RESUMEN

NK cells participate in ischemia reperfusion injury (IRI) and transplant rejection. Endogenous regulatory systems may exist to attenuate NK cell activation and cytotoxicity in IRI associated with kidney transplantation. A greater understanding of NK regulation will provide insights in transplant outcomes and could direct new therapeutic strategies. Kidney tubular epithelial cells (TECs) may negatively regulate NK cell activation by their surface expression of a complex family of C-type lectin-related proteins (Clrs). We have found that Clr-b and Clr-f were expressed by TECs. Clr-b was upregulated by inflammatory cytokines TNFα and IFNγ in vitro. Silencing of both Clr-b and Clr-f expression using siRNA resulted in increased NK cell killing of TECs compared to silencing of either Clr-b or Clr-f alone (p < 0.01) and when compared to control TECs (p < 0.001). NK cells treated in vitro with soluble Clr-b and Clr-f proteins reduced their capacity to kill TECs (p < 0.05). Hence, NK cell cytotoxicity can be inhibited by Clr proteins on the surface of TECs. Our study suggests a synergistic effect of Clr molecules in regulating NK cell function in renal cells and this may represent an important endogenous regulatory system to limit NK cell-mediated organ injury during inflammation.

RESUMEN

Cardiac ischemia results in anaerobic metabolism and lactic acid accumulation and with time, intracellular and extracellular acidosis. Ischemia and subsequent reperfusion injury (IRI) lead to various forms of programmed cell death. Necroptosis is a major form of programmed necrosis that worsens cardiac function directly and also promotes inflammation by the release of cellular contents. Potential effects of increasing acidosis on programmed cell death and their specific components have not been well studied. While apoptosis is caspase-dependent, in contrast, necroptosis is mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1/3). In our study, we observed that at physiological pH = 7.4, caspase-8 inhibition did not prevent TNFα-induced cell death in mouse cardiac vascular endothelial cells (MVECs) but promoted necroptotic cell death. As expected, necroptosis was blocked by RIPK1 inhibition. However, at pH = 6.5, TNFα induced an apoptosis-like pattern which was inhibited by caspase-8 inhibition. Interestingly phosphorylation of necroptotic molecules RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) was enhanced in an acidic pH environment. However, RIPK3 and MLKL phosphorylation was self-limited which may have limited their participation in necroptosis. In addition, an acidic pH promoted apoptosis-inducing factor (AIF) cleavage and nuclear translocation. AIF RNA silencing inhibited cell death, supporting the role of AIF in this cell death. In summary, our study demonstrated that the pH of the micro-environment during inflammation can bias cell death pathways by altering the function of necroptosis-related molecules and promoting AIF-mediated cell death. Further insights into the mechanisms by which an acidic cellular micro-environment influences these and perhaps other forms of regulated cell death, may lead to therapeutic strategies to attenuate IRI.

Asunto(s)

Apoptosis , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Factor de Necrosis Tumoral alfa , Animales , Concentración de Iones de Hidrógeno , Apoptosis/efectos de los fármacos , Necroptosis/efectos de los fármacos , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/metabolismo , Caspasa 8/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Células Cultivadas , Fosforilación , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología

RESUMEN

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.

Asunto(s)

Dinaminas , Rechazo de Injerto , Trasplante de Corazón , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Ratones , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Trasplante de Corazón/efectos adversos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Dinaminas/metabolismo , Dinaminas/genética , Mitocondrias/metabolismo , Células Endoteliales/metabolismo , Masculino , Ratones Endogámicos C57BL , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Transducción de Señal

RESUMEN

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.

Asunto(s)

Factor Inductor de la Apoptosis/metabolismo , Necroptosis , Peptidil-Prolil Isomerasa F/metabolismo , Animales , Factor Inductor de la Apoptosis/antagonistas & inhibidores , Factor Inductor de la Apoptosis/genética , Hipoxia de la Célula , Núcleo Celular/metabolismo , Peptidil-Prolil Isomerasa F/deficiencia , Peptidil-Prolil Isomerasa F/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Interferón gamma/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/citología , Modelos Biológicos , Necroptosis/efectos de los fármacos , Oxígeno/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

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.

Asunto(s)

Trasplante de Corazón , Receptor Toll-Like 3 , Animales , Apoptosis , Muerte Celular , Trasplante de Corazón/efectos adversos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Donantes de Tejidos , Receptor Toll-Like 3/metabolismo

RESUMEN

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.

Asunto(s)

Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Neoplasias Mamarias Experimentales/genética , MicroARNs/metabolismo , Regiones no Traducidas 3' , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Cultivadas , Citocinas/genética , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Inmunoterapia/métodos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo

RESUMEN

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.

Asunto(s)

Apoptosis , Caspasa 8/metabolismo , Células Epiteliales/enzimología , Enfermedades Renales/enzimología , Túbulos Renales/enzimología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Daño por Reperfusión/enzimología , Animales , Apoptosis/efectos de los fármacos , Caspasa 8/genética , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Enfermedades Renales/genética , Enfermedades Renales/patología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Transducción de Señal

RESUMEN

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.

Asunto(s)

Permeabilidad de la Membrana Celular , Células Endoteliales/patología , Rechazo de Injerto/etiología , Trasplante de Corazón/efectos adversos , Mitocondrias/patología , Necroptosis , Peptidil-Prolil Isomerasa F/metabolismo , Aloinjertos , Animales , Peptidil-Prolil Isomerasa F/genética , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Supervivencia de Injerto , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitocondrias/inmunología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Donantes de Tejidos , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

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.

Asunto(s)

Apoptosis , Citoplasma/metabolismo , Células Endoteliales/citología , Necrosis , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Concentración de Iones de Hidrógeno , Inflamación , Ratones , Proteínas Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo

RESUMEN

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.

Asunto(s)

Apoptosis , Linfocitos T CD4-Positivos/metabolismo , Citotoxicidad Inmunológica , Células Endoteliales/enzimología , Rechazo de Injerto/enzimología , Trasplante de Corazón/efectos adversos , Microvasos/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Aloinjertos , Animales , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/patología , Proteína Ligando Fas/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Granzimas/metabolismo , Activación de Linfocitos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/metabolismo , Microvasos/patología , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

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.

RESUMEN

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.

Asunto(s)

ARN no Traducido/metabolismo , Animales , Muerte Celular , Humanos , MicroARNs/metabolismo , Modelos Biológicos , ARN no Traducido/genética

RESUMEN

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.

Asunto(s)

Rechazo de Injerto , Trasplante de Riñón/efectos adversos , Células Asesinas Naturales/inmunología , Aloinjertos , Animales , Apoptosis , Túbulos Renales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK/análisis , Osteopontina/fisiología

RESUMEN

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.

Asunto(s)

Citocinas/genética , Interleucina-18/metabolismo , Interleucina-1/metabolismo , Riñón/inmunología , Riñón/lesiones , Daño por Reperfusión/inmunología , Animales , Línea Celular , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1/antagonistas & inhibidores , Interleucina-1/genética , Riñón/irrigación sanguínea , Túbulos Renales/inmunología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Receptores de Interleucina-18/genética , Daño por Reperfusión/genética , Daño por Reperfusión/patología

RESUMEN

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.

Asunto(s)

Trasplante de Corazón/efectos adversos , Interleucina-6/metabolismo , Células Asesinas Naturales/inmunología , Traslado Adoptivo , Aloinjertos , Animales , Linfocitos B/inmunología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/patología , Isquemia Fría/efectos adversos , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/inmunología , Rechazo de Injerto/etiología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Interleucina-6/deficiencia , Interleucina-6/genética , Células Asesinas Naturales/trasplante , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología , Donantes de Tejidos

RESUMEN

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.

Asunto(s)

Lesión Renal Aguda/genética , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Ácido Glicirrínico/farmacología , Proteína HMGB1/efectos de los fármacos , Riñón/efectos de los fármacos , Daño por Reperfusión/genética , Lesión Renal Aguda/metabolismo , Animales , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Riñón/metabolismo , Ratones , Daño por Reperfusión/metabolismo , Regulación hacia Arriba

RESUMEN

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.

Asunto(s)

Antígeno B7-1/genética , Antígenos CD40/genética , Silenciador del Gen , Rechazo de Injerto/prevención & control , Trasplante de Corazón , Animales , Secuencia de Bases , Cartilla de ADN , Citometría de Flujo , Rechazo de Injerto/genética , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

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.

Asunto(s)

Supervivencia de Injerto , Granzimas/antagonistas & inhibidores , Trasplante de Riñón , Túbulos Renales/patología , Proteínas de la Membrana/fisiología , Serpinas/fisiología , Aloinjertos , Animales , Células Cultivadas , Células Epiteliales/patología , Rechazo de Injerto , Granzimas/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C

RESUMEN

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αß(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVß2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quimerismo/efectos de los fármacos , Supresión Clonal/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Trasplante de Médula Ósea/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Supresión Clonal/efectos de los fármacos , Ciclofosfamida/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/inmunología , Inmunosupresores/uso terapéutico , Células Asesinas Naturales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Perforina/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Trasplante de Piel/inmunología , Linfocitos T Reguladores/efectos de los fármacos

RESUMEN

RNA interference (RNAi) is an endogenous mechanism of cellular RNA control through degradation of specific messenger RNA sequences. This process of gene silencing may be exploited by the use of small interfering RNA (siRNA) to mediate precise control of targeted cellular functions. The nature of transplantation leads invariably to tissue injury, as organs are damaged by the loss of blood supply and resultant ischemia associated with the procurement procedure. Upon reperfusion, an inflammatory program is activated, and subsequent injury results in delayed graft function and, potentially, organ failure. Many of the molecular components in ischemia-reperfusion injury (IRI) have been identified, but effective therapeutics are not currently available. Accumulating evidence supports a role for siRNA in controlling IRI, as siRNA is specific, relatively low in toxicity, and limited in duration of effect. The capacity of siRNA to control IRI-related transcription factors, cell death and apoptosis, complement factors, and oxidative stress molecules supports the concept that RNAi-based therapeutics represent a novel and promising strategy for the control of IRI. However, there are issues of RNAi strategies, including siRNA design, "off-target" effects, and delivery that merit consideration in approaching IRI with gene silencing. This review will provide an overview of current concepts in RNAi and the potential application to IRI in solid organ transplantation.

Asunto(s)

Trasplante de Órganos , ARN Interferente Pequeño/uso terapéutico , Daño por Reperfusión/prevención & control , Sistemas de Liberación de Medicamentos , Humanos , MicroARNs/fisiología , Trasplante de Órganos/fisiología , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/fisiología , Daño por Reperfusión/genética