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/patologíaRESUMEN
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 SeñalRESUMEN
BACKGROUND: Delayed graft function (DGF) increases the renal allograft failure risk. Late-onset Cytomegalovirus (CMV) infection's effect on the association between DGF and allograft failure has not been determined. METHODS: In this retrospective cohort, we included all renal allograft recipients at London Health Sciences Centre from January 1, 2014 to December 30, 2017, and continued clinical follow-up until February 28, 2020. We determined whether late-onset CMV infection affects the association between DGF and allograft failure in stratified and Cox proportional hazard analyses. RESULTS: Of 384 patients (median age [interquartile range]: 55 [43.3-63]; 38.7% female), 57 recipients (14.8%) were diagnosed with DGF. Patients with DGF were at a greater risk of CMV infection than patients without DGF (22.8% vs. 11.3%, p = .017). Late-onset CMV infection (odds ratio [OR]: 4.7, 95% CI: 2.07-10.68) and rejection (OR: 9.59, 95% CI: 4.15-22.16) significantly increased the risk of allograft failure in recipients with DGF. Patients with DGF had a significantly greater risk of graft failure than those without DGF (17.5% vs. 6.1%, p = .007). In the adjusted Cox hazard model, CMV infection significantly increased the risk of allograft failure (aHR: 3.19, 95% CI: 1.49-6.84). CONCLUSION: Late-onset CMV infection considerably increased the risk of graft failure in patients with DGF. A hybrid preventive model including prophylaxis followed by CMV-specific cell-mediated immunity monitoring may decrease the risk of allograft failure in recipients with DGF.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Riñón , Insuficiencia Renal , Humanos , Femenino , Masculino , Trasplante de Riñón/efectos adversos , Estudios Retrospectivos , Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/epidemiología , Riñón , Citomegalovirus , Progresión de la Enfermedad , AloinjertosRESUMEN
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/metabolismoRESUMEN
BACKGROUND: Solid organ transplant (SOT) recipients are at risk of Pneumocystis pneumonia (PCP). PCP is associated with significant morbidity and mortality. The effect of acute T cell-mediated rejection (TCMR) on post-transplant PCP has not been determined yet. METHODS: In this case-control study, we estimated the risk of PCP following acute TCMR during a lookback period of 180 days. We also determined the effects of contributing factors such as CMV infection. RESULTS: We compared 15 SOT (8 kidney, 4 heart, 2 liver, and 1 kidney-pancreas) recipients with PCP with 60 matched recipients who did not develop PCP (control group) during the study period (December 2013 to February 2016). PCP occurred after a complete course of prophylaxis (ie, late-onset PCP) in 60% of patients. Patients with PCP frequently required intensive care unit (ICU) admission (73.3%). Post-transplant PCP was associated with considerable allograft loss (53.4%) and mortality (26.7%). In the 6-month lookback period, acute TCMR (OR: 13.1, 95% CI: 3.2, 53.2), and CMV infection (OR: 15.1,95% CI: 4.0, 53.2.1) were significantly associated with post-transplant PCP. CONCLUSIONS: Post-transplant PCP is associated with substantial risk of ICU admission, allograft failure, and mortality. Anti-Pneumocystis prophylaxis for at least 6 months following acute TCMR may reduce the risk.
Asunto(s)
Trasplante de Órganos , Pneumocystis carinii , Neumonía por Pneumocystis , Estudios de Casos y Controles , Rechazo de Injerto/epidemiología , Rechazo de Injerto/etiología , Humanos , Trasplante de Órganos/efectos adversos , Neumonía por Pneumocystis/epidemiología , Neumonía por Pneumocystis/etiología , Receptores de TrasplantesRESUMEN
Renal transplant recipients remain at risk of delayed-onset cytomegalovirus (CMV) infection occurring beyond a complete course of prophylaxis. In this retrospective cohort, all 278 patients who received renal allografts from deceased donors from 2014 to 2016 were followed until September 1, 2019. We determined the effect of early-vs late-onset acute rejection (EAR vs LAR [ie, occurring beyond 12 months after transplantation]) on CMV infection and subsequently long-term allograft outcome. Median (IQR) duration of follow-up was 1186.0 (904.7-1531.2) days. Seventy patients including 49 patients with EAR and 21 with LAR received augmented immunosuppression. In the same interval, 40 patients developed CMV infection (36 patients beyond 90 days after transplantation [90%]). In logistic regression analysis, D+/R- CMV serostatus (OR: 5.5, 95% CI: 2.5-12.2) and LAR (OR: 7.9, 95% CI: 2.8-22.2) significantly increased the risk of CMV infection. In Cox proportional hazard model, delayed-onset CMV infection (HR: 2.51, 95% CI: 1.08-5.86) and LAR (HR: 5.46, 95% CI: 2.26-13.14) significantly increased the risk of allograft loss. Patients with LAR are at risk of late-onset CMV infection. Post-LAR, targeted prophylaxis may reduce the risk of CMV infection and subsequently allograft loss. Further studies are required to demonstrate the effect of targeted prophylaxis following LAR.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Riñón , Aloinjertos , Antivirales/uso terapéutico , Citomegalovirus , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/prevención & control , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Humanos , Trasplante de Riñón/efectos adversos , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Pneumocystis pneumonia (PCP) outbreaks may occur in solid organ transplant (SOT) patients. Transmissibility of Pneumocystis jirovecii among SOT and non-SOT patients has not been investigated. Ten SOT (ie, 4 heart, 4 kidney, 2 liver allograft recipients) and 11 non-SOT (ie, 7 HIV-infected, 3 hematologic malignancies, and 1 stem cell transplant) patients with PCP were admitted to London Health Sciences Center (LHSC) from October 2014 to August 2016. We investigated the course of illness and outcome of PCP in SOT and non-SOT patients. Post-transplant PCP was frequently an acute-onset disease (90% vs. 18.2%, p = .01) requiring ICU admission (70% vs. 20%, p = .03) and hemodialysis (60% vs. 0, p = .003). Mortality was more frequent in SOT patients (40% vs. 18.1%, p = .36). Multilocus sequence typing (MLST) demonstrated circulation of a single genotype of P. jirovecii among SOT patients. However, 8 different genotypes were detected from non-SOT patients. Reinstitution of prophylaxis successfully controlled post-transplant cluster until end of observation period in October 2019. No transmission was detected from non-SOT patients to SOT recipients. Detection of a single P. jirovecii genotype from all SOT recipients highlights the likelihood of nosocomial transmission. No source control method is recommended by current guidelines. Improvement of preventive strategies is required.
Asunto(s)
Infección Hospitalaria , Neumonía Asociada a la Atención Médica , Pneumocystis carinii , Neumonía por Pneumocystis , Aloinjertos , Infección Hospitalaria/epidemiología , Infección Hospitalaria/etiología , Genotipo , Humanos , Tipificación de Secuencias Multilocus , Pneumocystis carinii/genética , Neumonía por Pneumocystis/diagnóstico , Neumonía por Pneumocystis/epidemiología , Neumonía por Pneumocystis/etiologíaRESUMEN
Human leukocyte antigen (HLA) class I presentation pathway plays a central role in natural killer (NK) cell and cytotoxic T-cell activities against BK polyomavirus (BKPyV) DNAemia. We determined the risk of sustained BKPyV DNAemia in 175 consecutive renal transplant recipients considering the simultaneous effect of donor/recipient HLA class I antigens and pre- or post-transplant variables. Median (IQR) age was 53 (44-64) years, and 37% of patients were female. 40 patients (22.9%) developed sustained BKPyV DNAemia [median (IQR) viral load: 9740 (4350-17 125) copies/ml]. In the Cox proportional hazard analysis, HLA-A1 (HR: 3.06, 95% CI: 1.51-6.17) and HLA-B35-Cw4 (HR: 4.63, 95% CI: 2.12-10.14) significantly increased the risk of sustained BKPyV DNAemia, while 2 HLA-C mismatches provided a marginally protective effect (HR: 0.32, 95% CI: 0.10-0.98). HLA-Cw4 is a ligand for NK cell inhibitory receptor, and HLA-B35 is in strong linkage disequilibrium with the HLA-Cw4 allele. The association between HLA-B35-Cw4 expression and sustained BKPyV DNAemia supports the important role of cytotoxic T cells and NK cells that would normally control BKPyV activation through engagement with immunoglobulin-like killer receptors (KIRs). Further studies are required to investigate the effect of HLA-C alleles along with NK cell activity against BKPyV DNAemia.
Asunto(s)
Virus BK , Trasplante de Riñón , Infecciones por Polyomavirus , Adulto , Virus BK/genética , Femenino , Antígeno HLA-A1 , Humanos , Trasplante de Riñón/efectos adversos , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/etiología , Receptores de TrasplantesRESUMEN
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/farmacologíaRESUMEN
Targeting the renin-angiotensin system and optimizing tacrolimus exposure are both postulated to improve outcomes in renal transplant recipients (RTRs) by preventing interstitial fibrosis/tubular atrophy (IF/TA). In this multicenter, prospective, open-label controlled trial, adult de novo RTRs were randomized in a 2 × 2 design to low- vs standard-dose (LOW vs STD) prolonged-release tacrolimus and to angiotensin-converting enzyme inhibitors/angiotensin II receptor 1 blockers (ACEi/ARBs) vs other antihypertensive therapy (OAHT). There were 2 coprimary endpoints: the prevalence of IF/TA at month 6 and at month 24. IF/TA prevalence was similar for LOW vs STD tacrolimus at month 6 (36.8% vs 39.5%; P = .80) and ACEi/ARBs vs OAHT at month 24 (54.8% vs 58.2%; P = .33). IF/TA progression decreased significantly with LOW vs STD tacrolimus at month 24 (mean [SD] change, +0.42 [1.477] vs +1.10 [1.577]; P = .0039). Across the 4 treatment groups, LOW + ACEi/ARB patients exhibited the lowest mean IF/TA change and, compared with LOW + OAHT patients, experienced significantly delayed time to first T cell-mediated rejection. Renal function was stable from month 1 to month 24 in all treatment groups. No unexpected safety findings were detected. Coupled with LOW tacrolimus dosing, ACEi/ARBs appear to reduce IF/TA progression and delay rejection relative to reduced tacrolimus exposure without renin-angiotensin system blockade. ClinicalTrials.gov identifier: NCT00933231.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/administración & dosificación , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Trasplante de Riñón/métodos , Tacrolimus/administración & dosificación , Adulto , Aloinjertos , Atrofia , Preparaciones de Acción Retardada , Quimioterapia Combinada , Femenino , Fibrosis , Rechazo de Injerto/etiología , Rechazo de Injerto/inmunología , Humanos , Inmunosupresores/administración & dosificación , Riñón/patología , Riñón/fisiopatología , Trasplante de Riñón/efectos adversos , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/etiología , Pronóstico , Estudios Prospectivos , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Activación ViralRESUMEN
KEY MESSAGE: We demonstrate for the first time that a fully bioactive human IL-37, a newly discovered cytokine acting as a fundamental inhibitor of innate immunity, can be recombinantly produced in plant cells. Interleukin 37 (IL-37), a newly discovered member of the interleukin (IL)-1 family of cytokines, plays a pivotal role in limiting innate inflammation and suppressing acquired immune responses, thus holding high potential for treating a wide array of human inflammatory and autoimmune disorders. In this study, we have developed transgenic plants as a novel expression platform for production of human IL-37 (IL-37). Plant transformation vectors synthesizing various forms of the b isoform of IL-37, including an unprocessed full-length precursor form (proIL-37b), a mature form (matIL-37b) and an IL-37 fusion protein in which IL-37b was fused to soybean agglutinin (SBA-IL-37b), have been constructed and introduced into tobacco plants. The expression of all forms of IL-37b was driven by a strong constitutive 35S promoter. Transgenic tobacco plants were generated with each of these constructs. Depending on the form of IL-37b being produced, the expression level of proIL-37b reached approximately 1% of TSP, while matIL-37b expression was substantially lower (0.01% TSP). Fusion to SBA substantially increased the expression of matIL-37b, with the expression level of fusion protein accounting for 1% of TSP. Functional analysis using a cell-based in vitro assay showed that plant-made matIL-37b and proIL-37b are both biologically active, but plant-made matIL-37b exhibited significantly greater biological activity than proIL-37b. These results demonstrate that plants have great potential of being a green bioreactor for low-cost, large-scale production of biologically active IL-37.
Asunto(s)
Interleucina-1/metabolismo , Citocinas/metabolismo , Humanos , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Interleucina-1/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
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 SeñalRESUMEN
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/patologíaRESUMEN
The incretin hormone glucagon-like peptide-1 (GLP-1) is recognized as a promising candidate for the treatment of type 2 diabetes (T2D), with one of its mimetics, exenatide (synthetic exendin-4) having already been licensed for clinical use. We seek to further improve the therapeutic efficacy of exendin-4 (Ex-4) using innovative fusion protein technology. Here, we report the production in plants a fusion protein containing Ex-4 coupled with human transferrin (Ex-4-Tf) and its characterization. We demonstrated that plant-made Ex-4-Tf retained the activity of both proteins. In particular, the fusion protein stimulated insulin release from pancreatic ß-cells, promoted ß-cell proliferation, stimulated differentiation of pancreatic precursor cells into insulin-producing cells, retained the ability to internalize into human intestinal cells and resisted stomach acid and proteolytic enzymes. Importantly, oral administration of partially purified Ex-4-Tf significantly improved glucose tolerance, whereas commercial Ex-4 administered by the same oral route failed to show any significant improvement in glucose tolerance in mice. Furthermore, intraperitoneal (IP) injection of Ex-4-Tf showed a beneficial effect in mice similar to IP-injected Ex-4. We also showed that plants provide a robust system for the expression of Ex-4-Tf, producing up to 37 µg prEx-4-Tf/g fresh leaf weight in transgenic tobacco and 137 µg prEx-4-Tf/g freshweight in transiently transformed leaves of N. benthamiana. These results indicate that Ex-4-Tf holds substantial promise as a new oral therapy for type 2 diabetes. The production of prEx-4-Tf in plants may offer a convenient and cost-effective method to deliver the antidiabetic medicine in partially processed plant food products.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Nicotiana/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/uso terapéutico , Administración Oral , Animales , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Bacteriano/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Electroforesis en Gel de Poliacrilamida , Endocitosis/efectos de los fármacos , Enterocitos/citología , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Mucosa Gástrica/metabolismo , Vectores Genéticos/metabolismo , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Homeostasis/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones Endogámicos C57BL , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/farmacología , Nicotiana/genéticaRESUMEN
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.
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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 ArribaRESUMEN
INTRODUCTION AND OBJECTIVES: The ImmuKnow assay measures cell-mediated immunity by quantifying ATP release from CD4+ T-cells in peripheral blood. Herein, we hypothesized that this assay could predict complications associated with over-/under-immunosuppression in patients with kidney transplant (KT). METHODS: Sixty-seven patients undergoing KT were recruited prospectively and had ATP levels measured preoperatively, and at specified intervals over two months. Clinicians were blinded to ATP levels. Clinical events including rejection and infection/cancer were documented with a median follow-up of 21 months. Parameters including absolute ATP levels and changes in ATP patterns (slopes, delta) were analyzed. Association between ATP parameters and clinical outcomes was compared using the likelihood-ratio test and Kaplan-Meier curves. RESULTS: Absolute ATP values postoperatively had poor predictive value with regard to rejection or infection/malignancy. As well, changes in ATP values were poorly associated with complications. Importantly, patients with pre-transplant ATP values <300 ng/mL had significantly less rejection episodes vs. those with ATP values >300 ng/mL (p < 0.0001). CONCLUSIONS: For the first time, we have evidence that a preoperative ImmuKnow level can stratify patients with KT into low/high risk groups for rejection. Future studies used to assess the utility of this assay to design individualized immunosuppressive regimens are required.
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Adenosina Trifosfato/metabolismo , Rechazo de Injerto/diagnóstico , Inmunoensayo/métodos , Enfermedades Renales/complicaciones , Trasplante de Riñón/efectos adversos , Complicaciones Posoperatorias/diagnóstico , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Femenino , Estudios de Seguimiento , Rechazo de Injerto/etiología , Rechazo de Injerto/mortalidad , Humanos , Enfermedades Renales/cirugía , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/mortalidad , Pronóstico , Estudios Prospectivos , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
The immune system is regulated by a complex set of genetic, molecular, and cellular interactions. Rapid advances in the study of immunity and its network of interactions have been boosted by a spectrum of "omics" technologies that have generated huge amounts of data that have reached the status of big data (BD). With recent developments in artificial intelligence (AI), theoretical and clinical breakthroughs could emerge. Analyses of large data sets with AI tools will allow the formulation of new testable hypotheses open new research avenues and provide innovative strategies for regulating immunity and treating immunological diseases. This includes diagnosis and identification of rare diseases, prevention and treatment of autoimmune diseases, allergic disorders, infectious diseases, metabolomic disorders, cancer, and organ transplantation. However, ethical and regulatory challenges remain as to how these studies will be used to advance our understanding of basic immunology and how immunity might be regulated in health and disease. This will be particularly important for entities in which the complexity of interactions occurring at the same time and multiple cellular pathways have eluded conventional approaches to understanding and treatment. The analyses of BD by AI are likely to be complicated as both positive and negative outcomes of regulating immunity may have important ethical ramifications that need to be considered. We suggest there is an immediate need to develop guidelines as to how the analyses of immunological BD by AI tools should guide immune-based interventions to treat various diseases, prevent infections, and maintain health within an ethical framework.
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Enfermedades Autoinmunes , Hipersensibilidad , Humanos , Inteligencia Artificial , Macrodatos , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/terapia , Comunicación CelularRESUMEN
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
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.
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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 fármacosRESUMEN
PURPOSE: Organ cold storage and subsequent transplantation are associated with significant ischemia-reperfusion injury, leading to cell death, graft inflammation and decreased graft function. MATERIALS AND METHODS: CORM-3s reduce oxidative stress and prevent inflammation in kidneys stored at 4C and subsequently transplanted. Graft survival and function are markedly improved compared to kidneys preserved and stored in University of Wisconsin solution alone. We determined whether CORM-3 has direct antiapoptotic effects on in vitro preparations of human HUVECs exposed to anoxic conditions. We also determined whether direct administration of CORM-3 to renal grafts before and/or after cold storage would prevent renal damage during the transplantation process. RESULTS: CORM-3 supplementation led to a significantly increased frequency of live cells (mean ± SD 72.3% ± 1.9%, p <0.01), reduced apoptosis (14.9% ± 6.1%, p <0.01) and decreased mitochondrial transmembrane potential (40.2% ± 7.2%, p <0.05) in HUVECs exposed to 20 hours of cold storage compared to controls (11.6% ± 3.5%, 82.2% ± 2.3% and 78.2% ± 3.2%, respectively). In keeping with this antiapoptotic effect CORM-3 supplementation led to a mean 7.4 ± 2.1-fold up-regulation in Bcl-2 gene expression. CORM-3 supplementation in standard preservation solution was most beneficial at initial ischemic injury and before cold storage exposure. However, additional reflushing before vascular reperfusion showed an additive benefit to graft survival and function after transplantation. This was confirmed by decreased glomerular and tubular necrosis, and apoptosis in double flushed grafts. CONCLUSIONS: CORM-3 supplementation in standard University of Wisconsin solution has a significant impact on decreasing cellular and graft injury, and improving survival through its antiapoptotic effects, which are likely mediated through mitochondrial membrane stabilization.