Effect of salinity on denitrification, membrane fouling and bacterial community in a fixed-bed biofilm membrane reactor.

In this study, a fixed-bed biofilm membrane bioreactor was used to assess denitrification and carbon removal performance, membrane fouling, composition, and the dynamics of microbial communities across 10 salinity levels. As salinity levels increased (from 0 to 30 g/L), the removal efficiency of total nitrogen and chemical oxygen demand decreased from 98 and 86% in Phase I to 25 and 45% in Phase X, respectively. Beyond a salinity level of 10 g/L, membrane fouling accelerated considerably. The analysis of fouling resistance distribution suggested that soluble microbial products (SMPs) were the primary cause of this phenomenon. The irregularity in microbial community succession reflected the varying adaptability of different bacteria to different salinity levels. The relative abundance of Sulfuritalea, Lentimircobium, Thauera, and Pseudomonas increased from 20.2 to 47.7% as the experiments progressed. Extracellular polymeric substances-related analysis suggested that Azospirillum plays a positive role in preserving the structural integrity of the biofilm carrier. The SMP-related analysis showed a positive correlation between Lentimircobium, Thauera, Pseudomonas, and the SMP content. These results suggested that these three bacterial genera significantly promoted the release of SMP under salt stress, which in turn led to severe membrane fouling.

Recombinant apoptosis inhibitor of macrophage protein reduces delayed graft function in a murine model of kidney transplantation.

Reperfusion injury following cold and warm ischemia (IRI) is unavoidable during kidney transplantation and contributes to delayed graft function (DGF) and premature graft loss. Death of tubular epithelial cells (TECs) by necrosis during IRI releases pro-inflammatory mediators (e.g. HMGB1), propagating further inflammation (necroinflammation) and tissue damage. Kidney Injury Molecule-1 (KIM-1) is a phagocytic receptor upregulated on proximal TECs during acute kidney injury. We have previously shown that renal KIM-1 protects the graft against transplant associated IRI by enabling TECs to clear apoptotic and necrotic cells, and that recognition of necrotic cells by KIM-1 is augmented in the presence of the opsonin, apoptosis inhibitor of macrophages (AIM). Here, we tested whether recombinant AIM (rAIM) could be used to mitigate transplant associated IRI. We administered rAIM or vehicle control to nephrectomised B6 mice transplanted with a single B6 donor kidney. Compared to grafts in vehicle-treated recipients, grafts from rAIM-treated mice exhibited significantly less renal dysfunction, tubular cell death, tissue damage, tubular obstruction, as well as local and systemic inflammation. Both mouse and human rAIM enhanced the clearance of necrotic cells by murine and human TECs, respectively in vitro. These data support testing of rAIM as a potential therapeutic agent to reduce DGF following kidney transplantation.

Mitochondrial permeability regulates cardiac endothelial cell necroptosis and cardiac allograft rejection.

Transplantation is invariably associated with programmed cell death including apoptosis and necrosis, resulting in delayed graft function and organ rejection. We have demonstrated the contribution of necroptosis to mouse microvascular endothelial cell (MVEC) death and transplant rejection. Organ injury results in the opening of mitochondrial permeability transition pores (mPTPs), which can trigger apoptotic molecules release that ultimately results in cell death. The effect of mPTPs in the necroptotic pathway remains controversial; importantly, their role in transplant rejection is not clear. In this study, tumor necrosis factor-α triggered MVECs to undergo receptor-interacting protein kinase family (RIPK1/3)-dependent necroptosis. Interestingly, inhibition of mPTP opening could also inhibit necroptotic cell death. Cyclophilin-D (Cyp-D) is a key regulator of the mPTPs. Both inhibition and deficiency of Cyp-D protected MVECs from necroptosis (n = 3, P < .00001). Additionally, inhibition of Cyp-D attenuated RIPK3-downstream mixed-lineage kinase domain-like protein phosphorylation. In vivo, Cyp-D-deficient cardiac grafts showed prolonged survival in allogeneic BALB/c mice posttransplant compared with wild-type grafts (n = 7, P < .0001). Our study results suggest that the mPTPs may be important mechanistic mediators of necroptosis in cardiac grafts. There is therapeutic potential in targeting cell death via inhibition of the mPTP-regulating molecule Cyp-D to prevent cardiac graft rejection.

Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.

Ischemia-reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage-associated molecular patterns (eg, high-mobility group box-1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM-1 expression in KIM-1+/+ kidney grafts posttransplantation. Compared to recipients of KIM-1+/+ kidneys, recipients of KIM-1-/- kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM-1-/- grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM-1+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM-1-/- grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.

Daily therapy with a slow-releasing H2S donor GYY4137 enables early functional recovery and ameliorates renal injury associated with urinary obstruction.

OBJECTIVES: To assess the effects of slow-releasing H2S donor GYY4137 on post-obstructive renal function and injury following unilateral ureteral obstruction (UUO) by using the UUO and reimplantation (UUO-R) model in rats and to elucidate potential mechanisms by using an in vitro model of epithelial-mesenchymal transition (EMT). METHODS: Male Lewis rats underwent UUO at the left ureterovesical junction. From post-operative day (POD) 1-13, rats received daily intraperitoneal (IP) injection of phosphate buffered saline (PBS, 1 mL) or GYY4137 (200 µmol/kg/day in 1 mL PBS, IP). On POD 14, the ureter was reimplanted back into the bladder, followed by a right nephrectomy. Urine and serum samples were collected to monitor renal function. On POD 30, the left kidney was removed and tissue sections were stained with H&E, TUNEL, CD68, CD206, myeloperoxidase, and Masson's trichrome to determine cortical thickness, apoptosis, inflammation, and fibrosis. In our in vitro model of EMT, NRK52E cells were treated with 10 ng/mL TGF-ß1, 10 µM GYY4137 and/or 50 µM GYY4137. Western blot analysis was performed to determine the expression of E-cadherin, vimentin, Smad7 and TGF-ß1 receptor II (TßRII). RESULTS: GYY4137 led to a moderate decrease in post-obstructive serum creatinine, cystatin C and FENa. We also observed a trend towards a decrease in post-obstructive proteinuria following GYY4137 treatment. Histologically, we observed a significant decrease in apoptosis, inflammation, and fibrosis. Furthermore, our in vitro studies demonstrate that in the presence of TGF-ß1, GYY4137 significantly decreases vimentin and TßRII and significantly increases E-cadherin and Smad7. CONCLUSIONS: H2S may help to accelerate the recovery of renal function post-obstruction and attenuates renal injury associated with UUO. It is possible that H2S mitigates fibrosis by regulating the TGF-ß1-mediated EMT pathway. Taken together, our data suggest that H2S may be a potential novel therapy for improving renal function and limiting renal injury associated with obstructive uropathy.

Necroptosis Is Involved in CD4+ T Cell-Mediated Microvascular Endothelial Cell Death and Chronic Cardiac Allograft Rejection.

BACKGROUND: Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection. METHODS: We used major histocompatibility complex class II mismatched C57BL/6N (H-2; B6) or B6.RIP3 (H-2; RIP3) mice to B6.C-H-2 (H2-Ab1; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3 cardiac grafts. RESULT: CD4 T cell-mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4 T cell-mediated MVEC death involves TNFα, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4 T cells had attenuated MVEC death through granzyme B and FasL. CONCLUSIONS: CD4 T cell-mediated MVEC death involves in TNFα, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

GYY4137, a Slow-Releasing Hydrogen Sulfide Donor, Ameliorates Renal Damage Associated with Chronic Obstructive Uropathy.

PURPOSE: Chronic obstructive uropathy can cause irreversible kidney injury, atrophy and inflammation, which can ultimately lead to fibrosis. Epithelial-mesenchymal transition is a key trigger of fibrosis that is caused by up-regulation of TGF-ß1 (transforming growth factor-ß1) and ANGII (angiotensin II). H2S is an endogenously produced gasotransmitter with cytoprotective properties. We sought to elucidate the effects of the slow-releasing H2S donor GYY4137 on chronic ureteral obstruction and evaluate the potential mechanisms. MATERIALS AND METHODS: Following unilateral ureteral obstruction male Lewis rats were given daily intraperitoneal administration of phosphate buffered saline vehicle (obstruction group) or phosphate buffered saline plus 200 µmol/kg GYY4137 (obstruction plus GYY4137 group) for 30 days. Urine and serum samples were collected to determine physiological parameters of renal function and injury. Kidneys were removed on postoperative day 30 to evaluate histopathology and protein expression. Epithelial-mesenchymal transition in LLC-PK1 pig kidney epithelial cells was induced with TGF-ß1 and treated with GYY4137 to evaluate potential mechanisms via in vitro scratch wound assays. RESULTS: H2S treatment decreased serum creatinine and the urine protein-to-creatinine excretion ratio after unilateral ureteral obstruction. In addition, H2S mitigated cortical loss, inflammatory damage and tubulointerstitial fibrosis. Tissues showed decreased expression of epithelial-mesenchymal transition markers upon H2S treatment. Epithelial-mesenchymal transition progression in LLC-PK1 was alleviated upon in vitro administration of GYY4137. CONCLUSIONS: To our knowledge our findings demonstrate for the first time the protective effects of H2S in chronic obstructive uropathy. This may represent a potential therapeutic solution to ameliorate renal damage and improve the clinical outcomes of urinary obstruction.

IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury.

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia-reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1ß. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1ß and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation.

Natural killer cells play a critical role in cardiac allograft vasculopathy in an interleukin-6--dependent manner.

BACKGROUND: Approximately 50% of cardiac transplants fail in the long term, and currently, there are no specific treatments to prevent chronic rejection. In the clinic, donor cardiac graft ischemia time is limited to within a few hours and correlates with delayed graft function and organ failure. It is still unknown how ischemic injury negatively influences allograft function over the long term despite advances in immunosuppression therapy. METHODS: Allogeneic cardiac grafts were stored at 4 °C for 4 hr before being transplanted into T/B cell-deficient Rag(-/-) mice or T/B/natural killer (NK) cell-deficient γc(-/-)Rag(-/-) mice. Grafts were harvested 60 days after transplantation and indicators of chronic allograft vasculopathy (CAV) were quantified. RESULTS: We have found that cold ischemia of cardiac grafts induces CAV after transplantation into Rag1(-/-) mice. Interestingly, cold ischemia-induced CAV posttransplantation was not seen in T/B/NK cell-deficient γc(-/-)Rag(-/-) mice. However, cardiac grafts in γc(-/-)Rag(-/-) mice that received an adoptive transfer of NK cells developed CAV, supporting the role of NK cells in CAV development. Analysis of various cytokines that contribute to NK cell function revealed high interleukin (IL)-6 expression in cardiac grafts with CAV. In addition, IL-6-deficient cardiac grafts did not develop CAV after transplantation into allogeneic Rag(-/-) mice. CONCLUSION: These data demonstrate that cold ischemia and NK cells play critical roles in the development CAV. Natural killer cells and injured grafts may play a reciprocal role for CAV development in an IL-6-independent manner. Specific therapeutic strategies may be required to attenuate NK cell contribution to chronic cardiac rejection.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection.

BACKGROUND: While substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival. METHOD: Vectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed. RESULT: Allogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10-16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4+CD25+FoxP3+ regulatory T cell differentiation in vitro. CONCLUSION: This study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

Carbon monoxide-releasing molecules protect against ischemia-reperfusion injury during kidney transplantation.

Carbon monoxide (CO) can provide beneficial antiapoptotic and anti-inflammatory effects in the context of ischemia-reperfusion injury (IRI). Here we tested the ability of pretreating the kidney donor with carbon monoxide-releasing molecules (CORM) to prevent IRI in a transplant model. Isogeneic Brown Norway donor rats were pretreated with CORM-2 18 h before kidney retrieval. The kidneys were then cold-preserved for 26 h and transplanted into Lewis rat recipients that had undergone bilateral nephrectomy. Allografts from Brown Norway to Lewis rats were also performed after 6 h of cold ischemic time with low-dose tacrolimus treatment. All recipients receiving CORM-2-treated isografts survived the transplant process and had near-normal serum creatinine levels, whereas all control animals died of uremia by the third post-operative day. This beneficial effect was also seen in isografted Lewis recipients receiving kidneys perfused with CORM-3, indicating that CORMs have direct effects on the kidney. Pretreatment of human umbilical vein endothelial cells in culture with CORM-2 for 1 h significantly reduced cytokine-induced nicotinamide adenine dinucleotide phosphate-dependent production of superoxide, activation of the inflammation-relevant transcription factor nuclear factor-κB, upregulated expression of E-selectin and intercellular adhesion molecule-1 adhesion proteins, and leukocyte adhesion to the endothelial cells. Thus, CORM-2-derived CO protects renal transplants from IRI by modulating inflammation.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4(+)Foxp3(+) Treg cell accumulation.

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαß(+)CD3(+)CD4⁻CD8⁻NK1.1⁻ (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1-9) induced long-term heart allograft survival (101±31 vs. 39±13 days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4(+) Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12 days vs. 101±31 days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4(+)Foxp3(+) Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.

Prevention of chronic renal allograft rejection by soluble CD83.

BACKGROUND: Recombinant human soluble CD83 had previously exhibited significant immunosuppressive properties that involved interference with dendritic cell maturation in both mouse and humans, inhibition of autoimmunity in mice, and induction of antigen-specific mouse cardiac allograft tolerance when used in combination with other immunosuppressive drugs. Our current research focus turned to examining the effects of peritransplant soluble CD83 (sCD83) administration on prevention of chronic renal allograft rejection. METHODS: Fisher344-to-Lewis orthotopic rat renal transplants were performed with sequential recipient killing on postoperative days (PODs) 2, 14, and 140 to examine both the acute and chronic effects of peritransplant sCD83 treatment in rat recipients. RESULTS: Recipients treated with sCD83 exhibited a marked decrease in IgM and IgG deposition in the graft and antidonor antibody levels in the circulation, as early as POD14 and persisting until POD140. sCD83 treatment also reduced the infiltration of T cells and monocytes into the graft tissue and inhibited intragraft expression of MyD88 and inflammatory cytokine levels during the observation period. sCD83-treated grafts demonstrated normal histology beyond POD140, including dramatic reductions in tubular atrophy and interstitial fibrosis compared with untreated recipients. CONCLUSION: We have demonstrated that peritransplant treatment with recombinant sCD83 attenuates both innate and adaptive immune responses and leads to prevention of chronic rejection in a rat renal transplant model. Because sCD83 is of human origin, the therapeutic approach used in our rodent transplant model holds significant promise for clinical transplantation.

Immunosuppression involving soluble CD83 induces tolerogenic dendritic cells that prevent cardiac allograft rejection.

BACKGROUND: Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance. METHODS: We used the C3H-to-C57BL/6 mouse cardiac transplantation model that exhibits a combination of severe cell-mediated rejection and moderate antibody-mediated rejection and investigated whether sCD83 could augment a combination therapy consisting of Rapamycin (Rapa) and anti-CD45RB monoclonal antibody (α-CD45) to prolong allograft survival. RESULTS: Monotherapies consisting of Rapa and α-CD45 were incapable of preventing rejection. However, all treatments involving sCD83 were capable of (1) down-modulating expression of various DC surface molecules, such as major histocompatibility complex class II and costimulatory molecules, (2) reducing the allogeneic stimulatory capacity of the DCs, and (3) significantly inhibiting antidonor antibody responses. Most striking results were observed in the triple therapy-treated group, sCD83Rapaα-CD45, where cell-mediated rejection and antibody-mediated rejection were abrogated for over 100 days. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days, whereas third-party skin grafts were rejected. Success of triple therapy treatment was accompanied by enhancement of tolerogenic-DCs that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. CONCLUSIONS: Our study revealed that sCD83 is capable of attenuating DC maturation and function, and inducing donor-specific allograft tolerance, in the absence of toxicity. Thus, sCD83 seems to be a safe and valuable counterpart to current DC-modulating agents.

Novel small interfering RNA-containing solution protecting donor organs in heart transplantation.

BACKGROUND: Ischemia/reperfusion injury is a major factor in graft quality and subsequent function in the transplantation setting. We hypothesize that the process of RNA interference may be used to "engineer" a graft to suppress expression of genes associated with inflammation, apoptosis, and complement, which are believed to cause ischemia/reperfusion injury. Such manipulation of pathological gene expression may be performed by treatment of the graft ex vivo with small interfering RNA (siRNA) as part of the preservation procedure. METHODS AND RESULTS: Heart grafts from BALB/c mice were preserved in UW solution (control) or UW solution containing siRNAs targeting tumor necrosis factor-alpha, C3, and Fas genes (siRNA solution) at 4 degrees C for 48 hours and subsequently transplanted into syngeneic recipients. Tumor necrosis factor-alpha, C3, and Fas genes were elevated by ischemia/reperfusion injury after 48 hours of preservation in UW solution. Preservation in siRNA solution knocked down gene expression at the level of messenger RNA and protein in the grafts after transplantation. All grafts preserved in siRNA solution showed strong contraction, whereas grafts preserved in control solution demonstrated no detectable contraction by high-frequency ultrasound scanning. siRNA solution-treated organs exhibited improved histology and diminished neutrophil and lymphocyte infiltration compared with control solution-treated organs. Furthermore, the treated heart grafts retained strong beating up to the end of the observation period (>100 days), whereas all control grafts lost function within 8 days. CONCLUSIONS: Incorporation of siRNA into organ storage solution is a feasible and effective method of attenuating ischemia/reperfusion injury, protecting cardiac function, and prolonging graft survival.

Generation of therapeutic dendritic cells and regulatory T cells for preventing allogeneic cardiac graft rejection.

Tolerogenic dendritic cells (Tol-DCs) and regulatory T cells (Treg) are key factors in the induction and maintenance of transplantation tolerance. We previously demonstrated that ex vivo-isolated Tol-DCs promote Treg generation, and vice versa, in an in vitro co-culture system. Here we demonstrate the occurrence of such an immune regulatory feedback loop in vivo. Tol-DC generated in vitro by treatment with LF 15-0195 exhibited features of immature DC and express low levels of MHC class II, CD86 and CD40. These Tol-DCs were capable of augmenting CD4(+)CD25(+)CTLA4(+) and FoxP3(+) Treg cell numbers and activity in cardiac allograft recipients. On the other hand, Tol-DCs possessed an ability to generate Treg cells in vitro. The adoptive transfer of these in vitro-generated Treg cells resulted in an increase of Tol-DC in vivo, suggesting that an immune regulatory feedback loop, between Tol-DC and Treg, exists in vivo. Furthermore, the administration of in vitro-generated Tol-DCs or Treg cells prevented rejection of allografts. Co-administration of Tol-DC and Treg synergized efficacy of promoting allograft survival heart transplantation. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs and Treg.

Tolerogenic dendritic cells transferring hyporesponsiveness and synergizing T regulatory cells in transplant tolerance.

Dendritic cells are among the most potent antigen-presenting cells and are important in the development of both immunity and tolerance. Tolerogenic dendritic cell (Tol-DC) is a key factor in the induction and maintenance of tolerance during transplantation. However, the precise mechanism and direct evidence of in vivo immune modulation remain unclear. In the present study, we identified critical roles of immune modulation on transplant tolerance through interactions between Tol-DCs and regulatory T (Treg) cells. Tol-DCs remained in an immature state and were insensitive to maturation stimuli. Tol-DCs in tolerant recipients heightened the expression of indoleamine 2,3-dioxygenase (IDO) that induced allogeneic T-cell apoptosis. Adoptive transfer of Tol-DCs isolated from primary tolerant recipients resulted in augmentation of CD4(+)CD25(+)CTLA4(+) Treg cells and prolonged graft survival in secondary allogeneic heart transplantation and synergized with Treg cells to induce tolerance in secondary recipients. This study indicates that Tol-DC offers two functions during the process of tolerogenesis: suppression of anti-donor T-cell responses through production of IDO and interaction with Treg cells, which provides a framework for future research into tolerance induction.

Immune modulation and tolerance induction by RelB-silenced dendritic cells through RNA interference.

Dendritic cells (DC), the most potent APCs, can initiate the immune response or help induce immune tolerance, depending upon their level of maturation. DC maturation is associated with activation of the NF-kappaB pathway, and the primary NF-kappaB protein involved in DC maturation is RelB, which coordinates RelA/p50-mediated DC differentiation. In this study, we show that silencing RelB using small interfering RNA results in arrest of DC maturation with reduced expression of the MHC class II, CD80, and CD86. Functionally, RelB-silenced DC inhibited MLR, and inhibitory effects on alloreactive immune responses were in an Ag-specific fashion. RelB-silenced DC also displayed strong in vivo immune regulation. An inhibited Ag-specific response was seen after immunization with keyhole limpet hemocyanin-pulsed and RelB-silenced DC, due to the expansion of T regulatory cells. Administration of donor-derived RelB-silenced DC significantly prevented allograft rejection in murine heart transplantation. This study demonstrates for the first time that transplant tolerance can be induced by means of RNA interference using in vitro-generated tolerogenic DC.

Prolongation of cardiac allograft survival by inhibition of ERK1/2 signaling in a mouse model.

BACKGROUND: It has been demonstrated that in vitro the presence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling inhibitor suppresses T cell activation and Th1 development. However, pharmacological interference of ERK1/2 signaling by administration of its small molecule inhibitor has not been tested as a therapeutic target in the prevention of allograft rejection. METHODS: The immunosuppressive effect of targeting ERK1/2 signaling was tested on cardiac allograft survival in C57BL/6 (H-2b) to Balb/c (H-2d) murine model using PD98059 inhibitor. Phosphorylation/activation of ERK 1/2 and STAT6 proteins were assessed by Western blot. RESULTS: Blockade of ERK1/2 using PD98059 had significant immunosuppressive effect and prolonged survival of mouse cardiac allografts from 8.3+/-0.5 days (vehicle) to 12.6+/-1.3 days (100 mg/kg PD98059; P<0.0001). Combination therapy of PD98059 (100 mg/kg) with cyclosporine (CsA, 15 mg/kg for 20 days) additionally enhanced graft survival (34.4+/-1.2 days) compared to CsA (14.9+/-1.1 days; P<0.0001) or PD98059 monotherapy (P<0.0001). Attenuation of graft rejection by PD98059 correlated to reduction of intragraft ERK phosphorylation and leukocyte infiltration, and to increase in interleukin (IL)-4 or decrease in interferon-gamma production within the grafts. In vitro inhibition of ERK1/2 by PD98059 promoted Th2 differentiation by upregulation IL-4 production but not altering IL-4 stimulating STAT6 pathway. CONCLUSION: Targeting ERK1/2 signaling results in suppression of alloimmune responses by an unique mechanism that involves Th1/Th2 skewing, suggesting a therapeutic potential of inhibition of ERK1/2 signaling for transplant rejection, particularly in combination with CsA.

Endothelin-mediated oncofetal fibronectin expression in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy is a sclerotic process characterized by an increased extracellular matrix (ECM) protein deposition. Fibronectin (FN) is a major component of ECM. FN has been reported to undergo alternative splicing and produce several isoforms including the extra domain-B (ED-B) containing embryonic isoform. In the present study, we investigated ED-B FN expression in chronic allograft nephropathy and its relationship with endothelins (ET). METHODS: To establish chronic allograft nephropathy, allografts were performed between Fisher 344 --> Lewis rats. Allograft recipients were then randomly divided into two groups, allografts and allografts treated with ET receptor antagonist bosentan. Lewis --> Lewis recipients were used as isograft controls. Grafts were harvested at 30, 90 and 140 days for histological and gene expression analyses with respect to ED-B FN, ET-1 and transforming growth factor-beta1 (TGF-beta1) mRNA. ED-B FN protein levels were assessed by immunohistochemical analysis. Additionally, we analyzed human renal allograft biopsies. RESULTS: Our data demonstrates that rat chronic allograft nephropathy is associated with progressive upregulation of ED-B FN mRNA and protein. ET-1 and TGF-beta1 mRNA were also upregulated. Treatment of allograft recipient rats with bosentan prevented upregulation of ED-B FN and TGF-beta1. We further show that total FN, ED-B FN, ET-1 and TGF-beta1 mRNA expression were upregulated in human chronic allograft nephropathy specimens. CONCLUSION: Results obtained from both human and rat renal allograft tissues suggest that expression of ED-B FN is upregulated in chronic allograft nephropathy and such upregulation is mediated via ET-1 and its interaction with TGF-beta1.