Current asthma therapies fail to target airway remodeling that correlates with asthma severity driving disease progression that ultimately leads to loss of lung function. Macroautophagy (hereinafter "autophagy") is a fundamental cell-recycling mechanism in all eukaryotic cells; emerging evidence suggests that it is dysregulated in asthma. We investigated the interrelationship between autophagy and airway remodeling and assessed preclinical efficacy of a known autophagy inhibitor in murine models of asthma. Human asthmatic and nonasthmatic lung tissues were histologically evaluated and were immunostained for key autophagy markers. The percentage area of positive staining was quantified in the epithelium and airway smooth muscle bundles using ImageJ software. Furthermore, the autophagy inhibitor chloroquine was tested intranasally in prophylactic (3 wk) and treatment (5 wk) models of allergic asthma in mice. Human asthmatic tissues showed greater tissue inflammation and demonstrated hallmark features of airway remodeling, displaying thickened epithelium (P , 0.001) and reticular basement membrane (P , 0.0001), greater lamina propria depth (P , 0.005), and increased airway smooth muscle bundles (P , 0.001) with higher expression of Beclin-1 (P , 0.01) and ATG5 (autophagy-related gene 5) (P , 0.05) together with reduced p62 (P , 0.05) compared with nonasthmatic control tissues. Beclin-1 expression was significantly higher in asthmatic epithelium and ciliated cells (P , 0.05), suggesting a potential role of ciliophagy in asthma. Murine asthma models demonstrated effective preclinical efficacy (reduced key features of allergic asthma: airway inflammation, airway hyperresponsiveness, and airway remodeling) of the autophagy inhibitor chloroquine. Our data demonstrate cell context-dependent and selective activation of autophagy in structural cells in asthma. Furthermore, this pathway can be effectively targeted to ameliorate airway remodeling in asthma.

No abstract

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild–moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.

Increased iron levels and dysregulated iron homeostasis, or both, occur in several lung diseases. Here, the effects of iron accumulation on the pathogenesis of pulmonary fibrosis and associated lung function decline was investigated using a combination of murine models of iron overload and bleomycin-induced pulmonary fibrosis, primary human lung fibroblasts treated with iron, and histological samples from patients with or without idiopathic pulmonary fibrosis (IPF). Iron levels are significantly increased in iron overloaded transferrin receptor 2 (Tfr2) mutant mice and homeostatic iron regulator (Hfe) gene-deficient mice and this is associated with increases in airway fibrosis and reduced lung function. Furthermore, fibrosis and lung function decline are associated with pulmonary iron accumulation in bleomycin-induced pulmonary fibrosis. In addition, we show that iron accumulation is increased in lung sections from patients with IPF and that human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels. Significantly, we show that intranasal treatment with the iron chelator, deferoxamine (DFO), from the time when pulmonary iron levels accumulate, prevents airway fibrosis and decline in lung function in experimental pulmonary fibrosis. Pulmonary fibrosis is associated with an increase in Tfr1 + macrophages that display altered phenotype in disease, and DFO treatment modified the abundance of these cells. These experimental and clinical data demonstrate that increased accumulation of pulmonary iron plays a key role in the pathogenesis of pulmonary fibrosis and lung function decline. Furthermore, these data highlight the potential for the therapeutic targeting of increased pulmonary iron in the treatment of fibrotic lung diseases such as IPF.

The expression of the cell recognition molecule F3/Contactin (CNTN1) is generally associated with the functions of post-mitotic neurons. In the embryonic cortex, however, we find it expressed by proliferating ventricular zone (VZ) precursors. In contrast to previous findings in the developing cerebellum, F3/Contactin transgenic overexpression in the early cortical VZ promotes proliferation and expands the precursor pool at the expense of neurogenesis. At later stages, when F3/Contactin levels subside, however, neurogenesis resumes, suggesting that F3/Contactin expression in the VZ is inversely related to neurogenesis and plays a role in a feedback control mechanism, regulating the orderly progression of cortical development. The modified F3/Contactin profile therefore results in delayed corticogenesis, as judged by downregulation in upper and lower layer marker expression and by BrdU birth dating, indicating that, in this transgenic model, increased F3/Contactin levels counteract neuronal precursor commitment. These effects also occur in primary cultures and are reproduced by addition of an F3/Fc fusion protein to wild type cultures. Together, these data indicate a completely novel function for F3/Contactin. Parallel changes in the generation of the Notch Intracellular Domain and in the expression of the Hes-1 transcription factor indicate that activation of the Notch pathway plays a role in this phenotype, consistent with previous in vitro reports that F3/Contactin is a Notch1 ligand.

SUMMARYModulation of the sonic hedgehog (SHH) pathway is a crucial factor in cerebellar morphogenesis. Stimulation of granule neuron progenitor (GNP) proliferation is a central function of SHH signalling, but how this is controlled locally is not understood. We show that two sequentially expressed members of the contactin (CNTN) family of adhesion molecules, TAG1 and F3, act antagonistically to control SHH-induced proliferation: F3 suppresses SHH-induced GNP proliferation and induces differentiation, whereas TAG1 antagonises F3. Production of GNPs in TAG1-null mice is delayed and reduced. F3 and TAG1 colocalise on GNPs with the related L1-like adhesion molecule NrCAM, and F3 fails to suppress the SHH-induced proliferation of NrCAM-deficient GNPs. We show that F3 and SHH both primarily affect a group of intermediate GNPs (IPs), which, though actively dividing, also express molecules associated with differentiation, including -tubulin III (TuJ1) and TAG1. In vivo, intermediate progenitors form a discrete layer in the middle of the external germinal layer (mEGL), while F3 becomes expressed on the axons of postmitotic granule neurons as they leave the inner EGL (iEGL). We propose, therefore, that F3 acts as a localised signal in the iEGL that induces SHH-stimulated cells in the overlying mEGL to exit cell cycle and differentiate. By contrast, expression of TAG1 on GNPs antagonises this signal in the mEGL, preventing premature differentiation and sustaining GNP expansion in a paracrine fashion. Together, these findings indicate that CNTN and L1-like proteins play a significant role in modulating SHH-induced neuronal precursor proliferation.

The p53 gene in neuroblastoma tumors (NB) is rarely mutated but the protein accumulates in the cytoplasm. Because p53 can mediate the cytotoxic effects of chemotherapeutic agents, it is important to determine whether accumulation of p53 in the cytoplasm impairs p53 function. Data presented here indicate that hyperactive nuclear export of p53 suppresses etoposide-induced apoptosis but does not prevent growth arrest. We compared p53 function in a pair of NB subclones. Our data show etoposide induces complete trans-location of p53 to the nucleus and activation of apoptosis in the neuroblastic NB cell line SH-SY5Y (N-type), which expresses low levels of MDM2. However, in Schwann cell-like SH-EP1 cells (S-type), which have up to 10-fold higher levels of MDM2, p53 accumulates in the cytoplasm and the cells are extremely resistant to etoposide-induced apoptosis. Notably, when MDM2 expression is inhibited in S-type cells, with a phosphorothioated antisense oligonucleotide (AS5), then p53 accumulates in the nucleus and the SH-EP1 cells undergo apoptosis. Surprisingly, induction of p21 and G1-arrest are not attenuated in S-type cells, despite the predominantly cytoplasmic location of p53. Whereas, G1-arrest is attenuated in the SH-SY5Y cells, which have high levels of nuclear p53. Taken together, these findings suggest attenuation of G1-arrest is related to the differentiation status of neuroblastomas and occurs downstream of p53 nuclear accumulation. These results demonstrate for the first time that hyperactive nuclear export of p53 attenuates chemotherapy-induced apoptosis in NB cells, and our findings suggest that inhibitors of MDM2 may enhance the therapeutic efficacy of etoposide by promoting apoptosis rather than trans-differentiation.

Short-chain fatty acids (SCFAs), produced as by-products of dietary fiber metabolism by gut bacteria, have anti-inflammatory properties and could potentially be used for the treatment of inflammatory diseases, including asthma. The direct effects of SCFAs on inflammatory responses in primary human lung mesenchymal cells have not been assessed. We investigated whether SCFAs can protect against tumor necrosis factor (TNF)α-induced inflammation in primary human lung fibroblasts (HLFs) and airway smooth muscle (ASM) cells in vitro. HLFs and ASM cells were exposed to SCFAs, acetate (C2:0), propionate (C3:0), and butyrate (C4:0) (0.01–25 mM) with or without TNFα, and the release of proinflammatory cytokines, IL-6, and CXCL8 was measured using ELISA. We found that none of the SCFAs suppressed TNFα-induced cytokine release. On the contrary, challenge with supraphysiological concentrations (10–25 mM), as might be used therapeutically, of propionate or butyrate in combination with TNFα resulted in substantially greater IL-6 and CXCL8 release from HLFs and ASM cells than challenge with TNFα alone, demonstrating synergistic effects. In ASM cells, challenge with acetate also enhanced TNFα-induced IL-6, but not CXCL8 release. Synergistic upregulation of IL-6 and CXCL8 was mediated through the activation of free fatty acid receptor (FFAR)3, but not FFAR2. The signaling pathways involved were further examined using specific inhibitors and immunoblotting, and responses were found to be mediated through p38 MAPK signaling. This study demonstrates that proinflammatory, rather than anti-inflammatory effects of SCFAs are evident in lung mesenchymal cells.