BLTR1 Is Decreased in Steroid Resistant Pro-Inflammatory CD28nullCD8+ T Lymphocytes in Patients with COPD-The Spillover Hypothesis Explained?

INTRODUCTION: Pro-inflammatory CD8+ T cells are increased in the lungs and also in the peripheral circulation of both smokers and chronic obstructive pulmonary disease (COPD) patients. The reason for this is unclear but has been described as a spillover from cells in the lungs that may cause the systemic inflammation noted in COPD. We have recently shown an increase in steroid-resistant CD28nullCD8+ senescent lymphocytes in the lungs and peripheral blood in COPD. Leukotreine B4 (LB4) receptor 1 (BLTR1) is involved in recruitment of CD8+ T cells to sites of inflammation, and we hypothesized that it may be involved in the migration of these senescent lymphocytes from the lungs in COPD. METHODS: Via flow cytometry and Western blot BLTR1, IFNγ, and TNFα expression were measured in peripheral blood, BAL, and large proximal and small distal airway CD28±, CD8± T, and NKT-like cells from COPD patients and healthy control subjects (±prednisolone) following in vitro stimulation. Chemotaxis of leucocyte subsets was determined (±LB4 ± prednisolone). RESULTS: There was an increase in BLTR1-CD28nullCD8+ lymphocytes in the lungs and blood in patients with COPD compared with controls. BLTR1-CD28nullCD8+ T and NKT-like cells produce more IFN/TNF than BLTR+ cells and fail to migrate to LTB4. Treatment with 1 µM prednisolone in vitro resulted in upregulation of BLTR1 expression in pro-inflammatory CD28nullCD8+ cells and migration to LB4. CONCLUSIONS: Loss of BLTR1 is associated with an increased inflammatory potential of CD28nullCD8+ T cells and may allow these pro-inflammatory steroid-resistant cells to migrate to peripheral blood. Treatment strategies that upregulate BLTR1 may reduce systemic inflammation and associated co-morbidity in patients with COPD.

Reduction in Rubicon by cigarette smoke is associated with impaired phagocytosis and occurs through lysosomal degradation pathway.

BACKGROUND: A common feature of COPD is a defective lung macrophage phagocytic capacity that can contribute to chronic lung inflammation and infection. The precise mechanisms remain incompletely understood, although cigarette smoke is a known contributor. We previously showed deficiency of the LC3-associated phagocytosis (LAP) regulator, Rubicon, in macrophages from COPD subjects and in response to cigarette smoke. The current study investigated the molecular basis by which cigarette smoke extract (CSE) reduces Rubicon in THP-1, alveolar and blood monocyte-derived macrophages, and the relationship between Rubicon deficiency and CSE-impaired phagocytosis. METHODOLOGY: Phagocytic capacity of CSE-treated macrophages was measured by flow cytometry, Rubicon expression by Western blot and real time polymerase chain reaction, and autophagic-flux by LC3 and p62 levels. The effect of CSE on Rubicon degradation was determined using cycloheximide inhibition and Rubicon protein synthesis and half-life assessment. RESULTS: Phagocytosis was significantly impaired in CSE-exposed macrophages and strongly correlated with Rubicon expression. CSE-impaired autophagy, accelerated Rubicon degradation, and reduced its half-life. Lysosomal protease inhibitors, but not proteasome inhibitors, attenuated this effect. Autophagy induction did not significantly affect Rubicon expression. CONCLUSIONS: CSE decreases Rubicon through the lysosomal degradation pathway. Rubicon degradation and/or LAP impairment may contribute to dysregulated phagocytosis perpetuated by CSE.

Organotypic sinonasal airway culture systems are predictive of the mucociliary phenotype produced by bronchial airway epithelial cells.

Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff-Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1-32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1-14, converged over days 16-32, and were statistically similar over the entire culture period (global P < 0.001). This agreed with end-point (day 32) measures of tight junction protein abundance which were non-significant for each analyte (P > 0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFß and IL-1ß; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air-liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium.

Inhibition of LC3-associated phagocytosis in COPD and in response to cigarette smoke.

INTRODUCTION/RATIONALE: In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD. METHODS: Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence. RESULTS: Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis. CONCLUSION: LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

A Human Osteocyte Cell Line Model for Studying Staphylococcus aureus Persistence in Osteomyelitis.

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.

LC3-Associated Phagocytosis (LAP): A Potentially Influential Mediator of Efferocytosis-Related Tumor Progression and Aggressiveness.

One aim of cancer therapies is to induce apoptosis of tumor cells. Efficient removal of the apoptotic cells requires coordinated efforts between the processes of efferocytosis and LC3-associated phagocytosis (LAP). However, this activity has also been shown to produce anti-inflammatory and immunosuppressive signals that can be utilized by live tumor cells to evade immune defense mechanisms, resulting in tumor progression and aggressiveness. In the absence of LAP, mice exhibit suppressed tumor growth during efferocytosis, while LAP-sufficient mice show enhanced tumor progression. Little is known about how LAP or its regulators directly affect efferocytosis, tumor growth and treatment responses, and identifying the mechanisms involved has the potential to lead to the discovery of novel approaches to target cancer cells. Also incompletely understood is the direct effect of apoptotic cancer cells on LAP. This is particularly important as induction of apoptosis by current cytotoxic cancer therapies can potentially stimulate LAP following efferocytosis. Herein, we highlight the current understanding of the role of LAP and its relationship with efferocytosis in the tumor microenvironment with a view to presenting novel therapeutic strategies.

COPD-Related Modification to the Airway Epithelium Permits Intracellular Residence of Nontypeable Haemophilus influenzae and May Be Potentiated by Macrolide Arrest of Autophagy.

Introduction: COPD is an inflammatory airway pathology associated with recurrent infection by nontypeable Haemophilus influenzae (NTHi) that is not effectively managed by macrolide antibiotic therapy. We hypothesised that NTHi is able to reside intracellularly within COPD-derived airway epithelial cells (AEC), and that the factors contained in cigarette smoke when coupled with exposure to erythromycin or azithromycin arrest autophagy, the principle mechanism responsible for clearing intracellular bacteria (called "xenophagy"). Methods: Cultures of bronchial airway epithelial cells derived from control and COPD participants were differentiated at an air-liquid interface and exposed to macrolide antibiotics, 10% cigarette smoke-extract (CSE) and NTHi. Markers of autophagic flux and intracellular NTHi were assessed using Western blot analysis and transmission electron microscopy. Results: AEC treated with macrolide antibiotics or 10% CSE exhibited a block in autophagic flux as evidenced by a concomitant increase in LC3-II and Sequestosome abundance (vs control; both P < 0.01). While control AEC showed no clear evidence of intracellular NTHi, COPD-derived cultures exhibited abundant NTHi within the cytoplasm. Further, intracellular NTHi that were encapsulated within vesicles propagated from the apical epithelial layer to the basal cell layer. Discussion: Taken together, our findings indicate that COPD, cigarette smoke and macrolide antibiotics potentiate the susceptibility to persistent intracellular NTHi. A major mechanism for this is arresting normal autophagic flux in airway epithelial cells. Hence, structural modifications that mitigate this off-target effect of macrolides have significant potential to clear intracellular NTHi and thereby reduce the influence of this pathogen in the airways afflicted by COPD.

In vitro characteristics of an airway barrier-disrupting factor secreted by Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a major contributor to the pathophysiology of chronic rhinosinusitis (CRS). Previous research has shown that S. aureus-secreted products disrupt the airway barrier. METHODS: S. aureus ATCC 13565 and 25923 strains were grown at exponential, postexponential, and stationary phases. Microbial conditioned media (CM) was collected from the cultures and ultrafiltered (UF). Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was performed on the UF-CM. UF-CM was subjected to heat and protease treatment, size fractionation, and ultracentrifugation (UC) separation. Human nasal epithelial cells grown at air-liquid interface (HNEC-ALI) cultures were exposed to purified alpha hemolysin (Hla), staphylococcal enterotoxin A (SEA), lipoteichoic acid (LTA), and UF-CM. Barrier function outcomes were measured by transepithelial electrical resistance (TEER) and apparent permeability (Papp). UC fraction exposed cultures were subjected to immunofluorescence microscopy for tight junction (TJ) protein zonula occludens-1 (ZO-1). RESULTS: LC-ESI-MS/MS identified 107 proteins, with Hla being most abundant. Hla, SEA, and LTA did not alter the HNEC-ALI barrier as measured by TEER or Papp. Barrier disruption caused by UF-CM peaked in the postexponential phase, was sensitive to heat and protease treatment, >30-kDa in size, and enriched in the UC fraction. HNEC-ALI exposed to UF-CM and UC demonstrated loss of ZO-1 localization. CONCLUSION: These results suggest that the S. aureus factor responsible for TJ disruption in HNEC-ALI cultures is either a protein-macromolecule or a combination of secreted factors. The product is enriched in the UC fraction, suggesting it is associated with large structures such as membrane components or vesicles.

Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD.

BACKGROUND: Individuals with respiratory disease are being increasingly exposed to wildfire smoke as populations encroach further into forested regions and climate change continues to bring higher temperatures with lower rainfall. Frequent exposures have significant potential to accelerate conditions such as chronic obstructive pulmonary disease (COPD) which is characterised by an exaggerated inflammatory response to environmental stimuli. Here we employ models of human airway epithelium exposed to wildfire smoke-extract (WFSE) to examine modulation in airway epithelial cell (AEC) survival, fragility and barrier function. METHODS: Submerged cultures of small airway epithelial cells (SAEC) and differentiated air-liquid interface (ALI) cultures of primary bronchial AEC (bAEC) were treated for 1-24 h with 1-10% WFSE generated from plant species found in the Australian bushland. Autophagy (LC3-II and Sequestosome), apoptosis (Poly-(ADP)-Ribose Polymerase (PARP) cleavage) and tight junction proteins were measured using western blot. Barrier function was assessed via permeability of fluorescein tracers and measuring trans-epithelial electrical resistance. The production of IL-6 was assessed using ELISA. RESULTS: Primary epithelial models exposed to WFSE exhibited a significant blockade in autophagy as evidenced by an increase in LC3-II coupled with a concomitant elevation in Sequestosome abundance. These exposures also induced significant PARP cleavage indicative of apoptotic changes. ALI cultures of bAEC treated with 5% WFSE demonstrated barrier dysfunction with significant increases in paracellular molecular permeability and ionic conductance, and a reduction in the abundance of the tight junction proteins ZO-1 and Claudin-1. These cultures also exhibited increased IL-6 secretion consistent with the aberrant and pro-inflammatory repair response observed in the COPD airways. Further, blocks in autophagy and barrier disruption were significantly elevated in response to WFSE in comparison to similar exposures with cigarette smoke-extract. CONCLUSION: WFSE inhibits autophagic flux and induces barrier dysfunction in the airway epithelium. As autophagy is a central regulator of cellular repair, viability, and inflammation, targeting the block in autophagic flux may ameliorate the consequences of wildfire smoke-exposure for individuals with pre-existing respiratory conditions.

Bushfire smoke is pro-inflammatory and suppresses macrophage phagocytic function.

Bushfires are increasing in frequency and severity worldwide. Bushfire smoke contains organic/inorganic compounds including aldehydes and acrolein. We described suppressive effects of tobacco smoke on the phagocytic capacity of airway macrophages, linked to secondary necrosis of uncleared apoptotic epithelial cells, persistence of non-typeable H. influenzae (NTHi), and inflammation. We hypothesised that bushfire smoke extract (BFSE) would similarly impair macrophage function. THP-1 or monocyte-derived macrophages (MDM) were exposed to 1-10% BFSE prepared from foliage of 5 common Australian native plants (genus Acacia or Eucalyptus), or 10% cigarette smoke extract (CSE). Phagocytic recognition receptors were measured by flow cytometry; pro-inflammatory cytokines and caspase 1 by immunofluorescence or cytometric bead array; viability by LDH assay; and capsase-3/PARP by western blot. BFSE significantly decreased phagocytosis of apoptotic cells or NTHi by both THP-1 macrophages and MDM vs air control, consistent with the effects of CSE. BFSE significantly decreased MDM expression of CD36, CD44, SR-A1, CD206 and TLR-2 and increased active IL-1ß, caspase-1 and secreted IL-8. BFSE dose-dependently decreased THP-1 macrophage viability (5-fold increase in LDH at 10%) and significantly increased active caspase-3. BFSE impairs macrophage function to a similar extent as CSE, highlighting the need for further research, especially in patients with pre-existing lung disease.

Role of Basal Cells in Producing Persistent Lentivirus-Mediated Airway Gene Expression.

Cystic fibrosis (CF) lung disease is an ideal candidate for a genetic therapy. It has been shown previously that preconditioning with lysophosphatidylcholine (LPC) prior to lentiviral (LV) vector delivery results in long-term in vivo gene expression in the airway epithelium of CF mice. It was hypothesized that this outcome is largely due to transduction of airway basal cells that in turn pass the transgene onto their progeny. The aim of these studies was to confirm if the in vivo delivery of a human immunodeficiency virus type 1 (HIV-1) vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped LV vector following LPC airway conditioning results in transduction of mouse airway basal cells in situ and if the transgene is passed onto their progeny. Additionally, the study sought to determine the efficiency of in vitro transduction of human airway basal cells. First, normal mouse nasal airways were pretreated with LPC prior to delivery of a HIV-1 VSV-G pseudotyped LV vector carrying a LacZ marker gene (LV-LacZ). An epithelial ablation model utilizing polidocanol was then used to demonstrate that clonal outgrowth of linear and spotted clusters of transgene expressing ciliated, basal, and goblet cells occurs following transduction of basal cells. Second, human basal cells were cultured from primary bronchial epithelial cells, with identity confirmed by keratin 5 staining. High levels of transgene expression were found following LV-LacZ transduction. This study demonstrates the ability of the vector delivery protocol to transduce mouse airway basal cells, the LV vector to transduce human basal cells, and the likely role of these cells in maintaining long-term gene expression. These findings support and further develop the potential of LV gene transfer for persistent correction of CF airway disease.

Zinc deficiency as a codeterminant for airway epithelial barrier dysfunction in an ex vivo model of COPD.

There is now convincing evidence that the airway epithelium drives the pathogenesis of COPD. A major aspect of this is the disease-related reduction in barrier function that is potentiated by dysregulation of tight junction (TJ) protein complexes. However, a significant number of studies using in vitro smoke exposure models have not observed alterations in barrier permeability. We have previously shown that zinc (Zn) is an influential cytoprotective factor for the airway epithelium, and its depletion by cigarette smoke produces disease-related modifications consistent with inflammatory changes in COPD. We hypothesized that Zn deficiency is a significant co-stimulus with cigarette smoke extract (CSE) for potentiating the leaky barrier phenotype exhibited in COPD. We employed an ex vivo model of differentiated human airway epithelium exposed to Zn depletion and CSE to determine the contribution of Zn in maintaining normal epithelial permeability. Western blot analysis demonstrated a significant downregulation of the TJ proteins such as ZO-1 (-1.93-fold, P<0.05) and Claudin-1 (-3.37-fold, P<0.01) with the combination exposure. Assessment of barrier function via paracellular ionic conductance and tracer permeability also showed that Zn depletion was an important factor, which potentiated an increase in epithelial permeability (P<0.001 for both) compared to Zn depletion or CSE exposures in isolation. Visual inspection of the epithelium using transmission electron microscopy revealed a marked reduction in junction complexes between the adjacent airway epithelial cells treated with a combination of Zn depletion and CSE. These observations identify Zn deficiency as a significant codeterminant with CSE as a factor leading to an increase in airway epithelial permeability. Hence, as Zn dyshomeostasis has been reported in the airway epithelium exposed to chronic cigarette smoke and inflammation, targeting these phenomena may represent a promising strategy to ameliorate the leaky barrier phenotype that is synonymous with COPD.

Disrupted epithelial/macrophage crosstalk via Spinster homologue 2-mediated S1P signaling may drive defective macrophage phagocytic function in COPD.

INTRODUCTION: We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling. METHODS: Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling. RESULTS: Spns2 expression was significantly increased (p<0.05) in alveolar macrophages from current-smokers/COPD patients (n = 5) compared to healthy nonsmokers (n = 8) and non-smoker lung transplant patients (n = 4). Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p<0.001 in 3 experiments), primary nasal epithelial cells (p<0.01 in 2 experiments), and in smoke-exposed mice (p<0.001, n = 6 animals per group). Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells. CONCLUSION: Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure.

The uncoupling of autophagy and zinc homeostasis in airway epithelial cells as a fundamental contributor to COPD.

The proper regulation of zinc (Zn) trafficking proteins and the cellular distribution of Zn are critical for the maintenance of autophagic processes. However, there have been no studies that have examined Zn dyshomeostasis and the disease-related modulation of autophagy observed in the airways afflicted with chronic obstructive pulmonary disease (COPD). We hypothesized that dysregulated autophagy in airway epithelial cells (AECs) is related to Zn dysregulation in cigarette smoke (CS)-induced COPD. We applied a human ex vivo air-liquid interface model, a murine model of smoke exposure, and human lung tissues and investigated Zn, ZIP1, and ZIP2 Zn-influx proteins, autophagy [microtubule-associated 1A/1B-light chain-3 (LC3), Beclin-1], autophagic flux (Sequestosome), apoptosis [Bcl2; X-linked inhibitor of apoptosis (XIAP), poly (ADP)-ribose polymerase (PARP)], and inflammation [thymic stromal lymphopoietin (TSLP), regulated on activation, normal T cell expressed and secreted (RANTES), and IL-1ß]. Lung tissues from CS-exposed mice exhibit reduced free-Zn in AECs, with elevated ZIP1 and diminished ZIP2 expression. Interestingly, increased LC3 colocalized with ZIP1, suggesting an autophagic requirement for free-Zn to support its catabolic function. In human AECs, autophagy was initiated but was unable to efficiently degrade cellular debris, as evidenced by stable Beclin-1 and increased LC3-II, but with a concomitant elevation in Sequestosome. Autophagic dysfunction due to CS exposure coupled with Zn depletion also induced apoptosis, with the reduction of antiapoptotic and antiautophagic proteins Bcl2 and XIAP and PARP cleavage. This was accompanied by an increase in RANTES and TSLP, an activator of adaptive immunity. We conclude that the uncoupling of Zn trafficking and autophagy in AECs constitutes a fundamental disease-related mechanism for COPD pathogenesis and could provide a new therapeutic target.

Nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases.

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1ß (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1ß was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

BIRC3 single nucleotide polymorphism associate with asthma susceptibility and the abundance of eosinophils and neutrophils.

BACKGROUND AND OBJECTIVE: Aberrant apoptosis is a disease susceptibility mechanism relevant for asthma, whereby fragility of the airway epithelium and enhanced survival of inflammatory cells, contributes to its pathogenesis and prolongation. Cellular Inhibitor of Apoptosis Proteins (cIAP) suppress apoptosis, and participate in the immune response. In this study, single nucleotide polymorphisms (SNP) in the BIRC2 (codes cIAP1) and BIRC3 (cIAP2) genes were evaluated for an association with asthma. METHODS: Caucasian asthmatic (n = 203) and control (n = 198) subjects were selected from participants in the North West Adelaide Health Study. SNPs (n = 9) spanning the consecutively positioned BIRC2 and BIRC3 genes, were selected using a haplotype tagging approach. Alleles and haplotype associations were analysed by logistic regression, assuming an additive genetic model, and adjusted for gender and atopy. RESULTS: The frequency of the minor allele for the BIRC3 SNP rs3460 was significantly lower in asthmatics compared to the control cases (P = 0.046). BIRC3 SNPs rs7928663 and rs7127583 associated with a reduction in eosinophil and neutrophil abundance when assessed across the study population (multivariate P values = 0.002, and 0.005, respectively). Further, the frequency of a haplotype tagged by rs3460, rs7928663 and rs7127583 was reduced in the asthma sub group (P = 0.05), while the presence of the major allele for rs7928663 associated with an increased load of circulating eosinophils and neutrophils (multivariate P value = 0.001). CONCLUSIONS: Polymorphisms in the BIRC3 gene, but not BIRC2, are associated with a protective effect with regards to asthma susceptibility, and a reduced load of inflammatory cells.

Steroid resistance in COPD is associated with impaired molecular chaperone Hsp90 expression by pro-inflammatory lymphocytes.

BACKGROUND: Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. METHODS: Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 µM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. RESULTS: A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 µM prednisolone and 2.5 ng/mL cyclosporine A. CONCLUSIONS: Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.

Pro-phagocytic Effects of Thymoquinone on Cigarette Smoke-exposed Macrophages Occur by Modulation of the Sphingosine-1-phosphate Signalling System.

Oxidative stress, inflammation, increased bronchial epithelial cell apoptosis, and deficient phagocytic clearance of these cells (efferocytosis) by the alveolar macrophages are present in chronic obstructive pulmonary disease (COPD) and in response to cigarette smoke. We previously showed that the macrophage dysfunction is associated with changes to the sphingosine-1-phosphate (S1P) signalling system. We hypothesized that the antioxidant/anti-inflammatory agent, thymoquinone, would improve macrophage phagocytosis via modulation of the S1P system and protect bronchial epithelial cells from cigarette smoke or lipopolysaccharide (LPS)-induced apoptosis. Phagocytosis was assessed using flow cytometry, S1P mediators by Real-Time PCR, and apoptosis of 16HBE bronchial epithelial cells using flow cytometry and immunohistochemistry. Cigarette smoke and LPS decreased phagocytosis and increased S1P receptor (S1PR)-5 mRNA in THP-1 macrophages. Thymoquinone enhanced efferocytic/phagocytic ability, antagonized the effects of cigarette smoke extract and LPS on phagocytosis and S1PR5, and protected bronchial epithelial cells from cigarette smoke-induced apoptosis. Thymoquinone is worth further investigating as a potential therapeutic strategy for smoking-related lung diseases.

Cigarette smoke inhibits efferocytosis via deregulation of sphingosine kinase signaling: reversal with exogenous S1P and the S1P analogue FTY720.

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease.