Effects of Daytime Dry Fasting on Hydration, Glucose Metabolism and Circadian Phase: A Prospective Exploratory Cohort Study in Bahá'í Volunteers.

Background: Religiously motivated Bahá'í fasting (BF) is a form of intermittent dry fasting celebrated by abstaining from food and drinks during daylight hours every year in March for 19 consecutive days. Aim: To test the safety and effects of BF on hydration, metabolism, and the circadian clock. Methods: Thirty-four healthy Bahá'í volunteers (15 women) participated in this prospective, exploratory cohort study. Laboratory examinations were carried out in four study visits: before fasting (V0), in the third week of fasting (V1) as well as 3 weeks (V3) and 3 months (V4) after fasting. Data collection included blood and urine samples, anthropometric measurements and bioelectrical impedance analysis. At V0 and V1, 24- and 12-hour urine and serum osmolality were measured. At V0-V2, alterations in the circadian clock phase were monitored in 16 participants. Our study was augmented by an additional survey with 144 healthy Bahá'í volunteers filling out questionnaires and with subgroups attending metabolic measurements (n = 11) and qualitative interviews (n = 13), the results of which will be published separately. Results: Exploratory data analysis revealed that serum osmolality (n = 34, p < 0.001) and 24-hour urine osmolality (n = 34, p = 0.003) decreased during daytime fasting but remained largely within the physiological range and returned to pre-fasting levels during night hours. BMI (body mass index), total body fat mass, and resting metabolic rate decreased during fasting (n = 34, p < 0.001), while body cell mass and body water appeared unchanged. The circadian phase estimated by transcript biomarkers of blood monocytes advanced by 1.1 h (n = 16, p < 0.005) during fasting and returned to pre-fasting values 3 weeks after fasting. Most observed changes were not detectable anymore 3 months after fasting. Conclusions: Results indicate that BF (Bahá'í fasting) is safe, has no negative effects on hydration, can improve fat metabolism and can cause transient phase shifts of circadian rhythms. Trial Registration:https://www.clinicaltrials.gov/, identifier: NCT03443739.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Circadian rhythms in septic shock patients.

BACKGROUND: Despite the intensive efforts to improve the diagnosis and therapy of sepsis over the last decade, the mortality of septic shock remains high and causes substantial socioeconomical burden of disease. The function of immune cells is time-of-day-dependent and is regulated by several circadian clock genes. This study aims to investigate whether the rhythmicity of clock gene expression is altered in patients with septic shock. METHODS: This prospective pilot study was performed at the university hospital Charité-Universitätsmedizin Berlin, Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK). We included 20 patients with septic shock between May 2014 and January 2018, from whom blood was drawn every 4 h over a 24-h period to isolate CD14-positive monocytes and to measure the expression of 17 clock and clock-associated genes. Of these patients, 3 whose samples expressed fewer than 8 clock genes were excluded from the final analysis. A rhythmicity score SP was calculated, which comprises values between -1 (arrhythmic) and 1 (rhythmic), and expression data were compared to data of a healthy study population additionally. RESULTS: 77% of the measured clock genes showed inconclusive rhythms, i.e., neither rhythmic nor arrhythmic. The clock genes NR1D1, NR1D2 and CRY2 were the most rhythmic, while CLOCK and ARNTL were the least rhythmic. Overall, the rhythmicity scores for septic shock patients were significantly (p < 0.0001) lower (0.23 ± 0.26) compared to the control group (12 healthy young men, 0.70 ± 0.18). In addition, the expression of clock genes CRY1, NR1D1, NR1D2, DBP, and PER2 was suppressed in septic shock patients and CRY2 was significantly upregulated compared to controls. CONCLUSION: Molecular rhythms in immune cells of septic shock patients were substantially altered and decreased compared to healthy young men. The decrease in rhythmicity was clock gene-dependent. The loss of rhythmicity and down-regulation of clock gene expression might be caused by sepsis and might further deteriorate immune responses and organ injury, but further studies are necessary to understand underlying pathophysiological mechanisms. Trail registration Clinical trial registered with www.ClinicalTrials.gov (NCT02044575) on 24 January 2014.

A grid-cell based fecal sampling scheme reveals: land-use and altitude affect prevalence rates of Angiostrongylus vasorum and other parasites of red foxes (Vulpes vulpes).

In view of the role of foxes as a reservoir for Angiostrongylus vasorum, a nematode of the heart and lungs of dogs and foxes, its occurrence across Switzerland was investigated in foxes applying a standardized sampling scheme for fox fecal samples. In 72 study areas, which consisted of three 1-km2 grid cells, a total of 1481 samples were collected by walking transects following linear features in the terrain, and analyzed by a flotation-sieving method. The overall prevalence rate of A. vasorum in fecal samples was 8.8% (95% confidence interval, CI 7.4-10.3%), being significantly higher in the Swiss Plateau (11.4%, CI. 9.4-13.8%) compared with other bioregions, and more prevalent in areas with less than 50% of cultivated land (p = 0.043). Prevalence rates increased with decreasing altitudes being significantly higher below 400 m above sea level (20.6%, 95% CI 15.2-26.9%), while all samples collected above 900 m asl were negative. Eggs of Toxocara sp. (12.1%), Taeniidae (10.5%), Capillaria spp. (8.3%), Trichuris vulpis (5.5%), hookworms (5.3%), Toxascaris leonina (1.3%) and Strongyloides sp. (0.4%) were furthermore identified. Taeniid eggs were positively and Capillaria spp. negatively associated with the amount of cultivated land. The prevalence rates based on our fecal analyses were generally lower compared with previous studies from Switzerland which were based on fox necropsies. However, the grid cell-based sampling scheme with replicable transects for fox feces collection proved to be a suitable, non-invasive method for parasitic large-scale surveys providing information on the relative infectious pressure for the local dog population.

High-accuracy determination of internal circadian time from a single blood sample.

BACKGROUND: The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing. METHODS: To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes. RESULTS: We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost. CONCLUSION: The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock. FUNDING: This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

Principles for circadian orchestration of metabolic pathways.

Circadian rhythms govern multiple aspects of animal metabolism. Transcriptome-, proteome- and metabolome-wide measurements have revealed widespread circadian rhythms in metabolism governed by a cellular genetic oscillator, the circadian core clock. However, it remains unclear if and under which conditions transcriptional rhythms cause rhythms in particular metabolites and metabolic fluxes. Here, we analyzed the circadian orchestration of metabolic pathways by direct measurement of enzyme activities, analysis of transcriptome data, and developing a theoretical method called circadian response analysis. Contrary to a common assumption, we found that pronounced rhythms in metabolic pathways are often favored by separation rather than alignment in the times of peak activity of key enzymes. This property holds true for a set of metabolic pathway motifs (e.g., linear chains and branching points) and also under the conditions of fast kinetics typical for metabolic reactions. By circadian response analysis of pathway motifs, we determined exact timing separation constraints on rhythmic enzyme activities that allow for substantial rhythms in pathway flux and metabolite concentrations. Direct measurements of circadian enzyme activities in mouse skeletal muscle confirmed that such timing separation occurs in vivo.

Dictyostelium discoideum as a Novel Host System to Study the Interaction between Phagocytes and Yeasts.

The social amoeba Dictyostelium discoideum is a well-established model organism to study the interaction between bacteria and phagocytes. In contrast, research using D. discoideum as a host model for fungi is rare. We describe a comprehensive study, which uses D. discoideum as a host model system to investigate the interaction with apathogenic (Saccharomyces cerevisiae) and pathogenic (Candida sp.) yeast. We show that Dictyostelium can be co-cultivated with yeasts on solid media, offering a convenient test to study the interaction between fungi and phagocytes. We demonstrate that a number of D. discoideum mutants increase (atg1-, kil1-, kil2-) or decrease (atg6-) the ability of the amoebae to predate yeast cells. On the yeast side, growth characteristics, reduced phagocytosis rate, as well as known virulence factors of C. albicans (EFG1, CPH1, HGC1, ICL1) contribute to the resistance of yeast cells against predation by the amoebae. Investigating haploid C. albicans strains, we suggest using the amoebae plate test for screening purposes after random mutagenesis. Finally, we discuss the potential of our adapted amoebae plate test to use D. discoideum for risk assessment of yeast strains.

Dynamics of the circadian clock protein PERIOD2 in living cells.

In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclear-receptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.

Keratoplasty in patients with intellectual disability.

PURPOSE: The aim was to report the outcome of keratoplasty in patients with intellectual disability (ID). METHODS: All patients with ID who had undergone keratoplasty at the Eye Department, Red Cross Hospital, Munich, between January 1, 1991, and December 31, 2010, were identified and recalled for examination. In cases of missing data, the information contained in the patient's chart was analyzed and/or his ophthalmologist was contacted. RESULTS: A total of 38 eyes (29 patients) that had undergone 53 keratoplasty procedures were included in the analysis. In 20 patients, the general diagnosis was Down syndrome, 1 patient had ID as a result of brain tumor excision, and 8 patients had ID of an unknown origin. The indication for the primary keratoplasty (n = 35) was keratoconus in 33 patients and corneal opacity because of exposure in facial nerve palsy in 2 patients. In the 3 remaining patients, the first procedure at the study institution was a repeat keratoplasty. The median follow-up was 9 years (minimum 6 months, maximum 20 years). The graft survival rate of the primary graft was 86% (n = 30/35) from 27 months to 11 years. At the last follow-up visit, a clear graft was present in 89% of all the patients (n = 34/38). One eye underwent enucleation, and 3 other eyes remained with a cloudy graft after multiple interventions for rejection/corneal ulceration. CONCLUSIONS: The majority of the patients with ID clearly benefited from keratoplasty, although a few deleterious courses occurred. Keratoplasty in these individuals can be considered whenever patients can no longer comply with the visual demands of their particular individual life.

Esthetic evaluation of single-tooth implants in the anterior mandible.

OBJECTIVES: Single-tooth replacement of anterior mandibular teeth is frequently complicated by insufficient bucco-lingual bone width and limited mesio-distal space available for implant placement. The aim of the present study was to assess implant esthetics in the partially edentulous anterior mandible. MATERIAL AND METHODS: Esthetic evaluation of 43 anterior mandibular single-tooth implants in 15 women and 28 men was performed using esthetic indices (PES = Pink Esthetic Score, PI = Papilla Index, SES = Subjective Esthetic Score) as well as subjective patients' Visual Analogue Scale (VAS) ratings. Clinical and radiological parameters (implant and crown dimensions, pocket depth, bleeding on probing, plaque, keratinized mucosa, marginal bone level, and distance to adjacent teeth) were tested for influence. RESULTS: Implant esthetics were judged satisfactory (PES ≤10) in 42% of implants compared with a patient satisfaction rate of 87%. Correlation between objective indices (PES/PI: rs  = 0.62, PES/SES: rs  = -0.73, PI/SES: rs  = -0.48) was highly significant (P ≤ 0.001); however, no association to subjective patients' ratings could be observed. Type of prosthetic restoration (single crown vs. tulip-shaped double crowns), mesio-distal crown width as well as anatomic crown length significantly affected esthetic scores. Patients' judgment, by contrast, could not be associated to any prognostic factor. CONCLUSION: Subjective patient satisfaction with implant esthetics in the partially edentulous anterior mandible is high, however, remains hard to predict or objectively quantify.

The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease.

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like ßENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from ßENaC-Tg mice compared to wild-type littermates. In both CF patients and ßENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.

Chitin modulates innate immune responses of keratinocytes.

BACKGROUND: Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR) TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. CONCLUSIONS/SIGNIFICANCE: We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined.

Role of breast regression protein-39 in the pathogenesis of cigarette smoke-induced inflammation and emphysema.

The exaggerated expression of chitinase-like protein YKL-40, the human homologue of breast regression protein-39 (BRP-39), was reported in a number of diseases, including chronic obstructive pulmonary disease (COPD). However, the in vivo roles of YKL-40 in normal physiology or in the pathogenesis of specific diseases such as COPD remain poorly understood. We hypothesized that BRP-39/YKL-40 plays an important role in the pathogenesis of cigarette smoke (CS)-induced emphysema. To test this hypothesis, 10-week-old wild-type and BRP-39 null mutant mice (BRP-39(-/-)) were exposed to room air (RA) and CS for up to 10 months. The expression of BRP-39 was significantly induced in macrophages, airway epithelial cells, and alveolar Type II cells in the lungs of CS-exposed mice compared with RA-exposed mice, at least in part via an IL-18 signaling-dependent pathway. The null mutation of BRP-39 significantly reduced CS-induced bronchoalveolar lavage and tissue inflammation. However, CS-induced epithelial cell apoptosis and alveolar destruction were further enhanced in the absence of BRP-39. Consistent with these findings in mice, the tissue expression of YKL-40 was significantly increased in the lungs of current smokers compared with the lungs of ex-smokers or nonsmokers. In addition, serum concentrations of YKL-40 were significantly higher in smokers with COPD than in nonsmokers or smokers without COPD. These studies demonstrate a novel regulatory role of BRP-39/YKL-40 in CS-induced inflammation and emphysematous destruction. These studies also underscore that maintaining physiologic concentrations of YKL-40 in the lung is therapeutically important in preventing excessive inflammatory responses or emphysematous alveolar destruction.

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation.

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases.

BACKGROUND: Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases. METHODS: sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression. RESULTS: sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition. CONCLUSIONS: This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis.

Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39(-/-) mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39(-/-) animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.

Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63(+) granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation.

Acidic mammalian chitinase is secreted via an ADAM17/epidermal growth factor receptor-dependent pathway and stimulates chemokine production by pulmonary epithelial cells.

Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease.

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.