Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma.

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

Patient stratification and the unmet need in asthma.

Asthma is often described as an inflammatory disease of the lungs and in most patients symptomatic treatment with bronchodilators or inhaled corticosteroids is sufficient to control disease. Unfortunately there are a proportion of patients who fail to achieve control despite treatment with the best current treatment. These severe asthma patients have been considered a homogeneous group of patients that represent the unmet therapeutic need in asthma. Many novel therapies have been tested in unselected asthma patients and the effects have often been disappointing, particularly for the highly specific monoclonal antibody-based drugs such as anti-IL-13 and anti-IL-5. More recently, it has become clear that asthma is a syndrome with many different disease drivers. Clinical trials of anti-IL-13 and anti-IL-5 have focused on biomarker-defined patient groups and these trials have driven the clinical progression of these drugs. Work on asthma phenotyping indicates that there is a group of asthma patients where T helper cell type 2 (Th2) cytokines and inflammation predominate and these type 2 high (T2-high) patients can be defined by biomarkers and response to therapies targeting this type of immunity, including anti-IL-5 and anti-IL-13. However, there is still a subset of T2-low patients that do not respond to these new therapies. This T2-low group will represent the new unmet medical need now that the T2-high-targeting therapies have made it to the market. This review will examine the current thinking on patient stratification in asthma and the identification of the T2-high subset. It will also look at the T2-low patients and examine what may be the drivers of disease in these patients.

Impact of tobacco smoking on cytokine signaling via interleukin-17A in the peripheral airways.

There is excessive accumulation of neutrophils in the airways in chronic obstructive pulmonary disease (COPD) but the underlying mechanisms remain poorly understood. It is known that extracellular cytokine signaling via interleukin (IL)-17A contributes to neutrophil accumulation in the airways but nothing is known about the impact of tobacco smoking on extracellular signaling via IL-17A. Here, we characterized the impact of tobacco smoking on extracellular cytokine signaling via IL-17A in the peripheral airways in long-term smokers with and without COPD and in occasional smokers before and after short-term exposure to tobacco smoke. We quantified concentrations of IL-17A protein in cell-free bronchoalveolar lavage (BAL) fluid samples (Immuno-quantitative PCR) and cytotoxic T-cells (immunoreactivity for CD8+ and CD3+) in bronchial biopsies. Matrix metalloproteinase-8 and human beta defensin 2 proteins were also quantified (enzyme-linked immunosorbent assay) in the BAL samples. The concentrations of IL-17A in BAL fluid were higher in long-term smokers without COPD compared with nonsmoking healthy controls, whereas those with COPD did not differ significantly from either of the other groups. Short-term exposure to tobacco smoke did not induce sustained alterations in these concentrations in occasional smokers. Long-term smokers displayed higher concentrations of IL-17A than did occasional smokers. Moreover, these concentrations correlated with CD8+ and CD3+ cells in biopsies among long-term smokers with COPD. In healthy nonsmokers, BAL concentrations of matrix metalloproteinase-8 and IL-17A correlated, whereas this was not the case in the pooled group of long-term smokers with and without COPD. In contrast, BAL concentrations of human beta defensin 2 and IL-17A correlated in all study groups. This study implies that long-term but not short-term exposure to tobacco smoke increases extracellular cytokine signaling via IL-17A in the peripheral airways. In the smokers with COPD, this signaling may involve cytotoxic T-cells. Long-term exposure to tobacco smoke leads to a disturbed association of extracellular IL-17A signaling and matrix metalloproteinase-8, of potential importance for the coordination of antibacterial activity.

Increase in net activity of serine proteinases but not gelatinases after local endotoxin exposure in the peripheral airways of healthy subjects.

We tested the hypothesis that activation of the innate immune response induces an imbalance in the proteolytic homeostasis in the peripheral airways of healthy subjects, towards excess serine or gelatinase proteinase activity. During bronchoscopy, 18 healthy human subjects underwent intra-bronchial exposure to endotoxin and contra-lateral exposure to vehicle. Bronchoalveolar lavage (BAL) samples were harvested 24 or 48 hours (h) later. We quantified archetype proteinases, anti-proteinases, inflammatory BAL cells, and, importantly, total plus net proteinase activities using functional substrate assays. As expected, endotoxin exposure increased the concentrations of polymorphonuclear leukocytes (PMN's) and macrophages, of proteinases and the anti-proteinases tissue inhibitor of metalloproteinase-1, α-1-antitrypsin and, to a lesser extent, secretory leukoproteinase inhibitor, at both time points. Notably, at these time points, endotoxin exposure substantially increased the quantitative NE/SLPI ratio and the net serine proteinase activity corresponding to neutrophil elastase (NE). Endotoxin exposure also increased the total gelatinase activity corresponding to matrix metalloproteinase (MMP)-9; an activity dominating over that of MMP-2. However, endotoxin exposure had no impact on net gelatinolytic activity at 24 or 48 h after exposure. Thus, local activation of the innate immune response induces an imbalance towards increased net serine proteinase activity in the proteolytic homeostasis of the peripheral airways in healthy subjects. Hypothetically, this serine proteinase activity can contribute to tissue remodelling and hypersecretion via NE from PMN's, if it is triggered repeatedly, as might be the case in chronic inflammatory airway disorders.

Interleukin-22 produced by alveolar macrophages during activation of the innate immune response.

OBJECTIVE AND DESIGN: Interleukin (IL)-22 is important for mucosal host defense. Whereas previous studies focus on lymphocytes as sources of IL-22, we determined whether IL-22 is produced by inflammatory cells in the lungs other than T-lymphocytes during the activation of the innate immune response. MATERIAL, METHODS AND TREATMENT: Inflammatory cells in the lungs of Balb/c mice were primed by endotoxin (LPS, 10 µg) or peptidoglycan (PG, 40 µg) intranasally (3 days). After CD3 + cell depletion, lung homogenates were re-stimulated 24 h with LPS (100 ng/ml), PG (10 µg/ml), IL-23 (100 ng/ml) or vehicle. Human BAL macrophages were stimulated 24 h with PG (50 µg/ml) and IL-23 (100 ng/ml) or vehicle. The release of IL-22 was measured with ELISA and intracellular IL-22 with immunostaining. For statistics, either Dunnett or Students t test method was employed (n = 3-8). RESULTS: Re-stimulation in vitro increased concentrations of mouse IL-22 protein irrespective of priming in vivo. A majority of macrophages in mouse lung and BAL samples displayed immunostaining for IL-22. In analogy, human BAL macrophages released IL-22 protein, and a third of these cells displayed immunostaining for IL-22. CONCLUSIONS: Alveolar macrophages can produce and release IL-22 during the activation of the innate immune response and thereby constitute a potentially important regulator of mucosal host defence in the lungs.

Appearance of remodelled and dendritic cell-rich alveolar-lymphoid interfaces provides a structural basis for increased alveolar antigen uptake in chronic obstructive pulmonary disease.

RATIONALE: The alveolar pathology in chronic obstructive pulmonary disease (COPD) involves antigen-driven immune events. However, the induction sites of alveolar adaptive immune responses have remained poorly investigated. OBJECTIVES: To explore the hypothesis that interfaces between the alveolar lumen and lymphoid aggregates (LAs) provide a structural basis for increased alveolar antigen uptake in COPD lungs. METHODS: Lung samples from patients with mild (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I), moderate-severe (GOLD II-III), and very severe (GOLD IV) COPD were subjected to detailed histological assessments of adaptive immune system components. Never smokers and smokers without COPD served as controls. RESULTS: Quantitative histology, involving computerised three-dimensional reconstructions, confirmed a rich occurrence of alveolar-restricted LAs and revealed, for the first time, that the vast majority of vascular or bronchiolar associated LAs had alveolar interfaces but also an intricate network of lymphatic vessels. Uniquely to COPD lungs, the interface epithelium had transformed into a columnar phenotype. Accumulation of langerin (CD207)(+) dendritic cells occurred in the interface epithelium in patients with COPD but not controls. The antigen-capturing capacity of langerin(+) dendritic cells was confirmed by increased alveolar protrusions and physical T cell contact. Several of these immune remodelling parameters correlated with lung function parameters. CONCLUSIONS: Severe stages of COPD are associated with an emergence of remodelled and dendritic cell-rich alveolar-lymphoid interfaces. This novel type of immune remodelling, which predicts an increased capacity to respond to alveolar antigens, is suggested to contribute to aggravated inflammation in COPD.

Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.

It is now established that IL-17 has a broad pro-inflammatory potential in mammalian host defense, in inflammatory disease and in autoimmunity, whereas little is known about its anti-inflammatory potential and inhibitory feedback mechanisms. Here, we examined whether IL-17A can inhibit the extracellular release of IL-23 protein, the upstream regulator of IL-17A producing lymphocyte subsets, that is released from macrophages during pulmonary inflammation. We characterized the effect of IL-17A on IL-23 release in several models of pulmonary inflammation, evaluated the presence of IL-17 receptor A (RA) and C (RC) on human alveolar macrophages and assessed the role of the Rho family GTPase Rac1 as a mediator of the effect of IL-17A on the release of IL-23 protein. In a model of sepsis-induced pneumonia, intravenous exposure to Staphylococcus aureus caused higher IL-23 protein concentrations in cell-free bronchoalveolar lavage (BAL) samples from IL-17A knockout (KO) mice, compared with wild type (WT) control mice. In a model of Gram-negative airway infection, pre-treatment with a neutralizing anti-IL-17A Ab and subsequent intranasal (i.n.) exposure to LPS caused higher IL-23 and IL-17A protein concentrations in BAL samples compared with mice exposed to LPS, but pre-treated with an isotype control Ab. Moreover, i.n. exposure with IL-17A protein per se decreased IL- 23 protein concentrations in BAL samples. We detected IL-17RA and IL-17RC on human alveolar macrophages, and found that in vitro stimulation of these cells with IL-17A protein, after exposure to LPS, decreased IL-23 protein in conditioned medium, but not IL-23 p19 or p40 mRNA. This study indicates that IL-17A can partially inhibit the release of IL-23 protein during pulmonary inflammation, presumably by stimulating the here demonstrated receptor units IL-17RA and IL-17RC on alveolar macrophages. Hypothetically, the demonstrated mechanism may serve as negative feedback to protect from excessive IL-17A signaling and to control antibacterial host defense once it is activated.

Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

It has previously been shown that nano-meter sized vesicles (30-100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.

A gender difference in circulating neutrophils in malnourished patients with COPD.

BACKGROUND: Circulating markers of inflammation in chronic obstructive pulmonary disease (COPD) may correlate to disease progression and extrapulmonary complications such as malnourishment. However, surprisingly little is known about gender-related differences for circulating inflammatory markers in COPD. PURPOSE: To characterize differences in circulating markers of inflammation in malnourished female and male patients with COPD. SUBJECTS: Thirty female and 11 male patients with a clinical diagnosis of COPD and malnourishment were examined. A group of control subjects without evidence of COPD was recruited for comparison of some variables. METHODS: Blood samples were drawn, and the following parameters were studied: leukocytes and differential counts, C-reactive protein (CRP), tumor necrosis factor-α, interleukin (IL)-6 and IL-8, myeloperoxidase (MPO), neutrophil elastase (NE), intracellular adhesion molecule-1, vascular endothelial adhesion molecule-1, and E-selectin. RESULTS: The mean neutrophil concentration was significantly (P = 0.019) higher in female (4.5 × 10(9)/L) than in male patients with COPD (3.5 × 10(9)/L) and significantly higher than in female control subjects (3.1 × 10(9)/L) (P < 0.01, n = 85). The mean CRP values were considerably higher in female (4.9 mg/mL) than in male patients with COPD (1.5 mg/mL), but the difference was not statistically significant (P = 0.20). The mean concentrations of IL-6 and IL-8 tended to be higher in female than in male patients with COPD, but these differences did not reach statistical significance either (P > 0.05). Confounding factors (smoking, medication) could not explain the gender differences noted. The concentrations of MPO and NE displayed a strong correlation (r = 0.89; P < 0.01, n = 41) but revealed no gender differences. The latter was true for concentrations of adhesion molecules as well. CONCLUSIONS: Our study puts forward evidence of a gender-related difference in systemic inflammation in malnourished patients with COPD in terms of circulating neutrophils being more abundant in female patients. Among these female patients, there was also a trend toward an increase in two neutrophil-mobilizing cytokines. New and better-powered studies are warranted to confirm and characterize this potentially important phenomenon in greater detail.

Impact of interleukin-17 on macrophage phagocytosis of apoptotic neutrophils and particles.

There is now substantial evidence that the cytokine interleukin-17 orchestrates the accumulation of neutrophils in mammals and thereby contributes to host defense. However, the role of IL-17 in controlling neutrophil turnover is not fully understood. Here, we demonstrate that IL-17 stimulates the apoptosis of mouse neutrophils and, simultaneously, the release of the microbicidal compound, myeloperoxidase. IL-17 also stimulates mouse macrophages to phagocytose aged neutrophils and latex beads, and it induces an increase in a soluble form of the phagocytic receptor, lectin-like oxidized low-density lipoprotein receptor-1 as well. In contrast, IL-17 does not markedly increase the release of the archetype neutrophil-recruiting cytokine, macrophage inflammatory protein-2 in mouse macrophages. Importantly, IL-17 also stimulates the phagocytosis of latex beads in human monocyte-derived macrophages. Thus, IL-17 bears the potential to control both phagocytosis and neutrophil turnover during activation of host defense.

Effects of tobacco smoke on IL-16 in CD8+ cells from human airways and blood: a key role for oxygen free radicals?

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8(+) and CD4(+) cells as well as the CD4(+) chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8(+) cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8(+) cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8(+) cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a ß-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8(+) cells. OFR may play a key role in this context.

Cigarette smoke extract modulates respiratory defence mechanisms through effects on T-cells and airway epithelial cells.

Chronic obstructive pulmonary disease (COPD) is a disease primarily caused by cigarette smoking, which in turn has been shown to affect the susceptibility to and progression of airway infections. The question addressed in this study was how components from cigarette smoke could affect the defence mechanisms of T-cells and epithelial cells, and thereby contribute to the development of the COPD pathology. T-cells and monocytes were isolated from buffycoats from healthy donors and T-cell responses studied in response to cigarette smoke extract (CSE). Activation level (CD25 expression), proliferation (BrdU incorporation) and intracellular expression of the cytotoxic markers granzyme-b and TIA-1 were determined using flowcytometry. Normal human bronchial epithelial cells were obtained from Cambrex and differentiated in air-liquid interface cultures. After exposure to CSE barrier function (trans-epithelial electric resistance, TEER), MUC5AC and interleukin-8 production were measured. T-cell activation, proliferation and expression of the cytotoxic proteins granzyme-b and TIA-1 were significantly reduced in response to 0.5-1% of CSE. The epithelial cells were more resistant to CSE and responded at doses 20 times higher than T-cells. The expression of interleukin-8 and MUC5AC was significantly increased after exposure to 15% and 30% CSE and TEER was largely unaffected at 30% CSE but clearly reduced at 40% CSE. This study shows that mechanisms, in both T-cells and airway epithelial cells, involved in the defence against infectious agents are modulated by CSE.

Systemic CD4+ T-cell activation is correlated with FEV1 in smokers.

The inflammation of the lungs in chronic obstructive pulmonary disease (COPD) is characterised by increased numbers of macrophages, neutrophils and T-cells. Decline in lung function in these patients has been correlated to the number of CD8+ T-cells present in the lung as well as to a decline in the ratio of CD4+/CD8+ T-cells. Although systemic components are likely to be present, circulating lymphocyte populations in COPD patients have not been well characterised. This study aimed at correlating lung function to expression of five different T-cell activation markers on peripheral blood CD4+ and CD8+ T-cells in COPD patients and matched smokers. Furthermore, proportions of lymphocyte populations and degree of systemic T-cell activation in COPD patients were compared to that in smokers and never-smokers. Peripheral blood lymphocytes from six never-smokers, eight smokers and 17 smokers with COPD were analysed using flowcytometry. The number of lymphocytes per millilitre was higher in smokers than in never-smokers. No differences were found between the three groups in regard to proportions of lymphocyte populations, but the number of CD4+ T-cells in smokers was higher than in both never-smokers and COPD patients. The degree of T-cell activation was similar in all patient groups; however, a clear correlation between CD69 expression on CD4+ T-cells and lung function (FEV(1)% of predicted) was found when examining current smokers, with or without COPD. Elevated numbers of CD69+ CD4+ T-cells in blood thus seem to be protective against airway obstruction in smokers while still exposed to cigarette smoke, the main inducer of COPD.

C-36 peptide, a degradation product of alpha1-antitrypsin, modulates human monocyte activation through LPS signaling pathways.

alpha1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359-394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LPS), albeit with lower magnitude, by inducing monocyte cytokine (TNFalpha, IL-1beta) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-kappaB (NF-kappaB). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNFalpha release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit CD3/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways.

alphaEbeta7 expression on CD8+ T-cells in COPD BAL fluid and on TGF-beta stimulated T-cells in vitro.

The airway inflammation in patients with COPD shows increased numbers of CD8+ T-cells. Until now few studies have shown any functional data indicating a role for these cells in the pathogenesis of COPD. This paper focuses on a subset of CD8+ T-cells present in human lung, the intra-epithelial lymphocytes expressing the integrin alphaEbeta7, and their presence in bronchoalveolar lavage fluid from COPD patients. In this study we demonstrate that 64-89% of the CD8+ T-cells in bronchoalveolar lavage fluid from COPD patients are positive for CD103, the alpha subunit of alphaEbeta7. We also present an in vitro system in which it is possible to differentiate peripheral T-cells into a phenotype resembling the one found in bronchoalveolar lavage fluid, i.e., CD8+ CD103+. In this in vitro system we demonstrate that, in addition to TGF-beta1, cell-to-cell interaction between the T-cell and an antigen-presenting cell represented here by the monocyte, is crucial for a rapid, high and sustained expression of CD103. The signal provided by the monocytes is shown to be mediated through LFA-1 on the T-cell. Furthermore, differentiation of CD8+ T-cells by TGF-beta1 and monocytes results in down regulation of INF-gamma, TNF-alpha and GM-CSF production. IL-8 production is, however, retained in the alphaEbeta7 expressing cells. We see this work as an initiation on the quest for a functional characterization of one of the different types of CD8+ T cells present in COPD. In the longer perspective we hope this can lead to an increased understanding of how these cells can contribute to the disease pathology.