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.

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.

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.

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.

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.

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.

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.

Circulating fibrocytes as biomarker of prognosis in Hermansky-Pudlak syndrome.

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.

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.

Heterogeneity of lung mononuclear phagocytes during pneumonia: contribution of chemokine receptors.

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.

Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3.

We investigated possible cellular receptors for the human CXC chemokine platelet factor-4 variant/CXCL4L1, a potent inhibitor of angiogenesis. We found that CXCL4L1 has lower affinity for heparin and chondroitin sulfate-E than platelet factor-4 (CXCL4) and showed that CXCL10 and CXCL4L1 could displace each other on microvascular endothelial cells. Labeled CXCL4L1 also bound to CXCR3A- and CXCR3B-transfectants and was displaced by CXCL4L1, CXCL4, and CXCL10. The CXCL4L1 anti-angiogenic activity was blocked by anti-CXCR3 antibodies (Abs) in the Matrigel and cornea micropocket assays. CXCL4L1 application in CXCR3(-/-) or in wild-type mice treated with neutralizing anti-CXCR3 Abs, resulted in reduced inhibitory activity of CXCL4L1 on tumor growth and vascularization of Lewis lung carcinoma. Furthermore, CXCL4L1 and CXCL4 chemoattracted activated T cells, human natural killer cells, and human immature dendritic cells (DCs). Migration of DCs toward CXCL4 and CXCL4L1 was desensitized by preincubation with CXCL10 and CXCL11, inhibited by pertussis toxin, and neutralized by anti-CXCR3 Abs. Chemotaxis of T cells, natural killer cells, and DCs is likely to contribute to the antitumoral action. However, the in vivo data indicate that the angiostatic property of CXCL4L1 is equally important in retarding tumor growth. Thus, both CXCR3A and CXCR3B are implicated in the chemotactic and vascular effects of CXCL4L1.

Adenosine A(2B) receptor deficiency promotes host defenses against gram-negative bacterial pneumonia.

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.

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.

Neutrophils mediate maturation and efflux of lung dendritic cells in response to Aspergillus fumigatus germ tubes.

Invasive aspergillosis is a life-threatening complication of neutrophil deficiency or dysfunction. Neutropenia has previously been associated with enhanced influx of CD11b-expressing conventional dendritic cells to the lungs in response to Aspergillus species, but whether neutrophils directly modulate the function of dendritic cells in this infection is not known. We hypothesized that, in the setting of intrapulmonary challenge with Aspergillus, neutrophils promote the maturation and traffic of lung conventional dendritic cells to draining mediastinal lymph nodes. We report that neutropenia results in a marked accumulation of dendritic cells in the lungs of mice challenged with Aspergillus but greatly diminishes their egress to mediastinal lymph nodes independent of neutrophil microbicidal functions. Furthermore, the phenotype of lung dendritic cells was more immature in neutropenic animals than in nonneutropenic mice exposed to the microorganism. Consistent with this, coincubation with neutrophils greatly enhanced the upregulation of costimulatory molecules on dendritic cells exposed to Aspergillus in vitro, a process that was dependent on cell contact and the dendritic cell receptor DC-SIGN. Taken together, our data support an immunomodulatory cross talk between neutrophils and dendritic cells in the context of host response to Aspergillus that promotes the maturation and efflux of lung dendritic cells.

Plasma CXCL12 levels as a predictor of future stroke.

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.

Interferon-inducible CXC chemokines directly contribute to host defense against inhalational anthrax in a murine model of infection.

Chemokines have been found to exert direct, defensin-like antimicrobial activity in vitro, suggesting that, in addition to orchestrating cellular accumulation and activation, chemokines may contribute directly to the innate host response against infection. No observations have been made, however, demonstrating direct chemokine-mediated promotion of host defense in vivo. Here, we show that the murine interferon-inducible CXC chemokines CXCL9, CXCL10, and CXCL11 each exert direct antimicrobial effects in vitro against Bacillus anthracis Sterne strain spores and bacilli including disruptions in spore germination and marked reductions in spore and bacilli viability as assessed using CFU determination and a fluorometric assay of metabolic activity. Similar chemokine-mediated antimicrobial activity was also observed against fully virulent Ames strain spores and encapsulated bacilli. Moreover, antibody-mediated neutralization of these CXC chemokines in vivo was found to significantly increase host susceptibility to pulmonary B. anthracis infection in a murine model of inhalational anthrax with disease progression characterized by systemic bacterial dissemination, toxemia, and host death. Neutralization of the shared chemokine receptor CXCR3, responsible for mediating cellular recruitment in response to CXCL9, CXCL10, and CXCL11, was not found to increase host susceptibility to inhalational anthrax. Taken together, our data demonstrate a novel, receptor-independent antimicrobial role for the interferon-inducible CXC chemokines in pulmonary innate immunity in vivo. These data also support an immunomodulatory approach for effectively treating and/or preventing pulmonary B. anthracis infection, as well as infections caused by pathogenic and potentially, multi-drug resistant bacteria including other spore-forming organisms.

Neutropenia enhances lung dendritic cell recruitment in response to Aspergillus via a cytokine-to-chemokine amplification loop.

Current understanding of specific defense mechanisms in the context of neutropenic infections is limited. It has previously been reported that invasive aspergillosis, a prototypic opportunistic infection in neutropenic hosts, is associated with marked accumulation of inflammatory dendritic cells (DCs) in the lungs. Given recent data indicating that neutrophils can modulate immune responses independent of their direct microbial killing, we hypothesized that neutropenia impacts the host response to Aspergillus by determining the migration and phenotype of lung DCs. Inflammatory DCs, but not other DC subsets, were found to accumulate in the lungs of neutropenic hosts challenged with killed or live-attenuated Aspergillus as compared with nonneutropenic hosts, indicating that the accumulation was independent of neutrophil microbicidal activity. The mechanism of this accumulation in neutropenic hosts was found to be augmented influx of DCs, or their precursors, from the blood to the lungs. This effect was attributable to greatly elevated lung TNF expression in neutropenic as compared with nonneutropenic animals. This resulted in greater lung expression of the chemokine ligands CCL2 and CCL20, which, in turn, mediated enhanced recruitment of TNF-producing inflammatory DCs, resulting in a positive feedback cycle. Finally, in the context of neutropenic invasive aspergillosis, depletion of DCs resulted in impaired fungal clearance, indicating that this mechanism is protective for the host. These observations identify what we believe is a novel defense mechanism in invasive aspergillosis that is the result of alterations in DC traffic and phenotype and is specific to neutropenic hosts.

Pilot analysis of the plasma metabolite profiles associated with emphysematous Chronic Obstructive Pulmonary Disease phenotype.

The current pilot study examined the hypothesis that cigarette smokers who developed an emphysematous phenotype of Chronic Obstructive Pulmonary Disease (COPD) were associated with distinctive patterns in their corresponding metabolomics profile as compared to those who did not. Peripheral blood plasma samples were collected from 38 subjects with different phenotypes of COPD. They were categorized into three groups: healthy non-smokers (n=16), smokers without emphysema (n=8), and smokers with emphysema (n=14). Ultra High Performance Liquid Chromatography/quadrupole-Time-of-Flight Mass Spectrometry techniques were used to identify a large number of metabolite markers (3534). Unsupervised clustering analysis accurately separated the smokers with emphysema from others without emphysema and demonstrated potentials of this metabolomics data. Subsequently predictive models were created with a supervised learning set, and these predictive models were found to be highly accurate in identifying the subjects with the emphysematous phenotype of COPD with excellent sensitivity and specificity. Our methodology provides a preliminary model that differentiates an emphysematous COPD phenotype from other COPD phenotypes on the basis of the metabolomics profiles. These results also suggest that the metabolomics profiling could potentially guide the characterization of relevant metabolites that leads to an emphysematous COPD phenotype.

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.