mTORC2 signalling regulates M2 macrophage differentiation in response to helminth infection and adaptive thermogenesis.

Alternatively activated macrophages (M2) have an important function in innate immune responses to parasitic helminths, and emerging evidence also indicates these cells are regulators of systemic metabolism. Here we show a critical role for mTORC2 signalling in the generation of M2 macrophages. Abrogation of mTORC2 signalling in macrophages by selective conditional deletion of the adaptor molecule Rictor inhibits the generation of M2 macrophages while leaving the generation of classically activated macrophages (M1) intact. Selective deletion of Rictor in macrophages prevents M2 differentiation and clearance of a parasitic helminth infection in mice, and also abrogates the ability of mice to regulate brown fat and maintain core body temperature. Our findings define a role for mTORC2 in macrophages in integrating signals from the immune microenvironment to promote innate type 2 immunity, and also to integrate systemic metabolic and thermogenic responses.

Enhanced resolution of experimental ARDS through IL-4-mediated lung macrophage reprogramming.

Despite intense investigation, acute respiratory distress syndrome (ARDS) remains an enormous clinical problem for which no specific therapies currently exist. In this study, we used intratracheal lipopolysaccharide or Pseudomonas bacteria administration to model experimental acute lung injury (ALI) and to further understand mediators of the resolution phase of ARDS. Recent work demonstrates macrophages transition from a predominant proinflammatory M1 phenotype during acute inflammation to an anti-inflammatory M2 phenotype with ALI resolution. We tested the hypothesis that IL-4, a potent inducer of M2-specific protein expression, would accelerate ALI resolution and lung repair through reprogramming of endogenous inflammatory macrophages. In fact, IL-4 treatment was found to offer dramatic benefits following delayed administration to mice subjected to experimental ALI, including increased survival, accelerated resolution of lung injury, and improved lung function. Expression of the M2 proteins Arg1, FIZZ1, and Ym1 was increased in lung tissues following IL-4 treatment, and among macrophages, FIZZ1 was most prominently upregulated in the interstitial subpopulation. A similar trend was observed for the expression of macrophage mannose receptor (MMR) and Dectin-1 on the surface of alveolar macrophages following IL-4 administration. Macrophage depletion or STAT6 deficiency abrogated the therapeutic effect of IL-4. Collectively, these data demonstrate that IL-4-mediated therapeutic macrophage reprogramming can accelerate resolution and lung repair despite delayed use following experimental ALI. IL-4 or other therapies that target late-phase, proresolution pathways may hold promise for the treatment of human ARDS.

Hydroxymethylation as a Novel Environmental Biosensor.

Beyond the genome, epigenetics has become a promising approach in understanding the interactions between the gene and the environment. Epigenetic regulation includes DNA methylation, histone modifications, and non-coding RNAs. Among these, DNA methylation, which is the addition of a methyl group to the fifth base of cytosine to produce 5-methylcytosine (5-mC), is most commonly studied. Epigenetic regulation has changed given the discovery of 5-hydroxymethylcytosine (5-hmC), considered the "sixth base", and the nature of TET proteins to catalyze 5-mC oxidation to 5-hmC. 5-hydroxymethylation has been proposed to be a stable intermediate between methylation and demethylation and has raised questions about the functions of 5-hmC in gene regulation in cells, tissues, and organs in response to environmental exposure. Herein, we have provided an introduction to the chemistry of 5-hydroxymethylation, and the techniques for detection of 5-hydroxymethylation. In addition, we have reviewed current reports describing how 5-hmC responds to environmental factors, leading to the development of disease. And finally, we have discussed the potential use of 5-hmC in the study of disease development. All in all, it is our goal to provide innovative and convincing epigenetic studies for understanding the etiology of environmentally-related human disease, and translate these epigenetic findings into lifestyle recommendations and clinical practices to prevent and cure disease.

Foxp3+ regulatory T cells promote lung epithelial proliferation.

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3(+) regulatory T cells (Foxp3(+) T(reg) cells) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (lipopolysaccharide) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3(+) T(reg) cells in the lung during the course of resolution. To dissect the role that Foxp3(+) T(reg) cells exert on epithelial proliferation, we depleted Foxp3(+) T(reg) cells, which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3(+) T(reg) numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy, we found that Foxp3(+) T(reg) cells enhanced epithelial proliferation. Moreover, Foxp3(+) T(reg) cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3(+) T(reg) cells in repair of the lung epithelium.

Elastase-coupled beads as a tool for characterizing localized alveolar tissue destruction associated with the onset of emphysema.

Intratracheal elastase challenge of laboratory animals has long been established as a model for observing the physiological and morphological changes that result from alveolar destruction, the hallmark of emphysema. However, instillation of elastase suspended in buffer results in widespread inflammation and variable emphysematous lesions, which has made the identification of specific cellular and molecular events associated with the onset of emphysema difficult to define. Here we establish a bead-based elastase delivery system that induces localized tissue destruction, a key event in the initiation of emphysema. Elastase was coupled to bisacrylamide beads, which were shown to retain enzymatic activity prior to intratracheal administration in mice. C57BL/6 mice were given a single dose of 40,000 beads, which became distributed throughout the small airways and parenchyma of the lung. Elastase-coupled beads resulted in a quantifiable loss of alveolar tissue immediately surrounding the beads, an effect that was not observed with beads that lacked protein altogether or with beads containing elastase inactivated by an irreversible inhibitor. Furthermore, beads bound with active elastase elicited local recruitment of mononuclear cells, including macrophages, and polymorphonuclear neutrophils to the site of bead deposition, a feature consistent with the cellular infiltration observed following conventional solubilized elastase challenges. This work identifies a novel bead-based enzyme delivery system that also extends the elastase model of emphysema to permit the characterization of mechanisms that drive alveolar surface area loss following elastin degradation in focal emphysematous lesions.

HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs.

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.

Genetic variability of induction and emergence times for inhalational anaesthetics.

BACKGROUND AND OBJECTIVES: Anaesthetic requirements differ among inbred mouse strains. We tested the genetic influence on induction and arousal times to inhalational anaesthetics in two of these strains. METHODS: Five male C57BL/6J (B6) and five male C3H/HeJ (C3) mice were each exposed to five different concentrations of nitrous oxide (N2O) at five different levels of halothane. Time to sleep and arousal were assessed. Data were analysed by repeated measures of analysis of variance. RESULTS: Halothane, N2O and genetic strain, all were significant independent factors on the time to sleep, while only N2O was a significant independent factor on the time to arousal (P = 0.004). B6 mice took significantly longer to fall asleep compared to the C3 mice controlling for halothane and N2O concentrations (F-ratio = 36, P < 0.0001). The effect of N2O on time to arousal was only significant for the B6 strain (F-ratio = 10, P = 0.005), and not for the C3 strain (F-ratio = 0.8, P = 0.38). CONCLUSIONS: Genetics influences the time to sleep for anaesthetic agents in mice.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Effect of bronchial thermoplasty on airway distensibility.

Recent studies have reported that the application of thermal energy delivered through a bronchoscope (bronchial thermoplasty) impairs the ability of airways to narrow in response to methacholine. How such altered smooth muscle affects the response of airways to lung inflation may have important clinical implications, particularly as it relates to the abnormal response of asthmatic subjects to lung inflation and deep inspiration. The aim of this study was to examine whether bronchial thermoplasty affected airway distension with lung inflation in relaxed and contracted airways. A total of 230 airways were studied, ranging 2.5-15 mm, in six dogs. These airways were divided into two groups: an untreated (control) population and a bronchial thermoplasty-treated population. Prior to treatment, the airway pressure-area curves in the two groups of airways were identical. In contrast, the relaxed and contracted airway pressure-area curves in treated airways were shifted upward at all points, showing increased airway area at both 3 and 5 weeks post-treatment. In conclusion, these results show that reducing that amount of functional smooth muscle with bronchial thermoplasty leads to increased airway size in both relaxed and contracted states over a normal range of inflation pressures.

Radiofrequency ablation of airway smooth muscle for sustained treatment of asthma: preliminary investigations.

Bronchial thermoplasty is a procedure now being tested in humans for the treatment of asthma. Current studies focusing on safety are encouraging. The procedure, which causes extensive ablation of airway smooth muscle (ASM), is well tolerated, and there is a sustained reduction in airway responsiveness to methacholine. Two assumptions underlie the development of this procedure: 1) ASM is a vestigial tissue; and 2) that treatment directed at ASM alone will provide sustained symptomatic and physiological improvement in asthmatic humans. Even if this procedure is efficacious, it must be safe in the long-term. Current studies in animals and humans suggest that this is very likely to be the case. While bronchial thermoplasty may have a broad application, especially for patients who wish for a permanent amelioration of their symptoms or have difficulty adhering to medical regimens, the compelling use of this procedure is for patients who are inadequately controlled on current drug therapy or who cannot adhere to therapeutic regimens. The application of this procedure for the treatment of asthma is currently being considered by regulatory agencies, and study centres are currently disseminated throughout North America and Europe. Within the next 1-2 yrs, a profile of the potential role of this therapy in human asthma should be developed fully.

Influence of volatile anaesthetics on hypercapnoeic ventilatory responses in mice with blunted respiratory drive.

BACKGROUND: Subanaesthetic concentrations of volatile anaesthetics significantly affect the respiratory response to hypoxia and hypercapnoeia. Individuals with an inherited blunted respiratory drive are more affected than normal individuals. To test the hypothesis that subjects with blunted hypercapnoeic respiratory drive are diversely affected by different anaesthetics, we studied the effects of three volatile anaesthetics on the control of breathing in C3H/HeJ (C3) mice, characterized by a blunted hypercapnoeic respiratory response. METHODS: Using whole body plethysmography, we assessed respiratory rate (RR) and pressure amplitude in 11 male C3 mice at rest, during anaesthesia with isoflurane, sevoflurane or desflurane, and during recovery. To test respiratory drive, mice were exposed to 8% carbon dioxide. Data were analysed by two-way-analysis of variance with post hoc tests and Bonferroni correction. RESULTS: RR was unaffected during sevoflurane anaesthesia up to 1.0 MAC. Likewise, sevoflurane at 1.5 MAC affected RR less than either isoflurane (P=0.0014) or desflurane (P=0.0048). The increased RR to a carbon dioxide challenge was blocked by all three anaesthetics even at the lowest concentration, and remained depressed during recovery (P<0.0001). Tidal volume was unaffected by all three anaesthetics. CONCLUSIONS: In C3 mice, spontaneous ventilation was less affected during sevoflurane compared with either isoflurane or desflurane anaesthesia. However, the RR response to hypercapnoeia was abolished at 0.5 MAC for all the anaesthetic agents and remained depressed even at the end of recovery. Our data suggest that different volatile anaesthetics have varying effects on the control of breathing frequency but all block the respiratory response to carbon dioxide. Therefore, a genetic predisposition to a blunted carbon dioxide response represents a susceptibility factor that interacts with hypercapnoeic hypoventilation during maintenance of anaesthesia and in the emergence from anaesthesia, regardless of the agent used.

Heritable differences in respiratory drive and breathing pattern in mice during anaesthesia and emergence.

BACKGROUND: Postanaesthetic hypoxia and ischaemia can lead to postoperative morbidity and mortality. We studied the effect of isoflurane anaesthesia in two inbred mouse strains known for phenotypic differences in breathing pattern and respiratory drive during carbon dioxide challenge and their first-generation offspring (F(1)). METHODS: Using whole body plethysmography, we assessed respiratory rate (RR) and pressure amplitude (Amp) in male B6 (high responder to hypercapnia), C3 (low responder), and F(1) mice at rest, during anaesthesia with isoflurane, and during recovery from anaesthesia. At each time point, the magnitude and pattern of breathing were determined during hypercapnic challenge (FI(CO(2)) = 0.08). Data (mean (SD)) were analysed by generalized ANOVA with post hoc Bonferroni's correction (P<0.05). RESULTS: During isoflurane anaesthesia, strain differences between B6 and C3 mice in RR were obscured while differences in Amp persisted. In contrast to baseline RR responses to carbon dioxide were significantly reduced at 0.5 MAC (increase in RR: 175 (33) bpm, 147 (44) bpm, 127 (33) bpm, for B6, C3, and F(1) strains respectively) and completely blocked at 1.5 MAC (change in RR: -3 (10) bpm, -2 (1) bpm, -4 (5) bpm, for B6, C3, and F(1) strains, respectively). During recovery, B6 mice showed a significant increase in RR (77 (33) bpm; P<0.0001) as well as in Amp. This was not observed in either C3 (-22 (31) bpm) or F(1) mice (23 (51) bpm). CONCLUSION: Isoflurane anaesthesia abolished the strain differences in respiratory drive between B6, C3, and F(1) mice. However, during recovery from anaesthesia, significant strain variation in respiratory drive reappeared and was more pronounced compared with pre-anaesthetic levels. These results suggested, that genetic differences may have minimal contribution to decreased respiratory drive during anaesthesia, but may be a major risk factor for post-operative hypoventilation and the associated morbidity and mortality.

Airway response to deep inspiration: role of nitric oxide.

Deep inspirations (DI) have been shown to have both bronchoprotective and bronchodilator effects in healthy subjects. The bronchodilator effects of a DI appear to be impaired in asthmatics compared with healthy subjects. This study investigated the role of nitric oxide (NO) in the bronchodilator role of a DI. In five anaesthetised and ventilated dogs, high-resolution computed tomography was used to measure the changes in airway size after a small (25 cmH2O) and large (45 cmH2O) DI before and after administering NG-nitro-L-arginine methyl ester to block NO synthesis. The depth of the inspiratory manoeuvre during a deep inspiration determined the subsequent qualitative behaviour of the airway response. Inflation to relatively high pressure resulted in airway dilation, whereas one to lower pressure leads to airway constriction. When NG-nitro-L-arginine methyl ester was administered, both a large and a small deep inspiration resulted in subsequent airway constriction. These results support the idea that nitric oxide may be a potential bronchoprotective agent in the airways.

Genetic regulation of systemic angiogenesis in the mouse lung.

The work in this study takes advantage of a new experimental model in the mouse that completely isolates the angiogenic process from the direct effects of ischemia. The model also leads to lung angiogenesis that mimics the vascular source of many lung pathologies, and allows investigation of the temporal and spatial factors that can promote or inhibit angiogenesis. This work describes the expression patterns of genes relevant to pro-angiogenic signals and conditions in response to ischemia in the lung. The most notable changes were increases in the expression of genes involved in inflammation and tissue remodeling. In particular, the results confirm a important role of ELR+ CXC chemokines as proangiogenic signals. In addition, the experimental findings in this mouse lung model show that lung ischemia, rather than hypoxia, is the essential trigger for angiogenesis. Results from this model also suggest potential approaches for determining critical pathways and potential therapeutic strategies related to the control of angiogenesis.

Airway response to deep inspiration: role of inflation pressure.

Deep inspirations (DIs) have been shown to have both bronchoprotective and bronchodilator effects in healthy subjects; however, the bronchodilator effects of a DI appear to be impaired in asthmatic compared with healthy subjects. Because the ability to generate high transpulmonary pressures at total lung capacity depends on both the lung properties and voluntary effort, we wondered how the response of airways to DI might be altered if the maneuver were done with less than maximal inflation. The present work was undertaken to examine the effects of varying the magnitude of lung inflation during the DI maneuver on subsequent airway caliber. In five anesthetized and ventilated dogs during methacholine infusion, changes in airway size after DIs of increasing magnitude were measured over the subsequent 5-min period using high-resolution computed tomography. Results show that the magnitude of lung inflation is extremely important, leading to a qualitative change in the airway response. A large DI (45 cmH(2)O airway pressure) caused subsequent airway dilation, whereas smaller DIs (< or =35 cmH(2)O) caused bronchoconstriction. The precise mechanism underlying these observations is uncertain, but it seems to be related to an interaction between intrinsic properties of the contracted airway smooth muscle and the response to mild stretch.