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Diabetes is a global public health burden and is characterized clinically by relative or absolute insulin deficiency. Therapeutic agents that stimulate insulin secretion and improve insulin sensitivity are in high demand as treatment options. CD47 is a cell surface glycoprotein implicated in multiple cellular functions including recognition of self, angiogenesis, and nitric oxide signaling; however, its role in the regulation of insulin secretion remains unknown. Here, we demonstrate that CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic ß cells and that it can be pharmacologically exploited to boost insulin secretion in both models. CD47 depletion stimulated insulin granule exocytosis via activation of the Rho GTPase Cdc42 in ß cells and improved glucose clearance and insulin sensitivity in vivo. CD47 blockade enhanced syngeneic islet transplantation efficiency and expedited the return to euglycemia in streptozotocin-induced diabetic mice. Further, anti-CD47 antibody treatment delayed the onset of diabetes in nonobese diabetic (NOD) mice and protected them from overt diabetes. Our findings identify CD47 as a regulator of insulin secretion, and its manipulation in ß cells offers a therapeutic opportunity for diabetes and islet transplantation by correcting insulin deficiency.
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Diabetes Mellitus Experimental , Resistencia a la Insulina , Células Secretoras de Insulina , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Humanos , Ratones , Antígeno CD47/metabolismo , Diabetes Mellitus Experimental/terapia , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones Endogámicos NODRESUMEN
Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation and will limit future beta cell replacement strategies. We applied hyperspectral fluorescent microscopy to determine whether we could non-invasively detect islet damage induced by oxidative stress, hypoxia, cytokine injury, and warm ischaemia, and so predict transplant outcomes in a mouse model. In assessing islet spectral signals for NAD(P)H, flavins, collagen-I, and cytochrome-C in intact islets, we distinguished islets compromised by oxidative stress (ROS) (AUC = 1.00), hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), and warm ischaemia (AUC = 0.94) compared to islets harvested from pristine anaesthetised heart-beating mouse donors. Significantly, with unsupervised assessment we defined an autofluorescent score for ischaemic islets that accurately predicted the restoration of glucose control in diabetic recipients following transplantation. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. These data show that the pre-transplant hyperspectral imaging of islet autofluorescence has promise for predicting islet viability and transplant success.
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Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Animales , Ratones , Imágenes Hiperespectrales , Islotes Pancreáticos/diagnóstico por imagen , Citocinas , HipoxiaRESUMEN
AIMS/HYPOTHESIS: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. METHODS: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (ßp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (ßNEMOKO mice), as well as ßA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. RESULTS: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as ßp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in ßp65KO islets. CONCLUSIONS/INTERPRETATION: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.
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Intolerancia a la Glucosa , Factor de Transcripción ReIA , Animales , Humanos , Ratones , Cromatina , Glucosa , FN-kappa B/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
The evolutionarily recent dispersal of anatomically modern humans (AMH) out of Africa (OoA) and across Eurasia provides a unique opportunity to examine the impacts of genetic selection as humans adapted to multiple new environments. Analysis of ancient Eurasian genomic datasets (~1,000 to 45,000 y old) reveals signatures of strong selection, including at least 57 hard sweeps after the initial AMH movement OoA, which have been obscured in modern populations by extensive admixture during the Holocene. The spatiotemporal patterns of these hard sweeps provide a means to reconstruct early AMH population dispersals OoA. We identify a previously unsuspected extended period of genetic adaptation lasting ~30,000 y, potentially in the Arabian Peninsula area, prior to a major Neandertal genetic introgression and subsequent rapid dispersal across Eurasia as far as Australia. Consistent functional targets of selection initiated during this period, which we term the Arabian Standstill, include loci involved in the regulation of fat storage, neural development, skin physiology, and cilia function. Similar adaptive signatures are also evident in introgressed archaic hominin loci and modern Arctic human groups, and we suggest that this signal represents selection for cold adaptation. Surprisingly, many of the candidate selected loci across these groups appear to directly interact and coordinately regulate biological processes, with a number associated with major modern diseases including the ciliopathies, metabolic syndrome, and neurodegenerative disorders. This expands the potential for ancestral human adaptation to directly impact modern diseases, providing a platform for evolutionary medicine.
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Hombre de Neandertal , Humanos , Animales , África , Aclimatación , Arabia , Selección GenéticaRESUMEN
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
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Técnicas Biosensibles , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas Biosensibles/métodosRESUMEN
Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.
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Lesión Renal Aguda , FN-kappa B , Humanos , Ratones , Animales , FN-kappa B/metabolismo , Factores de Transcripción/genética , Ubiquitina , Estudio de Asociación del Genoma Completo , Ligasas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Lesión Renal Aguda/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Islets transplanted for type-1 diabetes have their viability reduced by warm ischemia, dimethyloxalylglycine (DMOG; hypoxia model), oxidative stress and cytokine injury. This results in frequent transplant failures and the major burden of patients having to undergo multiple rounds of treatment for insulin independence. Presently there is no reliable measure to assess islet preparation viability prior to clinical transplantation. We investigated deep morphological signatures (DMS) for detecting the exposure of islets to viability compromising insults from brightfield images. Accuracies ranged from 98 % to 68 % for; ROS damage, pro-inflammatory cytokines, warm ischemia and DMOG. When islets were disaggregated to single cells to enable higher throughput data collection, good accuracy was still obtained (83-71 %). Encapsulation of islets reduced accuracy for cytokine exposure, but it was still high (78 %). Unsupervised modelling of the DMS for islet preparations transplanted into a syngeneic mouse model was able to predict whether or not they would restore glucose control with 100 % accuracy. Our strategy for constructing DMS' is effective for the assessment of islet pre-transplant viability. If translated into the clinic, standard equipment could be used to prospectively identify non-functional islet preparations unable to contribute to the restoration of glucose control and reduce the burden of unsuccessful treatments.
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Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. ARTICLE HIGHLIGHTS: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.
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Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Porcinos , Humanos , Animales , Ratones , Trasplante de Islotes Pancreáticos/métodos , Supervivencia de Injerto , Islotes Pancreáticos/irrigación sanguínea , Diabetes Mellitus Tipo 1/cirugía , ColágenoRESUMEN
The inflammatory microenvironment of solid tumors creates a protumorigenic milieu that resembles chronic inflammation akin to a subverted wound healing response. Here, we investigated the effect of converting the tumor microenvironment from a chronically inflamed state to one of acute microbial inflammation by injecting microbial bioparticles directly into tumors. Intratumoral microbial bioparticle injection led to rapid and dramatic changes in the tumor immune composition, the most striking of which was a substantial increase in the presence of activated neutrophils. In situ photoconversion and intravital microscopy indicated that tumor neutrophils transiently switched from sessile producers of VEGF to highly motile neutrophils that clustered to make neutrophil-rich domains in the tumor. The neutrophil clusters remodeled tumor tissue and repressed tumor growth. Single-cell transcriptional analysis of microbe-stimulated neutrophils showed a profound shift in gene expression towards heightened activation and antimicrobial effector function. Microbe-activated neutrophils also upregulated chemokines known to regulate neutrophil and CD8+ T-cell recruitment. Microbial therapy also boosted CD8+ T-cell function and enhanced the therapeutic benefit of checkpoint inhibitor therapy in tumor-bearing mice and provided protection in a model of tumor recurrence. These data indicate that one of the major effector mechanisms of microbial therapy is the conversion of tumor neutrophils from a wound healing to an acutely activated cytotoxic phenotype, highlighting a rationale for broader deployment of microbial therapy in the treatment of solid cancers. SIGNIFICANCE: Intratumoral injection of microbial bioparticles stimulates neutrophil antitumor functions, suggesting pathways for optimizing efficacy of microbial therapies and paving the way for their broader utilization in the clinic.
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Neoplasias , Neutrófilos , Ratones , Animales , Neutrófilos/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Linfocitos T CD8-positivos , Inflamación/patología , Fenotipo , Infiltración Neutrófila , Microambiente TumoralAsunto(s)
Linfocitos T , Donantes de Tejidos , Humanos , Rechazo de Injerto , Antígenos HLA , Isoanticuerpos , Supervivencia de InjertoRESUMEN
The role of natural selection in shaping biological diversity is an area of intense interest in modern biology. To date, studies of positive selection have primarily relied on genomic datasets from contemporary populations, which are susceptible to confounding factors associated with complex and often unknown aspects of population history. In particular, admixture between diverged populations can distort or hide prior selection events in modern genomes, though this process is not explicitly accounted for in most selection studies despite its apparent ubiquity in humans and other species. Through analyses of ancient and modern human genomes, we show that previously reported Holocene-era admixture has masked more than 50 historic hard sweeps in modern European genomes. Our results imply that this canonical mode of selection has probably been underappreciated in the evolutionary history of humans and suggest that our current understanding of the tempo and mode of selection in natural populations may be inaccurate.
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Hominidae , Selección Genética , Animales , Humanos , Evolución Biológica , Genoma Humano , GenómicaRESUMEN
Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic ß-cells. Although ß-cell targeted autoimmune processes and ß-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports ß-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing ß-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced ß-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine ß-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of ß-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Animales , Humanos , Ratones , Supervivencia Celular , Desmogleínas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , EstreptozocinaRESUMEN
Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.
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Infertilidad Femenina , Animales , Femenino , Regulación de la Expresión Génica , Humanos , Infertilidad Femenina/genética , Inflamación/genética , Ratones , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: The cytokine IL-33 is an activator of innate lymphoid cells 2 (ILC2s) in innate immunity and allergic inflammation. B cell activating factor (BAFF) plays a central role in B cell proliferation and differentiation, and high levels of this protein cause excess antibody production, including IgA. BAFF-transgenic mice overexpress BAFF and spontaneously develop glomerulonephritis that resembles human IgA nephropathy. METHODS: We administered IL-33 or PBS to wild-type and BAFF-transgenic mice. After treating Rag1-deficient mice with IL-33, with or without anti-CD90.2 to preferentially deplete ILC2s, we isolated splenocytes, which were adoptively transferred into BAFF-transgenic mice. RESULTS: BAFF-transgenic mice treated with IL-33 developed more severe kidney dysfunction and proteinuria, glomerular sclerosis, tubulointerstitial damage, and glomerular deposition of IgA and C3. Compared with wild-type mice, BAFF-transgenic mice exhibited increases of CD19+ B cells in spleen and kidney and ILC2s in kidney and intestine, which were further increased by administration of IL-33. Administering IL-33 to wild-type mice had no effect on kidney function or histology, nor did it alter the number of ILC2s in spleen, kidney, or intestine. To understand the role of ILC2s, splenocytes were transferred from IL-33-treated Rag1-deficient mice into BAFF-transgenic mice. Glomerulonephritis and IgA deposition were exacerbated by transfer of IL-33-stimulated Rag1-deficient splenocytes, but not by ILC2 (anti-CD90.2)-depleted splenocytes. Wild-type mice infused with IL-33-treated Rag1-deficient splenocytes showed no change in kidney function or ILC2 numbers or distribution. CONCLUSIONS: IL-33-expanded ILC2s exacerbated IgA glomerulonephritis in a mouse model. These findings indicate that IL-33 and ILC2s warrant evaluation as possible mediators of human IgA nephropathy.
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Glomerulonefritis por IGA , Interleucina-33 , Animales , Factor Activador de Células B , Femenino , Proteínas de Homeodominio/genética , Humanos , Inmunidad Innata , Inmunoglobulina A , Interleucina-4 , Linfocitos , Masculino , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. METHODS: We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-κB activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. RESULTS: Forcing the expression of A20 in NPI with Ad5 vector blocked NF-κB activation by inhibiting IκBα phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-κB-dependent gene expression without adverse impact upon NPI maturation. CONCLUSION: We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.
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Células Secretoras de Insulina , Islotes Pancreáticos , Animales , Dependovirus , Terapia Genética , Vectores Genéticos , Xenoinjertos , Inflamación , Ratones , Porcinos , Trasplante Heterólogo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
The CRISPR-Cas9 and related systems offer a unique genome-editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome. Despite its molecular precision, temporal and spatial control of gene editing with the CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with the CRISPR-Cas9 system. We demonstrated its efficient protein release by respectively assessing the targeted knockout of the eGFP gene in human HEK293/GFP cells and the TNFAIP3 gene in TNFα-induced HEK293 cells. We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). These findings indicate that light-triggered liposomes may have new options for precise control of CRISPR-Cas9 release and editing.
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Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Liposomas/química , Animales , Embrión no Mamífero/metabolismo , Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Luz , Oxígeno Singlete/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.
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Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Pez Cebra/genética , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/genética , Secuencia Conservada , Evolución Molecular , Variación Genética , Humanos , Inflamación/etiología , Inflamación/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Animales , Modelos Genéticos , Mutación Missense , FN-kappa B/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/deficiencia , Pez Cebra/fisiología , Proteínas de Pez Cebra/deficienciaRESUMEN
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects but is restricted in its clinical application by a limiting supply of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signaling thresholds. Here, we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for ~45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon Tregs and was antigen specific, and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signaling, graft hyperinflammation, and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators, with no impact on glucose-stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signaling thresholds to favor immune tolerance and promotes islet allogeneic survival. Clinically, this would allow for reduced immunosuppression and support the use of alternate islet sources.
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Tolerancia Inmunológica/fisiología , Trasplante de Islotes Pancreáticos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Supervivencia de Injerto , Humanos , Trasplante HomólogoRESUMEN
Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.