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1.
BMC Biol ; 21(1): 8, 2023 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635667

RESUMO

BACKGROUND: Regulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged. RESULTS: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2. CONCLUSIONS: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.


Assuntos
Linfócitos T CD8-Positivos , Linfócitos T Reguladores , Ratos , Animais , Linfócitos T Reguladores/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo
2.
J Immunol ; 207(2): 421-435, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34233909

RESUMO

Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas de Membrana/imunologia , Animais , Endossomos/imunologia , Feminino , Genes MHC da Classe II/imunologia , Complexo de Golgi/imunologia , Imunidade Inata/imunologia , Mucosa Intestinal/imunologia , Canais Iônicos/imunologia , Linfócitos/imunologia , Lisossomos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th17/imunologia , Tretinoína/imunologia
3.
Gene Ther ; 29(9): 520-535, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35105949

RESUMO

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Cromatografia Líquida , Distrofina/genética , Distrofina/metabolismo , Complexo de Proteínas Associadas Distrofina/metabolismo , Terapia Genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Ratos , Espectrometria de Massas em Tandem
4.
Int J Mol Sci ; 24(1)2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36613633

RESUMO

To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Animals from a new transgenic rat line expressing the green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received a single oral bolus of miR-320-3p or miR-375-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) on D-12. The pups were weaned early (D-15), or regularly (D-30). The expression of relevant miRNA, mRNAs, chromatin complexes, and duodenal cell density were assessed at 8 h post-inoculation and on D-45. The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p < 0.05). On regular weaning, on D-45, miR-320-3p and 375-3p were found to be downregulated in the stomach and upregulated in the hypothalamus (p < 0.001), whereas miR-320-3p was upregulated in the duodenum. After early weaning, miR-320-3p and miR-375-3p were downregulated in the stomach and the duodenum, but upregulated in the hypothalamus and the hippocampus. Combination of miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the female brain stem, miR-320-3p, miR-504, and miR-16-5p levels were all upregulated. Investigating the oral miRNA-320-3p loads in the duodenal cell lineage paved the way for designing new therapeutics to avoid unexpected long-term impacts on the brain.


Assuntos
Aminoglicosídeos , MicroRNAs , Animais , Feminino , Ratos , Antibacterianos , Encéfalo/metabolismo , Cromatina , Lactação , MicroRNAs/administração & dosagem , Desmame
5.
Circ Res ; 125(7): 678-695, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31347976

RESUMO

RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K+ channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.


Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/genética , Mutação com Perda de Função , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Potenciais de Ação , Animais , Pressão Sanguínea , Feminino , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Ratos , Ratos Sprague-Dawley , Survivina/genética , Survivina/metabolismo , Vasoconstrição , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
6.
Am J Respir Cell Mol Biol ; 63(1): 118-131, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32209028

RESUMO

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.


Assuntos
Pneumopatia Veno-Oclusiva/metabolismo , Pneumopatia Veno-Oclusiva/patologia , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/metabolismo , Pulmão/patologia , Mutação/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/metabolismo
7.
FASEB J ; 33(12): 13492-13502, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31560858

RESUMO

Hereditary aceruloplasminemia (HA), related to mutations in the ceruloplasmin (Cp) gene, leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but it is not found in hepatic and splenic macrophages in humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA. A clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) knockout of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized. In Cp-/- rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly as in humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp-/- rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin-bound iron level was found. Copper was decreased in the spleen, whereas manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA, encouraging the search for additional mechanisms.-Kenawi, M., Rouger, E., Island, M.-L., Leroyer, P., Robin, F., Remy, S., Tesson, L., Anegon, I., Nay, K., Derbré, F., Brissot, P., Ropert, M., Cavey, T., Loréal, O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.


Assuntos
Ceruloplasmina/deficiência , Modelos Animais de Doenças , Distúrbios do Metabolismo do Ferro/complicações , Sobrecarga de Ferro/patologia , Ferro/metabolismo , Macrófagos/patologia , Doenças Neurodegenerativas/complicações , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Ceruloplasmina/antagonistas & inibidores , Ceruloplasmina/genética , Feminino , Ferro/análise , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/patologia , Sobrecarga de Ferro/etiologia , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Masculino , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Ratos , Ratos Sprague-Dawley , Homologia de Sequência , Baço/metabolismo , Baço/patologia
8.
Liver Transpl ; 25(1): 98-110, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358068

RESUMO

Hepatocyte transplantation (HT) has emerged as a promising alternative to orthotopic liver transplantation, yet liver preconditioning is needed to promote hepatocyte engraftment. A method of temporary occlusion of the portal flow called reversible portal vein embolization (RPVE) has been demonstrated to be an efficient method of liver preconditioning. By providing an additional regenerative stimulus, repeated reversible portal vein embolization (RRPVE) could further boost liver engraftment. The aim of this study was to determine the efficiency of liver engraftment of transplanted hepatocytes after RPVE and RRPVE in a rat model. Green fluorescent protein-expressing hepatocytes were isolated from transgenic rats and transplanted into 3 groups of syngeneic recipient rats. HT was associated with RPVE in group 1, with RRPVE in group 2, and with sham embolization in the sham group. Liver engraftment was assessed at day 28 after HT on liver samples after immunostaining. Procedures were well tolerated in all groups. RRPVE resulted in increased engraftment rate in total liver parenchyma compared with RPVE (3.4% ± 0.81% versus 1.4% ± 0.34%; P < 0.001). In conclusion, RRPVE successfully enhanced hepatocyte engraftment after HT and could be helpful in the frame of failure of HT due to low cell engraftment.


Assuntos
Embolização Terapêutica/métodos , Hepatócitos/transplante , Veia Porta/cirurgia , Condicionamento Pré-Transplante/métodos , Procedimentos Cirúrgicos Vasculares/métodos , Animais , Fígado/cirurgia , Masculino , Modelos Animais , Ratos , Ratos Transgênicos
9.
Genome Res ; 24(8): 1371-83, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24989021

RESUMO

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.


Assuntos
Marcação de Genes , Engenharia Genética , Animais , Sequência de Bases , Células Cultivadas , Enzimas de Restrição do DNA/biossíntese , Enzimas de Restrição do DNA/genética , Feminino , Hipoxantina Fosforribosiltransferase/genética , Masculino , Microinjeções , Ratos Sprague-Dawley , Ratos Transgênicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reparo de DNA por Recombinação , Zigoto
10.
J Autoimmun ; 81: 44-55, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28342735

RESUMO

Developing protocols aimed at inhibiting effector T cells would be key for the treatment of T cell-dependent autoimmune diseases including type 1 autoimmune diabetes (T1D) and multiple sclerosis (MS). While heme oxygenase-1 (HO-1) inducers are clinically approved drugs for non-immune-related diseases, they do have immunosuppressive properties when administered systemically in rodents. Here we show that HO-1 inducers inhibit antigen-specific effector T cells when injected intradermally together with the T cell cognate antigens in mice. This phenomenon was observed in both a CD8+ T cell-mediated model of T1D and in a CD4+ T cell-dependent MS model. Intradermal injection of HO-1 inducers induced the recruitment of HO-1+ monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1+MoDCs, effector T-cells exhibited a lower velocity and a reduced ability to migrate towards chemokine gradients resulting in impaired accumulation to the inflamed organ. Intradermal co-injection of a clinically approved HO-1 inducer and a specific antigen to non-human primates also induced HO-1+ MoDCs to accumulate in dermal draining LN and to suppress delayed-type hypersensitivity. Therefore, in both mice and non-human primates, HO-1 inducers delivered locally inhibited effector T-cells in an antigen-specific manner, paving the way for repositioning these drugs for the treatment of immune-mediated diseases.


Assuntos
Antígenos/imunologia , Heme Oxigenase-1/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Imunização , Camundongos , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/imunologia , Papio anubis , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
11.
Transgenic Res ; 26(5): 703-708, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28780744

RESUMO

On May 11th and 12th 2017 was held in Nantes, France, the international meeting "Advances in transgenic animal models and techniques" ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.


Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes/tendências , Modelos Animais , Animais , Humanos
12.
J Immunol ; 195(10): 5035-44, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26432892

RESUMO

Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-ß1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3(+)CD4(+)CD25(+) regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic.


Assuntos
Linfócitos B Reguladores/imunologia , Antígenos CD40/imunologia , Granzimas/imunologia , Transplante de Coração , Plasmócitos/imunologia , Transdução de Sinais/imunologia , Tolerância ao Transplante , Aloenxertos , Animais , Antígenos CD/imunologia , Citocinas/imunologia , Isoantígenos/imunologia , Masculino , Ratos , Linfócitos T Reguladores/imunologia
13.
Circulation ; 131(11): 1006-18, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25593290

RESUMO

BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.


Assuntos
Transdiferenciação Celular , Células Endoteliais/patologia , Hipertensão Pulmonar/patologia , Mesoderma/patologia , Actinas/biossíntese , Actinas/genética , Animais , Biomarcadores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/biossíntese , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipóxia/complicações , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Monocrotalina/toxicidade , Mutação , RNA Mensageiro/biossíntese , Ratos , Sirolimo/farmacologia , Remodelação Vascular , Vimentina/biossíntese , Vimentina/genética
14.
Calcif Tissue Int ; 96(5): 430-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25694361

RESUMO

Bone repair is an important concept in tissue engineering, and the ability to repair bone in hypotrophic conditions such as that of irradiated bone, represents a challenge for this field. Previous studies have shown that a combination of bone marrow and (BCP) was effective to repair irradiated bone. However, the origin and role played by each cell type in bone healing still remains unclear. In order to track the grafted cells, the development of an animal model that is immunotolerant to an allograft of bone marrow would be useful. Furthermore, because the immune system interacts with bone turnover, it is of critical importance to demonstrate that immunosuppressive drugs do not interfere with bone repair. After a preliminary study of immunotolerance, cyclosporin-A was chosen to be used in immunosuppressive therapy. Ten rats were included to observe qualitative and quantitative bone repair 8 days and 6 weeks after the creation of bone defects. The defects were filled with an allograft of bone marrow alone or in association with BCP under immunosuppressive treatment (cyclosporin-A). The results showed that there was no significant interaction of cyclosporin-A with osseous regeneration. The use of this new immunotolerant rat model of bone marrow allograft in future studies will provide insight on how the cells within the bone marrow graft contribute to bone healing, especially in irradiated conditions.


Assuntos
Transplante de Medula Óssea/métodos , Ciclosporina/farmacologia , Modelos Animais de Doenças , Terapia de Imunossupressão/métodos , Imunossupressores/farmacologia , Engenharia Tecidual/métodos , Aloenxertos , Animais , Osso e Ossos/lesões , Osso e Ossos/cirurgia , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Varredura , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transplante Homólogo/métodos
15.
Methods ; 69(1): 102-7, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24583114

RESUMO

The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.


Assuntos
Técnicas de Inativação de Genes , Mutagênese Sítio-Dirigida/métodos , Animais , Reparo do DNA por Junção de Extremidades , Desoxirribonucleases/química , Desoxirribonucleases/genética , Transferência Embrionária , Embrião de Mamíferos , Feminino , Recombinação Homóloga , Microinjeções , Ratos , Ratos Sprague-Dawley
16.
Eur J Immunol ; 43(1): 209-18, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23065740

RESUMO

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)-treated DCs loaded with pancreatic ß-cell peptides protect mice from disease. This protection is peptide-restricted, independent of IL-10 secretion by DCs and of CD4(+) T cells. Although no differences were observed in autoreactive CD8(+) T-cell function from gCO-treated versus untreated DC-immunized groups, gCO-treated DCs strongly inhibited accumulation of autoreactive CD8(+) T cells in the pancreas. Interestingly, induction of ß1-integrin was curtailed when CD8(+) T cells were primed with gCO-treated DCs, and the capacity of these CD8(+) T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO-treated DCs appears to be an original strategy to control autoimmune disease.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Monóxido de Carbono/farmacologia , Células Dendríticas/efeitos dos fármacos , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/imunologia , Integrina beta1/biossíntese , Pâncreas/imunologia , Animais , Autoantígenos/imunologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Tolerância Imunológica , Integrina beta1/genética , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia
17.
Eur J Immunol ; 43(11): 2832-44, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23852701

RESUMO

Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe(2+) , and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion.


Assuntos
Apresentação de Antígeno/imunologia , Monóxido de Carbono/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Monóxido de Carbono/farmacologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endocitose/imunologia , Endossomos/efeitos dos fármacos , Heme Oxigenase-1/imunologia , Heme Oxigenase-1/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Lisossomos/efeitos dos fármacos , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia
18.
FASEB J ; 27(2): 703-11, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23150522

RESUMO

Despite the recent availability of gene-specific nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), there is still a need for new tools to modify the genome of different species in an efficient, rapid, and less costly manner. One aim of this study was to apply, for the first time, engineered meganucleases to mutate an endogenous gene in animal zygotes. The second aim was to target the mouse and rat recombination activating gene 1 (Rag1) to describe, for the first time, Rag1 knockout immunodeficient rats. We microinjected a plasmid encoding a meganuclease for Rag1 into the pronucleus of mouse and rat zygotes. Mutant animals were detected by PCR sequencing of the targeted sequence. A homozygous RAG1-deficient rat line was generated and immunophenotyped. Meganucleases were efficient, because 3.4 and 0.6% of mouse and rat microinjected zygotes, respectively, generated mutated animals. RAG1-deficient rats showed significantly decreased proportions and numbers of immature and mature T and B lymphocytes and normal NK cells vs. littermate wild-type controls. In summary, we describe the use of engineered meganucleases to inactivate an endogenous gene with efficiencies comparable to those of ZFNs and TALENs. Moreover, we generated an immunodeficient rat line useful for studies in which there is a need for biological parameters to be analyzed in the absence of immune responses.


Assuntos
Técnicas de Inativação de Genes/métodos , Genes RAG-1 , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Animais , Sequência de Bases , DNA/administração & dosagem , DNA/genética , Endonucleases/genética , Endonucleases/metabolismo , Marcação de Genes/métodos , Engenharia Genética/métodos , Transplante de Coração/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Transplante Homólogo
19.
Mol Ther Methods Clin Dev ; 30: 30-47, 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37746247

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

20.
iScience ; 26(4): 106399, 2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-37034986

RESUMO

CRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 µM of Cas9, equimolar Cas9/gRNA ratio and 2 µM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models.

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