Search

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.

Although IL-34 and CSF-1 share actions as key mediators of monocytes/macrophages survival and differentiation, they also display differences that should be identified to better define their respective roles in health and diseases. IL-34 displays low sequence homology with CSF-1 but has a similar general structure and they both bind to a common receptor CSF-1R, although binding and subsequent intracellular signaling shows differences. CSF-1R expression has been until now mainly described at a steady state in monocytes/macrophages and myeloid dendritic cells, as well as in some cancers. IL-34 has also 2 other receptors, protein-tyrosine phosphatase zeta (PTPζ) and CD138 (Syndecan-1), expressed in some epithelium, cells of the central nervous system (CNS), as well as in numerous cancers. While most, if not all, of CSF-1 actions are mediated through monocyte/macrophages, IL-34 has also other potential actions through PTPζ and CD138. Additionally, IL-34 and CSF-1 are produced by different cells in different tissues. This review describes and discusses similarities and differences between IL-34 and CSF-1 at steady state and in pathological situations and identifies possible ways to target IL-34, CSF-1, and its receptors.

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.

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.

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.

Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal Na retention leading to oedema. This Na retention is usually attributed to epithelial sodium channel (ENaC) activation following plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped 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 co-expressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that expression of the truncated ASIC2b in 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 in any of the patients with other renal diseases. We have, therefore, identified a novel variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.

SummaryIntracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORγt+ cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen 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. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4+ T cell priming.

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

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. ispartof: AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY vol:63 issue:1 pages:118-131 ispartof: location:United States status: published

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.

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.

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

International audience; 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.

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.

Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (CS) that decrease inflammation and immune responses present in patients and animal models. CS have however limited efficacy and important and numerous side effects. Therefore, there is a need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease doses of CS. We first assessed the status of immune system of dystrophin-deficient rats (Dmdmdx) that closely reproduce the phenotype of DMD patients. Dmdmdx rats showed increased leukocyte infiltration in skeletal and cardiac muscles, containing mostly macrophages but also T cells, and increased expression of several cytokines. Anti-CD45RC Monoclonal antibody (Mab) treatment induced immune tolerance in models of organ transplantation and GVHD (Graft Versus Host Disease). We observed that muscles and blood of DMD patients contained T CD4+ and CD8+ expressing high levels of CD45RChigh cells. Treatment of young Dmdmdx rats with anti-CD45RC MAb corrected skeletal muscle strength associated to a depletion of effectors CD45RChigh T cells with no obvious side-effects. Prednisolone treatment of Dmdmdx rats similarly increased skeletal muscle strength and was also associated to a depletion of effectors CD45RChigh cells but resulted in severe weight loss.Overall, Dmdmdx rats display important immune inflammatory response and thus represent a useful model to analyze new anti-inflammatory and tolerogenic treatments for DMD. As an example, a new treatment with anti-CD45RC antibodies improved muscle strength in Dmdmdx rats as prednisolone did but without side effects. Anti-CD45RC therapy could complement other therapies in DMD patients.

National audience

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 Francois Bonamy, CNRS, Region 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.

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 recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

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.

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 CD24intCD38+CD27+IgD−IgM+/low subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-β1–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.