RESUMO
Development of acute respiratory distress syndrome (ARDS) in influenza A virus (IAV)-infected mice is associated with inhibition of ATII (alveolar type II) epithelial cell de novo phosphatidylcholine synthesis, and administration of the phosphatidylcholine precursor cytidine 5'-diphosphocholine (CDP-choline) attenuates IAV-induced acute respiratory distress syndrome in mice. We hypothesized inhibition of phosphatidylcholine synthesis would also impact the function of ATII cell mitochondria. To test this hypothesis, adult C57BL/6 mice of both sexes were inoculated intranasally with 10,000 pfu/mouse influenza A/WSN/33 (H1N1). Control mice were mock-infected with virus diluent. Mice were treated with saline vehicle or CDP-choline (100 µg/mouse i.p.) once daily from 1 to 5 days postinoculation (dpi). ATII cells were isolated by a standard lung digestion protocol at 6 dpi for analysis of mitochondrial function. IAV infection increased uptake of the glucose analog fludeoxyglucose F 18 by the lungs and caused a switch from oxidative phosphorylation to aerobic glycolysis as a primary means of ATII cell ATP synthesis by 6 dpi. Infection also induced ATII cell mitochondrial depolarization and shrinkage, upregulation of PGC-1α, decreased cardiolipin content, and reduced expression of mitofusin 1, OPA1, DRP1, complexes I and IV of the electron transport chain, and enzymes involved in cardiolipin synthesis. Daily CDP-choline treatment prevented the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis resulting from IAV infection but did not fully reverse the glycolytic shift. CDP-choline also did not prevent the alterations in mitochondrial protein expression resulting from infection. Taken together, our data show ATII cell mitochondrial dysfunction after IAV infection results from impaired de novo phospholipid synthesis, but the glycolytic shift does not.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Síndrome do Desconforto Respiratório , Animais , Cardiolipinas , Citidina Difosfato Colina , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , FosfatidilcolinasRESUMO
Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant-induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM-induced lung injury than the wild-type mice. Treatment of wild-type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM-induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM-induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant-induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant-induced lung injury.
Assuntos
Lesão Pulmonar Aguda , Mecloretamina , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/genética , Animais , Células Endoteliais/metabolismo , Pulmão/metabolismo , Mecloretamina/uso terapêutico , Mecloretamina/toxicidade , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
There is an urgent need for new drugs for patients with acute respiratory distress syndrome (ARDS), including those with coronavirus disease (COVID-19). ARDS in influenza-infected mice is associated with reduced concentrations of liponucleotides (essential precursors for de novo phospholipid synthesis) in alveolar type II (ATII) epithelial cells. Because surfactant phospholipid synthesis is a primary function of ATII cells, we hypothesized that disrupting this process could contribute significantly to the pathogenesis of influenza-induced ARDS. The goal of this study was to determine whether parenteral liponucleotide supplementation can attenuate ARDS. C57BL/6 mice inoculated intranasally with 10,000 plaque-forming units/mouse of H1N1 influenza A/WSN/33 virus were treated with CDP (cytidine 5'-diphospho)-choline (100 µg/mouse i.p.) ± CDP -diacylglycerol 16:0/16:0 (10 µg/mouse i.p.) once daily from 1 to 5 days after inoculation (to model postexposure influenza prophylaxis) or as a single dose on Day 5 (to model treatment of patients with ongoing influenza-induced ARDS). Daily postexposure prophylaxis with CDP-choline attenuated influenza-induced hypoxemia, pulmonary edema, alterations in lung mechanics, impairment of alveolar fluid clearance, and pulmonary inflammation without altering viral replication. These effects were not recapitulated by the daily administration of CTP (cytidine triphosphate) and/or choline. Daily coadministration of CDP-diacylglycerol significantly enhanced the beneficial effects of CDP-choline and also modified the ATII cell lipidome, reversing the infection-induced decrease in phosphatidylcholine and increasing concentrations of most other lipid classes in ATII cells. Single-dose treatment with both liponucleotides at 5 days after inoculation also attenuated hypoxemia, altered lung mechanics, and inflammation. Overall, our data show that liponucleotides act rapidly to reduce disease severity in mice with severe influenza-induced ARDS.
Assuntos
Células Epiteliais Alveolares/metabolismo , Citidina Difosfato Colina/farmacologia , Diglicerídeos de Citidina Difosfato/farmacologia , Vírus da Influenza A Subtipo H1N1/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Síndrome do Desconforto Respiratório/prevenção & controle , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , COVID-19/patologia , Camundongos , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , SARS-CoV-2/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.
Assuntos
Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/fisiologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Metabolismo dos Lipídeos , Metaboloma , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Surfactantes Pulmonares/metabolismo , Células Epiteliais Alveolares/virologia , Animais , Líquido da Lavagem Broncoalveolar , Separação Celular , Colesterol/metabolismo , Citidina Difosfato Colina , Camundongos Endogâmicos C57BL , Fosfolipídeos/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors have gained an extensive record of safety and efficacy in animal models of human disease. Infrequent reports of genotoxicity have been limited to specific vectors associated with excess hepatocellular carcinomas (HCC) in mice. In order to understand potential mechanisms of genotoxicity, and identify patterns of insertion that could promote tumor formation, we compared a self-complementary AAV (scAAV) vector designed to promote insertional activation (scAAV-CBA-null) to a conventional scAAV-CMV-GFP vector. HCC-prone C3H/HeJ mice and severe combined immunodeficiency (SCID) mice were infected with vector plus secondary treatments including partial hepatectomy (HPX) and camptothecin (CPT) to determine the effects of cell cycling and DNA damage on tumor incidence. Infection with either vector led to a significant increase in HCC incidence in male C3H/HeJ mice. Partial HPX after infection reduced HCC incidence in the cytomegalovirus-green fluorescent protein (CMV-GFP)-infected mice, but not in the cognate chicken ß-actin (CBA)-null infected group. Tumors from CBA-null infected, hepatectomized mice were more likely to contain significant levels of vector DNA than tumors from the corresponding CMV-GFP-infected group. Most CBA-null vector insertions recovered from tumors were associated with known proto-oncogenes or tumor suppressors. Specific patterns of insertion suggested read-through transcription, enhancer effects, and disruption of tumor suppressors as likely mechanisms for genotoxicity.
Assuntos
Carcinoma Hepatocelular/virologia , Dependovirus/genética , Neoplasias Hepáticas Experimentais/virologia , Mutagênese Insercional , Integração Viral , Animais , Sequência de Bases , Camptotecina , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Dano ao DNA , Dependovirus/fisiologia , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Fator 3 de Crescimento de Fibroblastos/genética , Vetores Genéticos , Genoma Viral , Hepatectomia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas p21(ras)/genética , Proto-Oncogenes , Proteína SOS1/genética , Ativação TranscricionalRESUMO
BACKGROUND: New drugs for veterinary patients with acute respiratory distress syndrome (ARDS) are urgently needed. Early or late postinfection treatment of influenza-infected mice with the liponucleotide cytidine diphosphocholine (CDP-choline) resulted in decreased hypoxemia, pulmonary edema, lung dysfunction, and inflammation without altering viral replication. These findings suggested CDP-choline could have benefit as adjunctive treatment for ARDS in veterinary patients (VetARDS). OBJECTIVES: Determine if parenterally administered CDP-choline can attenuate mild VetARDS in dogs with aspiration pneumonia. ANIMALS: Dogs admitted to a veterinary intensive care unit (ICU) for aspiration pneumonia. METHODS: Subjects were enrolled in a randomized, double-blinded, placebo-controlled trial of treatment with vehicle (0.1 mL/kg sterile 0.9% saline, IV; n = 8) or CDP-choline (5 mg/kg in 0.1 mL/kg 0.9% saline, IV; n = 9) q12h over the first 48 hours after ICU admission. RESULTS: No significant differences in signalment or clinical findings were found between placebo- and CDP-choline-treated dogs on admission. All dogs exhibited tachycardia, tachypnea, hypertension, hypoxemia, hypocapnia, lymphopenia, and neutrophilia. CDP-choline administration resulted in rapid, progressive, and clinically relevant increases in oxygenation as determined by pulse oximetry and ratios of arterial oxygen partial pressure (Pa O2 mmHg) to fractional inspired oxygen (% Fi O2 ) and decreases in alveolar-arterial (A-a) gradients that did not occur in placebo (saline)-treated animals. Treatment with CDP-choline was also associated with less platelet consumption over the first 48 hours, but had no detectable detrimental effects. CONCLUSIONS AND CLINICAL IMPORTANCE: Ctyidine diphosphcholine acts rapidly to promote gas exchange in dogs with naturally occurring aspiration pneumonia and is a potential adjunctive treatment in VetARDS patients.
Assuntos
Doenças do Cão , Pneumonia Aspirativa , Síndrome do Desconforto Respiratório , Animais , Cães , Citidina , Citidina Difosfato Colina/uso terapêutico , Doenças do Cão/tratamento farmacológico , Hipóxia/tratamento farmacológico , Hipóxia/veterinária , Pulmão , Oxigênio/uso terapêutico , Pneumonia Aspirativa/veterinária , Síndrome do Desconforto Respiratório/veterinária , Solução SalinaRESUMO
Influenza A virus (IAV) infection alters lung epithelial cell metabolism in vitro by promoting a glycolytic shift. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. A/WSN/33 IAV-inoculated C57BL/6 mice were treated daily from 1 day post-inoculation (d.p.i.) with 2-deoxy-d-glucose (2-DG) to inhibit glycolysis and with the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate (DCA) to promote flux through the TCA cycle. To block OXPHOS, mice were treated every other day from 1 d.p.i. with the Complex I inhibitor rotenone (ROT). 2-DG significantly decreased nocturnal activity, reduced respiratory exchange ratios, worsened hypoxemia, exacerbated lung dysfunction, and increased humoral inflammation at 6 d.p.i. DCA and ROT treatment normalized oxygenation and airway resistance and attenuated IAV-induced pulmonary edema, histopathology, and nitrotyrosine formation. None of the treatments altered viral replication. These data suggest that a shift to glycolysis is host-protective in influenza.
Assuntos
Células Epiteliais/metabolismo , Glicólise , Vírus da Influenza A Subtipo H1N1/patogenicidade , Lesão Pulmonar/virologia , Pulmão/metabolismo , Animais , Feminino , Pulmão/química , Pulmão/virologia , Lesão Pulmonar/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Tirosina/análogos & derivados , Tirosina/análise , Tirosina/metabolismo , Replicação ViralRESUMO
The cotton rat (Sigmodon hispidus) is an excellent small animal model for human respiratory viral infections such as human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). These respiratory viral infections, as well as other pulmonary inflammatory diseases such as asthma, are associated with lung mechanic disturbances. So far, the pathophysiological effects of viral infection and allergy on cotton rat lungs have not been measured, although this information might be an important tool to determine the efficacy of vaccine and drug candidates. To characterize pulmonary function in the cotton rat, we established forced oscillation technique in uninfected, RSV infected and HDM sensitized cotton rats, and characterized pulmonary inflammation, mucus production, pulmonary edema, and oxygenation. There was a gender difference after RSV infection, with females demonstrating airway hyper-responsiveness while males did not. Female cotton rats 2dpi had a mild increase in pulmonary edema (wet: dry weight ratios). At day 4 post infection, female cotton rats demonstrated mild pulmonary inflammation, no increase in mucus production or reduction in oxygenation. Pulmonary function was not significantly impaired after RSV infection. In contrast, cotton rats sensitized to HDM demonstrated airway hyper-responsiveness with a significant increase in pulmonary inflammation, increase in baseline tissue damping, and a decrease in baseline pulmonary compliance. In summary, we established baseline data for forced oscillation technique and other respiratory measures in the cotton rat and used it to analyze respiratory diseases in cotton rats.
Assuntos
Testes de Função Respiratória , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Animais , Antígenos/imunologia , Feminino , Complacência Pulmonar , Masculino , Pyroglyphidae/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Caracteres Sexuais , SigmodontinaeRESUMO
Alveolar type II (ATII) cells are essential to lung function and a primary site of influenza A virus (IAV) replication. Effects of IAV infection on ATII cell microRNA (miR) expression have not been comprehensively investigated. Infection of C57BL/6 mice with 10,000 or 100 pfu/mouse of IAV A/WSN/33 (H1N1) significantly altered expression of 73 out of 1908 mature murine miRs in ATII cells at 2 days post-infection (d.p.i.) and 253 miRs at 6 d.p.i. miR-155-5p (miR-155) showed the greatest increase in expression within ATII cells at both timepoints and the magnitude of this increase correlated with inoculum size and pulmonary edema severity. Influenza-induced lung injury was attenuated in C57BL/6-congenic miR-155-knockout mice without affecting viral replication. Attenuation of lung injury was dependent on deletion of miR-155 from stromal cells and was recapitulated in ATII cell-specific miR-155-knockout mice. These data suggest that ATII cell miR-155 is a potential therapeutic target for IAV-induced ARDS.
Assuntos
Células Epiteliais Alveolares/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/genética , Influenza Humana/mortalidade , MicroRNAs/genética , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/mortalidade , Células Epiteliais Alveolares/virologia , Animais , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/virologiaRESUMO
Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the United States. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model for pathogenesis study. In this study, we developed lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. A LAB strain (Lactococcus lactis) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector and was secreted into media supernatants. To test whether LAB-based HuNoV vaccine candidate is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. LAB DNA and VP1 were detected in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces respectively at day 13 post-inoculation, and further increased at later time points. After being challenged with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less, or no, HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that a LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.
Assuntos
Infecções por Caliciviridae/prevenção & controle , Gastroenterite/prevenção & controle , Lactococcus lactis , Norovirus/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , DNA Bacteriano/isolamento & purificação , Fezes/microbiologia , Gastroenterite/virologia , Trato Gastrointestinal/microbiologia , Vetores Genéticos , Vida Livre de Germes , Norovirus/genética , Suínos , Proteínas Estruturais Virais/genéticaRESUMO
Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.
Assuntos
Carboxipeptidases/metabolismo , Leishmania major/metabolismo , Peptídeos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Carboxipeptidases/química , Carboxipeptidases/genética , Leishmania major/genética , Peptídeos/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Especificidade por SubstratoRESUMO
Macrophages and their monocyte precursors mediate innate immune responses and can promote a spectrum of phenotypes from pro-inflammatory to pro-resolving. Currently, there are few markers that allow for robust dissection of macrophage phenotype. We recently identified CD38 as a marker of inflammatory macrophages in murine in vitro and in vivo models. However, it is unknown whether CD38 plays a similar marker and/or functional role in human macrophages and inflammatory diseases. Here, we establish that CD38 transcript and protein are robustly induced in human macrophages exposed to LPS (±IFN-γ) inflammatory stimuli, but not with the alternative stimulus, IL-4. Pharmacologic and/or genetic CD38 loss-of-function significantly reduced the secretion of inflammatory cytokines IL-6 and IL-12p40 and glycolytic activity in human primary macrophages. Finally, monocyte analyses in systemic lupus erythematosus patients revealed that, while all monocytes express CD38, high CD38 expression in the non-classical monocyte subpopulation is associated with disease. These data are consistent with an inflammatory marker role for CD38 in human macrophages and monocytes.
RESUMO
Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 µm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling.IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.
Assuntos
Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Fímbrias Bacterianas/metabolismo , Haemophilus influenzae/fisiologia , Simulação por Computador , Processamento de Imagem Assistida por Computador , Microscopia ConfocalRESUMO
The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16-24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated.
Assuntos
Morte Celular , Vesículas Extracelulares/imunologia , Monócitos/imunologia , Técnicas de Cultura de Células , Sobrevivência Celular , Sistemas de Liberação de Medicamentos/métodos , Células HEK293 , Humanos , Fagocitose , Células U937RESUMO
Previous studies have demonstrated that Leishmania donovani attenuates STAT1-mediated signaling in macrophages; however it is not clear whether other species of Leishmania, which cause cutaneous disease, also interfere with macrophage IFN-gamma signaling. Therefore, we determined the effect of Leishmania major and Leishmania mexicana infection on STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7 macrophages. We found that both L. major and L. mexicana suppressed IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-gammaRbeta (beta subunit of interferon gamma receptor) expression, reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana infection on Jak1, Jak2 and STAT1 activation was more profound when compared with L. major. Although tyrosine phosphorylation of STAT1alpha was decreased in IFN-gamma stimulated macrophages infected with L. major or L. mexicana, those infected with L. mexicana showed a significant increase in phosphorylation of the dominant negative STAT1beta. These findings indicate that L. major and L. mexicana attenuate STAT1-mediated IFN-gamma signaling in macrophages. Furthermore, they also demonstrate that L. mexicana preferentially enhances tyrosine phosphorylation of dominant negative STAT1beta, which may be one of the several survival mechanisms used by this parasite to evade the host defense mechanisms.
Assuntos
Proteínas de Ligação a DNA/imunologia , Interferon gama/biossíntese , Leishmania major/imunologia , Leishmania mexicana/imunologia , Macrófagos/imunologia , Transativadores/imunologia , Tirosina/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 2 , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais/imunologia , Transativadores/metabolismo , Regulação para Cima/imunologiaRESUMO
OBJECTIVES/HYPOTHESIS: The hypothesis is that signature bacterial proteins can be identified in sinus secretions via high-throughput, proteomic based techniques. Nontypeable Haemophilus influenzae (NTHI) is the most common bacterial pathogen associated with sinusitis and serves as proof of principle pathogen for identifying biomarkers. STUDY DESIGN: In vitro and in vivo studies using proteomic-based analysis of cultures of NTHI and a novel, experimental chinchilla polymicrobial sinusitis model. METHODS: Nano-liquid chromatography /tandem mass spectrometry (nano-LC-MS/MS) was performed to annotate the secretome from an NTHI biofilm. A model of NTHI-induced sinusitis was developed in a chinchilla, and NTHI proteins were detected in chinchilla secretions. A reference standard RT-PCR-based assay was adapted to allow for sensitivity and specificity testing of the identified signature biomarkers in human patients. RESULTS: Outer membrane proteins P2 (OMP-P2) and P5 (OMP-P5) were identified as promising candidates for the detection of NTHI biofilms and positively detected in nasopharyngeal secretions of chinchillas experimentally infected with NTHI. An RT-PCR based test for the presence of NTHI biofilms demonstrated 100% sensitivity and 100% specificity when tested against eight unique strains commonly found in human bacterial rhinosinusitis. CONCLUSIONS: Proteomic analysis was successful in identifying signature proteins for possible use as a biomarker for chronic rhinosinusitis (CRS). OMP-P2 and OMP-P5 were validated as promising candidates and were positively detected from nasopharyngeal secretions from chinchillas experimentally infected with NTHI. Collectively, these data support the use of OMP-P2 and OMP-P5 as biomarkers for a human clinical trial to develop a point-of-care medical diagnostic test to assist in the diagnosis and treatment of CRS.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/classificação , Rinite/diagnóstico , Sinusite/diagnóstico , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Biomarcadores/metabolismo , Chinchila , Doença Crônica , Modelos Animais de Doenças , Infecções por Haemophilus/genética , Haemophilus influenzae/genética , Humanos , Técnicas In Vitro , Assistência ao Paciente , Proteômica , Melhoria de Qualidade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Reprodutibilidade dos Testes , Rinite/microbiologia , Sensibilidade e Especificidade , Sinusite/microbiologia , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: Signal transducer and activator of transcription (STAT) 1 signaling pathway mediates biological functions of interferon (IFN) gamma, which is a key cytokine-regulating T helper 1 (Thl) differentiation. Although constitutive activation of STAT1 has been reported in the airway epithelium of patients with chronic asthma, its in vivo role in the pathogenesis of allergic rhinitis is not clear. We determined the role of STAT1 in the pathogenesis of allergic rhinitis in vivo using STAT1 gene-deficient (STAT1-/-) mice and a murine model of Schistosoma mansoni egg antigen (SEA)-induced allergic rhinitis. METHODS: STATI -/- BALB/c and wild-type (WT) mice were sensitized by intranasal administration of SEA, and their immunologic responses were examined. RESULTS: STATI-1- mice showed impaired nasal eosinophilia and markedly reduced histamine-induced nasal hyperresponsiveness after SEA sensitization. Moreover, levels of Th2-associated SEA-specific IgG1 and IgE antibodies were lower in STAT1-/- mice. Anti-CD3stimulated nasal lymphocytes from STAT1-/-mice also produced less amounts of Th2-associated cytokines IL-4, IL-5, IL-10, and IL-13 compared with WT mice, but both produced comparable levels of IFN-gamma. CONCLUSION: These results show that STAT1 is involved in the pathogenesis of SEA-induced allergic rhinitis in BALB/c mice. Furthermore, they reveal a surprising role of STAT1 in induction of nasal eosinophilia, and Th2-type cytokine production from nasal lymphocytes during allergic rhinitis.
Assuntos
Alérgenos , Antígenos de Helmintos , Rinite Alérgica Perene/genética , Rinite Alérgica Perene/imunologia , Fator de Transcrição STAT1/genética , Schistosoma mansoni/imunologia , Células Th2/imunologia , Alérgenos/administração & dosagem , Animais , Antígenos de Helmintos/administração & dosagem , Citocinas/biossíntese , Modelos Animais de Doenças , Eosinofilia/imunologia , Feminino , Imunização , Imunoglobulina A/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Inflamação/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Testes de Provocação NasalRESUMO
CXC chemokine receptor 3 (CXCR3) ligands CXCL9 and CXCL10 are produced at high levels in mice and humans infected with Leishmania donovani, but their contribution to host resistance against L. donovani is not clear. Here, using CXCR3(-/-) mice, we demonstrate that, although CXCR3 regulates early immune cell trafficking and hepatic inflammation during L. donovani infection, it is not essential for immunity against L. donovani, unlike L. major. CXCR3(-/-) C57BL/6 mice show a delayed onset of hepatic inflammation and granuloma formation after L. donovani infection. However, they mount an efficient T helper cell type 1 response, recruit T cells to the liver, and control parasite growth as efficiently as do CXCR3(+/+) C57BL/6 mice.
Assuntos
Inflamação/imunologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Fígado , Receptores de Quimiocinas/metabolismo , Animais , Cricetinae , Citocinas/metabolismo , Feminino , Granuloma/imunologia , Granuloma/parasitologia , Humanos , Inflamação/parasitologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Fígado/imunologia , Fígado/parasitologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR3 , Baço/imunologia , Baço/parasitologiaRESUMO
To compare the roles of programmed death 1 ligand 1 (PD-L1) and PD-L2 in regulating immunity to infection, we investigated responses of mice lacking PD-L1 or PD-L2 to infection with Leishmania mexicana. PD-L1(-/-) and PD-L2(-/-) mice exhibited distinct disease outcomes following infection with L. mexicana. In comparison to susceptible WT mice, PD-L1(-/-) mice showed resistance to L. mexicana, as demonstrated by reduced growth of cutaneous lesions and parasite burden. In contrast, PD-L2(-/-) mice developed exacerbated disease with increased parasite burden. Host resistance to L. mexicana is partly associated with the development of a Th1 response and down-regulation of the Th2 response. Both PD-L1(-/-) and PD-L2(-/-) mice produced levels of IFN-gamma similar to WT mice. However, the development of IL-4-producing cells was reduced in PD-L1(-/-) mice, demonstrating a role for PD-L1 in regulating Th cell differentiation. This inadequate Th2 response may explain the increased resistance of PD-L1(-/-) mice. Although no alterations in Th1/Th2 skewing were observed in PD-L2(-/-) mice, PD-L2(-/-) mice exhibited a marked increase in L. mexicana-specific antibody production. Increased Leishmania-specific IgG production may suppress the healing response through FcgammaR ligation on macrophages. Taken together, our results demonstrate that PD-L1 and PD-L2 have distinct roles in regulating the immune response to L. mexicana.
Assuntos
Antígeno B7-1/imunologia , Leishmaniose Cutânea/imunologia , Glicoproteínas de Membrana/imunologia , Peptídeos/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Antígeno B7-H1 , Southern Blotting , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Leishmania mexicana/imunologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Transgênicos , Peptídeos/deficiência , Proteína 2 Ligante de Morte Celular Programada 1 , Células Th1/citologia , Células Th2/citologiaRESUMO
The interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR-/-) mice. TCCR-/- mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR-/- mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4(+) T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR-/- mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4(+) T-cell activity, it is not required for the resolution of hepatic immunopathology.