Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

A protective role for B-1 cells and oxidation-specific epitope IgM in lung fibrosis.

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.

Sirolimus suppresses circulating fibrocytes in idiopathic pulmonary fibrosis in a randomized controlled crossover trial.

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.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation.

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

Aspergillus Utilizes Extracellular Heme as an Iron Source During Invasive Pneumonia, Driving Infection Severity.

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.

Systemic Fibrocyte Levels and Keloid Expression of the Chemoattractant CXCL12 Are Upregulated Compared With Patients With Normal Scar.

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.

Neutrophil-Derived Tumor Necrosis Factor Drives Fungal Acute Lung Injury in Chronic Granulomatous Disease.

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.

Circulating fibrocyte levels correlate with infarct size in patients with ST elevation myocardial infarction treated with primary percutaneous coronary intervention.

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.

Circulating fibrocytes as prognostic biomarkers of autoimmune interstitial lung disease.

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 6.25 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 100 000 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.

Circulating fibrocytes traffic to the lung in murine acute lung injury and predict outcomes in human acute respiratory distress syndrome: a pilot study.

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.

Effect of Modifier Structure on the Activation of Leukotriene A4 Hydrolase Aminopeptidase Activity.

Activation of the leukotriene A4 hydrolase (LTA4H) aminopeptidase (AP) activity with 4-methoxydiphenylmethane (4MDM) promoted resolution of neutrophil infiltration in a murine cigarette smoke-induced model for emphysematous chronic obstructive pulmonary disease. Recently, 4-(4-benzylphenyl)thiazol-2-amine (ARM1) was published as a ligand for LTA4H with potential anti-inflammatory properties. To investigate the effect of modifier structure on enzyme kinetics of LTA4H, a series of analogues bearing structural features of ARM1 and 4MDM were synthesized using trifluoroborate Suzuki coupling reactions. Following, the 2.8 Å X-ray crystal structure of LTA4H complexed with 4-OMe-ARM1, a 4MDM-ARM1 hybrid molecule, was determined. Kinetic analysis showed that ARM1 and related analogues lowered affinity for the enzyme-substrate complex, resulting in a change of mechanism from hyperbolic mixed predominately catalytic activation (HMx(Sp < Ca)A) as observed for 4MDM to a predominately specific activation (HMx(Sp > Ca)A) mechanism. 4-OMe-ARM1 was then shown to dose responsively reduce LTB4 production in human neutrophils.

Leukotriene A4 Hydrolase Activation and Leukotriene B4 Production by Eosinophils in Severe Asthma.

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.

Hepcidin-mediated iron sequestration protects against bacterial dissemination during pneumonia.

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.

Circulating fibrocytes as biomarkers of impaired lung function in adults with sickle cell disease.

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.

M-CSF Mediates Host Defense during Bacterial Pneumonia by Promoting the Survival of Lung and Liver Mononuclear Phagocytes.

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.

Circulating fibrocytes as predictors of adverse events in unstable angina.

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.

Increased circulating fibrocytes are associated with higher reticulocyte percent in children with sickle cell anemia.

BACKGROUND: Interstitial lung disease is common in patients with sickle cell anemia (SCA). Fibrocytes are circulating cells implicated in the pathogenesis of pulmonary fibrosis and airway remodeling in asthma. In this study, we tested the hypotheses that fibrocyte levels are: (1) increased in children with SCA compared to healthy controls, and (2) associated with pulmonary disease. PROCEDURE: Cross-sectional cohort study of children with SCA who participated in the Sleep Asthma Cohort Study. RESULTS: Fibrocyte levels were obtained from 45 children with SCA and 24 controls. Mean age of SCA cases was 14 years and 53% were female. In children with SCA, levels of circulating fibrocytes were greater than controls (P < 0.01). The fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on the majority of cells and CCR2 and CCR7 expressed on a smaller subset. Almost half of fibrocytes demonstrated α-smooth muscle actin activation. Increased fibrocyte levels were associated with a higher reticulocyte count (P = 0.03) and older age (P = 0.048) in children with SCA. However, children with increased levels of fibrocytes were not more likely to have asthma or lower percent predicted forced expiratory volume in 1 sec/forced vital capacity (FEV1 /FVC) or FEV1 than those with lower fibrocyte levels. CONCLUSIONS: Higher levels of fibrocytes in children with SCA compared to controls may be due to hemolysis. Longitudinal studies may be able to better assess the relationship between fibrocyte level and pulmonary dysfunction.

Number, activation, and differentiation of circulating fibrocytes correlate with asthma severity.

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.

Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity.

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.