RESUMEN
The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.
Asunto(s)
Antiinfecciosos , Criptococosis , Cryptococcus neoformans , Criptococosis/microbiología , Humanos , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Fagocitosis , Fagosomas/microbiologíaRESUMEN
Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Caspasa 3/uso terapéutico , Células Endoteliales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Apoptosis , Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4+ T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4+ T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4+ T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4+ T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4+ T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Metilación de ADN , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Neumonía/inmunología , Animales , Animales Recién Nacidos , Linfocitos T CD4-Positivos/metabolismo , Islas de CpG , Epigénesis Genética , Infecciones por Escherichia coli/genética , Ratones , Ratones Endogámicos C57BL , Neumonía/genética , Activación TranscripcionalRESUMEN
RATIONALE: A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility. OBJECTIVES: This study evaluated the diagnostic performance of a molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults. METHODS: The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts. MEASUREMENTS AND MAIN RESULTS: In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity. CONCLUSIONS: SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).
Asunto(s)
Cuidados Críticos/métodos , Unidades de Cuidados Intensivos , Sepsis/diagnóstico , Prueba Bactericida de Suero/métodos , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico , Adulto , Anciano , Estudios de Cohortes , Enfermedad Crítica , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Estudios Prospectivos , Curva ROC , Estudios Retrospectivos , Sensibilidad y Especificidad , Sepsis/sangre , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Estados UnidosRESUMEN
Neonates have greater morbidity/mortality from lower respiratory tract infections (LRTI) compared to older children. Lack of conditioning of the pulmonary immune system due to limited environmental exposures and/or infectious challenges likely contributes to the increase susceptibility in the neonate. In this study, we sought to gain insights into the nature and dynamics of the neonatal pulmonary immune response to LRTI using a murine model. METHODS: Wildtype (WT) and Ccr2-/- C57BL/6 neonatal and juvenile mice received E. coli or PBS by direct pharyngeal aspiration. Flow cytometry was used to measure immune cell dynamics and identify cytokine-producing cells. Real-time PCR and ELISA were used to measure cytokine/chemokine expression. RESULTS: Innate immune cell recruitment in response to E. coli-induced LRTI was delayed in the neonatal lung compared to juvenile lung. Lung clearance of bacteria was also significantly delayed in the neonate. Ccr2-/- neonates, which lack an intact CCL2-CCR2 axis, had higher mortality after E. coli challenged than Ccr2+/+ neonates. A greater percentage of CD8+ T cells and monocytes from WT neonates challenged with E. coli produced TNF compared to controls. CONCLUSION: The pulmonary immune response to E. coli-induced LRTI differed significantly between neonatal and juvenile mice. Neonates were more susceptible to increasing doses of E. coli and exhibited greater mortality than juveniles. In the absence of an intact CCL2-CCR2 axis, susceptibility to LRTI-induced mortality was further increased in neonatal mice. Taken together these findings underscore the importance of age-related differences in the innate immune response to LRTI during early stages of postnatal life.
Asunto(s)
Quimiocina CCL2/inmunología , Infecciones por Escherichia coli/inmunología , Inmunidad Innata , Pulmón/microbiología , Receptores CCR2/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Factores de Edad , Animales , Animales Recién Nacidos , Bronquios/microbiología , Quimiocina CCL2/deficiencia , Quimiocinas/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Escherichia coli , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/mortalidad , Inflamación , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CCR2/deficienciaRESUMEN
Lymphocytes have been shown to modulate angiogenesis. Our previous work showed that T regulatory (Treg) cell depletion prevented angiogenesis. In the present study, we sought to examine T-cell populations during lung angiogenesis and subsequent angiostasis. In a mouse model of ischemia-induced systemic angiogenesis in the lung, we examined the time course (0-35 d) of neovascularization and T-cell phenotypes within the lung after left pulmonary artery ligation (LPAL). T cells increased and reached a maximum by 10 days after LPAL and then progressively decreased, suggestive of a modulatory role during the early phase of new vessel growth. Because others have shown IFN-γ to be angiostatic in tumor models, we focused on this effector T-cell cytokine to control the magnitude of angiogenesis. Results showed that IFN-γ protein is secreted at low levels after LPAL and that mice required Treg depletion to see the full effect of effector T cells. Using Foxp3(DTR) and diphtheria toxin to deplete T regulatory cells, increased numbers of effector T cells (CD8(+)) and/or increased capacity to secrete the prominent angiostatic cytokine IFN-γ (CD4(+)) were seen. In vitro culture of mouse systemic and pulmonary microvascular endothelial cells with IFN-γ showed increased endothelial cell apoptosis. CD8(-/-) mice and IFN-γR(-/-) mice showed enhanced angiogenesis compared with wild-type mice, confirming that, in this model, IFN-γ limits the extent of systemic neovascularization in the lung.
Asunto(s)
Células Endoteliales/inmunología , Isquemia/inmunología , Pulmón/irrigación sanguínea , Pulmón/inmunología , Neovascularización Fisiológica , Linfocitos T Reguladores/inmunología , Animales , Apoptosis , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patología , Isquemia/fisiopatología , Pulmón/metabolismo , Pulmón/patología , Activación de Linfocitos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptores de Interferón/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Receptor de Interferón gammaRESUMEN
Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis.
Asunto(s)
Células Endoteliales/fisiología , Células Epiteliales/fisiología , Citometría de Flujo/métodos , Animales , Linaje de la Célula , Supervivencia Celular , Pulmón/citología , Masculino , Ratones Endogámicos C57BLRESUMEN
Overwhelming lung inflammation frequently occurs following exposure to both direct infectious and noninfectious agents and is a leading cause of mortality worldwide. In that context, immunomodulatory strategies may be used to limit severity of impending organ damage. We sought to determine whether priming the lung by activating the immune system, or immunological priming, could accelerate resolution of severe lung inflammation. We assessed the importance of alveolar macrophages, regulatory T cells, and their potential interaction during immunological priming. We demonstrate that oropharyngeal delivery of low-dose LPS can immunologically prime the lung to augment alveolar macrophage production of IL-10 and enhance resolution of lung inflammation induced by a lethal dose of LPS or by Pseudomonas bacterial pneumonia. IL-10-deficient mice did not achieve priming and were unable to accelerate lung injury resolution. Depletion of lung macrophages or regulatory T cells during the priming response completely abrogated the positive effect of immunological priming on resolution of lung inflammation and significantly reduced alveolar macrophage IL-10 production. Finally, we demonstrated that oropharyngeal delivery of synthetic CpG-oligonucleotides elicited minimal lung inflammation compared with low-dose LPS but nonetheless primed the lung to accelerate resolution of lung injury following subsequent lethal LPS exposure. Immunological priming is a viable immunomodulatory strategy used to enhance resolution in an experimental acute lung injury model with the potential for therapeutic benefit against a wide array of injurious exposures.
Asunto(s)
Macrófagos Alveolares/inmunología , Neumonía/inmunología , Linfocitos T Reguladores/inmunología , Vacunación/métodos , Animales , Citocinas/biosíntesis , Citometría de Flujo , Interleucina-10/inmunología , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/prevención & controlAsunto(s)
Infecciones por Coronavirus/terapia , Inmunoterapia/métodos , Neumonía Viral/terapia , Síndrome de Dificultad Respiratoria/terapia , Linfocitos T Reguladores/fisiología , Anciano , Betacoronavirus , COVID-19 , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Síndrome de Dificultad Respiratoria/virología , SARS-CoV-2RESUMEN
Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4(+) lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4(+) CD25(+) forkhead homeobox protein-3 (Foxp3)(+) cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3(+) Treg cells can be depleted with diphtheria toxin (DT; Foxp3(DTR)), we unexpectedly found that Foxp3(+) Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3(gfp) controls). Adoptive transfer studies using CD4(+) CD25(+) splenocytes from congenic CD45.1 mice into Foxp3(+) Treg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3(gfp) controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-ß, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII](int), CD11C(+)) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Isquemia/metabolismo , Pulmón/irrigación sanguínea , Neovascularización Fisiológica , Linfocitos T Reguladores/metabolismo , Traslado Adoptivo , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Animales , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Isquemia/genética , Isquemia/inmunología , Isquemia/fisiopatología , Macrófagos/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Factores de TiempoRESUMEN
Neonates and infants have a higher morbidity and mortality associated with lower respiratory tract illnesses compared with older children. To identify age-related and longitudinal differences in the cellular immune response to acute lung injury (ALI), neonatal and juvenile mice were given Escherichia coli LPS using a novel, minimally invasive aspiration technique. Neonatal and juvenile mice received between 3.75 and 7.5 mg/kg LPS by intrapharyngeal aspiration. Airway and lung cells were isolated and characterized by flow cytometry, cytokine/chemokine mRNA expression from lung homogenates was quantified, and lung morphometry and injury scores were performed. LPS-treated neonatal mice underwent adoptive transfer with adult T regulatory cells (Tregs). After LPS aspiration, lung monocytes isolated from neonatal mice had a predominant M2 phenotype, whereas lung monocytes from juvenile mice displayed a mixed M1/M2 phenotype. At 72 hours after LPS exposure, neonatal lungs were slower to resolve inflammation and expressed lower mRNA levels of CCL2, CCL5, CXCL10, and IL-10. Juvenile, but not neonatal, mice demonstrated a significant increase in airway Tregs after LPS exposure. Adoptive transfer of adult Tregs into LPS-challenged neonatal mice resulted in reduced lung inflammation and improved weight gain. These findings underscore several vulnerabilities in the neonatal immune response to LPS-induced ALI. Most striking was the deficiency in airway Tregs after LPS aspiration. Adoptive transfer of adult Tregs mitigated LPS-induced ALI in neonatal mice, highlighting the importance of age-related differences in Tregs and their response to ALI during early postnatal development.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Animales Recién Nacidos/inmunología , Lipopolisacáridos/inmunología , Pulmón/inmunología , Traslado Adoptivo/métodos , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocina CCL2/inmunología , Quimiocina CCL5/inmunología , Quimiocina CXCL10/inmunología , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Interleucina-10/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Embarazo , Linfocitos T Reguladores/inmunologíaRESUMEN
Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4(+)CD25(hi)Foxp3(+) Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3(DTR)) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 10(5) DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS.
Asunto(s)
Lesión Pulmonar Aguda/tratamiento farmacológico , Azacitidina/análogos & derivados , Metilasas de Modificación del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Pulmón/efectos de los fármacos , Neumonía/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/virología , Traslado Adoptivo , Animales , Azacitidina/farmacología , Células Cultivadas , Quimiotaxis de Leucocito , Metilasas de Modificación del ADN/metabolismo , Decitabina , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Subtipo H1N1 del Virus de la Influenza A , Lipopolisacáridos , Pulmón/enzimología , Pulmón/inmunología , Pulmón/virología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Neumonía/inducido químicamente , Neumonía/enzimología , Neumonía/inmunología , Neumonía/virología , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Linfocitos T Reguladores/virología , Factores de TiempoRESUMEN
OBJECTIVE: Myocardial angiogenesis is presumed to play a role in RV adaptation to PH, though definitive evidence and functional correlations are lacking. We aimed to use definitive methods to correlate RV angiogenesis, hypertrophy, and function in a murine PH model. METHODS: Mice were exposed to CH for 21 days to induce PH and RV remodeling. We used unbiased stereology and flow cytometry to quantify angiogenesis and myocyte hypertrophy, and pressure-volume loops to measure RV function. RESULTS: Within seven days, RV-specific increases in total capillary length (10,576 ± 2574 cm vs. 6822 ± 1379 cm; p = 0.02), surface area (10 ± 3.3 cm(2) vs. 4.9 ± 1.5 cm(2) ; p = 0.01), and volume (0.0013 ± 0.0005 cm(3) vs. 0.0006 ± 0.0001 cm(3) ; p = 0.02) were observed, and RV EC proliferation increased nearly 10-fold. Continued exposure led to progressive RVH without additional angiogenesis. RV function was preserved, but activation of hypoxia-dependent gene expression was observed in both ventricles after 21 days. CONCLUSIONS: Early RV remodeling in CH-PH is associated with RV angiogenesis and preserved RV function. Continued CH-PH is associated with RVH but not angiogenesis, leading to biventricular activation of hypoxia-dependent gene expression.
Asunto(s)
Ventrículos Cardíacos/fisiopatología , Hipertensión Pulmonar/fisiopatología , Hipoxia/fisiopatología , Neovascularización Patológica/fisiopatología , Remodelación Ventricular , Animales , Masculino , RatonesRESUMEN
Acute respiratory distress syndrome (ARDS) is a devastating disease with distinct pathological stages. Fundamental to ARDS is the acute onset of lung inflammation as a part of the body's immune response to a variety of local and systemic stimuli. In patients surviving the inflammatory and subsequent fibroproliferative stages, transition from injury to resolution and recovery is an active process dependent on a series of highly coordinated events regulated by the immune system. Experimental animal models of acute lung injury (ALI) reproduce key components of the injury and resolution phases of human ARDS and provide a methodology to explore mechanisms and potential new therapies. Macrophages are essential to innate immunity and host defense, playing a featured role in the lung and alveolar space. Key aspects of their biological response, including differentiation, phenotype, function, and cellular interactions, are determined in large part by the presence, severity, and chronicity of local inflammation. Studies support the importance of macrophages to initiate and maintain the inflammatory response, as well as a determinant of resolution of lung inflammation and repair. We will discuss distinct roles for lung macrophages during early inflammatory and late resolution phases of ARDS using experimental animal models. In addition, each section will highlight human studies that relate to the diverse role of macrophages in initiation and resolution of ALI and ARDS.
Asunto(s)
Macrófagos Alveolares/fisiología , Neumonía/inmunología , Lesión Pulmonar Aguda/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Neumonía/fisiopatologíaRESUMEN
Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS-/- mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS-/- mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS-/- mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS-/- mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.
Asunto(s)
Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/terapia , Monocitos/enzimología , Monocitos/inmunología , Óxido Nítrico Sintasa de Tipo II/uso terapéutico , Lesión Pulmonar Aguda/inmunología , Animales , Antígeno B7-2/biosíntesis , Línea Celular , Línea Celular Transformada , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/uso terapéutico , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Óxido Nítrico Sintasa de Tipo II/deficienciaRESUMEN
Alveolar macrophages (AMs) act as gatekeepers of the lung's immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) Early Growth Response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA-sequencing, ATAC-sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes, and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmarker in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.