Respiration supports intraphagosomal filamentation and escape of Candida albicans from macrophages.

IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets.

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal α1 -helix for lysosomal swelling and successful intoxication.

The spectrin cytoskeleton integrates endothelial mechanoresponses.

Physiological blood flow induces the secretion of vasoactive compounds, notably nitric oxide, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. Here, we demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrins modulate the cell surface density of hyaluronan and sense and translate shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive PIEZO1 channels are thought to reside. Accordingly, shear-induced PIEZO1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced endothelial nitric oxide synthase stimulation were similarly dependent on spectrin. We conclude that the apical spectrin network is not only required for shear sensing but also transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK.

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Bridging the Gap Between RCTs and RWE Through Endpoint Selection.

This commentary is authored by several industry real-world evidence (RWE) experts, with support from IQVIA, as part of the 'RWE Leadership Forum': a group of Industry Leaders who have come together as non-competitive partners to understand and respond to RWD/E challenges and opportunities with a single expert voice. Here, the forum discusses the value in bridging the industry disconnect between RTCs and RWE, with a view to promoting the use of RWE in the RCT environment. RCT endpoints are explored along several axes including their clinical relevance and their measure of direct patient benefit, and then compared with their real-world counterparts to identify suitable paths, or gaps, for assimilating RWE endpoints into the RCT environment.

Mapping the electrostatic profiles of cellular membranes.

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately -35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Inactivation of Rho GTPases by Burkholderia cenocepacia Induces a WASH-Mediated Actin Polymerization that Delays Phagosome Maturation.

Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.

Understanding the challenge of comparative effectiveness research in focal epilepsy: A review of network meta-analyses and real-world evidence on antiepileptic drugs.

OBJECTIVE: Head-to-head randomized controlled trials (RCTs) are the gold standard for assessing comparative treatment effects. In the absence of direct comparisons between all possible antiepileptic drugs (AEDs), however, clinical decision-making in focal (partial onset) epilepsy relies on alternative evidence borne from indirect comparisons including network meta-analyses (NMAs) and from real-world evidence (RWE) studies. We review NMAs and observational RWE studies comparing AEDs in the adjunctive setting to compare the robustness of these methods and to formulate recommendations for future evidence development. METHODS: A literature review identified NMAs and RWE studies comparing AEDs for the adjunctive treatment of focal seizures published between January 2008 and October 2018. NMAs were evaluated for robustness using a framework based on guidelines from the National Institute for Health and Care Excellence Decision Support Unit and the International Society for Pharmacoeconomics and Outcomes Research. RWE studies were evaluated using the GRACE checklist. RESULTS: From a total of 1993 records, 11 NMAs and six RWE studies were eligible. Key limitations identified in the NMAs include nonsystematic selection of RCTs, unexplored heterogeneity between included RCTs in terms of study and patient characteristics, and selection of AEDs and AED doses or dosing strategies that are not reflective of clinical practice. The main limitations of RWE studies concern sample size, design, and analysis methods. Approximately 90% of comparisons between individual AEDs were nonsignificant in the NMAs. None of the RWE studies adjusted for baseline differences between comparator groups; therefore, they lack the validity to make comparative conclusions. SIGNIFICANCE: Current NMAs and RWE studies provide only nominal comparative evidence for AED treatments in focal epilepsy, and should be used with caution for decision-making due to their methodological limitations. To overcome these hurdles, adherence to methodological guidelines and concerted efforts to collect relevant outcome data in the real world are needed.

Strengthening pharma's contract with society: the value of trusted partnerships between pharma and healthcare facilitated by real-world data.

This White Paper is authored by 11 industry real-world evidence (RWE) experts, with support from IQVIA, as part of the 'RWE Leadership Forum': a group of industry leaders who come together as noncompetitive partners to understand and respond to internal or external RWD/E challenges and opportunities with a single expert voice. Herein we aim to clarify the rules of engagement between pharma and healthcare in order to establish trust-based partnerships, which will unlock unique value for society, including the medical community and the ultimate beneficiary, the patient.

P38 MAPK and glucocorticoid receptor crosstalk in bronchial epithelial cells.

p38 MAPK inhibition may have additive and synergistic anti-inflammatory effects when used with corticosteroids. We investigated crosstalk between p38 MAPK inhibitors and corticosteroids in bronchial epithelial cells to investigate synergistic effects on cytokine production and the molecular mechanisms involved. Effects of the p38 MAPK inhibitor BIRB-796 and dexamethasone alone and in combination on LPS, polyI:C or TNFα -induced IL-6, CXCL8 and RANTES were assessed in 16HBEs (human epithelial cell line) and on TNFα-induced IL-6 and CXCL8 in primary human epithelial cells from asthma patients and healthy controls. 16HBEs were used to assess effects of BIRB-796 alone and in combination with dexamethasone on glucocorticoid receptor (GR) activity by reporter gene assay, expression of GR target genes and nuclear localisation using Western blot. The effects of BIRB-796 on TNFα stimulated phosphorylation of p38 MAPK and GR at serine (S) 226 by Western blot. Epithelial levels of phosphorylated p38 MAPK and GR S226 were determined by immunohistochemistry in bronchial biopsies from asthma patients and healthy controls. BIRB-796 in combination with dexamethasone increased inhibition of cytokine production in a synergistic manner. Combination treatment significantly increased GR nuclear localisation compared to dexamethasone alone. BIRB-796 inhibited TNFα-induced p38 MAPK and GR S226 phosphorylation. Phosphorylated GR S226 and p38 MAPK levels were increased in bronchial epithelium of more severe asthma patients. Molecular crosstalk exists between p38 MAPK activation and GR function in human bronchial epithelial cells, which alters GR activity. Combining a p38 MAPK inhibitor and a corticosteroid may demonstrate therapeutic potential in severe asthma. KEY MESSAGES: • Combination of corticosteroid and p38 inhibitor in human bronchial epithelial cells • Combination increased cytokine inhibition synergistically and nuclear GR • p38 MAPK inhibition reduced TNFα-induced phosphorylation of GR at S226 but not S211 • Phosphorylated GRS226 and p38 is increased in bronchial epithelium in severe asthma • Combining a p38 inhibitor and a corticosteroid may be effective in asthma treatment.

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.

Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl- and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes.

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae.

Can Matching-Adjusted Indirect Comparison Methods Mitigate Placebo Response Differences Among Patient Populations in Adjunctive Trials of Brivaracetam and Levetiracetam?

BACKGROUND AND OBJECTIVE: Patients with focal seizures recruited into adjunctive antiepileptic drug (AED) trials have become more refractory and severe over time; concurrently, placebo responses have increased. To attempt to account for heterogeneity among trials, propensity-score weighted patient-level data were used to indirectly compare placebo responses reported in brivaracetam and levetiracetam trials. METHODS: Patient-level data from randomised, placebo-controlled brivaracetam (recruited 2007-2014) and levetiracetam (1993-1998) trials were pooled. Consistent inclusion/exclusion criteria were applied and outcomes were defined consistently. Potentially confounding baseline characteristics were adjusted for using propensity score weighting. Weighting success was assessed using placebo response. RESULTS: In total, 707 and 473 active drug and 399 and 253 placebo patients comprised the brivaracetam and levetiracetam groups, respectively. Before weighting, several baseline variables were significantly different between groups; after weighting, prior vagal nerve stimulation, co-morbid depression and co-morbid anxiety remained different. Before weighting, median seizure frequency reduction was 21.7 and 3.9% in the brivaracetam and levetiracetam placebo arms, respectively; after weighting, median reduction was 15.0 and 6.0%. The comparison of non-randomised groups could be biased by unobserved confounding factors and region of residence. Lifetime AED history was unavailable in the brivaracetam trials and excluded from analysis. CONCLUSIONS: Placebo responses remained different between brivaracetam and levetiracetam trials after propensity score weighting, indicating the presence of residual confounding factors associated with placebo response in these trials. It therefore remains problematic to conduct reliable indirect comparisons of brivaracetam and levetiracetam given the current evidence base, which may apply to comparisons between other AED trials.

COPD monocytes demonstrate impaired migratory ability.

BACKGROUND: Increased lung macrophage numbers in COPD may arise from upregulation of blood monocyte recruitment into the lungs. CCR5 is a monocyte chemokine receptor regulated by interleukin-6 (IL-6); the concentration of CCR5 ligands are known to be elevated in COPD lungs. The objective of this study was to investigate mechanisms of monocyte recruitment to the lung in COPD, including the role of CCR5 signalling. METHODS: Ninety one COPD patients, 29 smokers (S) and 37 non-smokers (NS) underwent sputum induction, plasma sampling (to measure IL-6 and soluble IL-6 receptor [sIL-6R] by immunoassay), monocyte characterization (by flow cytometry) and monocyte isolation for cell migration and quantitative polymerase chain reaction studies. Lung tissue was used for immunohistochemistry. RESULTS: Plasma IL-6 and sIL-6R levels were increased in COPD. Greater proportions of COPD CD14++CD16+ monocytes expressed CCR5 compared to controls. Monocyte stimulation with IL-6 and sIL-6R increased CCR5 gene expression. COPD monocytes demonstrated impaired migration towards sputum supernatant compared to NS (% migration, 4.4 vs 11.5, respectively; p < 0.05). Pulmonary microvessels showed reduced monocyte recruitment (% marginated cells) in COPD compared to NS, (9.3% vs 83.1%, respectively). The proportion of replicating Ki67+ alveolar macrophages was reduced in COPD compared to NS. All alveolar macrophages from COPD and S expressed the anti-apoptosis marker BCL2; this protein was not present in non-smokers or COPD ex-smokers. CONCLUSION: COPD monocytes show decreased migratory ability despite increased CCR5 expression. Increased COPD lung macrophage numbers may be due to delayed apoptosis.

In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.

Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (Kp,cell) of all drugs (5 µM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH4Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro Kp,cell ranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in Kp,cell was observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean Kp,cell of clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH4Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro Kp,cell obtained in the presence of NH4Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine Kp,cell observed in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.

Anti-inflammatory potential of PI3Kδ and JAK inhibitors in asthma patients.

BACKGROUND: Phosphatidylinositol 3-kinase delta (PI3Kδ) and Janus-activated kinases (JAK) are both novel anti-inflammatory targets in asthma that affect lymphocyte activation. We have investigated the anti-inflammatory effects of PI3Kδ and JAK inhibition on cytokine release from asthma bronchoalveolar lavage (BAL) cells and T-cell activation, and measured lung PI3Kδ and JAK signalling pathway expression. METHOD: Cells isolated from asthma patients and healthy subjects were treated with PI3Kδ or JAK inhibitors, and/or dexamethasone, before T-cell receptor stimulation. Levels of IFNγ, IL-13 and IL-17 were measured by ELISA and flow cytometry was used to assess T-cell activation. PI3Kδ, PI3Kγ, phosphorylated protein kinase B (pAKT) and Signal Transducer and Activator of Transcription (STAT) protein expression were assessed by immunohistochemistry in bronchial biopsy tissue from asthma patients and healthy subjects. PI3Kδ expression in BAL CD3 cells was measured by flow cytometry. RESULTS: JAK and PI3Kδ inhibitors reduced cytokine levels from both asthma and healthy BAL cells. Combining dexamethasone with either a JAK or PI3Kδ inhibitor showed an additive anti-inflammatory effect. JAK and PI3Kδ inhibitors were shown to have direct effects on T-cell activation. Immunohistochemistry showed increased numbers of PI3Kδ expressing cells in asthma bronchial tissue compared to controls. Asthma CD3 cells in BAL expressed higher levels of PI3Kδ protein compared to healthy cells. CONCLUSIONS: Targeting PI3Kδ or JAK may prove effective in reducing T-cell activation and the resulting cytokine production in asthma.

Neutral sphingomyelinase-2, acid sphingomyelinase, and ceramide levels in COPD patients compared to controls.

BACKGROUND: Increased pulmonary ceramide levels are suggested to play a causative role in lung diseases including COPD. Neutral sphingomyelinase-2 (nSMase-2) and acid SMase (aSMase), which hydrolyze sphingomyelin to produce ceramide, are activated by a range of cellular stresses, including inflammatory cytokines and pathogens, but notably cigarette smoke appears to only activate nSMase-2. Our primary objective was to investigate nSMase-2 and aSMase protein localization and quantification in lung tissue from nonsmokers (NS), smokers (S), and COPD patients. In addition, various ceramide species (C16, C18, and C20) were measured in alveolar macrophages from COPD patients versus controls. MATERIALS AND METHODS: Patients undergoing surgical resection for suspected or confirmed lung cancer were recruited, and nSMase-2 and aSMase protein was investigated in different areas of lung tissue (small airways, alveolar walls, subepithelium, and alveolar macrophages) by immunohistochemistry. Ceramide species were measured in alveolar macrophages from COPD patients and controls by mass spectrometry. RESULTS: nSMase-2 and aSMase were detected in the majority of small airways. There was a significant increase in nSMase-2 immunoreactivity in alveolar macrophages from COPD patients (54%) compared with NS (31.7%) (P<0.05), and in aSMase immunoreactivity in COPD (68.2%) and S (69.5%) alveolar macrophages compared with NS (52.4%) (P<0.05). aSMase labeling was also increased in the subepithelium and alveolar walls of S compared with NS. Ceramide (C20) was significantly increased in alveolar macrophages from COPD patients compared with controls. CONCLUSION: nSMase-2 and aSMase are both increased in COPD alveolar macrophages at the protein level; this may contribute toward the elevated ceramide (C20) detected in alveolar macrophages from COPD patients.

Additive anti-inflammatory effects of corticosteroids and phosphodiesterase-4 inhibitors in COPD CD8 cells.

BACKGROUND: CD8 lymphocytes play an important role in the pathogenesis of COPD. Corticosteroids and phosphodiesterase 4 (PDE4) inhibitors are anti-inflammatory drugs used for COPD treatment. Little is known of the combined effect of these drugs on COPD CD8 cells. We studied the effect of corticosteroid combined with PDE4 inhibitors on cytokine release form circulating and pulmonary CD8 cells, and on glucocorticoid (GR) nuclear translocation. METHODS: The effect of dexamethasone alone and in combination with the PDE4 inhibitors roflumilast and GSK256066 on cytokine release from circulating and pulmonary CD8 cells was measured. The effect of the compounds on nuclear translocation of GR and cyclic AMP-responsive element-binding protein (CREB) was studied using immunofluorescence. RESULTS: Dexamethasone inhibited cytokine release from COPD CD8 cells in a concentration dependent manner. PDE4 inhibitors enhanced this anti-inflammatory effect in an additive manner. PDE4 inhibitors did not increase corticosteroid induced GR nuclear translocation. PDE4 inhibitors, but not corticosteroid, increased phospho-CREB nuclear translocation. CONCLUSION: The combination of corticosteroids and PDE4 inhibitors results in an additive anti-inflammatory effect in COPD CD8 cells. This enhanced anti-inflammatory effect could translate to important clinical benefits for patients with COPD.