The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

Nicotinamide mononucleotide as a therapeutic agent to alleviate multi-organ failure in sepsis.

BACKGROUND: Sepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms. METHODS: Sepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents. RESULTS: We demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition. CONCLUSION: NAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis.

MLKL-mediated necroptosis is a target for cardiac protection in mouse models of type-1 diabetes.

BACKGROUND: Cardiomyocyte death contributes to cardiac pathology of diabetes. Studies have shown that the RIPK3/MLKL necroptosis signaling is activated in diabetic hearts. Deletion of RIPK3 was reported to attenuate myocardial injury and heart dysfunction in streptozocin (STZ)-induced diabetic mice, suggesting a potential role of necroptosis in diabetic cardiomyopathy. This study characterized cardiomyocyte necroptosis in diabetic hearts and investigated whether MLKL-mediated necroptosis is a target for cardiac protection in diabetes. METHODS: Type 1 diabetes was induced in RIPK3 knockout, MLKL knockout and wild-type mice. Akita Type-1 diabetic mice were injected with shRNA for MLKL. Myocardial function was assessed by echocardiography. Immuno-histological analyses determined cardiomyocyte death and fibrosis in the heart. Cultured adult mouse cardiomyocytes were incubated with high glucose in the presence of various drugs. Cell death and phosphorylation of RIPK3 and MLKL were analysed. RESULTS: We showed that the levels of phosphorylated RIPK3 and MLKL were higher in high glucose-stimulated cardiomyocytes and hearts of STZ-induced type-1 diabetic mice, akita mice and type-1 diabetic monkeys when compared to non-diabetic controls. Inhibition of RIPK3 by its pharmacological inhibitor or gene deletion, or MLKL deletion prevented high glucose-induced MLKL phosphorylation and attenuated necroptosis in cardiomyocytes. In STZ-induced type-1 diabetic mice, cardiomyocyte necroptosis was present along with elevated cardiac troponin I in serum and MLKL oligomerization, and co-localized with phosphorylated MLKL. Deletion of RIPK3 or MLKL prevented MLKL phosphorylation and cardiac necroptosis, attenuated serum cardiac troponin I levels, reduced myocardial collagen deposition and improved myocardial function in STZ-injected mice. Additionally, shRNA-mediated down-regulation of MLKL reduced cardiomyocyte necroptosis in akita mice. Interestingly, incubation with anti-diabetic drugs (empagliflozin and metformin) prevented phosphorylation of RIPK3 and MLKL, and reduced cell death in high glucose-induced cardiomyocytes. CONCLUSIONS: We have provided evidence that cardiomyocyte necroptosis is present in diabetic hearts and that MLKL-mediated cardiomyocyte necroptosis contributes to diabetic cardiomyopathy. These findings highlight MLKL-mediated necroptosis as a target for cardiac protection in diabetes.

Cyclophilin D Regulates the Nuclear Translocation of AIF, Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury.

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.

Toll-like receptor 3 is an endogenous sensor of cell death and a potential target for induction of long-term cardiac transplant survival.

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll-like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self-RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3-induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase-3 and mixed lineage kinase domain-like protein were inhibited in TLR3-/- cardiac grafts compared with C57BL/6 grafts posttransplant (n = 5, p < .001). Importantly, TLR3-/- cardiac grafts had prolonged survival in allogeneic BALB/c mice (mean survival = 121 ± 67 vs. 31 ± 6 days of C57BL/6 grafts, n = 7, p = .002). In summary, our study suggests that TLR3 is an important cell death inducer in ECs and cardiac grafts and thus a potential therapeutic target in preventing cardiac transplant rejection.

miR-149-3p reverses CD8+ T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells.

Blockade of inhibitory receptors (IRs) is one of the most effective immunotherapeutic approaches to treat cancer. Dysfunction of miRNAs is a major cause of aberrant expression of IRs and contributes to the immune escape of cancer cells. How miRNAs regulate immune checkpoint proteins in breast cancer remains largely unknown. In this study, downregulation of miRNAs was observed in PD-1-overexpressing CD8+ T cells using miRNA array analysis of mouse breast cancer homografts. The data reveal that miR-149-3p was predicted to bind the 3'UTRs of mRNAs encoding T-cell inhibitor receptors PD-1, TIM-3, BTLA and Foxp1. Treatment of CD8+ T cells with an miR-149-3p mimic reduced apoptosis, attenuated changes in mRNA markers of T-cell exhaustion and downregulated mRNAs encoding PD-1, TIM-3, BTLA and Foxp1. On the other hand, T-cell proliferation and secretion of effector cytokines indicative of increased T-cell activation (IL-2, TNF-α, IFN-γ) were upregulated after miR-149-3p mimic treatment. Moreover, the treatment with a miR-149-3p mimic promoted the capacity of CD8+ T cells to kill targeted 4T1 mouse breast tumour cells. Collectively, these data show that miR-149-3p can reverse CD8+ T-cell exhaustion and reveal it to be a potential antitumour immunotherapeutic agent in breast cancer.

Increased calpain-1 in mitochondria induces dilated heart failure in mice: role of mitochondrial superoxide anion.

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.

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.

Loss of receptor interacting protein kinases 3 and caspase-8 augments intrinsic apoptosis in tubular epithelial cell and promote kidney ischaemia-reperfusion injury.

BACKGROUND: Ischaemia-reperfusion injury (IRI) is associated with programmed cell death that promotes inflammation and organ dysfunction. Necroptosis is mediated by members of receptor interacting protein kinases (RIPK1/3). Inhibition of RIPK1/3 provides a pro-survival benefit in kidney IRI. Caspase-8 initiates apoptosis and contributes to IRI. We studied whether inhibiting both RIPK3 and caspase-8 would provide an additional benefit in kidney IRI. METHODS: A clamp was applied to the left kidney pedicle for 45 min followed by right kidney nephrectomy. Kidney and serum from wild type, RIPK3-/- , and RIPK3-/- caspase-8-/- double knockout (DKO) mice were collected post-IRI for assessment of injury. Tubular epithelial cells (TEC) isolated from wild type, RIPK3-/- , and DKO mice were treated with interferons-γ and interleukin-1ß to induce apoptotic death. RESULTS: Kidney IRI of DKO mice did not show improvement over RIPK3-/- mice. We have found that DKO triggered 'intrinsic' apoptosis in TEC in response to interleukin-1ß and interferons-γ. Up-regulation of the B-cell lymphoma 2 (Bcl-2)-associated death promoter, the Bcl-2-homologous antagonist killer and Bcl-2-associated X protein and enhanced activation of caspase-3 and 9 were found in DKO TEC. TEC infected with Murine cytomegalovirus that encodes multiple cell death inhibitors resist to death. CONCLUSION: We show that the deletion of both RIPK3 and caspase-8 does not provide additive benefit in IRI or TEC death and may enhance injury by up-regulation of intrinsic apoptosis. This suggests blocking multiple death pathways may be required for the prevention of kidney IRI clinically.

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem.

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.

Intracellular pH Regulates TRAIL-Induced Apoptosis and Necroptosis in Endothelial Cells.

During ischemia or inflammation of organs, intracellular pH can decrease if acid production exceeds buffering capacity. Thus, the microenvironment can expose parenchymal cells to a reduced extracellular pH which can alter pH-dependent intracellular functions. We have previously shown that while silencing caspase-8 in an in vivo ischemia reperfusion injury (IRI) model results in improved organ function and survival, removal of caspase-8 function in a donor organ can paradoxically result in enhanced receptor-interacting protein kinase 1/3- (RIPK1/3-) regulated necroptosis and accelerated graft loss following transplantation. In our current study, TRAIL- (TNF-related apoptosis-inducing ligand-) induced cell death in vitro at neutral pH and caspase-8 inhibition-enhanced RIPK1-dependent necroptotic death were confirmed. In contrast, both caspase-8 inhibition and RIPK1 inhibition attenuated cell death at a cell pH of 6.7. Cell death was attenuated with mixed lineage kinase domain-like (MLKL) silencing, indicating that MLKL membrane rupture, a distinctive feature of necroptosis, occurs regardless of pH. In summary, there is a distinct regulatory control of apoptosis and necroptosis in endothelial cells at different intracellular pH. These results highlight the complexity of modulating cell death and therapeutic strategies that may need to consider different consequences on cell death dependent on the model.

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.

Prevention of allograft rejection in heart transplantation through concurrent gene silencing of TLR and Kinase signaling pathways.

Toll-like receptors (TLRs) act as initiators and conductors responsible for both innate and adaptive immune responses in organ transplantation. The mammalian target of rapamycin (mTOR) is one of the most critical signaling kinases that affects broad aspects of cellular functions including metabolism, growth, and survival. Recipients (BALB/c) were treated with MyD88, TRIF and mTOR siRNA vectors, 3 and 7 days prior to heart transplantation and 7, 14 and 21 days after transplantation. After siRNA treatment, recipients received a fully MHC-mismatched C57BL/6 heart. Treatment with mTOR siRNA significantly prolonged allograft survival in heart transplantation. Moreover, the combination of mTOR siRNA with MyD88 and TRIF siRNA further extended the allograft survival; Flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40, CD86 expression, upregulation of PD-L1 expression in splenic dendritic cells in MyD88, TRIF and mTOR treated mice. There is significantly upregulated T cell exhaustion in T cells isolated from tolerant recipients. This study is the first demonstration of preventing immune rejection of allogeneic heart grafts through concurrent gene silencing of TLR and kinase signaling pathways, highlighting the therapeutic potential of siRNA in clinical transplantation.

Regulatory non-coding RNA: new instruments in the orchestration of cell death.

Non-coding RNA (ncRNA) comprises a substantial portion of primary transcripts that are generated by genomic transcription, but are not translated into protein. The possible functions of these once considered 'junk' molecules have incited considerable interest and new insights have emerged. The two major members of ncRNAs, namely micro RNA (miRNA) and long non-coding RNA (lncRNA), have important regulatory roles in gene expression and many important physiological processes, which has recently been extended to programmed cell death. The previous paradigm of programmed cell death only by apoptosis has recently expanded to include modalities of regulated necrosis (RN), and particularly necroptosis. However, most research efforts in this field have been on protein regulators, leaving the role of ncRNAs largely unexplored. In this review, we discuss important findings concerning miRNAs and lncRNAs that modulate apoptosis and RN pathways, as well as the miRNA-lncRNA interactions that affect cell death regulation.

Natural Killer Cells Mediate Long-term Kidney Allograft Injury.

BACKGROUND: Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell-mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied. METHODS: A "parent to F1" kidney transplant model was used to study NK cell-mediated transplant rejection. RESULTS: The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 µmol/L to 37.2 ± 5.9 µmol/L, P < 0.001) at 60 days after transplantation. B6Rag-to-CB6F1Rag (B6RagxBALB/cRag) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 µmol/L vs 17.5 ± 5.1 µmol/L in nontransplant Rag mice, P < 0.05). Importantly, depletion of NK cells in Rag1 recipients inhibited kidney injury (24.6 ± 5.5 µmol/L, P < 0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 µmol/L, P < 0.001). CONCLUSIONS: Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

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.

Glycyrrhizic acid ameliorates HMGB1-mediated cell death and inflammation after renal ischemia reperfusion injury.

BACKGROUND: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. METHODS/RESULTS: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. CONCLUSIONS: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.

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.

Serine protease inhibitor-6 inhibits granzyme B-mediated injury of renal tubular cells and promotes renal allograft survival.

BACKGROUND: Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. METHODS AND RESULTS: We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B-mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2) into nephrectomized recipients (H-2). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 µmol/L; n=5; P<0.01), consistent with observed tubular injury and extensive mononuclear cell infiltration. Loss of donor kidney SPI-6 shortened graft survival time (20±19 vs. 66±33 days; n=8-10; P<0.001). CONCLUSION: Our data show for the first time that resistance of kidney TEC to cytotoxic T-cell granzyme B-induced death in vitro and in vivo is mediated by the expression of SPI-6. We suggest that SPI-6 is an important endogenous mechanism to prevent rejection injury from perforin or granzyme B effectors and enhanced PI-9 or SPI-6 expressions by TEC may provide protection from diverse forms of inflammatory kidney injury and promote long-term allograft survival.