KiT-GENIE, the French genetic biobank of kidney transplantation.

KiT-GENIE is a monocentric DNA biobank set up to consolidate the very rich and homogeneous DIVAT French cohort of kidney donors and recipients (D/R) in order to explore the molecular factors involved in kidney transplantation outcomes. We collected DNA samples for kidney transplantations performed in Nantes, and we leveraged GWAS genotyping data for securing high-quality genetic data with deep SNP and HLA annotations through imputations and for inferring D/R genetic ancestry. Overall, the biobank included 4217 individuals (n = 1945 D + 2,272 R, including 1969 D/R pairs), 7.4 M SNPs and over 200 clinical variables. KiT-GENIE represents an accurate snapshot of kidney transplantation clinical practice in Nantes between 2002 and 2018, with an enrichment in living kidney donors (17%) and recipients with focal segmental glomerulosclerosis (4%). Recipients were predominantly male (63%), of European ancestry (93%), with a mean age of 51yo and 86% experienced their first graft over the study period. D/R pairs were 93% from European ancestry, and 95% pairs exhibited at least one HLA allelic mismatch. The mean follow-up time was 6.7 years with a hindsight up to 25 years. Recipients experienced biopsy-proven rejection and graft loss for 16.6% and 21.3%, respectively. KiT-GENIE constitutes one of the largest kidney transplantation genetic cohorts worldwide to date. It includes homogeneous high-quality clinical and genetic data for donors and recipients, hence offering a unique opportunity to investigate immunogenetic and genetic factors, as well as donor-recipient interactions and mismatches involved in rejection, graft survival, primary disease recurrence and other comorbidities.

Carbon monoxide decreases endosome-lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells.

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

Preclinical studies on specific gene therapy for recessive retinal degenerative diseases.

Inherited retinal diseases are non-lethal and have a wide level of genetic heterogeneity. Many of the genes involved have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because they mediate long-term transgene expression in a variety of retinal cell types and elicit minimal immune responses. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial. In this manuscript, we focus on recombinant AAV-mediated specific gene therapy for recessive retinal degenerative diseases we describe the preclinical studies for the treatment of retinal degeneration caused by retinal pigmented epithelium (RPE) cells or photoreceptor defects and the immune response induced by retinal rAAV gene transfer.

Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity.

Heme oxygenase-1 (HO-1) exerts its functions via the catabolism of heme into carbon monoxide (CO), Fe(2+), 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.

Lentivirus mediated HO-1 gene transfer enhances myogenic precursor cell survival after autologous transplantation in pig.

Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation.

IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells.

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.

Over-expression of heme oxygenase-1 by adenoviral gene transfer improves pregnancy outcome in a murine model of abortion.

Mammalian pregnancy is a complex phenomenon allowing the maternal immune system to support its allogeneic fetus. Physiological pathways protecting the fetus from rejection are thought to be comparable with those leading to allograft acceptance. Heme oxygenase (HO)-1 is known to protect locally against rejection in transplantation models due to its anti-oxidant, anti-inflammatory and cytoprotective functions. Based on previous data on low HO-1 levels in placenta from mice undergoing abortion, we hypothesized that an up-regulation of HO-1 during pregnancy would avoid fetal rejection in the murine abortion combination CBA/J x DBA/2J, using BALB/c-mated CBA/J as normal controls. We injected pregnant mice undergoing abortion with 1 x 10(5) PFU of an adenoviral vector containing HO-1 and GFP (AdHO-1/GFP), and compared the pregnancy outcome with PBS- or 1 x 10(5) AdEGFP-treated abortion-prone mice and with PBS-treated normal pregnant mice. The abortion rate diminished significantly after adenoviral gene transfer of AdHO-1/GFP. The systemic and local IL-4/IFN-gamma ratio was augmented in AdHO-1-treated mice compared to abortion-prone mice. Interestingly, the HO-1 treatment up-regulated the ratio IL-10/TNF-alpha in spleen but not in decidual lymphocytes. HO-1-treated mice further showed diminished apoptosis rate and increased Bag-1 mRNA levels at the materno-fetal interface. Thus, we propose HO-1 as a key regulator of pregnancy success. HO-1 would exert its action by locally up-regulating the Th2/Th1 cytokine ratio and by further protecting tissues from apoptosis.

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase.

Heme oxygenase-1 (HO-1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF-7 and T47D) express HO-1. When overexpressed, we found this enzyme to have anti-proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1-MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO-1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti-proliferative HO-1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO-1. We also describe that HO-1 inhibits IDO at the post-translational level through heme starvation. In vivo, we show that rat normal breast expresses HO-1 and IDO. In contrast, N-nitrosomethylurea-induced breast adenocarcinomas only express IDO. In conclusion, we show that HO-1/IDO cross-regulation modulates apoptosis and proliferation in rat and human breast cancer cells.

Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression.

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.

Gene transfer of heme oxygenase-1 and carbon monoxide delivery inhibit chronic rejection.

The hallmark of chronic rejection is the occlusion of the artery lumen by intima hyperplasia as a consequence of leukocyte infiltration and vascular smooth muscle cell (VSMC) migration and proliferation. Heme oxygenase-1 (HO-1) is a tissue protective molecule which degrades heme into carbon monoxide (CO), free iron and biliverdin. We analyzed the effects of HO-1 gene transfer into the vessel wall using an adenoviral vector (AdHO-1) and of CO delivery in a model of chronic allogeneic aorta rejection in rats. Carbon monoxide treatment was achieved by a new pharmacological approach in transplantation using methylene chloride (MC), which releases CO after degradation. AdHO-1-mediated gene transfer into aorta endothelial cells (ECs) or CO delivery resulted in a significant reduction in intimal thickness compared to untreated or noncoding adenovirus-treated controls. Aortas transduced with AdHO-1 or treated with CO showed a reduction in the number of leukocytes as well as in the expression of adhesion molecules, costimulatory molecules and cytokines, with the gene transfer treatment displaying a more pronounced effect than the CO treatment. Conversely, CO inhibited VSMC accumulation in the intima more efficiently than AdHO-1 treatment. Gene transfer of HO-1 and pharmacological manipulation of CO are novel approaches to the analysis and treatment of chronic rejection.