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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.

AimTo investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning.MethodsAnimals from a new transgenic rat line expressing green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received at Day-12, near neural diversification, a single oral bolus of mir-320-3p or miR-375-3p, embedded in DiOleyl-Succinyl-Paromomycin (DOSP), and were further early (Day-15) or regularly (Day-30) weaned. Relevant miRNA (miR-320-3p, miR-375-3p, miR-375-5p, miR-16-5p, miR-132-3p, miR-504), polr3d, hspb6, inflammation, enteroendocrine, and circadian clock-related mRNAs, chromatin complexes, and duodenal cell density were assayed at 8h post-inoculation and at Day-45.ResultsThe miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (pConclusionAddressing oral miRNA-320-3p loads to duodenal cell lineage is paving the way for the design of new therapeutics, manipulating long term consequences of early life stress.

BackgroundCD4+and CD8+regulatory T cells (Treg) in diverse species include different subsets from different origins. In all species, CD8+Treg have been poorly characterized. CD4+and CD8+Treg in rats have only partially been characterized and there is no rat model in which FOXP3+Treg are genetically tagged.ResultsWe generated a rat transgenic line using the CRISPR/Cas9 system in which EGFP was inserted in frame on the 3’ end of theFoxp3gene using a 2A self-cleaving peptide. EGFP was exclusively expressed by CD4+and CD8+T cells in similar proportion as observed with anti-FOXP3 antibodies. CD4+EGFP+Treg were 5-10 times more frequent than CD8+EGFP+Treg. CD4+and CD8+EGFP+Treg expressed both the CD25highCD127lowCD45RClow/-markers. 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 resulted in the induction of EGFP+Treg. Preferential expansion of CD4+and CD8+EGFP+Treg could be detected upon in vivo administration of a low dose of IL-2.ConclusionsThis new and uniqueFoxp3-EGFPrat line constitutes a useful model to identify and isolate viable natural and induced CD4+and CD8+Treg. Additionally, it allows to identify new molecules expressed in CD8+Treg that may allow to better define their phenotype and function not only in rats but also in other species.

The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2μM and 0.4μM of Cas9, coupled with 2μM of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal sodium retention leading to edema. This sodium retention is usually attributed to epithelial sodium channel (ENaC) activation after plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped (CC) rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We then identified a truncated variant of acid-sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when coexpressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that the expression of the truncated ASIC2b in the kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but not in any of the patients with other renal diseases. We have therefore identified a variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.

International audience; 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.

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 β1-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 DMDsupmdx/suprats, 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.

Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD. Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal. These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD., Summary: We characterized the cardiovascular abnormalities of Dmdmdx rats, demonstrating that Dmdmdx rats show similar cardiac and vascular endothelial function impairments to Duchenne muscular dystrophy patients, representing a model of the dystrophic heart.

Introduction: Pulmonary veno-occlusive disease (PVOD) is an orphan fatal disease. The identification of biallelic inactivating mutations of EIF2AK4 (encoding GCN2) as the major genetic cause of PVOD has clarified the nosology of the disease. On the other hand, BMP/BMPR2 axis is a major axis of pulmonary arterial hypertension pathobiology, a close but better understood form of severe pulmonary hypertension (PH) Objective: To investigate the functional relationship between GCN2 and the BMP/BMPR2 axis. Methods: We investigated GCN2 and the BMP/BMPR2 axis in human and animal models of PVOD, and in cultured human pulmonary microvascular endothelial cells (hPMEC) with interventional experiments (siRNA and pharmacological inhibitors/activators) Results: Pulmonary GCN2 protein expression was decreased in all forms of PVOD. We showed that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling in hPMEC. This molecular relationship was confirmed in vivo, in the lungs of a newly created transgenic rat model knock out for Eif2ak4. We found a 4 fold decrease in SMAD1/5/9 phosphorylation in KO rats. We showed this regulation may be mediated through GCN2-dependent repression of chordin, an extracellular antagonist of BMP signaling. GCN2 inhibition induced a dramatic increase in hPMEC proliferation, which is highly relevant of PVOD genesis. BMP9 treatment was able to block this exuberant proliferation Conclusion: GCN2 loss-of-function negatively regulates SMAD1/5/9 phosphorylation. Despite this dampened BMP signaling, exogenous BMP9 was still able to reverse GCN2 inhibition-induced hPMEC proliferation. BMP9 may hence be considered as potential therapeutic options for PVOD

Background Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments. Methods We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. Results As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful. Conclusions Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.

International audience; 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 þ T cell-mediated model of T1D and in a CD4 þ T cell-dependent MS model. Intradermal injection of HO-1 in-ducers induced the recruitment of HO-1 þ monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1 þ 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 þ 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.

International audience; Corticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD). Even though they decrease inflammation, they have limited efficacy and are associated with significant side effects. There is therefore the need for new protolerogenic treatments to replace CS. Dystrophin-deficient rats (Dmd mdx ) closely resemble the pathological phenotype of DMD patients. We performed the first Immunophenotyping of Dmd mdx rats and showed leukocyte infiltration in skeletal and cardiac muscles, which consisted mostly of macrophages and T cells including CD45RChigh T cells. Muscles of DMD patients also contain elevated CD45RChigh T cells. We treated Dmd mdx rats with an anti-CD45RC MAb used in previous studies to deplete CD45RChigh T cells and induce immune tolerance in models of organ transplantation. Treatment of young Dmd mdx rats with anti-CD45RC MAb corrected skeletal muscle strength and was associated with depletion of CD45RChigh T cells with no side effects. Treatment of young Dmd mdx rats with prednisolone resulted in increase in skeletal muscle strength but also severe growth retardation. In conclusion, anti-CD45RC MAb treatment has potential in the treatment of DMD and might eventually result in reduction or elimination of CS use.

Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations inWe 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.Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 ofWe established the first

Introduction Mitral valve prolapse (MVP) affects 3% of the population and is characterized by a myxomatous MV remodeling. The pathophysiological mechanisms involved in MVP remain elusive and the only therapeutic option is to perform MV surgery. The first causing mutation in MVP has been identified on the FLNA gene. We recently generated a KI rat for the FLNA-P637Q mutation that develops MVP. A recent RNA-seq analysis revealed an upregulation of genes involved in immune response and inflammation pathways in the valves of KI rats compared to WT. Objective The aim of this study was then to identify and quantify the immune cells present in MVs and assess their role in the development and/or progression of the disease. Methods Flow cytometry was used to quantify the hematopoietic cells (CD45), resident macrophages (CD206), myeloid lineage (CD11b), endothelial progenitors (CD34) and endothelial cells (CD31) in the MV of 3-weeks old KI and WT rats. The location of those cells within the MV tissues was analysed by immunofluorescence on cryosectioned MVs. Results Cytometry results showed a higher proportion of CD45+ and CD206+ cells in the KI rats compared to WT (20% vs. 14% and 28% vs. 16%, respectively) but similar proportion of CD31+ (14%). The CD45 and CD206 immunofluorescences performed on WT animals showed a clear localization of the positive cells on the edge of the atrial side of the leaflet, in the sub-endothelium layer. In KI rats, CD45+ and CD206+ cells were diffusely located: close to the atrial side of the MV leaflet, but also into the medial third of the leaflet. Conclusion This preliminary study showed that recruitment of immune cells in MVP could be an important player in the pathophysiological process leading the disease. To elucidate the specific roles of this pathway, the temporal recruitment of immune cells and their specific activity need to be studied over the course of the disease progression.

Introduction Pulmonary veno-occlusive disease (PVOD) occurs in humans as heritable form due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2), or as a sporadic form. The pathobiology of this orphan fatal disease is currently unknown. However, the identification of EIF2AK4 as the major genetic cause of PVOD has clarified the nosology of the disease. On the other hand, BMP/BMPR2 axis is a major axis of pulmonary arterial hypertension pathobiology, a close but better understood form of severe pulmonary hypertension (PH). Objective To investigate the functional relationship between GCN2 and the BMP/BMPR2 axis. Methods We investigated GCN2 and the BMP/BMPR2 axis in human and animal models of PVOD, and in cultured human pulmonary microvascular endothelial cells (hPMEC) with interventional experiments (siRNA and pharmacological inhibitors/activators). Results Pulmonary GCN2 protein expression was decreased in all forms of PVOD. We showed that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling in hPMEC. This molecular relationship was confirmed in vivo, in the lungs of a newly created transgenic rat model knock out for Eif2ak4 (Δ152Ex1/Δ152Ex1). We found a 4 fold decrease in SMAD1/5/9 phosphorylation in KO rats. Since those rats don’t have spontaneous PH, the decrease in SMAD1/5/9 phosphorylation is not the mere consequence of high pulmonary artery pressures. We showed this regulation may be mediated through GCN2-dependent repression of chordin, an extracellular antagonist of BMP signaling. GCN2 inhibition induced a dramatic increase in hPMEC proliferation, which is highly relevant of PVOD genesis. BMP9 treatment was able to block this exuberant proliferation. Conclusion GCN2 loss-of-function negatively regulates SMAD1/5/9 phosphorylation. Despite this dampened BMP signaling, exogenous BMP9 was still able to reverse GCN2 inhibition-induced hPMEC proliferation. BMP9 may hence be considered as potential therapeutic options for PVOD.

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 β-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 β1-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.

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.

International audience; The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

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.

The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Heme oxygenase-1 (HO-1) exerts its functions via the catabolism of heme into carbon monoxide (CO), Fe2+, and biliverdin, as well as by depletion of free heme. We have recently described that overexpression of HO-1 is associated with the tolerogenic capacity to dendritic cells (DCs) stimulated by LPS. In this study, we demonstrate that treatment of human monocyte-derived DCs with CO blocks TLR3 and 4-induced phenotypic maturation, secretion of proinflammatory cytokines, and alloreactive T cell proliferation, while preserving IL-10 production. Treatment of DCs with biliverdin, bilirubin, and deferoxamine or replenishing intracellular heme stores had no effect on DC maturation. HO-1 and CO inhibited LPS-induced activation of the IFN regulatory factor 3 pathway and their effects were independent of p38, ERK, and JNK MAPK. HO-1 and CO treatment also inhibited mouse DC maturation in vitro and mouse DC immunogenic properties in vivo, as shown by adoptive cell transfer in a transgenic model of induced diabetes. Thus, for the first time, our data show that CO treatment inhibits DC immunogenicity induced by TLR ligands and that blockade of IFN regulatory factor 3 is associated with this effect.

We have previously shown that human monocyte-derived dendritic cells (DC) express indoleamine 2,3-dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS-treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF-alpha + poly(I:C)-induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock-down using short-hairpin RNA also led to diminished LPS-induced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPS-treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-kappaB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4(+)CD25(high) regulatory T cells in an IDO-dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4(+)CD25(high) regulatory T cells.

International audience

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.

The laboratory rat (R. norvegicus) is a very important experimental animal in several fields of biomedical research. This review describes the various techniques that have been used to generate transgenic rats: classical DNA microinjection and more recently described techniques such as lentiviral vector-mediated DNA transfer into early embryos, sperm-mediated transgenesis, embryo cloning by nuclear transfer and germline mutagenesis. It will also cover techniques associated to transgenesis such as sperm cryopreservation, embryo freezing and determination of zygosity. The availability of several technologies allowing genetic manipulation in the rat coupled to genomic data will allow biomedical research to fully benefit from the rat as an experimental animal.

Heme oxygenase-1 (HO-1) is an intracellular enzyme that degrades heme and inhibits immune responses and inflammation in vivo. In most cell types, HO-1 is inducible by inflammatory stimuli and oxidative stress. Here we demonstrate that human monocyte-derived immature dendritic cells (iDCs) and several but not all freshly isolated rat splenic DC subsets and rat bone marrow-derived iDCs, spontaneously express HO-1. HO-1 expression drastically decreases during human and rat DC maturation induced in vitro. In human tissues, iDCs also express HO-1, whereas mature DCs do not. Induction of HO-1 expression with cobalt protoporphyrin (CoPP) in human and rat DCs inhibits lipopolysaccharide (LPS)-induced phenotypic maturation and secretion of proinflammatory cytokines, resulting in the inhibition of alloreactive T-cell proliferation. CoPP-treated DCs, however, retain the ability to produce the anti-inflammatory cytokine interleukin 10 (IL-10). Reactive oxygen species induced by LPS in DCs were inhibited by induction of HO-1. In conclusion, we identify, for the first time, the capacity of HO-1 to block maturation of DCs and to inhibit proinflammatory and allogeneic immune responses while preserving IL-10 production. This novel immune function for HO-1 may be of interest for the inhibition of immune responses in autoimmune diseases, transplantation, and other conditions involving activation of the immune system. (Blood. 2005;106:1694-1702)

Elevated expression of heme oxygenase-1 (HO-1), an intracellular enzyme that degrades heme into carbon monoxide (CO), biliverdine and free iron, has anti-inflammatory and antiapoptotic effects in diverse models. Here, we analyzed the effects of specific overexpression of HO-1 following adenovirus-mediated (AdHO-1) gene transfer in an acute cardiac allograft rejection model. The intragraft (i.g.) injection of AdHO-1 into cardiac allografts, as well as intramuscular (i.m.) or intravenous (i.v.) administration, prolonged allograft survival with, respectively, 13.3, 62.5 and 80% of the grafts surviving long term (>100 days), whereas control grafts were rejected with acute kinetics. HO-1 overexpression was associated with inhibited allogeneic responses in MLRs using graft-infiltrating leukocytes and splenocytes, but not with lymph node cells. The inhibition of splenocyte proliferation was mediated by soluble factors and was dependent on the presence of APCs, since purified T cells proliferated normally. i.v. but not i.g. AdHO-1 administration decreased the number of graft-infiltrating leukocytes, cytokine mRNA accumulation and apoptosis in transplanted hearts, whereas i.v. and i.g. AdHO-1 did not modify normal immune responses against cognate antigens, indicating that there was no general immunosuppression. These results indicate that HO-1 overexpression prolongs the survival of vascularized allografts by promoting tolerogenic mechanisms acting on allogeneic cellular immune responses.

Neuropeptide Y (NPY), a peptide widely expressed in the brain, acts through the protein G-coupled receptors Y1, Y2 and Y5. In the adult rat, this peptide modulates many important functions such as the control of energy balance and anxiety. Its involvement in brain development has been less investigated. In the present study, we have analysed the expression of Y1 and Y2 in the developing rat cerebellum using RNase protection assay. Both receptors were detected in the embryo but at very low levels. Their expression then increased, reaching a peak at postnatal day 10. At later stages, we observed a down-regulation of both Y1 and Y2 mRNA levels. This pattern of expression was delayed in hypothyroid rats, suggesting that the regulation of NPY receptors was strictly related to cerebellar development stages. In situ hybridisation and immunohistochemistry analyses revealed specific localisations of the receptors. Y1 was exclusively expressed by Purkinje cells while Y2 was found mostly in granule cells of the internal granule cell layer. These observations argue in favour of specific roles for Y1 and Y2 in the developing cerebellum. In an initial attempt to characterise these roles, we have determined the number of apoptotic cells in the developing cerebellum of Y2−/− mice and analysed the effects of NPY on primary cultures of cerebellar granule neurones. Our data showed that the absence of Y2 did not increase cell death in the internal granule cell layer of the developing cerebellum, and that NPY by itself did not prevent the death of differentiated granule cells cultured in serum-free medium. However, we found that co-treatment of the cells by NPY and neuromediators such as NMDA or GABA strongly promoted the survival of granule neurones. Taken together, these observations suggest an involvement of the NPY receptors in cerebellar ontogenesis that remains to be demonstrated in vivo.

A large array of evidence supports the involvement of infiltrating T lymphocytes in the rejection process of intracerebral neuronal xenografts. Little is known, however, about the molecular mechanisms that drive the recruitment of this cell type. In the present work, we used real-time RT-PCR methodology to investigate the kinetics of cytokine gene expression during the infiltration of fetal porcine neurons (PNEU) implanted into the striatum of LEW.1A rats. T lymphocyte infiltration was followed by measuring the intracerebral levels of transcripts encoding the beta chain of the T cell receptor. These transcripts remained barely detectable until the fourth week (28 days) postimplantation, when a sudden accumulation occurred. Their kinetics, which support previous immunohistochemical observations, indicate that alphabetaT lymphocyte recruitment occurs rapidly after a delay of several weeks in this experimental model. Infiltration of PNEU grafts by T lymphocytes was accompanied by a concomitant, dramatic augmentation of transcripts coding for monocyte chemotactic protein-1 and RANTES (for regulated on activation, normal T cell expressed and secreted), two chemokines targeting this cell type, among others. Likewise, a sudden accumulation of transcripts of proinflammatory lymphokines [interleukin (IL)-1alpha, tumor necrosis factor-alpha, IL-6] as well as Th1 cytokines (IL-2, interferon-gamma) was also detected. In contrast, IL-4, -10, and -13 mRNA remained barely detectable at the different time points. No significant changes were noticed for IL-12 or transforming growth factor-beta transcripts. These data support the concept that T lymphocyte infiltration of PNEU grafts is actively promoted by a local production of chemokines and proinflammatory lymphokines and is based on a Th1 polarization.

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.

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.

In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well established that DNA methylation is an important mechanism involved in gene expression, DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD) after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to study whether DNA methylation was involved in the control of PCD. We found that 1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2′-deoxycytidine. This effect remains detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene remains unaffected. These results suggest that a change in DNA methylation pattern could suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even genes inducing change in the differentiated state of these cells.

International audience; 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 recom-binant 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.

Mesenchymal stem cells (MSCs) have attracted attention for their potential use in regenerative medicine such as brain transplantation. As MSCs are considered to be hypoimmunogenic, transplanted MSCs should not trigger a strong host inflammatory response. To verify this hypothesis, we studied the brain immune response after transplantation of human or rat MSCs into the rat striatum and MSC fate at days 5, 14, 21 and 63 after transplantation. Flow cytometry analysis indicated that both MSCs express CD90 and human leucocyte antigen (MHC) class I, but no MHC class II molecules. They do not express CD45 or CD34 antigens. However, MSC phenotype varies with passage number. Human MSCs have mRNAs for interleukin (IL)-6, IL-8, IL-12, tumour necrosis factor (TNF)-alpha and TGF-beta(1), whereas rat MSCs express IL-6-, IL-10-, IL-12- and TGF-beta(1)-mRNAs. The quantification shows higher levels of mRNAs for the anti-inflammatory molecules IL-6 and TGF-beta(1) than for pro-inflammatory cytokines IL-8 and IL-12; ELISA analysis showed no IL-12 whereas TGF-beta(1) and IL-6 were detected. Transplant size did not significantly vary between 14 and 63 days after transplantation, indicating an absence of immune rejection of the grafts. Very few mast cells and moderate macrophage and microglial infiltrations, observed at day 5 remained stable until day 63 after transplantation in both rat and human MSC grafts. The observations of very few dendritic cells, T alphabeta-cells, and no T gammadelta-lymphocytes, all three being associated with Tp rejection in the brain, support the contention that MSCs are hypoimmunogenic. Our results suggest that MSCs are of great interest in regenerative medicine in a (xeno)transplantation setting.

Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. HO-1 is induced by its substrate and by other stimuli, including agents involved in oxidative stress and proinflammatory cytokines as well as several anti-inflammatory stimuli. A growing body of evidence points toward the capacity of this molecule to inhibit immune reactions and the pivotal role of HO-1 in inflammatory diseases. We will first review the physiological role of HO-1 as determined by the analysis of HO-1-deficient individuals. This will be followed by an examination of the effect of HO-1 within immunopathological contexts such as immune disorders (autoimmunity and allergy) or infections. A section will be devoted to the use of an HO-1 inducer as an immunosuppressive molecule in transplantation. Finally, we will review the molecular basis of HO-1 actions on different immune cells.

Here we describe an efficient technique to generate transgenic rats by microinjection of short DNA fragments. We have focused on optimal conditions for superovulation of prepubescent females Sprague-Dawley (CD) strains to have good quality embryos, pseudopregnant females, zygotes preparation, optimal conditions for microinjection and embryo transfer into foster mothers.

Lentiviral vectors are now well recognized as good vehicles for gene delivery. This is because they can efficiently transduce both dividing and post-mitotic cells, and stably integrate into the host genome allowing for long-term expression of the transgene. Their potential utility for the generation of transgenic animals has been recognized as an attractive and promising alternative to the conventional DNA-microinjection method which lacks efficiency. The initial success of lentiviral transgenesis in mice considerably broadened its use in other species, in which classical transgenic techniques are difficult, such as in the rat.In this chapter, we describe detailed procedures for both the production of human immunodeficiency virus-1 (HIV-1)-derived lentiviral vectors and for the generation of transgenic rats by injection of these vectors into the perivitelline space of fertilized one-cell eggs.

Lentiviral vectors are now well recognized as good vehicles for gene delivery. This is because they can efficiently transduce both dividing and post-mitotic cells, and stably integrate into the host genome allowing for long-term expression of the transgene. Their potential utility for the generation of transgenic animals has been recognized as an attractive and promising alternative to the conventional DNA-microinjection method which lacks efficiency. The initial success of lentiviral transgenesis in mice considerably broadened its use in other species, in which classical transgenic techniques are difficult, such as in the rat.In this chapter, we describe detailed procedures for both the production of human immunodeficiency virus-1 (HIV-1)-derived lentiviral vectors and for the generation of transgenic rats by injection of these vectors into the perivitelline space of fertilized one-cell eggs.

Here we describe an efficient technique to generate transgenic rats by microinjection of short DNA fragments. We have focused on optimal conditions for superovulation of prepubescent females Sprague-Dawley (CD) strains to have good quality embryos, pseudopregnant females, zygotes preparation, optimal conditions for microinjection and embryo transfer into foster mothers.

The genetic dissection of physiological and pathological traits in laboratory model organisms is accelerated by the ability to engineer loss-of-function mutations at investigator-specified loci. This chapter describes the use of zinc-finger nucleases (ZFNs) for the targeted disruption of endogenous rat genes directly in the embryo. ZFNs can specifically disrupt target genes in cultured rat cells and in embryos from inbred and outbred strains, leading to permanently genetically modified animals. This technology allows for the rapid, targeted modification of the rat genome.

It is extremely useful to define a rapid and accurate method for identifying homozygous and heterozygous transgenic animals prior to setting up breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact. Southern-blotting is a means of identifying zygosity, but such a method is time consuming and produces a high level of ambiguous results. Some years ago, we described the rapid, precise, non-ambiguous, and high-throughput identification of zygosity in transgenic animals by real-time PCR. This technique allows us to make a clear-cut identification of transgenic rats, transgenic mice, and double-transgenic pigs. Since 2002, however, several authors have made improvements to this method. The following paper describes the ease with which zygosity is determined using real-time PCR.