RESUMO
Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.
Assuntos
Monócitos , Nanopartículas , RNA Mensageiro , Monócitos/metabolismo , Nanopartículas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Camundongos , Humanos , Polieletrólitos/química , Macrófagos/metabolismo , Poliaminas/química , Tamanho da Partícula , Diferenciação Celular , Técnicas de Transferência de Genes , Células Dendríticas/metabolismo , Eletricidade Estática , PolímerosRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Metilação de DNA , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Pneumonia/imunologia , Animais , Animais Recém-Nascidos , Linfócitos T CD4-Positivos/metabolismo , Ilhas de CpG , Epigênese Genética , Infecções por Escherichia coli/genética , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/genética , Ativação TranscricionalRESUMO
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).
Assuntos
Cuidados Críticos/métodos , Unidades de Terapia Intensiva , Sepse/diagnóstico , Teste Bactericida do Soro/métodos , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Adulto , Idoso , Estudos de Coortes , Estado Terminal , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Estudos Prospectivos , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade , Sepse/sangue , Síndrome de Resposta Inflamatória Sistêmica/sangue , Estados UnidosRESUMO
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.
Assuntos
Células Endoteliais/fisiologia , Células Epiteliais/fisiologia , Citometria de Fluxo/métodos , Animais , Linhagem da Célula , Sobrevivência Celular , Pulmão/citologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Macrófagos Alveolares/imunologia , Pneumonia/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodos , Animais , Citocinas/biossíntese , Citometria de Fluxo , Interleucina-10/imunologia , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/prevenção & controleAssuntos
Infecções por Coronavirus/terapia , Imunoterapia/métodos , Pneumonia Viral/terapia , Síndrome do Desconforto Respiratório/terapia , Linfócitos T Reguladores/fisiologia , Idoso , Betacoronavirus , COVID-19 , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2RESUMO
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.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Isquemia/metabolismo , Pulmão/irrigação sanguínea , Neovascularização Fisiológica , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Isquemia/genética , Isquemia/imunologia , Isquemia/fisiopatologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Fatores de TempoRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Azacitidina/análogos & derivados , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pulmão/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Linfócitos T Reguladores/efeitos dos fármacos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/virologia , Transferência Adotiva , Animais , Azacitidina/farmacologia , Células Cultivadas , Quimiotaxia de Leucócito , Metilases de Modificação do DNA/metabolismo , Decitabina , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Vírus da Influenza A Subtipo H1N1 , Lipopolissacarídeos , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/virologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Linfócitos T Reguladores/virologia , Fatores de TempoRESUMO
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.
Assuntos
Ventrículos do Coração/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Neovascularização Patológica/fisiopatologia , Remodelação Ventricular , Animais , Masculino , CamundongosRESUMO
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.
Assuntos
Macrófagos Alveolares/fisiologia , Pneumonia/imunologia , Lesão Pulmonar Aguda/imunologia , Animais , Modelos Animais de Doenças , Humanos , Pneumonia/fisiopatologiaRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/terapia , Monócitos/enzimologia , Monócitos/imunologia , Óxido Nítrico Sintase Tipo II/uso terapêutico , Lesão Pulmonar Aguda/imunologia , Animais , Antígeno B7-2/biossíntese , Linhagem Celular , Linhagem Celular Transformada , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/uso terapêutico , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Óxido Nítrico Sintase Tipo II/deficiênciaRESUMO
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.
RESUMO
(1) Background: SeptiCyte RAPID is a molecular test for discriminating sepsis from non-infectious systemic inflammation, and for estimating sepsis probabilities. The objective of this study was the clinical validation of SeptiCyte RAPID, based on testing retrospectively banked and prospectively collected patient samples. (2) Methods: The cartridge-based SeptiCyte RAPID test accepts a PAXgene blood RNA sample and provides sample-to-answer processing in ~1 h. The test output (SeptiScore, range 0-15) falls into four interpretation bands, with higher scores indicating higher probabilities of sepsis. Retrospective (N = 356) and prospective (N = 63) samples were tested from adult patients in ICU who either had the systemic inflammatory response syndrome (SIRS), or were suspected of having/diagnosed with sepsis. Patients were clinically evaluated by a panel of three expert physicians blinded to the SeptiCyte test results. Results were interpreted under either the Sepsis-2 or Sepsis-3 framework. (3) Results: Under the Sepsis-2 framework, SeptiCyte RAPID performance for the combined retrospective and prospective cohorts had Areas Under the ROC Curve (AUCs) ranging from 0.82 to 0.85, a negative predictive value of 0.91 (sensitivity 0.94) for SeptiScore Band 1 (score range 0.1-5.0; lowest risk of sepsis), and a positive predictive value of 0.81 (specificity 0.90) for SeptiScore Band 4 (score range 7.4-15; highest risk of sepsis). Performance estimates for the prospective cohort ranged from AUC 0.86-0.95. For physician-adjudicated sepsis cases that were blood culture (+) or blood, urine culture (+)(+), 43/48 (90%) of SeptiCyte scores fell in Bands 3 or 4. In multivariable analysis with up to 14 additional clinical variables, SeptiScore was the most important variable for sepsis diagnosis. A comparable performance was obtained for the majority of patients reanalyzed under the Sepsis-3 definition, although a subgroup of 16 patients was identified that was called septic under Sepsis-2 but not under Sepsis-3. (4) Conclusions: This study validates SeptiCyte RAPID for estimating sepsis probability, under both the Sepsis-2 and Sepsis-3 frameworks, for hospitalized patients on their first day of ICU admission.
RESUMO
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.
Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Benzilaminas , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Ciclamos , Fibroblastos/imunologia , Fibroblastos/patologia , Compostos Heterocíclicos/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/imunologia , Miofibroblastos/patologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/patologiaRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Macrófagos Alveolares/metabolismo , Oxigênio/toxicidade , Receptor A2A de Adenosina/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Animais , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Quimiocinas/metabolismo , Técnicas de Inativação de Genes , Mediadores da Inflamação/fisiologia , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigenoterapia , Fenetilaminas/farmacologia , Receptor A2A de Adenosina/genéticaRESUMO
RATIONALE: Acute lung injury (ALI) is a debilitating condition associated with severe skeletal muscle weakness that persists in humans long after lung injury has resolved. The molecular mechanisms underlying this condition are unknown. OBJECTIVES: To identify the muscle-specific molecular mechanisms responsible for muscle wasting in a mouse model of ALI. METHODS: Changes in skeletal muscle weight, fiber size, in vivo contractile performance, and expression of mRNAs and proteins encoding muscle atrophy-associated genes for muscle ring finger-1 (MuRF1) and atrogin1 were measured. Genetic inactivation of MuRF1 or electroporation-mediated transduction of miRNA-based short hairpin RNAs targeting either MuRF1 or atrogin1 were used to identify their role in ALI-associated skeletal muscle wasting. MEASUREMENTS AND MAIN RESULTS: Mice with ALI developed profound muscle atrophy and preferential loss of muscle contractile proteins associated with reduced muscle function in vivo. Although mRNA expression of the muscle-specific ubiquitin ligases, MuRF1 and atrogin1, was increased in ALI mice, only MuRF1 protein levels were up-regulated. Consistent with these changes, suppression of MuRF1 by genetic or biochemical approaches prevented muscle fiber atrophy, whereas suppression of atrogin1 expression was without effect. Despite resolution of lung injury and down-regulation of MuRF1 and atrogin1, force generation in ALI mice remained suppressed. CONCLUSIONS: These data show that MuRF1 is responsible for mediating muscle atrophy that occurs during the period of active lung injury in ALI mice and that, as in humans, skeletal muscle dysfunction persists despite resolution of lung injury.
Assuntos
Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Proteínas Musculares/genética , Atrofia Muscular/genética , Ubiquitina-Proteína Ligases/genética , Animais , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Regulação para Baixo , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Força Muscular/fisiologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Domínios RING Finger/genética , Distribuição Aleatória , Sensibilidade e Especificidade , Proteínas com Motivo TripartidoRESUMO
Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.