RESUMO
We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction efficiencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.
Assuntos
Dependovirus , Epigênese Genética , Fator IX , Vetores Genéticos , Macaca mulatta , Animais , Fator IX/genética , Fator IX/metabolismo , Dependovirus/genética , Camundongos , Humanos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Hemofilia B/genética , Hemofilia B/terapia , Transdução Genética , Terapia Genética/métodosRESUMO
Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.
RESUMO
Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans.
Assuntos
Proteínas do Capsídeo , Capsídeo , Humanos , Camundongos , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Transdução Genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Cromatina/genética , Cromatina/metabolismo , Epigênese GenéticaRESUMO
Adeno-associated viruses (AAVs) are commonly used for in vivo gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nε-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. We were able to identify AAV vectors that would tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging efficiency. We functionalized the Nε-AAVs through conjugation with DNA (AS1411) or RNA (E3) aptamers or with a folic acid moiety (FA). E3-, AS1411-, and FA-AAVs showed on average a 3- to 9-fold increase in transduction compared with their non-conjugated counterparts in different cancer cell lines. Using specific competitors, we established ligand-specific transduction. In vivo studies confirmed the selective uptake of FA-AAV and AS1411-AAV without off-target transduction in peripheral organs. Overall, the high versatility of these novel Nε-AAVs might pave the way to tailoring gene therapy vectors toward specific types of cells both for ex vivo and in vivo applications.
RESUMO
A limitation for recombinant adeno-associated virus (rAAV)-mediated gene transfer into the central nervous system (CNS) is the low penetration of vectors across the human blood-brain barrier (BBB). High doses of intravenously delivered vector are required to reach the CNS, which has resulted in varying adverse effects. Moreover, selective transduction of various cell types might be important depending on the disorder being treated. To enhance BBB penetration and improve CNS cell selectivity, we screened an AAV capsid-shuffled library using an in vitro transwell BBB system with separate layers of human endothelial cells, primary astrocytes and/or human induced pluripotent stem cell-derived cortical neurons. After multiple passages through the transwell, we identified chimeric AAV capsids with enhanced penetration and improved transduction of astrocytes and/or neurons compared with wild-type capsids. We identified the amino acids (aa) from regions 451-470 of AAV2 associated with the capsids selected for neurons, and a combination of aa from regions 413-496 of AAV-rh10 and 538-598 of AAV3B/LK03 associated with capsids selected for astrocytes. A small interfering RNA screen identified several genes that affect transcytosis of AAV across the BBB. Our work supports the use of a human transwell system for selecting enhanced AAV capsids targeting the CNS and may allow for unraveling the underlying molecular mechanisms of BBB penetration.
RESUMO
Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.
Assuntos
Sistemas CRISPR-Cas , Vetores Genéticos , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Endonucleases/genética , Edição de Genes/métodos , Recombinação Homóloga , Humanos , Camundongos , Vidarabina/análogos & derivadosRESUMO
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by â¼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to â¼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by â¼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by â¼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.
Assuntos
Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Dependovirus/genética , Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , RecQ Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Células HeLa , Células-Tronco Hematopoéticas/citologia , Recombinação Homóloga , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
Assuntos
Furanos/química , Luciferases/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Animais , Ensaios Enzimáticos/métodos , Especificidade por SubstratoRESUMO
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional ß cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived ß cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
Assuntos
Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Células Cultivadas , Diabetes Mellitus , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Biblioteca Gênica , Técnicas de Transferência de Genes , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
miR-122 is a highly expressed liver microRNA that is activated perinatally and aids in regulating cholesterol metabolism and promoting terminal differentiation of hepatocytes. Disrupting expression of miR-122 can re-activate embryo-expressed adult-silenced genes, ultimately leading to the development of hepatocellular carcinoma (HCC). Here we interrogate the liver transcriptome at various time points after genomic excision of miR-122 to determine the cellular consequences leading to oncogenesis. Loss of miR-122 leads to specific and progressive increases in expression of imprinted clusters of microRNAs and mRNA transcripts at the Igf2 and Dlk1-Dio3 loci that could be curbed by re-introduction of exogenous miR-122. mRNA targets of other abundant hepatic microRNAs are functionally repressed leading to widespread hepatic transcriptional de-regulation. Together, this reveals a transcriptomic framework for the hepatic response to loss of miR-122 and the outcome on other microRNAs and their cognate gene targets.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fator de Crescimento Insulin-Like II , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Iodeto Peroxidase/metabolismo , Neoplasias Hepáticas/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.
Assuntos
Proteínas do Capsídeo/genética , Dependovirus/genética , Engenharia Genética , Vetores Genéticos/genética , Fígado/metabolismo , Transdução Genética , Animais , Proteínas do Capsídeo/química , Feminino , Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Modelos Moleculares , Conformação ProteicaRESUMO
Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.
Assuntos
Pequeno RNA não Traduzido/genética , RNA de Transferência de Leucina/genética , Proteínas Ribossômicas/biossíntese , Ribossomos/genética , Ribossomos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pareamento de Bases , Sequência de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Masculino , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Pequeno RNA não Traduzido/antagonistas & inibidores , RNA de Transferência de Leucina/antagonistas & inibidores , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/efeitos dos fármacos , Especificidade por Substrato/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.
Assuntos
Dependovirus/genética , Resistência Microbiana a Medicamentos/genética , Engenharia Genética/métodos , Vetores Genéticos/química , Plasmídeos/química , alfa 1-Antitripsina/genética , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Dependovirus/metabolismo , Feminino , Inativação Gênica , Vetores Genéticos/metabolismo , Humanos , Canamicina/farmacologia , Fígado/metabolismo , Fígado/virologia , Camundongos , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Vírus do Sarcoma de Rous/genética , Vírus do Sarcoma de Rous/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transgenes , alfa 1-Antitripsina/metabolismoRESUMO
We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.
Assuntos
Regiões 5' não Traduzidas/genética , Dependovirus/genética , Éxons/genética , Genes Reporter/fisiologia , Vetores Genéticos/genética , Íntrons/genética , Transgenes/fisiologia , Animais , Replicação do DNA , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Recombinação GenéticaRESUMO
Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.
Assuntos
Repressão Epigenética/genética , Hepatopatias/etiologia , MicroRNAs/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/efeitos adversos , Alanina Transaminase/metabolismo , Animais , Northern Blotting , Dependovirus/genética , Técnicas de Silenciamento de Genes , Vetores Genéticos , Células HEK293 , Humanos , Hepatopatias/genética , Hepatopatias/metabolismo , Camundongos , Camundongos Knockout , Isoformas de RNA , RNA Mensageiro/metabolismo , Terapêutica com RNAi/efeitos adversos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , alfa 1-Antitripsina/metabolismoRESUMO
Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8-13 times more frequently than control vectors, providing an estimate that 23-39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells.
Assuntos
DNA Ribossômico/genética , Dependovirus/genética , Vetores Genéticos/genética , Transgenes , Integração Viral , Animais , Southern Blotting , Clonagem Molecular , Fator IX/genética , Fator IX/metabolismo , Feminino , Dosagem de Genes , Expressão Gênica , Loci Gênicos , Humanos , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Plasmídeos , Recombinação Genética , Análise de Sequência de DNA , Transdução GenéticaRESUMO
Human exposure to air pollutants, including ambient particulate matter, has been proposed as a mechanism for the rise in allergic disorders. Diesel exhaust particles, a major component of ambient particulate matter, induce sensitization to neoallergens, but the mechanisms by which sensitization occur remain unclear. We show that diesel exhaust particles upregulate thymic stromal lymphopoietin in human bronchial epithelial cells in an oxidant-dependent manner. Thymic stromal lymphopoietin induced by diesel exhaust particles was associated with maturation of myeloid dendritic cells, which was blocked by anti-thymic stromal lymphopoietin antibodies or silencing epithelial cell-derived thymic stromal lymphopoietin. Dendritic cells exposed to diesel exhaust particle-treated human bronchial epithelial cells induced Th2 polarization in a thymic stromal lymphopoietin-dependent manner. These findings provide new insight into the mechanisms by which diesel exhaust particles modify human lung mucosal immunity.
Assuntos
Poluentes Atmosféricos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Emissões de Veículos , Acetilcisteína/farmacologia , Anticorpos/farmacologia , Brônquios/citologia , Brônquios/imunologia , Brônquios/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Citocinas/farmacologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Tamanho da Partícula , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Linfopoietina do Estroma do TimoRESUMO
Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice. Am J Physiol Lung Cell Mol Physiol 293: , 2007. First published August 17, 2007; - Mechanical ventilation (MV) with O(2)-rich gas offers life-saving treatment for extremely premature infants with respiratory failure but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2- to 4-day-old mice with 40% O(2) for 8 h, compared with unventilated control pups, reduced lung expression of genes that regulate lung septation and angiogenesis (VEGF-A and its receptor, VEGF-R2; PDGF-A; and tenascin-C). MV with air for 8 h yielded similar results for PDGF-A and tenascin-C but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4- to 6-day-old mice with 40% O(2) for 24 h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A, and tenascin-C and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40% O(2) for 24 h, lung volume was similar to unventilated controls, whereas distal air space size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24 h of MV with 40% O(2). These molecular, cellular, and structural changes occurred without detectable lung inflammation as evaluated by histology and assays for proinflammatory cytokines, myeloperoxidase activity, and water content in lung. Thus lengthy MV of newborn mice with O(2)-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.
Assuntos
Biomarcadores/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Oxigênio/metabolismo , Alvéolos Pulmonares/patologia , Respiração Artificial , Animais , Animais Recém-Nascidos , Citocinas/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Oxigenoterapia , Alvéolos Pulmonares/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Alveolar epithelial cells are among the first cells to encounter inhaled particles or organisms. These cells likely participate in the initiation and modulation of the inflammatory response by production of chemokines. However, there is little information on the extent or regulation of chemokine production by these cells. Rat type II cells were studied under differentiated and dedifferentiated conditions to determine their ability to express and secrete CXC chemokines. Both differentiated and dedifferentiated type II cells secreted MIP-2, MCP-1, and CINC-2 in response to a cytokine mixture of IL-1beta, TNF-alpha, and IFN-gamma or to IL-1beta alone. The cytokine mixture also induced iNOS expression and nitrite secretion. Both differentiated and dedifferentiated type II cells expressed CINC-1 (GRO), CINC-2alpha, CINC-3 (MIP-2), and MCP-1 mRNA, and their expression was increased by the cytokine mixture or by IL-1beta alone. However, CINC-2beta, a splice variant of CINC-2, was only expressed under differentiated conditions stimulated by KGF and was not increased by the cytokine mixture or by IL-1beta. In situ hybridization of normal lung and lung instilled with Ad-KGF demonstrated that CINC-2beta was expressed by alveolar and bronchiolar epithelial cells in vivo. We conclude that CINC-2beta is regulated differently from most other chemokines and that its expression is related to the state of alveolar type II cell differentiation.
Assuntos
Quimiocinas CXC/metabolismo , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/genética , Citocinas/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Keratinocyte growth factor (KGF) is a mitogen for rat type II cells and also stimulates differentiation in vitro. Administration of KGF also protects the lung from a variety of injuries and subsequent development of fibrosis. Because transforming growth factor (TGF)-beta has been shown to inhibit epithelial cell proliferation and surfactant protein gene expression in other systems and is thought to be a major effector in pulmonary fibrosis, we sought to determine if TGF-beta would antagonize the effects of KGF in primary cultures of alveolar type II cells. Type II cells were cultured on a matrix of type I collagen and Matrigel in the presence or absence of KGF and/or TGF-beta. KGF alone greatly stimulated proliferation and increased cyclin-dependent kinase (cdk) 2 kinase activity and Retinoblastoma susceptibility gene product (Rb) phosphorylation. Cyclin D1, cdk2, and cdc25A protein levels were increased, and p15(Ink4b) and p27(Kip1) protein levels were decreased. TGF-beta markedly inhibited alveolar epithelial cell proliferation induced by KGF. TGF-beta inhibited cdk2 enzyme activity and Rb phosphorylation and increased p15(Ink4b) protein levels. TGF-beta also inhibited differentiation induced by KGF as measured by secretion of surfactant protein-A into the apical media. In summary, TGF-beta inhibits the proliferative effect of KGF in vitro and may be a biologic antagonist of KGF.