RESUMEN
CD4+ forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are essential in maintaining immune tolerance and suppressing excessive immune responses. Tregs also contribute to tissue repair processes distinct from their roles in immune suppression. For these reasons, Tregs are candidates for targeted therapies for inflammatory and autoimmune diseases, and in diseases where tissue damage occurs. MT-2 cells, an immortalized Treg-like cell line, offer a model to study Treg biology and their therapeutic potential. In the present study, we use clustered regularly interspaced palindromic repeats (CRISPR)-mediated knockdown of FOXP3 in MT-2 cells to understand the transcriptional and functional changes that occur when FOXP3 is lost and to compare MT-2 cells with primary human Tregs. We demonstrate that loss of FOXP3 affects the transcriptome of MT-2 cells and that FOXP3's potential downstream targets include a wide range of transcripts that participate in the cell cycle, promote growth and contribute to inflammatory processes, but do not wholly simulate previously reported human primary Treg transcriptional changes in the absence of FOXP3. We also demonstrate that FOXP3 regulates cell cycling and proliferation, expression of molecules crucial to Treg function and MT-2 cell-suppressive activities. Thus, MT-2 cells offer opportunities to address regulatory T-cell functions in vitro.
Asunto(s)
Terapia de Inmunosupresión , Linfocitos T Reguladores , Humanos , Línea Celular , Tolerancia Inmunológica , Factores de Transcripción Forkhead/metabolismoRESUMEN
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
Sex as a biological variable is an essential element of preclinical research. Sex-specific differences in lung volume, alveolar number, body weight, and the relationship between lung and body weight result in important questions about generating equivalent injuries in males and females so that comparisons in their responses can be assessed. Few studies compare stimulus dosing methods for murine lung models investigating immune responses. To examine sex-specific effects, we explored several dosing techniques for three stimuli, LPS, Streptococcus pneumoniae, and influenza A, on survival, injury parameters in bronchoalveolar lavage (BAL), and immune cell numbers in single-cell lung suspensions after injury. These data demonstrate that body weight-based dosing produced fewer differences between sexes when compared with injury initiated with inocula containing the same number of organisms. Comparison of the lung and body weights showed that females had a greater lung-to-body weight ratio than males. However, in LPS-induced injury, adjusting the dose for sex differences in this ratio in addition to body weight provided no new information about sex differences compared with dosing by body weight alone, most likely due to the variability in measures of the immune response. Studies evaluating BAL volumes revealed that smaller but more lavages resulted in greater returns and lower protein concentrations, particularly in the smaller female lungs. Thus, designing dosing and measurement methods that generate equivalent injuries facilitates comparison of immune responses between sexes. Continued development of methods for both induction and evaluation of injury will likely facilitate identification of sex differences in immune responses.
Asunto(s)
Lipopolisacáridos , Neumonía , Ratones , Femenino , Masculino , Animales , Lipopolisacáridos/farmacología , Pulmón , Recuento de Células , Peso CorporalRESUMEN
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
By enhancing tissue repair and modulating immune responses, Foxp3+ regulatory T cells (Tregs) play essential roles in resolution from lung injury. The current study investigated the effects that Tregs exert directly or indirectly on the transcriptional profiles of type 2 alveolar epithelial (AT2) cells during resolution in an experimental model of acute lung injury. Purified AT2 cells were isolated from uninjured mice or mice recovering from LPS-induced lung injury, either in the presence of Tregs or in Treg-depleted mice, and transcriptome profiling identified differentially expressed genes. Depletion of Tregs resulted in altered expression of 49 genes within AT2 cells during resolution, suggesting that Tregs present in this microenvironment influence AT2-cell function. Biological processes from Gene Ontology enriched in the absence of Tregs included those describing responses to IFN. Neutralizing IFN-γ in Treg-depleted mice reversed the effect of Treg depletion on inflammatory macrophages and B cells by preventing the increase in inflammatory macrophages and the decrease in B cells. Our results provide insight into the effects of Tregs on AT2 cells. Tregs directly or indirectly impact many AT2-cell functions, including IFN type I and II-mediated signaling pathways. Inhibition of IFN-γ expression and/or function may be one mechanism through which Tregs accelerate resolution after acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Células Epiteliales Alveolares/inmunología , Interferón gamma/inmunología , Pulmón/inmunología , Linfocitos T Reguladores/inmunología , Transcriptoma/inmunología , Animales , Linfocitos B/inmunología , Femenino , Factores de Transcripción Forkhead/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunologíaRESUMEN
Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Inmunomodulación/genética , Factores de Transcripción/deficiencia , Alelos , Animales , Biomarcadores , Sistemas CRISPR-Cas , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Edición Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Masculino , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Foxp3+ regulatory T cells (Tregs) play essential roles in immune homeostasis and repair of damaged lung tissue. We hypothesized that patients whose lung injury resolves quickly, as measured by time to liberation from mechanical ventilation, have a higher percentage of Tregs amongst CD4+ T cells in either airway, bronchoalveolar lavage (BAL) or peripheral blood samples. METHODS: We prospectively enrolled patients with ARDS requiring mechanical ventilation and collected serial samples, the first within 72 h of ARDS diagnosis (day 0) and the second 48-96 h later (day 3). We analyzed immune cell populations and cytokines in BAL, tracheal aspirates and peripheral blood, as well as cytokines in plasma, obtained at the time of bronchoscopy. The study cohort was divided into fast resolvers (FR; n = 8) and slow resolvers (SR; n = 5), based on the median number of days until first extubation for all participants (n = 13). The primary measure was the percentage of CD4+ T cells that were Tregs. RESULTS: The BAL of FR contained more Tregs than SR. This finding did not extend to Tregs in tracheal aspirates or blood. BAL Tregs expressed more of the full-length FOXP3 than a splice variant missing exon 2 compared to Tregs in simultaneously obtained peripheral blood. CONCLUSION: Tregs are present in the bronchoalveolar space during ARDS. A greater percentage of CD4+ cells were Tregs in the BAL of FR than SR. Tregs may play a role in the resolution of ARDS, and enhancing their numbers or functions may be a therapeutic target.
Asunto(s)
Síndrome de Dificultad Respiratoria , Lavado Broncoalveolar , Líquido del Lavado Bronquioalveolar , Humanos , Respiración Artificial , Síndrome de Dificultad Respiratoria/terapia , Linfocitos T ReguladoresRESUMEN
Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3+ regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.
Asunto(s)
Células Epiteliales Alveolares/citología , Factor 7 de Crecimiento de Fibroblastos/fisiología , Linfocitos T Reguladores/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Traslado Adoptivo , Células Epiteliales Alveolares/patología , Anfirregulina/biosíntesis , Anfirregulina/genética , Animales , División Celular , Técnicas de Cocultivo , Toxina Diftérica/toxicidad , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/genética , Factores de Transcripción Forkhead/análisis , Regulación de la Expresión Génica/inmunología , Humanos , Lipopolisacáridos/toxicidad , Pulmón/citología , Depleción Linfocítica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Neumonectomía , Complicaciones Posoperatorias/inmunología , Complicaciones Posoperatorias/metabolismo , Complicaciones Posoperatorias/patología , Linfocitos T Reguladores/clasificación , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplanteRESUMEN
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 & controlRESUMEN
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
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
Regulatory T cells, characterized by their expression of the transcription factor Forkhead box P3, are indispensable in maintaining immune homeostasis. The respiratory system is constantly exposed to many environmental challenges, making it susceptible to various insults and infections. Regulatory T cells play essential roles in maintaining homeostasis in the lung and promoting repair after injury. Regulatory T cell function dysregulation can lead to inflammation, tissue damage, or aberrant repair. Research on regulatory T cell mechanisms in the lung has unveiled their influence on lung inflammation and repair mechanisms. In this review, our goal is to highlight the advances in regulatory T cell biology with respect to lung injury and resolution. We further provide a perspective that a deeper understanding of regulatory T cell interactions in the lung microenvironment in health and disease states offers opportunities for therapeutic interventions as treatments to promote lung health.
Asunto(s)
Lesión Pulmonar , Humanos , Lesión Pulmonar/terapia , Linfocitos T Reguladores , Pulmón/metabolismo , Inflamación/metabolismo , Homeostasis , Factores de Transcripción Forkhead/metabolismoRESUMEN
Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality. Exacerbating factors increasing the risk of ARDS remain unknown. Supplemental oxygen is often necessary in both mild and severe lung disease. The potential effects of supplemental oxygen may include augmentation of lung inflammation by inhibiting anti-inflammatory pathways in alveolar macrophages. We sought to determine oxygen-derived effects on the anti-inflammatory A2A adenosinergic (ADORA2A) receptor in macrophages, and the role of the ADORA2A receptor in lung injury. Wild-type (WT) and ADORA2A(-/-) mice received intratracheal lipopolysaccharide (IT LPS), followed 12 hours later by continuous exposure to 21% oxygen (control mice) or 60% oxygen for 1 to 3 days. We measured the phenotypic endpoints of lung injury and the alveolar macrophage inflammatory state. We tested an ADORA2A-specific agonist, CGS-21680 hydrochloride, in LPS plus oxygen-exposed WT and ADORA2A(-/-) mice. We determined the specific effects of myeloid ADORA2A, using chimera experiments. Compared with WT mice, ADORA2A(-/-) mice exposed to IT LPS and 60% oxygen demonstrated significantly more histologic lung injury, alveolar neutrophils, and protein. Macrophages from ADORA2A(-/-) mice exposed to LPS plus oxygen expressed higher concentrations of proinflammatory cytokines and cosignaling molecules. CGS-21680 prevented the oxygen-induced augmentation of lung injury after LPS only in WT mice. Chimera experiments demonstrated that the transfer of WT but not ADORA2A(-/-) bone marrow cells into irradiated ADORA2A(-/-) mice reduced lung injury after LPS plus oxygen, demonstrating myeloid ADORA2A protection. ADORA2A is protective against lung injury after LPS and oxygen. Oxygen after LPS increases macrophage activation to augment lung injury by inhibiting the ADORA2A pathway.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Macrófagos Alveolares/metabolismo , Oxígeno/toxicidad , Receptor de Adenosina A2A/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/inmunología , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Animales , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Quimiocinas/metabolismo , Técnicas de Inactivación de Genes , Mediadores de Inflamación/fisiología , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia por Inhalación de Oxígeno , Fenetilaminas/farmacología , Receptor de Adenosina A2A/genéticaRESUMEN
Acute lung injury (ALI) causes significant morbidity and mortality. Fibroproliferation in ALI results in worse outcomes, but the mechanisms governing fibroproliferation remain poorly understood. Regulatory T cells (Tregs) are important in lung injury resolution. Their role in fibroproliferation is unknown. We sought to identify the role of Tregs in ALI fibroproliferation, using a murine model of lung injury. Wild-type (WT) and lymphocyte-deficient Rag-1(-/-) mice received intratracheal LPS. Fibroproliferation was characterized by histology and the measurement of lung collagen. Lung fibrocytes were measured by flow cytometry. To dissect the role of Tregs in fibroproliferation, Rag-1(-/-) mice received CD4(+)CD25(+) (Tregs) or CD4(+)CD25(-) Tcells (non-Tregs) at the time of LPS injury. To define the role of the chemokine (C-X-C motif) ligand 12 (CXCL12)-CXCR4 pathway in ALI fibroproliferation, Rag-1(-/-) mice were treated with the CXCR4 antagonist AMD3100 to block fibrocyte recruitment. WT and Rag-1(-/-) mice demonstrated significant collagen deposition on Day 3 after LPS. WT mice exhibited the clearance of collagen, but Rag-1(-/-) mice developed persistent fibrosis. This fibrosis was mediated by the sustained epithelial expression of CXCL12 (or stromal cell-derived factor 1 [SDF-1]) that led to increased fibrocyte recruitment. The adoptive transfer of Tregs resolved fibroproliferation by decreasing CXCL12 expression and subsequent fibrocyte recruitment. Blockade of the CXCL12-CXCR4 axis with AMD3100 also decreased lung fibrocytes and fibroproliferation. These results indicate a central role for Tregs in the resolution of ALI fibroproliferation by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis. A dissection of the role of Tregs in ALI fibroproliferation may inform the design of new therapeutic tools for patients with ALI.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Bencilaminas , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Ciclamas , Fibroblastos/inmunología , Fibroblastos/patología , Compuestos Heterocíclicos/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/inmunología , Miofibroblastos/patología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Chronic intermittent hypoxia (CIH) inhibits plasma lipoprotein clearance and adipose lipoprotein lipase (LPL) activity in association with upregulation of an LPL inhibitor angiopoietin-like protein 4 (Angptl4). We hypothesize that CIH inhibits triglyceride (TG) uptake via Angptl4 and that an anti-Angptl4-neutralizing antibody would abolish the effects of CIH. Male C57BL/6J mice were exposed to four weeks of CIH or intermittent air (IA) while treated with Ab (30 mg/kg ip once a week). TG clearance was assessed by [H(3)]triolein administration retroorbitally. CIH delayed TG clearance and suppressed TG uptake and LPL activity in all white adipose tissue depots, brown adipose tissue, and lungs, whereas heart, liver, and spleen were not affected. CD146+ CD11b- pulmonary microvascular endothelial cells were responsible for TG uptake in the lungs and its inhibition by CIH. Antibody to Angptl4 decreased plasma TG levels and increased TG clearance and uptake into adipose tissue and lungs in both control and CIH mice to a similar extent, but did not reverse the effects of CIH. The antibody reversed the effects of CIH on LPL in adipose tissue and lungs. In conclusion, CIH inactivates LPL by upregulating Angptl4, but inhibition of TG uptake occurs predominantly via an Angptl4/LPL-independent mechanism.