normal liver HL7702 cells (IC50 > 100 µM).

Bcl-2 hijacks the arsenic trioxide resistance in SH-SY5Y cells.

Aresenic trioxide (ATO) is proven to be active against leukaemia cells by inducing apoptosis and differentiation. Even though ATO could effectively induce remissions of leukaemia cells, the drug resistance was observed occasionally. To further dissect the mechanism of ATO resistance, we selected the ATO-resistant SH-SY5Y cells and found that Bcl-2 controlled the sensitivity of ATO in SH-SY5Y cells. We report that necroptosis, autophagy, NF-ƘB and MAPK signalling pathway are not involved in ATO-induced apoptosis. Moreover, the ATO-resistant cells showed distinct mitochondrial morphology compared with that of ATO-sensitive cells. Intriguingly, nude mice-bearing ATO-sensitive cells derived xenograft tumours are more sensitive to ATO treatment compared with that of ATO-resistant cells. These data demonstrate that cancer cells can acquire the ATO-resistance ability by increasing the Bcl-2 expression.

Novel immunosuppressive effect of FK506 by upregulation of PD-L1 via FKBP51 in heart transplantation.

The calcineurin inhibitor-FK506-is a first-line immunosuppressant that regulates T cell secretion of IL-2 and other cytokines. However, the mechanism of its protective effect on target cells and its role on tumour recurrence and interaction with anti-tumour immune checkpoint inhibitors, such as PD-L1 blocking, are still unclear. Here, in a murine heart transplantation model, we observed the upregulation of programmed death-ligand 1 (PD-L1) expression by FK506 in both dendritic cells (DCs) and allografts. Blocking PD-L1 during FK506 treatment increased IFN-γ and TNF-α expression, enhanced CD4+ and CD8+ T cell proliferation, and suppressed Treg differentiation. Moreover, PD-L1 decreased T cell infiltration and induced T cell apoptosis in both the spleen and graft. PD-L1 was not only required in FK506-mediated immunosuppression but also upregulated by FK506. Treatment with SAFit2, a FKBP51 selective inhibitor, reduced the expression of PD-L1 on DCs and the grafts and interfered with the immunosuppressive effect of FK506, suggesting that the mechanism depends on FK506-binding protein (FKBP) 51 expression. Overall, our results add new insights into the role of FK506, not only on T cell cytokine secretion but also on co-inhibitory molecular regulation and target cell immune privilege.

Clinical Safety and Efficacy of Locoregional Therapy Combined with Adoptive Transfer of Allogeneic γδ T Cells for Advanced Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

PURPOSE: To investigate the safety and efficacy of locoregional therapy plus adoptive transfer of allogeneic gamma delta (γδ) T cells for patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). METHODS: Thirty patients with HCC and 29 patients with ICC were randomly assigned to receive locoregional therapy (HCC, Group A, n = 15; ICC, Group C, n = 15) or locoregional therapy plus γδ T cell therapy (HCC, Group B, n = 15; ICC, Group D, n = 14). Groups A and C only received locoregional ablation (cryoablation or irreversible electroporation), whereas Groups B and D received locoregional therapy followed by adoptive transfer of allogeneic γδ T cells. The primary endpoints were safety, distant progression-free survival (PFS), local PFS, and overall survival (OS). RESULTS: The median distant PFS was significantly longer in the combined treatment groups than the locoregional treatment groups (HCC: 8 vs 4 months, P = .04; ICC: 8 vs 4 months, P = .021). There was no significant difference in local PFS between the 2 treatment modalities. Patients with HCC in the combined treatment group had a longer OS (median OS: 13 vs 8 months, P = .029). However, there was no significant difference in OS in patients with ICC between the 2 treatment modalities (median OS: 9.5 vs 8 months, P = .546). All adverse events were manageable with no significant difference in incidence between groups. CONCLUSIONS: The novel combination of locoregional ablation with adoptive transfer of allogeneic γδ cells was safe, with encouraging clinical efficacy against HCC and ICC.

Effects of different sperm sources on clinical outcomes in intracytoplasmic sperm injection cycles.

The aim was to investigate the influences of different sperm sources on clinical outcome and neonatal outcome of patients with intracytoplasmic sperm injection. We retrospectively analysed patients who underwent intracytoplasmic sperm injection in our reproductive centre from 2011 to 2020. We screened data on assisted reproductive outcomes from four groups of sources: testicular sperm, epididymal sperm, ejaculated sperm and donor sperm for analysis and divided the non-ejaculated group from the ejaculated group to explore their impact on clinical outcomes and neonatal outcomes. A total of 2139 cycles were involved in this study. There were significant differences in fertilisation rate (77.0% vs. 73.6%, p < .001), cleavage rate (97.4% vs. 94.4%, p < .001) and high-quality embryo rate (52.8% vs. 49.9%, p < .001) between the ejaculated and non-ejaculated sperm groups. There were no significant differences amongst the four groups in biochemical pregnancy rate, clinical pregnancy rate, abortion rate, live birth rate, male-female ratio and single-twin ratio. Different sperm sources did not affect the length, weight or physical defects of newborns amongst the groups. Sperm source did not affect pregnancy and neonatal outcomes of intracytoplasmic sperm injection in general.

Nickel sulfate exposure induces ovarian inflammation and fibrosis and decreases oocyte quality in mice.

Nickel is a heavy metal element extensively distributed in the environment and widely used in modern life. Divalent nickel is one of the most widespread forms of nickel and has been reported as toxic to various tissues. However, whether exposure to divalent nickel negatively affects ovarian homeostasis and oocyte quality remains unclear. In this study, we found that 3 weeks of nickel sulfate exposure affected body growth and decreased the weight and coefficient of the ovary, and increased atretic follicles exhibiting enhanced apoptosis in granulosa cells. Further studies have found that nickel sulfate triggered ovarian fibrosis and inflammation via transforming growth factor-ß1 and nuclear factor-κB pathways, and reduced oocyte development ability. In addition, nickel sulfate increased the level of reactive oxygen species, which induced DNA damage and early apoptosis. Moreover, it was found that nickel sulfate caused damage to the mitochondria showing aberrant morphology, distribution and membrane potential while decreased levels of histone methylation. To summarize, our results indicated that nickel sulfate exposure triggered ovarian fibrosis and inflammation and caused structural and functional disorders of mitochondria in oocytes, which consequently disturbed ovarian homeostasis and follicle development and decreased oocyte quality.

Live birth after cervical ectopic uterus transplantation in mice.

An ideal animal model is a prerequisite for the basic research of uterus transplantation. This study aimed to develop a new cervical ectopic uterus transplantation mice model, which was established by vascular anastomosis of the right common iliac artery and vein of the donor with the right common carotid artery and external jugular vein of the recipient, respectively, using the cuff method. The survival status of the transplanted uterus was assessed by macroscopic observation and histological examination after surgery, and the function of the graft uterus was tested by verifying whether the pregnancy is possible. A total of 40 transplants were performed, of which only 1 failed due to donor hemorrhage. After 26 transplants, the total operation time reduced to 52.4 ± 3.8 minutes, of which the total ischemia time took 6.6 ± 1.1 minutes. Sixty days after transplantation, all the graft uteri had a good blood supply and spontaneous contraction. The histology showed no significant difference between the transplanted uterus and the native. Embryo transfer experiments have proven that the transplanted uterus has uterine function. In conclusion, this new model is an effective and simple mice model for the studies of the scientific issues related to uterus transplantation.

Thalidomide with blockade of co-stimulatory molecules prolongs the survival of alloantigen-primed mice with cardiac allografts.

BACKGROUND: Miscellaneous memory cell populations that exist before organ transplantation are crucial barriers to transplantation. In the present study, we used a skin-primed heart transplantation model in mouse to evaluate the abilities of Thalidomide (TD), alone or in combination with co-stimulatory blockade, using monoclonal antibodies (mAbs) against memory T cells and alloantibodies to prolong the second cardiac survival. RESULTS: In the skin-primed heart transplantation model, TD combined with mAbs significantly prolonged the second cardiac survival, accompanied by inhibition of memory CD8+ T cells. This combined treatment enhanced the CD4+Foxp3+ regulatory T cells ratio in the spleen, restrained the infiltration of lymphocytes into the allograft, and suppressed the allo-response of spleen T cells in the recipient. The levels of allo-antibodies also decreased in the recipient serum. In addition, we detected low levels of the constitutions of the lytic machinery of cytotoxic cells, which cause allograft damage. CONCLUSIONS: Our study indicated a potential synergistic action of TD in combination with with mAbs to suppress the function of memory T cells and increase the survival of second allografts in alloantigen-primed mice.

Combination of N, N'-dicyclohexyl-N-arachidonic acylurea and tacrolimus prolongs cardiac allograft survival in mice.

Current immunosuppressive agents for organ transplantation are not ideal because of their strong toxicity and adverse effects. Hence, there is an urgent need to develop novel immunosuppressive agents. The compound N, N'-dicyclohexyl-N-arachidonic acylurea (DCAAA) is a novel highly unsaturated fatty acid from the traditional Chinese medicinal plant Radix Isatidis. In this study, we systematically investigated the toxicity, immunosuppressive effect and mechanisms underlying the activity of DCAAA. The toxicity tests showed that DCAAA treatment did not lead to red blood cell hemolysis and did not affect the liver and kidney functions in mice. The lymphocyte transformation test showed that DCAAA treatment inhibited lymphocyte proliferation in a dose-dependent manner. An in vivo cardiac allotransplantation experiment showed that DCAAA treatment could suppress the immune rejection and significantly prolong the survival of cardiac allografts in recipient mice by reducing the proportion of CD4+ T cells in the spleen and grafts, concentration of interferon-γ in the supernatant and serum and infiltration of inflammatory cells into the grafts. Moreover, a combination treatment with DCAAA and tacrolimus had a synergistic effect in preventing acute rejection of heart transplants. In vitro molecular biology experiments showed that DCAAA treatment inhibited activation of the T-cell receptor-mediated phosphoinostide 3-kinase-protein kinase B pathway, thereby arresting cell cycle transition from the G1 to the S phase, and inhibiting lymphocyte proliferation. Overall, our study reveals a novel, low-toxicity immunosuppressive agent that has the potential to reduce the toxic side effects of existing immunosuppressive agents when used in combination with them.

Arsenic trioxide ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice by inducing CD4+ T cell apoptosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by severe white matter demyelination. Because of its complex pathogenesis, there is no definite cure for MS. Experimental autoimmune encephalomyelitis (EAE) is an ideal animal model for the study of MS. Arsenic trioxide (ATO) is an ancient Chinese medicine used for its therapeutic properties with several autoimmune diseases. It is also used to inhibit acute immune rejection due to its anti-inflammatory and immunosuppressive properties. However, it is unclear whether ATO has a therapeutic effect on EAE, and the underlying mechanisms have not yet been clearly elucidated. In this study, we attempted to assess whether ATO could be used to ameliorate EAE in mice. METHODS: ATO (0.5 mg/kg/day) was administered intraperitoneally to EAE mice 10 days post-immunization for 8 days. On day 22 post-immunization, the spinal cord, spleen, and blood were collected to analyze demyelination, inflammation, microglia activation, and the proportion of CD4+ T cells. In vitro, for mechanistic studies, CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice and treated with ATO and then used for an apoptosis assay, JC-1 staining, imaging under a transmission electron microscope, and western blotting. RESULTS: ATO delayed the onset of EAE and alleviated the severity of EAE in mice. Treatment with ATO also attenuated demyelination, alleviated inflammation, reduced microglia activation, and decreased the expression levels of IL-2, IFN-γ, IL-1ß, IL-6, and TNF-α in EAE mice. Moreover, the number and proportion of CD4+ T cells in the spinal cord, spleen, and peripheral blood were reduced in ATO-treated EAE mice. Finally, ATO induced CD4+ T cell apoptosis via the mitochondrial pathway both in vitro and in vivo. Additionally, the administration of ATO had no adverse effect on the heart, liver, or kidney function, nor did it induce apoptosis in the spinal cord. CONCLUSIONS: Overall, our findings indicated that ATO plays a protective role in the initiation and progression of EAE and has the potential to be a novel drug in the treatment of MS.

Generation of bioartificial hearts using decellularized scaffolds and mixed cells.

BACKGROUND: Patients with end-stage heart failure must receive treatment to recover cardiac function, and the current primary therapy, heart transplantation, is plagued by the limited supply of donor hearts. Bioengineered artificial hearts generated by seeding of cells on decellularized scaffolds have been suggested as an alternative source for transplantation. This study aimed to develop a tissue-engineered heart with lower immunogenicity and functional similarity to a physiological heart that can be used for heart transplantation. MATERIALS AND METHODS: We used sodium dodecyl sulfate (SDS) to decellularize cardiac tissue to obtain a decellularized scaffold. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and identified by flow cytometric labeling of their surface markers. At the same time, the multi-directional differentiation of MSCs was analyzed. The MSCs, endothelial cells, and cardiomyocytes were allowed to adhere to the decellularized scaffold during perfusion, and the function of tissue-engineered heart was analyzed by immunohistochemistry and electrocardiogram. RESULTS: MSCs, isolated from rats differentiated into cardiomyocytes, were seeded along with primary rat cardiomyocytes and endothelial cells onto decellularized rat heart scaffolds. We first confirmed the pluripotency of the MSCs, performed immunostaining against cardiac markers expressed by MSC-derived cardiomyocytes, and completed surface antigen profiling of MSC-derived endothelial cells. After cell seeding and culture, we analyzed the performance of the bioartificial heart by electrocardiography but found that the bioartificial heart exhibited abnormal electrical activity. The results indicated that the tissue-engineered heart lacked some cells necessary for the conduction of electrical current, causing deficient conduction function compared to the normal heart. CONCLUSION: Our study suggests that MSCs derived from rats may be useful in the generation of a bioartificial heart, although technical challenges remain with regard to generating a fully functional bioartificial heart.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

Knockdown of TIPE2 increases the proliferation in lipopolysaccharide-stimulated gastric cancer cells.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant diseases with high morbidity and mortality, especially in Asian countries. During the GC developing progress, TIPE2, a member of TNF-alpha induced protein 8-like (TNFAIP8L) family, may play important roles. However, the molecular mechanisms of TIPE2 contributing to cell proliferation and tumor growth are poorly understood in GC. We performed flow cytometry to detect the cell cycle of TIPE2-knockdown GC cells under lipopolysaccharide (LPS) stimulation. METHODS: We measured TIPE2 expression in tumor samples from 46 human GC patients at mRNA level by Realtime PCR and in 68 pairs of GC tissues at protein level by immunohistochemistry. We established stable TIPE2 knockdown SGC7901 and BGC823 cell lines and performed CCK-8 and EdU proliferation assays under the stimulation of LPS. And then we analyzed AKT, IκBα and ERK phosphorylation levels, as well as cycle related proteins CDK4 and CyclinD3 in the stable TIPE2 knockdown SGC7901 and BGC823 cells. RESULTS: Our present studies indicated that the expression of TIPE2 was significantly decreased in tumor tissues compared to distant mucosa tissues in human GC patients. TIPE2 inhibited proliferation stimulated by LPS in SGC7901 and BGC823 cells. Silencing of TIPE2 significantly decreased cell G0/G1 phase ratio and increased G2/M phase. TIPE2 knockdown SGC7901 and BGC823 cells declined AKT and IκBα phosphorylation. TIPE2's action on GC cell cycle was. CONCLUSIONS: Our results demonstrated that TIPE2 is a novel tumor suppressor gene that inhibits GC growth may mediated via AKT and IκBα phosphorylated activation. We revealed that TIPE2 may effectively interdict neoplasm development, which has potential clinical application values for GC targeted therapies.

A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.

Xenotransplantation is an effective way to solve the problem of donor shortage in clinical transplantation. However, clinical use of xenotransplantation is currently limited due to immunological challenges such as acute vascular rejection and cell-mediated rejection. To finally surpass this immunological barrier, more preclinical research is needed into the molecular mechanisms of rejection and the possible effects of new immunosuppressants. Our aim was to create a refined, highly reproducible protocol to establish the most suitable rat-to-mouse heterotopic heart transplantation model using the cuff technique.

Preparation and characterization of small-diameter decellularized scaffolds for vascular tissue engineering in an animal model.

BACKGROUND: The development of a suitable extracellular matrix (ECM) scaffold is the first step in vascular tissue engineering (VTE). Synthetic vascular grafts are available as an alternative to autologous vessels in large-diameter arteries (>8 mm) and medium-diameter arteries (6-8 mm). In small-diameter vessels (<6 mm), synthetic vascular grafts are of limited use due to poor patency rates. Compared with a vascular prosthesis, natural tissue ECM has valuable advantages. Despite considerable progress in recent years, identifying an optimal protocol to create a scaffold for use in small-diameter (<6 mm) fully natural tissue-engineered vascular grafts (TEVG), remains elusive. Although reports on different decellularization techniques have been numerous, combination of and comparison between these methods are scarce; therefore, we have compared five different decellularization protocols for making small-diameter (<6 mm) ECM scaffolds and evaluated their characteristics relative to those of fresh vascular controls. RESULTS: The protocols differed in the choice of enzymatic digestion solvent, the use of non-ionic detergent, the durations of the individual steps, and UV crosslinking. Due to their small diameter and ready availability, rabbit arteria carotis were used as the source of the ECM scaffolds. The scaffolds were subcutaneously implanted in rats and the results were evaluated using various microscopy and immunostaining techniques. CONCLUSIONS: Our findings showed that a 2 h digestion time with 1× EDTA, replacing non-ionic detergent with double-distilled water for rinsing and the application of UV crosslinking gave rise to an ECM scaffold with the highest biocompatibility, lowest cytotoxicity and best mechanical properties for use in vivo or in situ pre-clinical research in VTE in comparison.

Recombinant IL-33 prolongs leflunomide-mediated graft survival by reducing IFN-γ and expanding CD4(+)Foxp3(+) T cells in concordant heart transplantation.

Interleukin (IL)-33 is a novel IL-1 family member, and its administration has been associated with promotion of T helper type-2 (Th2) cell activity and cytokines, particularly IL-4 and IL-5 in vivo. Recently, IL-33 was shown to increase CD4(+)Foxp3(+) regulatory T cells (Tregs) and to suppress levels of the Th1-type cytokine IFN-γ in allogeneic heart transplantation in mice. Therefore, we hypothesized that IL-33 and leflunomide (Lef) could prolong graft survival in the concordant mouse-to-rat heart transplantation model. In this model, xenografts undergo acute humoral xenograft rejection (AHXR) typically on day 3 or cell-mediated rejection approximately on day 7 if AHXR is inhibited by Lef treatment. Recipients were treated with Lef (n=6), IL-33 (n=6), IL-33 combined with Lef (n=6), or left untreated (n=6) for survival studies. Heart grafts were monitored until they stopped beating. Mouse heterotopic grafts were performed, and recipients were sacrificed on days 2 and 7 for histological and flow cytometric analyses. The combination of IL-33 and Lef significantly prolonged the grafts from 17.3±2.3 to 2.8±0.4 days, compared to untreated controls. IL-33 administration with Lef, while facilitating Th2-associated cytokines (IL-4 on day 2 but not day 7), also decreased IFN-γ on day 2 and day 7, compared with Lef treatment only. Furthermore, IL-33 with Lef administration caused an expansion of suppressive CD4(+)Foxp3(+) Tregs in rats. The IL-33 and Lef combination therapy resulted in significantly prolonged graft survival, associated with markedly decreased Th1 cells and increased IL-10 levels. In addition, the combination therapy significantly decreased the percentage of CD-45(+) B cells on days 2 and 7, compared with monotherapy. These findings reveal a new immunoregulatory property of IL-33. Specifically, it facilitates regulatory cells, particularly functional CD4(+)Foxp3(+) Tregs that underlie IL-33-mediated cardiac xenograft survival. Moreover, it can decrease Th1 cells and cytokine expression of Th1 T cells in xenograft recipients, for example IFN-γ.