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Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.
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BACKGROUNDFibrocytes are BM-derived circulating cells that traffic to the injured lungs and contribute to fibrogenesis. The mTOR inhibitor, sirolimus, inhibits fibrocyte CXCR4 expression, reducing fibrocyte traffic and attenuating lung fibrosis in animal models. We sought to test the hypothesis that short-term treatment with sirolimus reduces the concentration of CXCR4+ circulating fibrocytes in patients with idiopathic pulmonary fibrosis (IPF).METHODSWe conducted a short-term randomized double-blind placebo-controlled crossover pilot trial to assess the safety and tolerability of sirolimus in IPF. Participants were randomly assigned to sirolimus or placebo for approximately 6 weeks, and after a 4-week washout, they were assigned to the alternate treatment. Toxicity, lung function, and the concentration of circulating fibrocytes were measured before and after each treatment.RESULTSIn the 28 study participants, sirolimus resulted in a statistically significant 35% decline in the concentration of total fibrocytes, 34% decline in CXCR4+ fibrocytes, and 42% decline in fibrocytes expressing α-smooth muscle actin, but no significant change in these populations occurred on placebo. Respiratory adverse events occurred more frequently during treatment with placebo than sirolimus; the incidence of adverse events and drug tolerability did not otherwise differ during therapy with drug and placebo. Lung function was unaffected by either treatment, with the exception of a small decline in gas transfer during treatment with placebo.CONCLUSIONAs compared with placebo, short-term treatment with sirolimus resulted in reduction of circulating fibrocyte concentrations in participants with IPF, with an acceptable safety profile.TRIAL REGISTRATIONClinicalTrials.gov, accession no. NCT01462006.FUNDINGNIH R01HL098329 and American Heart Association 18TPA34170486.
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Fibrosis Pulmonar Idiopática , Sirolimus , Estados Unidos , Animales , Sirolimus/efectos adversos , Estudios Cruzados , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Fibroblastos/metabolismoRESUMEN
BACKGROUND: Depriving microbes of iron is critical to host defense. Hemeproteins, the largest source of iron within vertebrates, are abundant in infected tissues in aspergillosis due to hemorrhage, but Aspergillus species have been thought to lack heme import mechanisms. We hypothesized that heme provides iron to Aspergillus during invasive pneumonia, thereby worsening the outcomes of the infection. METHODS: We assessed the effect of heme on fungal phenotype in various in vitro conditions and in a neutropenic mouse model of invasive pulmonary aspergillosis. RESULTS: In mice with neutropenic invasive aspergillosis, we found a progressive and compartmentalized increase in lung heme iron. Fungal cells cultured under low iron conditions took up heme, resulting in increased fungal iron content, resolution of iron starvation, increased conidiation, and enhanced resistance to oxidative stress. Intrapulmonary administration of heme to mice with neutropenic invasive aspergillosis resulted in markedly increased lung fungal burden, lung injury, and mortality, whereas administration of heme analogs or heme with killed Aspergillus did not. Finally, infection caused by fungal germlings cultured in the presence of heme resulted in a more severe infection. CONCLUSIONS: Invasive aspergillosis induces local hemolysis in infected tissues, thereby supplying heme iron to the fungus, leading to lethal infection.
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Aspergilosis , Neumonía , Animales , Aspergillus , Aspergillus fumigatus , Hemo , Hierro , RatonesRESUMEN
BACKGROUND: Fibrocytes are bone marrow mesenchymal precursors with a surface phenotype compatible with leukocytes, fibroblasts, and hematopoietic progenitors that have been shown to traffic to wound healing sites in response to described chemokine pathways. Keloids are focal fibrotic responses to cutaneous trauma characterized by disordered collagen, which may be associated with elevated systemic fibrocyte levels and/or wound bed chemokine expression. METHODS: Blood specimens from patients with longstanding keloids and those who form grossly normal scars were assayed by fluorescence activated cell sorting analysis for fibrocytes (CD45+, Col I+). The expression of the fibrocyte chemotactic cell surface marker CXCR4, intracellular markers of fibroblast differentiation (pSMAD2/3), and plasma levels of the CXCR4 cognate CXCL12 were compared. Keloid specimens and grossly normal scars were excised, and local expression of CXCL12 was assayed. RESULTS: Keloid-forming patients demonstrated a significantly greater number of circulating fibrocytes (17.4 × 105 cells/mL) than control patients (1.01 × 105 cells/mL, P = 0.004). The absolute number of fibrocytes expressing CXCR4 was significantly greater (P = 0.012) in keloid-forming patients. Systemic CXCL12 levels were insignificantly greater in keloid-forming patients than controls. Keloid specimens had significantly greater CXCL12 expression (529.3 pg/mL) than normal scar (undetectable). CONCLUSIONS: Systemic fibrocyte levels and the CXCR4/CXCL12 biologic axis responsible for fibrocyte trafficking to areas of regional fibrosis were both upregulated in patients who form keloids compared with controls. Keloids persistently expressed CXLC12, which serves both as the main chemoattractant for fibrocytes and a downstream mediator for local inflammation, suggesting a role for this biologic axis in keloid formation and possibly recurrence.
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Quimiocina CXCL12 , Fibroblastos , Queloide , Diferenciación Celular , Factores Quimiotácticos , Cicatriz , Fibroblastos/patología , Fibrosis , Humanos , Queloide/patologíaRESUMEN
Chronic granulomatous disease (CGD) results from deficiency of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and impaired reactive oxygen species (ROS) generation. This leads to impaired killing of Aspergillus and, independently, a pathologic hyperinflammatory response to the organism. We hypothesized that neutrophil-derived ROS inhibit the inflammatory response to Aspergillus and that acute lung injury in CGD is due to failure of this regulation. Mice with gp91phox deficiency, the most common CGD mutation, had more severe lung injury, increased neutrophilinfiltration, and increased lung tumor necrosis factor (TNF) after Aspergillus challenge compared with wild-types. Neutrophils were surprisingly the predominant source of TNF in gp91phox-deficient lungs. TNF neutralization inhibited neutrophil recruitment in gp91phox-deficient mice and protected from lung injury. We propose that, in normal hosts, Aspergillus stimulates TNF-dependent neutrophil recruitment to the lungs and neutrophil-derived ROS limit inflammation. In CGD, in contrast, recruited neutrophils are the dominant source of TNF, promoting further neutrophil recruitment in a pathologic positive-feedback cycle, resulting in progressive lung injury.
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Lesión Pulmonar Aguda/etiología , Hongos/genética , Enfermedad Granulomatosa Crónica , Neutrófilos/inmunología , Factor de Necrosis Tumoral alfa , Animales , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/inmunología , Ratones , Ratones Noqueados , NADPH Oxidasas/inmunología , Especies Reactivas de Oxígeno , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
STUDY OBJECTIVE: Infarct size is a strong predictor of outcomes after ST elevation myocardial infarction (STEMI). Circulating fibrocytes are bone marrow-derived progenitor cells associated with fibrotic processes. We tested whether fibrocytes correlate with infarct size in STEMI patients treated with primary percutaneous coronary intervention (PCI). DESIGN: Prospective observational study. SETTING: Academic medical center. PARTICIPANTS: Subjects with STEMI treated with primary PCI. INTERVENTIONS: Peripheral blood draw and cardiac magnetic resonance imaging (CMR). MAIN OUTCOME MEASURE: Correlation of fibrocyte levels with infarct size. METHODS: Peripheral blood fibrocytes were quantified at discharge from STEMI hospitalization and at 6 months follow-up using flow cytometry. Infarct size was determined within 2 weeks of discharge and at 6 months follow-up using late gadolinium enhancement on CMR. RESULTS: Among 14 patients (median age 54 years, 79% men) with STEMI, there was a statistically significant positive correlation between fibrocyte levels at 6 months and 6-month infarct size on CMR (r = 0.58, p = 0.031). In addition, there was positive correlation between peak troponin I level (r = 0.85, p < 0.001), and white blood cell count (r = 0.55, p = 0.042) during the hospital stay and 6-month infarct size on CMR. CONCLUSIONS: Circulating fibrocytes measured 6 months after STEMI positively correlate with 6-month infarct size assessed by CMR.
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BACKGROUND: Autoimmunity is a common cause of pulmonary fibrosis and can present either as a manifestation of an established connective tissue disease or as the recently described entity of interstitial pneumonia with autoimmune features. The rate of progression and responsiveness to immunosuppression in these illnesses are difficult to predict. Circulating fibrocytes are bone marrow-derived progenitor cells that home to injured tissues and contribute to lung fibrogenesis. We sought to test the hypothesis that the blood fibrocyte concentration predicts outcome and treatment responsiveness in autoimmune interstitial lung diseases. METHODS: We compared the concentration of circulating fibrocytes in 50 subjects with autoimmune interstitial lung disease and 26 matched healthy controls and assessed the relationship between serial peripheral blood fibrocyte concentrations and clinical outcomes over a median of 6.25â years. RESULTS: As compared to controls, subjects with autoimmune interstitial lung disease had higher circulating concentrations of total fibrocytes, the subset of activated fibrocytes, and fibrocytes with activation of PI3K/AKT/mTOR, transforming growth factor-ß (TGF-ß) receptor and interleukin (IL)-4/IL-13 receptor signalling pathways. Over the follow-up period, there were episodes of marked elevation in the concentration of circulating fibrocytes in subjects with autoimmune interstitial lung disease but not controls. Initiation of immunosuppressive therapy was associated with a decline in the concentration of circulating fibrocytes. For each 100â000â cells·mL-1 increase in peak concentration of circulating fibrocytes, we found a 5% increase in odds of death or lung function decline. CONCLUSION: In patients with autoimmune interstitial lung disease, circulating fibrocytes may represent a biomarker of outcome and treatment response.
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BACKGROUND: Fibrosis is an integral component of the pathogenesis of acute lung injury and is associated with poor outcomes in patients with acute respiratory distress syndrome (ARDS). Fibrocytes are bone marrow-derived cells that traffic to injured tissues and contribute to fibrosis; hence their concentration in the peripheral blood has the potential to serve as a biomarker of lung fibrogenesis. We therefore sought to test the hypothesis that the concentration and phenotype of circulating fibrocytes in patients with ARDS predicts clinical outcomes. METHODS: For the animal studies, C57Bl/6 mice were infected with experimental Klebsiella pneumoniae in a model of acute lung injury; one-way ANOVA was used to compare multiple groups and two-way ANOVA was used to compare two groups over time. For the human study, 42 subjects with ARDS and 12 subjects with pneumonia (without ARDS) were compared to healthy controls. Chi-squared or Fisher's exact test were used to compare binary outcomes. Survival data was expressed using a Kaplan-Meier curve and compared by log-rank test. Univariable and multivariable logistic regression were used to predict death. RESULTS: In mice with acute lung injury caused by Klebsiella pneumonia, there was a time-dependent increase in lung soluble collagen that correlated with sequential expansion of fibrocytes in the bone marrow, blood, and then lung compartments. Correspondingly, when compared via cross-sectional analysis, the initial concentration of blood fibrocytes was elevated in human subjects with ARDS or pneumonia as compared to healthy controls. In addition, fibrocytes from subjects with ARDS displayed an activated phenotype and on serial measurements, exhibited intermittent episodes of markedly elevated concentration over a median of 1 week. A peak concentration of circulating fibrocytes above a threshold of > 4.8 × 106 cells/mL cells correlated with mortality that was independent of age, ratio of arterial oxygen concentration to the fraction of inspired oxygen, and vasopressor requirement. CONCLUSIONS: Circulating fibrocytes increase in a murine model of acute lung injury and elevation in the number of these cells above a certain threshold is correlated with mortality in human ARDS. Therefore, these cells may provide a useful and easily measured biomarker to predict outcomes in these patients.
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Lesión Pulmonar Aguda/patología , Células de la Médula Ósea/patología , Pulmón/patología , Síndrome de Dificultad Respiratoria/mortalidad , Síndrome de Dificultad Respiratoria/patología , Lesión Pulmonar Aguda/etiología , Adulto , Animales , Biomarcadores , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Persona de Mediana Edad , Proyectos Piloto , Pronóstico , Síndrome de Dificultad Respiratoria/etiologíaRESUMEN
Asthma is associated with the overproduction of leukotrienes (LTs), including LTB4. Patients with severe asthma can be highly responsive to 5-lipoxygenase (5-LO) inhibition, which blocks production of both the cysteinyl LTs and LTB4. Production of LTB4 has traditionally been ascribed to neutrophils, mononuclear phagocytes, and epithelial cells, and acts as a chemoattractant for inflammatory cells associated with asthma. The source of LTB4 is unclear, especially in eosinophilic asthma. We speculated that the benefit of 5-LO inhibition could be mediated in part by inhibition of eosinophil-derived LTB4. LTB4 concentrations were assayed in BAL fluid from patients with severe asthma characterized by isolated neutrophilic, eosinophilic, and paucigranulocytic inflammation. Expression of LTA4 hydrolase (LTA4H) by airway eosinophils was determined by immunohistochemistry (IHC). Subsequently, peripheral blood eosinophils were activated and secreted LTB4 was quantified by enzyme immunoassay. Blood eosinophil LTA4H expression was determined by flow cytometry, qPCR, and IHC. LTB4 concentrations were elevated in BAL fluid from patients with severe asthma, including those with isolated eosinophilic inflammation, and these eosinophils displayed LTA4H via IHC. LTA4H expression by blood eosinophils was confirmed by flow cytometry, IHC, and qPCR. Robust LTB4 production by blood eosinophils was observed in response to some, but not all, stimuli. We demonstrated that eosinophils express LTA4H transcripts and protein, and can be stimulated to secrete LTB4. We speculate that in many patients with asthma, eosinophil-derived LTB4 is increased, and this may contribute to the efficacy of 5-LO inhibition.
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Asma/patología , Eosinófilos/metabolismo , Epóxido Hidrolasas/metabolismo , Leucotrieno B4/biosíntesis , Araquidonato 5-Lipooxigenasa/metabolismo , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Niño , Preescolar , Femenino , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Neutrófilos/citologíaRESUMEN
Gram-negative pneumonia is a dangerous illness, and bacterial dissemination to the bloodstream during the infection is strongly associated with death. Antibiotic resistance among the causative pathogens has resulted in diminishing treatment options against this infection. Hepcidin is the master regulator of extracellular iron availability in vertebrates, but its role in the context of host defense is undefined. We hypothesized that hepcidin-mediated depletion of extracellular iron during Gram-negative pneumonia protects the host by limiting dissemination of bacteria to the bloodstream. During experimental pneumonia, hepcidin was induced in the liver in an IL-6-dependent manner and mediated a rapid decline in plasma iron. In contrast, hepcidin-deficient mice developed a paradoxical increase in plasma iron during infection associated with profound susceptibility to bacteremia. Incubation of bacteria with iron-supplemented plasma enhanced bacterial growth in vitro, and systemic administration of iron to WT mice similarly promoted increased susceptibility to bloodstream infection. Finally, treatment with a hepcidin analogue restored hypoferremia in hepcidin-deficient hosts, mediated bacterial control, and improved outcomes. These data show hepcidin induction during pneumonia to be essential to preventing bacterial dissemination by limiting extracellular iron availability. Hepcidin agonists may represent an effective therapy for Gram-negative infections in patients with impaired hepcidin production or signaling.
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Hepcidinas/fisiología , Hierro/metabolismo , Klebsiella pneumoniae/crecimiento & desarrollo , Neumonía Bacteriana/microbiología , Animales , Disponibilidad Biológica , Líquido del Lavado Bronquioalveolar , Humanos , Klebsiella pneumoniae/aislamiento & purificación , RatonesRESUMEN
Lung injury and fibrosis are common in patients with sickle cell disease (SCD). Fibrocytes, a population of circulating, bone marrow-derived cells, have been linked to development and progression of tissue fibrogenesis and have been implicated in the development of lung fibrosis in preclinical models of SCD. We tested the hypothesis that the levels and activation state of circulating fibrocytes during steady state are associated with abnormal pulmonary function in adults with SCD. In a prospective cohort of steady-state adults with SCD and healthy age- and race-matched control participants, we measured the concentration and activation state of circulating fibrocytes and assessed pulmonary phenotype with pulmonary function tests (PFTs), a respiratory questionnaire, 6-minute walk test, high-resolution chest computed tomography scan, and echocardiogram. Seventy-one adults with SCD and 26 healthy African American control participants were examined. Compared with control participants, patients with SCD demonstrated higher levels of circulating fibrocytes, a significant proportion of which expressed the activation marker α-smooth muscle actin. Within patients with SCD, elevated absolute concentrations of circulating fibrocytes were strongly and independently associated with impaired lung physiology, as measured by PFTs. We conclude that elevated circulating fibrocytes are associated with lung disease in adults with SCD during steady state, consistent with a role for these cells in pathogenesis of lung fibrosis in this disease. Circulating fibrocytes may represent a novel biomarker for progressive pulmonary fibrosis in patients with SCD.
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Gram-negative bacterial pneumonia is a common and dangerous infection with diminishing treatment options due to increasing antibiotic resistance among causal pathogens. The mononuclear phagocyte system is a heterogeneous group of leukocytes composed of tissue-resident macrophages, dendritic cells, and monocyte-derived cells that are critical in defense against pneumonia, but mechanisms that regulate their maintenance and function during infection are poorly defined. M-CSF has myriad effects on mononuclear phagocytes but its role in pneumonia is unknown. We therefore tested the hypothesis that M-CSF is required for mononuclear phagocyte-mediated host defenses during bacterial pneumonia in a murine model of infection. Genetic deletion or immunoneutralization of M-CSF resulted in reduced survival, increased bacterial burden, and greater lung injury. M-CSF was necessary for the expansion of lung mononuclear phagocytes during infection but did not affect the number of bone marrow or blood monocytes, proliferation of precursors, or recruitment of leukocytes to the lungs. In contrast, M-CSF was essential to survival and antimicrobial functions of both lung and liver mononuclear phagocytes during pneumonia, and its absence resulted in bacterial dissemination to the liver and hepatic necrosis. We conclude that M-CSF is critical to host defenses against bacterial pneumonia by mediating survival and antimicrobial functions of mononuclear phagocytes in the lungs and liver.
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Infecciones por Klebsiella/inmunología , Hígado/inmunología , Pulmón/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Sistema Mononuclear Fagocítico/inmunología , Fagocitos/inmunología , Neumonía Bacteriana/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Médula Ósea/inmunología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/inmunología , Hígado/citología , Hígado/microbiología , Hígado/patología , Pulmón/citología , Pulmón/microbiología , Pulmón/patología , Factor Estimulante de Colonias de Macrófagos/deficiencia , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Monocitos/inmunología , Monocitos/microbiología , Neumonía Bacteriana/microbiologíaRESUMEN
Half of the patients who present with unstable angina (UA) develop recurrent symptoms over the subsequent year. Identification of patients destined to develop such adverse events would be clinically valuable, but current tools do not allow for this discrimination. Fibrocytes are bone marrow-derived progenitor cells that co-express markers of leukocytes and fibroblasts and are released into the circulation in the context of tissue injury. We hypothesized that, in patients with UA, the number of circulating fibrocytes predicts subsequent adverse events. We enrolled 55 subjects with UA, 18 with chronic stable angina, and 22 controls and correlated their concentration of circulating fibrocytes to clinical events (recurrent angina, myocardial infarction, revascularization, or death) over the subsequent year. Subjects with UA had a >2-fold higher median concentration of both total and activated fibrocytes compared with subjects with chronic stable angina and controls. In UA subjects, the concentration of total fibrocytes identified those who developed recurrent angina requiring revascularization (time-dependent area under the curve 0.85) and was superior to risk stratification using thrombolysis in myocardial infarction risk score and N-terminal pro B-type natriuretic peptide levels (area under the curve, 0.53 and 0.56, respectively, P < 0.001). After multivariable adjustment for thrombolysis in myocardial infarction predicted death, MI, or recurrent ischemia, total fibrocyte level was associated with recurrent angina (hazard ratio, 1.016 per 10,000 cells/mL increase; 95% confidence interval, 1.007-1.024; P < 0.001). Circulating fibrocytes are elevated in patients with UA and successfully risk stratify them for adverse clinical outcomes. Fibrocytes may represent a novel biomarker of outcome in this population.
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Angina Inestable/patología , Movimiento Celular , Fibroblastos/patología , Adulto , Anciano , Angina Inestable/sangre , Estudios de Casos y Controles , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Recurrencia , Factor de Crecimiento Transformador beta1/sangreRESUMEN
BACKGROUND: A biomarker that predicts poor asthma control would be clinically useful. Fibrocytes are bone marrow-derived circulating progenitor cells that have been implicated in tissue fibrosis and T(H)2 responses in asthmatic patients. OBJECTIVE: We sought to test the hypothesis that the concentration and activation state of peripheral blood fibrocytes correlates with asthma severity. METHODS: By using fluorescence-activated cell sorting analysis, fibrocytes (CD45(+) and collagen 1 [Col1](+)) were enumerated and characterized in the buffy coats of fresh peripheral blood samples from 15 control subjects and 40 asthmatic patients. RESULTS: Concentrations of peripheral blood total (CD45(+)Col1(+)), activated (the TGF-ß transducing protein phosphorylated SMAD2/3 [p-SMAD2/3](+) or phosphorylated AKT [p-AKT](+)), and differentiated (α-smooth muscle actin [α-SMA](+)) fibrocytes were increased in asthmatic patients compared with control subjects. The increase in total and CD45(+)Col1(+)CXCR4(+) fibrocytes was primarily seen in patients with severe asthma (Global Initiative for Asthma steps 4-5) as opposed to those with milder asthma (Global Initiative for Asthma steps 1-3). In addition, numbers of circulating α-SMA(+) and α-SMA(+)CXCR4(+) fibrocytes were increased in asthmatic patients experiencing an asthma exacerbation in the preceding 12 months. A significant correlation (P < .05) was observed between CD45(+)Col1(+)CXCR4(+) fibrocytes and the activation phenotypes CD45(+)Col1(+)p-SMAD2/3(+) and CD45(+)Col1(+)p-AKT(+). CONCLUSION: There was correlation between circulating fibrocyte subsets and asthma severity, and there was an increased number of activated/differentiated fibrocytes in circulating blood of asthmatic patients experiencing an exacerbation in the preceding 12 months.
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Asma/sangre , Asma/diagnóstico , Recuento de Células , Diferenciación Celular , Células del Tejido Conectivo/citología , Células del Tejido Conectivo/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Superficie/metabolismo , Biomarcadores , Estudios de Casos y Controles , Niño , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.
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Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Quimiocina CXCL10/metabolismo , Inmunidad Innata/inmunología , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Sitios de Unión , Dihidrolipoamida Deshidrogenasa/genética , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli , Técnicas de Inactivación de Genes , Humanos , Unión Proteica , Piruvato Deshidrogenasa (Lipoamida)/genéticaRESUMEN
RATIONALE: The rate of progression of most interstitial lung diseases (ILD) is unpredictable. Fibrocytes are circulating bone marrow-derived cells that have been implicated in the pathogenesis of lung fibrosis. Hermansky-Pudlak syndrome (HPS), a genetic cause of ILD in early adulthood, allows for study of biomarkers of ILD in a homogeneous population at near-certain risk of developing fibrotic lung disease. OBJECTIVES: To test the hypothesis that, in subjects with HPS, the number or phenotype of circulating fibrocytes predicts progression and outcome of ILD. METHODS: We measured circulating fibrocyte counts and chemokine levels in a cohort of subjects with HPS and healthy control subjects and correlated the results to disease outcome. MEASUREMENTS AND MAIN RESULTS: In a cross-sectional analysis, peripheral blood fibrocyte concentrations were markedly elevated in a subset of subjects with HPS who had ILD but not subjects without lung disease or normal control subjects. The blood concentration of fibrocytes expressing the chemokine receptor CXCR4 correlated significantly with the plasma concentration of the CXCR4 ligand, CXCL12. In a longitudinal study, we found marked episodic elevations in circulating fibrocyte counts over a median follow-up period of 614 days. Elevations in both maximal values and final values of peripheral blood CXCR4(+) fibrocyte concentration were strongly associated with death from ILD. CONCLUSIONS: CXCR4(+) fibrocyte concentration may be useful as a biomarker for outcome of ILD in subjects with HPS.
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Síndrome de Hermanski-Pudlak/diagnóstico , Células Madre Mesenquimatosas/fisiología , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Femenino , Síndrome de Hermanski-Pudlak/sangre , Síndrome de Hermanski-Pudlak/mortalidad , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pronóstico , Receptores CXCR4/sangre , Receptores CXCR4/fisiología , Análisis de SupervivenciaRESUMEN
UNLABELLED: Chemokines have been implicated as key contributors of non-small cell lung cancer (NSCLC) metastasis. However, the role of CXCR7, a recently discovered receptor for CXCL12 ligand, in the pathogenesis of NSCLC is unknown. To define the relative contribution of chemokine receptors to migration and metastasis, we generated human lung A549 and H157 cell lines with stable knockdown of CXCR4, CXCR7, or both. Cancer cells exhibited chemotaxis to CXCL12 that was enhanced under hypoxic conditions, associated with a parallel induction of CXCR4, but not CXCR7. Interestingly, neither knockdown cell line differed in the rate of proliferation, apoptosis, or cell adherence; however, in both cell lines, CXCL12-induced migration was abolished when CXCR4 signaling was abrogated. In contrast, inhibition of CXCR7 signaling did not alter cellular migration to CXCL12. In an in vivo heterotropic xenograft model using A549 cells, expression of CXCR4, but not CXCR7, on cancer cells was necessary for the development of metastases. In addition, cancer cells knocked down for CXCR4 (or both CXCR4 and CXCR7) produced larger and more vascular tumors as compared with wild-type or CXCR7 knockdown tumors, an effect that was attributable to cancer cell-derived CXCR4 out competing endothelial cells for available CXCL12 in the tumor microenvironment. These results indicate that CXCR4, not CXCR7, expression engages CXCL12 to mediate NSCLC metastatic behavior. IMPLICATIONS: Targeting CXCR4-mediated migration and metastasis may be a viable therapeutic option in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Quimiocina CXCL12/farmacología , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia/genética , Receptores CXCR4/genética , Animales , Apoptosis/genética , Adhesión Celular/genética , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL11/farmacología , Quimiotaxis/genética , Células Endoteliales/patología , Femenino , Humanos , Ratones , Ratones SCID , Interferencia de ARN , ARN Interferente Pequeño , Receptores CXCR/genética , Microambiente TumoralRESUMEN
Bacterial pneumonia is a common and dangerous illness. Mononuclear phagocytes, which comprise monocyte, resident and recruited macrophage, and dendritic cell subsets, are critical to antimicrobial defenses, but the dynamics of their recruitment to the lungs in pneumonia is not established. We hypothesized that chemokine-mediated traffic of mononuclear phagocytes is important in defense against bacterial pneumonia. In a mouse model of Klebsiella pneumonia, circulating Ly6C(hi) and, to a lesser extent, Ly6C(lo) monocytes expanded in parallel with accumulation of inflammatory macrophages and CD11b(hi) dendritic cells and plasmacytoid dendritic cells in the lungs, whereas numbers of alveolar macrophages remained constant. CCR2 was expressed by Ly6C(hi) monocytes, recruited macrophages, and airway dendritic cells; CCR6 was prominently expressed by airway dendritic cells; and CX3CR1 was ubiquitously expressed by blood monocytes and lung CD11b(hi) dendritic cells during infection. CCR2-deficient, but not CCL2-, CX3CR1-, or CCR6-deficient animals exhibited worse outcomes of infection. The absence of CCR2 had no detectable effect on neutrophils but resulted in reduction of all subsets of lung mononuclear phagocytes in the lungs, including alveolar macrophages and airway and plasmacytoid dendritic cells. In addition, absence of CCR2 skewed the phenotype of lung mononuclear phagocytes, abrogating the appearance of M1 macrophages and TNF-producing dendritic cells in the lungs. Taken together, these data define the dynamics of mononuclear phagocytes during pneumonia.
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Quimiocina CCL2/fisiología , Monocitos/inmunología , Fagocitos/inmunología , Neumonía/inmunología , Receptores CCR2/fisiología , Receptores CCR6/fisiología , Receptores de Quimiocina/fisiología , Animales , Receptor 1 de Quimiocinas CX3C , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/patogenicidad , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Masculino , Ratones , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Peroxidasa/metabolismo , Fagocitos/metabolismo , Fagocitos/patología , Neumonía/metabolismo , Neumonía/patologíaRESUMEN
BACKGROUND AND PURPOSE: The chemokine ligand CXCL12 is constitutively expressed in the bone marrow and other tissues including the brain endothelium and is responsible for regulating the trafficking of bone marrow progenitor cells. CXCL12 has been shown to play a significant role in animal models of ischemic stroke but its role in human stroke is unclear. The aim of this study was to test the hypothesis that elevated circulating baseline CXCL12 levels are associated with subsequent stroke. METHODS: We prospectively collected demographic and angiographic data from consecutive patients referred for elective coronary angiography. Before coronary angiography a peripheral blood sample was collected for subsequent measurement of CXCL12. One-year stroke risk was calculated using the Framingham Risk Profile. Clinical follow-up was performed at 6 months and 1 year. RESULTS: Of 206 subjects enrolled, 10 (4.9%) sustained an ischemic stroke over the 1 year follow-up. There were no significant differences in baseline clinical characteristics or angiographic findings. However, median CXCL12 levels were significantly higher in those who sustained an ischemic stroke compared with those who did not (10 856 pg/mL versus 2241 pg/mL, P=0.007). The time to stroke distribution between subjects with baseline CXCL12 levels≥10 421 pg/mL and those with baseline CXCL12 levels<10 421 pg/mL was significantly different (log rank P<0.001). The weighted Cox proportional hazard model demonstrated that baseline CXCL12 levels≥10 421 pg/mL were significantly associated with ischemic stroke at follow-up (hazard ratio, 15.29; 95% CI, 3.05-76.71). CONCLUSIONS: Plasma CXCL12 levels may represent a novel biomarker of future ischemic stroke.
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Quimiocina CXCL12/sangre , Quimiocina CXCL12/inmunología , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/inmunología , Anciano , Biomarcadores/sangre , Angiografía Coronaria/estadística & datos numéricos , Bases de Datos Factuales/estadística & datos numéricos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Factores de Riesgo , Accidente Cerebrovascular/sangreRESUMEN
RATIONALE: Activation of the adenosine A(2B) receptor (A(2B)R) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. OBJECTIVES: To test the hypothesis that absence of adenosine A(2B) receptor signaling promotes host defense against bacterial pneumonia. METHODS: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A(2B)R. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. MEASUREMENTS AND MAIN RESULTS: A(2B)R(-/-) mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow-derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A(2B)R(-/-) and wild-type mice, but A(2B)R(-/-) neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A(2B)R(-/-) mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. CONCLUSIONS: These data suggest that the absence of A(2B)R signaling enhances antimicrobial activity in gram-negative bacterial pneumonia.