Jean-Pierre Louboutin

Preterminal host dendritic cells in irradiated mice prime CD8+ T cell–mediated acute graft-versus-host disease

To understand the relationship between host antigen-presenting cells (APCs) and donor T cells in initiating graft-versus-host disease (GVHD), we followed the fate of host dendritic cells (DCs) in irradiated C57BL/6 (B6) recipient mice and the interaction of these cells with minor histocompatibility antigen- (miHA-) mismatched CD8+ T cells from C3H.SW donors. Host CD11c+ DCs were rapidly activated and aggregated in the T cell areas of the spleen within 6 hours of lethal irradiation. By 5 days after irradiation, <1% of host DCs were detectable, but the activated donor CD8+ T cells had already undergone as many as seven divisions. Thus, proliferation of donor CD8+ T cells preceded the disappearance of host DCs. When C3H.SW donor CD8+ T cells were primed in vivo in irradiated B6 mice or ex vivo by host CD11c+ DCs for 24-36 hours, they were able to proliferate and differentiate into IFN-gamma-producing cells in beta(2)-microglobulin-deficient (beta(2)m(-/-)) B6 recipients and to mediate acute GVHD in beta(2)m(-/-) --> B6 chimeric mice. These results indicate that, although host DCs disappear rapidly after allogeneic bone marrow transplantation, they prime donor T cells before their disappearance and play a critical role in triggering donor CD8+ T cell-mediated GVHD.

Toll-Like Receptor 4 Mediates Innate Immune Responses to Haemophilus influenzae Infection in Mouse Lung

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1β, IL-6, TNF-α, macrophage-inflammatory protein (MIP)-1α, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1β, IL-6, TNF-α, MIP-1α, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-α and MIP-1α expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-κB pathway in these cells based on the findings of decreased overall IκB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.

Human Airway Epithelial Cells Sense Pseudomonas aeruginosa Infection via Recognition of Flagellin by Toll-Like Receptor 5

ABSTRACT Pseudomonas aeruginosa, an opportunistic respiratory pathogen that infects the majority of patients with cystic fibrosis, initiates host inflammatory responses through interaction with airway epithelial cells. The Toll-like receptors (TLRs) are a family of pathogen pattern recognition receptors that play key roles in host innate immunity. In this study we aimed to determine whether TLRs mediate the interaction between P. aeruginosa and airway epithelial cells. Individual murine TLRs (TLR1 to TLR9) and dual combinations of these TLRs that activate an NF-κB-driven luciferase reporter in response to PAO1 were screened in HEK 293 cells. TLR5, TLR2, a combination of TLR1 and TLR2, or a combination of TLR2 and TLR6 responded to PAO1. Another P. aeruginosa strain, strain PAK, activated TLR5 similarly, while the isogenic flagellin-deficient strain PAK/fliC and the flagellum-free bacterium Haemophilus influenzae failed to activate TLR5. Reverse transcription-PCR was used to probe the presence of multiple TLRs (including TLR5) in primary human airway epithelial cells (HAECs). Immunostaining with TLR5 antibodies showed that TLR5 was expressed in HAECs and on the apical surface of the human trachea epithelium. In HAECs, PAO1, PAK, and Burkholderia cepacia, but not flagellin-deficient strain PAK/fliC or a B. cepacia fliC mutant, activated the NF-κB reporter. Dominant negative TLR5 specifically blocked the response to P. aeruginosa but not to the response to lipoteichoic acid, a specific ligand of TLR2. We also determined that MyD88, IRAK, TRAF6, and Toll-interacting protein (Tollip), but not TIRAP, were involved in the TLR-mediated response to P. aeruginosa in HAECs. These findings demonstrate that the airway epithelial receptor TLR5 senses P. aeruginosa through its flagellin protein, which may have an important role in the initiation of the host inflammatory reaction to clear the invading pathogen.

APCs in the Liver and Spleen Recruit Activated Allogeneic CD8+ T Cells to Elicit Hepatic Graft-Versus-Host Disease

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8+ T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8+ T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8+ T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8+ T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.

Gene transfer into skeletal muscle using novel AAV serotypes.

Skeletal muscle is an interesting target for gene delivery because of its mass and because the vectors can be delivered in a noninvasive way. Adeno‐associated virus (AAV) vectors are capable of transducing skeletal muscle fibers and achieving stable and safe transgene expression. To date, most animal experiments using AAV have been based on AAV serotype 2, but some recent studies have demonstrated that AAV1 is more efficient than AAV2/2 in transducing muscle fibers. Recently, novel AAVs (AAV7 and AAV8) were isolated from rhesus macaques.

Gene therapy with novel adeno-associated virus vectors substantially diminishes atherosclerosis in a murine model of familial hypercholesterolemia.

Familial hypercholesterolemia is an inherited disease caused by mutations in the LDL receptor gene leading to severe hypercholesterolemia and atherosclerosis. The LDL receptor is predominantly expressed in the liver, making it a preferred target organ for somatic gene therapy. We recently isolated a new family of vectors based on adeno‐associated viruses (AAVs) isolated from nonhuman primates, which enable efficient and stable transgene expression following in vivo gene delivery to liver.

Preexisting Immunity to Adenovirus in Rhesus Monkeys Fails To Prevent Vector-Induced Toxicity

ABSTRACT In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.

Correction of the Dystrophic Phenotype by In Vivo Targeting of Muscle Progenitor Cells

Successful gene therapy for most inherited diseases will require stable expression of the therapeutic gene. This can be addressed with integrating or self-replicating viruses by targeting postmitotic cells that have a long lifetime or stem cells that can replenish defective tissue with corrected cells. In this study, we explore the possibility of targeting a muscle stem cell population in situ through in vivo administration of vector. To develop this concept, we selected a mouse model of muscular dystrophy (mdx mice) that undergoes rapid turnover of muscle fibers. In vivo targeting of muscle progenitor cells, notably satellite cells, with a pseudotyped lentiviral vector encoding the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of mdx mice. This study shows that progenitor cells can be genetically engineered in vivo and subsequently proliferate into terminally differentiated tissue carrying the genetic graft in a way that stably corrects function.

Transduction of Human Islets with Pseudotyped Lentiviral Vectors

Type I diabetes is caused by an autoimmune-mediated elimination of insulin-secreting pancreatic islets. Genetic modification of islets offers a powerful molecular tool for improving our understanding of islet biology. Moreover, efficient genetic engineering of islets could allow for evaluation of new strategies aimed at preventing islet destruction. The present study evaluated the ability of a human immunodeficiency virus (HIV)-based lentiviral vector pseudotyped with various viral envelopes to target human islets ex vivo, with the goal of improving efficiency while minimizing toxicity. Transfer of the enhanced green fluorescent protein reporter gene in human islets was first evaluated with an HIV-based vector pseudotyped with the vesicular stomatitis virus (VSV), murine leukemia virus, Ebola, rabies, Mokola, or lymphocytic choriomeningitis virus (LCMV) envelope glycoprotein to optimize transduction efficiency. Results indicated that LCMV-pseudotyped vector transduced insulin-secreting beta cells with the highest efficiency. Moreover, toxicity associated with transduction of islets was found to be lower with LCMV-pseudotyped vector than with VSV-G-pseudotyped vector, the second most efficient vector for islet transduction. Overall, our study describes an improved methodology for achieving safe and efficient gene transfer into cells of human islets.

Intrathecal Gene Therapy Corrects CNS Pathology in a Feline Model of Mucopolysaccharidosis I

Enzyme replacement therapy has revolutionized the treatment of the somatic manifestations of lysosomal storage diseases (LSD), although it has been ineffective in treating central nervous system (CNS) manifestations of these disorders. The development of neurotrophic vectors based on novel serotypes of adeno-associated viruses (AAV) such as AAV9 provides a potential platform for stable and efficient delivery of enzymes to the CNS. We evaluated the safety and efficacy of intrathecal delivery of AAV9 expressing α-l-iduronidase (IDUA) in a previously described feline model of mucopolysaccharidosis I (MPS I). A neurological phenotype has not been defined in these animals, so our analysis focused on the biochemical and histological CNS abnormalities characteristic of MPS I. Five MPS I cats were dosed with AAV9 vector at 4-7 months of age and followed for 6 months. Treated animals demonstrated virtually complete correction of biochemical and histological manifestations of the disease throughout the CNS. There was a range of antibody responses against IDUA in this cohort which reduced detectable enzyme without substantially reducing efficacy; there was no evidence of toxicity. This first demonstration of the efficacy of intrathecal gene therapy in a large animal model of a LSD should pave the way for translation into the clinic.