Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

New insights and current tools for genetically engineered (GE) sheep and goats.

Genetically engineered sheep and goats represent useful models applied to proof of concepts, large-scale production of novel products or processes, and improvement of animal traits, which is of interest in biomedicine, biopharma, and livestock. This disruptive biotechnology arose in the 80s by injecting DNA fragments into the pronucleus of zygote-staged embryos. Pronuclear microinjection set the transgenic concept into people's mind but was characterized by inefficient and often frustrating results mostly because of uncontrolled and/or random integration and unpredictable transgene expression. Somatic cell nuclear transfer launched the second wave in the late 90s, solving several weaknesses of the previous technique by making feasible the transfer of a genetically modified and fully characterized cell into an enucleated oocyte, capable of cell reprogramming to generate genetically engineered animals. Important advances were also achieved during the 2000s with the arrival of new techniques like the lentivirus system, transposons, RNA interference, site-specific recombinases, and sperm-mediated transgenesis. We are now living the irruption of the third technological wave in which genome edition is possible by using endonucleases, particularly the CRISPR/Cas system. Sheep and goats were recently produced by CRISPR/Cas9, and for sure, cattle will be reported soon. We will see new genetically engineered farm animals produced by homologous recombination, multiple gene editing in one-step generation and conditional modifications, among other advancements. In the following decade, genome edition will continue expanding our technical possibilities, which will contribute to the advancement of science, the development of clinical or commercial applications, and the improvement of people's life quality around the world.

IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis.

Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

Autologous dendritic cells prolong allograft survival through Tmem176b-dependent antigen cross-presentation.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.

IgG response to intracerebral xenotransplantation: specificity and role in the rejection of porcine neurons.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

Cell therapy with autologous tolerogenic dendritic cells induces allograft tolerance through interferon-gamma and epstein-barr virus-induced gene 3.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαß(+) CD3(+) CD4(-) CD8(-) NKRP1(-) cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

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.

Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates.

Clinical translation of dendritic cell (DC)-based cell therapy requires preclinical studies in nonhuman primates (NHPs). The aim of this work was to establish the in vitro conditions for generation of NHP tolerogenic DCs (Tol-DCs), as well as to analyze the molecular mechanisms by which these cells could control an immune response. Two populations of NHP bone marrow-derived DCs (BMDCs) were obtained: adherent and nonadherent. Although both populations displayed a quite similar phenotype, they were very different functionally. We characterized the adherent BMDCs as Tol-DCs that were poor stimulators of T cells and actively inhibited T-cell proliferation, whereas the nonadherent population displayed immunogenic properties in vitro. Interestingly, the anti-inflammatory and immunosuppressive enzyme heme oxygenase-1 (HO-1) was up-regulated in Tol-DCs, compared to the immunogenic BMDCs. We demonstrated that HO-1 mediates the immunosuppressive properties of Tol-DCs in vitro (in NHPs and rats) and that HO-1 is involved in the in vivo tolerogenic effect of Tol-DCs in a rat model of allotransplantation. In conclusion, here we characterized the in vitro generation of NHP Tol-DCs. Furthermore, we showed for the first time that HO-1 plays a role in the active inhibition of T-cell responses by rat and NHP Tol-DCs.

Progress and prospects: genetic engineering in xenotransplantation.

In this review, we summarize the work published over the last 2 years using genetic modifications of animals in the field of xenotransplantation. Genetic engineering of the donor has become a powerful tool in xenotransplantation, both for the inactivation of one particular porcine gene and for the addition of human genes with the goal of overcoming xenogeneic barriers. We summarize the work relative to the knockout of the alpha1,3-galactosyltransferase gene, followed by genetic engineering aimed at reducing the humoral and cellular immune response, complement activation and coagulation. Finally, we report on the genetic modification of pigs to reduce porcine endogenous retrovirus infection risk in the xenogeneic context.

Role of IFNgamma in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

Induction of long-term cardiac allograft survival by heme oxygenase-1 gene transfer.

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.

Cartilaginous metaplasia and calcification in aortic allograft is associated with transforming growth factor beta 1 expression.

BACKGROUND: Calcification of homografts and vascular conduits is poorly understood. Mechanisms leading to calcification were studied in a rat model of aortic allografts. METHODS: Rat aortas from Lew1W (RT1(u)) were transplanted into Lew1A (RT1(a)). Animals were killed at 30 days and 180 days, and aortic grafts were removed and analyzed for histologic and immunohistologic studies. RESULTS: Intimal surface increased progressively over 6 months and was the site of important modifications. Intimal cellular population changed from a leukocyte (CD45, OX1-OX30)- and macrophage (CD68, ED-1)-based population at 30 days to predominantly alpha-smooth muscle actin-expressing cells at 180 days. At 180 days, allografts were characterized by an abundant extracellular matrix composed of collagen and elastic fibers associated with extensive calcification (von Kossa staining) located in the intima and media. Osteoblastic activity was present in calcified lesion as shown by alkaline phosphatase activity. At 180 days, numerous chondrocytes (protein S100-positive and alpha-smooth muscle actin-negative) were present focally in the media. However, double immunostaining revealed that a cellular population within the media with a chondrocyte-like morphology was alpha-smooth muscle actin-positive and S100-negative. Active form of transforming growth factor beta1 was expressed from 30 to 80 days in the medial and intimal layers. CONCLUSIONS: These observations suggest that alpha-smooth muscle actin-positive cells within aortic allografts are eventually transformed to a chondrocyte-like structure, leading to vascular cartilaginous metaplasia associated with the expression of transforming growth factor beta1 and could be a potential pathway leading to extensive vascular wall calcification in allografts through endochondral ossification.

Generation of heme oxygenase-1-transgenic rats.

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2K(b) promoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

Inhibition of graft arteriosclerosis development in rat aortas following heme oxygenase-1 gene transfer.

Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into tree end products: biliverdin, free iron and carbon monoxide. This enzyme has recently been shown to have anti-inflammatory and tissue protective effects. HO-1 expression is involved in organ protection in pathological situations, and immunosuppressive treatments resulting in indefinite graft survival without chronic rejection have been associated with HO-1 expression by cells of the vessel wall. The aim of this study was to analyze the effect of specific HO-1 overexpression. We used a recombinant adenovirus coding for human HO-1 cDNA in a rat aorta chronic rejection model, 30 days after transplantation. Control groups included rats non treated or treated with a non-coding adenovirus Addl324. We first demonstrated that AdHO-1 was efficiently expressed in endothelial cells in vitro, and in rat aortas ex vivo after adenovirus gene transfer. We found that intimal thickening in AdHO-1 treated aortas (10.8 +/- 3.8%, n=5) was significantly decreased compared to untreated (21.2 +/- 5.6%, n = 5) or Addl324-treated (21.1 +/- 1.2%, n = 4) aortas. Immunohistology showed that treatment with AdHO-1 resulted in a significant reduction in leukocyte infiltration and a decreasing number of VSMC in the intima, compared to Addl324-treated aortas. However, this effect of HO-1 on chronic rejection did not imply modifications on numbers of apoptotic cells in the graft or of alloantibody levels. We have demonstrated, for the first time, that specific HO-1 overexpression following gene transfer of HO-1 inhibited chronic rejection by reducing leukocyte and VSMC infiltration of the aorta intima.

Genetic engineering in allotransplantation of vascularized organs.

Transplantation offers a unique opportunity for gene transfer into allografts before grafting. After organ retrieval, the cold ischemic period renders organs available for manipulation and gene transfer. Local expression of protective or immunomodulatory molecules within the graft environment offers a better local bioavailability of bioreagents and potentially less systemic side effects. Protection against ischemia-reperfusion injury, acute and/or chronic rejection without significant side effects would be a major breakthrough in transplant research. However, protocols of transfection adapted to the transplant setting and control of gene expression must be clearly evaluated before going to clinical trials. The first part of this review deals with gene transfer techniques into the allograft, emphasizing particular transplant conditions that are encountered and that must be respected when designing protocols for gene transfer experiments. The second part deals with specific therapeutic strategies to protect and prolong allograft survival.

Lethal hepatitis after gene transfer of IL-4 in the liver is independent of immune responses and dependent on apoptosis of hepatocytes: a rodent model of IL-4-induced hepatitis.

The putative role of IL-4 in human and animal models of hepatitis has not yet been directly determined. We now report that direct expression of IL-4 in the liver of rats or mice using recombinant adenoviruses coding for rat or mouse IL-4 (AdrIL-4 and AdmIL-4, respectively) results in a lethal, dose-dependent hepatitis. The hepatitis induced by IL-4 was characterized by hepatocyte apoptosis and a massive monocyte/macrophage infiltrate. IL-4-induced hepatitis was independent of T cell-mediated immune responses. Hepatitis occurred even after gene transfer of IL-4 into nude rats, CD8-depleted rats, cyclosporine A-treated rats, or recombinase-activating gene 2(-/-) immunodeficient mice. Peripheral depletion of leukocytes using high doses of cyclophosphamide, and/or the specific depletion of liver macrophages with liposome-encapsulated dichloromethylene diphosphonate in rats did not block lethal IL-4-induced hepatitis. Direct transduction of hepatocytes with adenoviruses was not essential, since injection of AdrIL-4 into the hind limb induced an identical hepatitis. Finally, primary rat hepatocytes in culture also showed apoptosis when cultured in the presence of rIL-4. IL-4-dependent hepatitis was associated with increases in the intrahepatic levels of IFN-gamma, TNF-alpha, and Fas ligand. Administration of AdmIL-4 to IFN-gamma, TNF-alpha receptor type I, or TNF-alpha receptor type II knockout mice also resulted in lethal hepatitis, whereas a moderate protection was observed in Fas-deficient lpr mice. IL-4-dependent hepatocyte apoptosis could be abolished by treatment with caspase inhibitory peptides. Our results thus demonstrate that IL-4 causes hepatocyte apoptosis, which is only partially dependent on the activation of Apo-1-Fas signaling and is largely independent of any immune cells in the liver.