RESUMO
KEY MESSAGE: A stable Agrobacterium-mediated transformation system was constructed for B. juncea, and BjuLKP2 was overexpressed, leading to plant yellowing. A stable and efficient transformation system is necessary to verify gene functions in plants. To establish an Agrobacterium-mediated transformation system for B. juncea, various factors, including the explant types, hormone combination and concentration, infection time and concentration, were optimized. Eventually, a reliable system was established, and two BjuLKP2 overexpression (OE) lines, which displayed yellowing of cotyledons, shoot tips, leaves and flower buds, as well as a decrease in total chlorophyll content, were generated. qRT-PCR assays revealed significant upregulation of five chlorophyll synthesis genes and downregulation of one gene in the BjuLKP2 OE line. Furthermore, antioxidant capacity assays revealed reduced activities of APX, CAT and SOD, while POD activity increased in the BjuLKP2 OE26. Additionally, the kinetic determination of chlorophyll fluorescence induction suggested a decrease in the photosynthetic ability of BjuLKP2 OE26. GUS assays revealed the expression of BjuLKP2 in various tissues, including the roots, hypocotyls, cotyledons, leaf vasculature, trichomes, sepals, petals, filaments, styles and stigma bases, but not in seeds. Scanning electron revealed alterations in chloroplast ultrastructure in both the sponge and palisade tissue. Collectively, these findings indicate that BjuLKP2 plays a role in plant yellowing through a reduction in chlorophyll content and changes in chloroplasts structure.
Assuntos
Clorofila , Regulação da Expressão Gênica de Plantas , Mostardeira , Agrobacterium/genética , Clorofila/metabolismo , Mostardeira/genética , Fotossíntese , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transformação GenéticaRESUMO
Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5-tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 µM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 µM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.
Assuntos
Deficiência de alfa 1-Antitripsina , alfa-Defensinas , Humanos , Monócitos/metabolismo , alfa-Defensinas/metabolismo , Macrófagos/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Macrófagos Alveolares/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by a hepatic accumulation of mutant alpha-1 antitrypsin (ZAAT). Individuals with AATD are prone to develop a chronic liver disease that remains undiagnosed until late stage of the disease. Here, we sought to characterize the liver pathophysiology of a human transgenic mouse model for AATD with a manifestation of liver disease compared with normal transgenic mice model. Male and female transgenic mice for normal (Pi*M) and mutant variant (Pi*Z) human alpha-1 antitrypsin at 3 and 6 mo of age were subjected to this study. The progression of hepatic ZAAT accumulation, hepatocyte injury, steatosis, liver inflammation, and fibrotic features were monitored by performing an in vivo study. We have also performed a Next-Gene transcriptomic analysis of the transgenic mice liver tissue 16 h after lipopolysaccharide (LPS) administration to delineate liver inflammatory response in Pi*Z mice as compared with Pi*M. Our results show hepatic ZAAT accumulation, followed by hepatocyte ballooning and liver steatosis developed at 3 mo in Pi*Z mice compared with the mice carrying normal variant of human alpha-1 antitrypsin. We observed higher levels of hepatic immune cell infiltrations in both 3- and 6-mo-old Pi*Z mice compared with Pi*M as an indication of liver inflammation. Liver fibrosis was observed as accumulation of collagen in 6-mo-old Pi*Z liver tissues compared with Pi*M control mice. Furthermore, the transcriptomic analysis revealed a dysregulated liver immune response to LPS in Pi*Z mice compared with Pi*M. Of particular interest for translational work, this study aims to establish a mouse model of AATD with a strong manifestation of liver disease that will be a valuable in vivo tool to study the pathophysiology of AATD-mediated liver disease. Our data suggest that the human transgenic mouse model of AATD could provide a suitable model for the evaluation of therapeutic approaches and preventive reagents against AATD-mediated liver disease.NEW & NOTEWORTHY We have characterized a mouse model of human alpha-1 antitrypsin deficiency with a strong manifestation of liver disease that can be used as an in vivo tool to test preventive and therapeutic reagents. Our data explores the altered immunophenotype of alpha-1 antitrypsin-deficient liver macrophages and suggests a relationship between acute inflammation, immune response, and fibrosis.
Assuntos
Fígado Gorduroso , Deficiência de alfa 1-Antitripsina , Masculino , Feminino , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Lipopolissacarídeos , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Camundongos Transgênicos , Modelos Animais de Doenças , InflamaçãoRESUMO
Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.
Assuntos
Hepatócitos/citologia , Deficiência de alfa 1-Antitripsina/patologia , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transporte Proteico , Proteólise , Análise de Sequência de RNA , alfa 1-Antitripsina/química , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
BACKGROUND: Coagulase-negative staphylococci (CoNS) are a group of opportunistic pathogens, which are widely spread in the environment. Animal breeding is an important source of pathogen spreading. However, the concentration and characteristics of CoNS in the bioaerosols of henhouses are unclear. RESULTS: In this study, we showed that CoNS were significantly increased in bioaerosols of henhouses during the first 60 days, and reached 2.0 × 106 CFU/m3, which account for 75.4% of total bacteria. One hundred and two CoNS isolates from bioaerosols and nasal swabs of farmers were further identified, covering seven species. Among these, 41.2% isolates were Staphylococcus sciuri, which was the predominant species, followed by S. equorum, S. saprophyticus, S. haemolyticus, S. xylosus, S. arlettae and S. gallinarum. There were high rates of resistance to oxacillin in CoNS (49.0%), which were defined as Methicillin-Resistant CoNS (MRCoNS), and 36.3% isolates contained resistance gene mecA. Bioaerosol infection models showed that, chickens exposed to aerosolized S. sciuri had significant induction of inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8 and IL-10 at 5 days post-infection (dpi) in lungs and at 7 dpi in spleens. CONCLUSIONS: We reported a high concentration of CoNS in henhouses, and S. sciuri was the preponderant CoNS species. Antibiotic resistance analysis and bioaerosols infection of CoNS further highlighted its hazards on resistance and immunological challenge. These results suggested that, CoNS in bioaerosols could be one serious factor in the henhouses for not only poultry industry but also public health.
Assuntos
Galinhas/microbiologia , Mucosa Nasal/microbiologia , Staphylococcus/isolamento & purificação , Animais , China , Farmacorresistência Bacteriana , Fazendeiros , Abrigo para Animais , Humanos , Interleucinas/metabolismo , Oxacilina/farmacologiaRESUMO
Pulmonary hypertension complicates the care of many patients with chronic lung diseases (defined as Group 3 pulmonary hypertension), yet the mechanisms that mediate the development of pulmonary vascular disease are not clearly defined. Despite being the most prevalent form of pulmonary hypertension, to date there is no approved treatment for patients with disease. Myeloid-derived suppressor cells (MDSCs) and endothelial cells in the lung express the chemokine receptor CXCR2, implicated in the evolution of both neoplastic and pulmonary vascular remodeling. However, precise cellular contribution to lung disease is unknown. Therefore, we used mice with tissue-specific deletion of CXCR2 to investigate the role of this receptor in Group 3 pulmonary hypertension. Deletion of CXCR2 in myeloid cells attenuated the recruitment of polymorphonuclear MDSCs to the lungs, inhibited vascular remodeling, and protected against pulmonary hypertension. Conversely, loss of CXCR2 in endothelial cells resulted in worsened vascular remodeling, associated with increased MDSC migratory capacity attributable to increased ligand availability, consistent with analyzed patient sample data. Taken together, these data suggest that CXCR2 regulates MDSC activation, informing potential therapeutic application of MDSC-targeted treatments.
Assuntos
Células Endoteliais/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Células Supressoras Mieloides/metabolismo , Fibrose Pulmonar/metabolismo , Receptores de Interleucina-8B/genética , Transdução de Sinais , Animais , Bleomicina/administração & dosagem , Comunicação Celular , Movimento Celular , Células Endoteliais/patologia , Feminino , Expressão Gênica , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/etiologia , Hipóxia/genética , Hipóxia/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/patologia , Cultura Primária de Células , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Receptores de Interleucina-8B/deficiência , Remodelação VascularRESUMO
Alpha-1-antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha-1-antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER-associated degradation and autophagy. We hypothesized that alpha-1-antitrypsin (AAT)-interacting proteins play critical roles in quality control of human AAT. Using co-immunoprecipitation, we identified ERdj3, an ER-resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin-EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3-mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease. J. Cell. Biochem. 118: 3090-3101, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteólise , alfa 1-Antitripsina/metabolismo , Linhagem Celular , Retículo Endoplasmático/genética , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , alfa 1-Antitripsina/genéticaRESUMO
Preeclampsia is a kind of disease that severely harms the health of pregnant women and infants. To better understand the molecular mechanisms involved in preeclampsia, we used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to construct a comparative peptidomic profiling of human serum between normal and preeclamptic pregnancies. A total of 201 peptides were confidently identified, with 21 up-regulated and three down-regulated. Further analysis indicated that these differentially expressed peptides correlate with enzyme regulator activity, biological regulation, and coagulation cascades occurring during pathological changes of preeclampsia. The identification of key peptides in serum may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early onset severe PE (sPE). J. Cell. Biochem. 118: 4341-4348, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Peptídeos/sangue , Pré-Eclâmpsia/sangue , Proteômica , Adulto , Biomarcadores/sangue , Feminino , Humanos , GravidezRESUMO
AIMS: This study aimed to identify the different expression of circular RNAs (circRNAs) in the placental tissues of pregnant women with preeclampsia (PE) and to provide a new avenue of research regarding the pathological mechanisms of PE. METHODS: In this study, we collected 40 placental tissues from PE patients and 35 placental tissues from gestational age-matched patients who gave premature birth. Arraystar circRNA Microarray Technology (KANGCHEN, Shanghai, China) was used to analyze the differential expression of circRNAs. According to the basic content of circRNAs in the two groups and their fold changes and due to the practicability of the designed divergent primers of each candidate circRNA, we selected three up-regulated circRNAs, hsa_circRNA_100782, hsa_circRNA_102682 and hsa_circRNA_104820, to validate the data. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was utilized to estimate the Ct values in both groups. We further evaluated the differences with a paired t-test and a receiver operating characteristic (ROC) curve. RESULTS: Many circRNAs were found to be differentially expressed in PE placental tissues versus their controls; of these, 143 circRNAs were up-regulated and 158 were down-regulated. The expression levels of hsa_circRNA_100782 (p < 0.05), hsa_circRNA_102682 (p < 0.05), and hsa_circRNA_104820 (p < 0.0001) were validated as significantly up-regulated in the experimental group compared with the controls. Finally, we performed a literature comparison to forecast the possible mechanisms of circRNA function during PE. CONCLUSION: circRNA expression significantly differed in placental PE tissues compared with controls. According to the circRNA microarray results and the existing papers, circRNAs may contribute to the pathogenesis of PE by acting as miRNA sponges; this possibility requires additional investigation in future studies.
Assuntos
MicroRNAs/genética , Trabalho de Parto Prematuro/genética , Placenta/metabolismo , Pré-Eclâmpsia/genética , RNA/genética , Adulto , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Trabalho de Parto Prematuro/metabolismo , Trabalho de Parto Prematuro/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Placenta/fisiopatologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/fisiopatologia , Gravidez , RNA/metabolismo , RNA Circular , Curva ROC , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Exosomes are extracellular vesicles that are involved in several biological processes. The roles of proteins from human umbilical cord blood exosomes in the pathogenesis of preeclampsia remains poorly understood. METHODS: In this study, we used high-resolution LC-MS/MS technologies to construct a comparative proteomic profiling of human umbilical cord blood exosomes between normal and preeclamptic pregnancies. RESULTS: A total of 221 proteins were detected in human umbilical cord blood exosomes, with 14 upregulated and 15 downregulated proteins were definitively identified between preeclamptic and control pregnancies. Further bioinformatics analysis (Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis) indicated that these differentially expressed proteins correlate with enzyme regulator activity, binding, extracellular region, cell part, biological regulation, cellular process and complement and coagulation cascades occurring during pathological changes of preeclampsia. CONCLUSION: Our results show significantly altered expression profiles of proteins in human umbilical cord blood exosomes between normal and preeclampsia pregnancies. These proteins may be involved in the etiology of preeclampsia.
Assuntos
Exossomos/metabolismo , Sangue Fetal/metabolismo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Proteômica , Espectrometria de Massas em Tandem/métodos , Adulto , Cromatografia Líquida , Cromossomos Humanos/metabolismo , Biologia Computacional , Feminino , Ontologia Genética , Humanos , Nanopartículas , Pré-Eclâmpsia/patologia , Gravidez , Mapas de Interação de Proteínas , Proteoma/metabolismoRESUMO
Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P < .0001 and P < .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.
Assuntos
Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , alfa 1-Antitripsina/farmacologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Encéfalo/fisiopatologia , Citoproteção , Modelos Animais de Doenças , Endotelina-1 , Humanos , Infarto da Artéria Cerebral Média/induzido quimicamente , Infarto da Artéria Cerebral Média/diagnóstico , Infarto da Artéria Cerebral Média/fisiopatologia , Injeções Intravenosas , Imageamento por Ressonância Magnética , Masculino , Microinjeções , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Ratos Sprague-Dawley , Limiar Sensorial/efeitos dos fármacos , Fatores de Tempo , alfa 1-Antitripsina/administração & dosagemRESUMO
BACKGROUND: Obesity has been established as a significant risk factor for osteoarthritis. Anti-obesity medications (AOMs) have demonstrated efficacy in weight management. However, potential impact on osteoarthritis risk remains uncertain. METHODS: This retrospective cohort study used Kythera data from NOV2022 to JULY2024. Patients with obesity using AOMs were identified through diagnosis and prescription claims for tirzepatide, semaglutide, or liraglutide between 1NOV2023 and 31JAN2024, with a 6-month follow-up to assess OA risk. OA risk, analyzed using Cox regression and propensity score matching, controlled for comorbidities and sociodemographic factors. RESULTS: There were 39,394 patients living with obesity using AOM (23,933 semaglutide 12,854 tirzepatide, 2,607 liraglutide) and 72,405 without AOM use. The adjusted osteoarthritis risk was 27% % lower in AOM users than in non-users (hazard ratio (HR) = 073, 95% CI (0.67-0.79), p < 0.01). Among AOMs, tirzepatide was associated with a significantly lower osteoarthritis (OA) risk compared to semaglutide (HR = 0.57, 95% CI: 0.50-0.65, p < 0.0001). Liraglutide was linked to a significantly higher OA risk vs tirzepatide (HR = 1.63, 95% CI: 1.23-2.15, p = 0.0007). CONCLUSIONS: AOM use was associated with a significantly lower risk of OA and may be an effective obesity management intervention.
RESUMO
Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.
Assuntos
Dependovirus/imunologia , Imunidade/imunologia , Transgenes/imunologia , alfa 1-Antitripsina/imunologia , Transferência Adotiva , Animais , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Dependovirus/classificação , Dependovirus/genética , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunização , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Especificidade da Espécie , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção , Transgenes/genética , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genéticaRESUMO
α-1 antitrypsin (AAT) is a serine protease inhibitor that plays a pivotal role in maintaining lung homeostasis. The most common AAT allele associated with AAT deficiency (AATD) is PiZ. Z-AAT accumulates in cells due to misfolding, causing severe AATD. The major function of AAT is to neutralize neutrophil elastase in the lung. It is generally accepted that loss of antiprotease function is a major cause of COPD in individuals with AATD. However, it is now being recognized that the toxic gain-of-function effect of Z-AAT in macrophage likely contributes to lung disease. In the present study, we determined that TLR7 signaling is activated in Z-MDMs, and the expression level of NLRP3, one of the targets of TLR7 signaling, is significantly higher in Z- compared with M-MDMs. We also determined that the level of endosomal Alu RNA is significantly higher in Z-compared with M-MDMs. Alu RNA is a known endogenous ligand that activates TLR7 signaling. Z-AAT likely induces the expression of Alu elements in MDMs and accelerates monocyte death, leading to the higher level of endosomal Alu RNA in Z-MDMs. Taken together,this study identifies a mechanism responsible for the toxic gain of function of Z-AAT macrophages.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Deficiência de alfa 1-Antitripsina , Humanos , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , RNA , Receptor 7 Toll-Like/genética , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
Alpha-1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full-length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R-like ER kinase and inositol-requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro-apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Deficiência de alfa 1-Antitripsina , Animais , Carcinoma Hepatocelular/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Resposta a Proteínas não Dobradas/genética , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
BACKGROUND & AIMS: The adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential applications in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) with respect to morphology, immune-phenotype, and multiple differentiation capability. In the present study, we investigated the feasibility of AT-MSC-based liver gene delivery for the treatment of alpha 1-antitrypsin deficiency. METHODS: Mouse AT-MSCs were transduced by rAAV vectors and transplanted into the mouse liver. RESULTS: We showed that AT-MSCs can be transduced by recombinant adeno-associated viral vector serotype 1 (rAAV1-CB-hAAT). After transplanting to the mouse liver, ex vivo transduced AT-MSCs expressed the transgene product, human alpha 1-antitrypsin (hAAT). Importantly, serum levels of hAAT were sustained and no anti-hAAT antibody was detected in any recipients. CONCLUSIONS: These results demonstrated that AT-MSCs can be transduced by rAAV vectors, engrafted into recipient livers, contribute to liver regeneration, and serve as a platform for transgene expression without eliciting an immune response. AT-MSC-based gene therapy presents a novel approach for the treatment of liver diseases, such as AAT deficiency.
Assuntos
Tecido Adiposo/citologia , Terapia Genética/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Animais , Anticorpos/sangue , Dependovirus/genética , Sobrevivência de Enxerto/imunologia , Humanos , Fígado/citologia , Fígado/fisiologia , Regeneração Hepática/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transgenes/genética , Transgenes/imunologia , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/imunologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/imunologiaRESUMO
Adult stem cell-based gene therapy holds several unique advantages including avoidance of germline or other undesirable cell transductions. We have previously shown that liver progenitor (oval) cells can be used as a platform for liver gene delivery of human alpha1-antitrypsin (hAAT). However, this cell source cannot be used in humans for autologous transplantation. In the present study, we tested the feasibility of bone marrow (BM) cell-based liver gene delivery of hAAT. In vitro studies showed that BM cells can be transduced by lentiviral vector (Lenti-CB-hAAT) and recombinant adeno-associated viral vectors (rAAV1-CB-hAAT, and rAAV8-CB-hAAT). Transplantation studies showed that transplanted BM cells homed into liver, differentiated into hepatocytes and expressed hAAT in the liver. Importantly, we showed that transplantation of rAAV8-CB-hAAT vector-transduced BM cells resulted in sustained levels of hAAT in the systemic circulation of recipient mice. These results demonstrated that rAAV vector-mediated BM cell-based liver gene therapy is feasible for the treatment of AAT deficiency and implies a novel therapy for the treatment of liver diseases.
Assuntos
Fígado/metabolismo , alfa 1-Antitripsina/metabolismo , Adenoviridae/genética , Albuminas/genética , Albuminas/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética/métodos , Cromossomo Y/metabolismo , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genéticaRESUMO
BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) secondary to chronic lung disease (World Health Organization Group 3 PH) is deadly, with lung transplant being the only available long-term treatment option. Myeloid-derived cells are known to affect progression of both pulmonary fibrosis and PH, although the mechanism of action is unknown. Therefore, we investigated the effect of myeloid cell proliferation induced by emergency myelopoiesis on development of PH and therapy directed against programmed death-ligand 1 (PD-L1), expressed by myeloid cells in prevention of pulmonary vascular remodelling. EXPERIMENTAL APPROACH: LysM.Cre-DTR ("mDTR") mice were injected with bleomycin (0.018 U·g-1 , i.p.) while receiving either vehicle or diphtheria toxin (DT; 100 ng, i.p.) to induce severe PH. Approximately 4 weeks after initiation of bleomycin protocol, right ventricular pressure measurements were performed and tissue samples collected for histologic assessment. In a separate experiment, DT-treated mice were given anti-PD-L1 antibody (αPD-L1; 500 µg, i.p.) preventive treatment before bleomycin administration. KEY RESULTS: Mice undergoing induction of emergency myelopoiesis displayed more severe PH, right ventricular remodelling and pulmonary vascular muscularization compared to controls, without a change in lung fibrosis. This worsening of PH was associated with increased pulmonary myeloid-derived suppressor cell (MDSC), particularly polymorphonuclear MDSC (PMN-MDSC). Treatment with αPD-L1 normalized pulmonary pressures. PD-L1 expression was likewise found to be elevated on circulating PMN-MDSC from patients with interstitial lung disease and PH. CONCLUSIONS AND IMPLICATIONS: PD-L1 is a viable therapeutic target in PH, acting through a signalling axis involving MDSC. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.
Assuntos
Células Supressoras Mieloides , Fibrose Pulmonar , Animais , Bleomicina , Humanos , Camundongos , Mielopoese , Fibrose Pulmonar/induzido quimicamente , Remodelação VascularRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT). METHODS: DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-induced interleukin (IL)-6 production from mouse fibroblast cells were also determined. RESULTS: Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition. CONCLUSIONS: The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.
Assuntos
Artrite Experimental/tratamento farmacológico , Doxiciclina/uso terapêutico , Terapia Genética/métodos , alfa 1-Antitripsina/uso terapêutico , Animais , Anticorpos/metabolismo , Antirreumáticos/uso terapêutico , Artrite Experimental/genética , Artrite Experimental/patologia , Dependovirus/efeitos dos fármacos , Dependovirus/genética , Humanos , Interleucina-6/metabolismo , Articulações/efeitos dos fármacos , Articulações/patologia , Camundongos , Células NIH 3T3 , Tetraciclina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , alfa 1-Antitripsina/imunologiaRESUMO
Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms.