Suzanne L. Traves

Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2.

Leukocyte migration is critical to maintaining host defense, but uncontrolled cellular infiltration into tissues can lead to chronic inflammation. In the lung, such diseases include chronic obstructive pulmonary disease (COPD), a debilitating, respiratory condition characterized by progressive and largely irreversible airflow limitation for which cigarette smoking is the major risk factor. COPD is associated with an increased inflammatory cell influx including increased macrophage numbers in the airways and tissue. Alveolar macrophages develop from immigrating blood monocytes and have the capacity to cause the pathological changes associated with COPD. This study addressed the hypothesis that increased macrophage numbers in COPD are a result of increased recruitment of monocytes from the circulation. Chemotaxis assays of peripheral blood mononuclear cells (PBMC)/monocytes from nonsmokers, smokers, and COPD patients demonstrated increased chemotactic responses for cells from COPD patients when compared with controls toward growth‐related oncogene (GRO)α and neutrophil‐activating peptide (NAP)‐2 but not toward monocyte chemoattractant protein, interleukin‐8, or epithelial‐derived NAP(ENA)‐78. The enhanced chemotactic response toward GROα and NAP‐2 was not mediated by differences in expression of their cellular receptors, CXCR1 or CXCR2. Receptor expression studies using flow cytometry indicated that in COPD, monocyte expression of CXCR2 is regulated differently from nonsmokers and smokers, which may account for the enhanced migration toward GROα and NAP‐2. The results highlight the potential of CXCR2 antagonists as therapy for COPD and demonstrate that an enhanced PBMC/monocyte response to specific CXC chemokines in these patients may contribute to increased recruitment and activation of macrophages in the lungs.

Th17 Cells in Airway Diseases

Chronic inflammation is a key feature of many airway diseases. Leukocyte accumulation in the lung has the capacity to mediate many aspects of the pathophysiology of such diseases including asthma and chronic obstructive pulmonary disease (COPD). Until recently, the CD4+ lymphocyte component of these inflammatory influxes was thought to consist of Th1 or Th2 type cells, however a third group of cells termed Th17 have been identified. These cells follow a distinct differentiation profile requiring TGFbeta and IL-6 leading to the expression of the Th17 selective transcription factor, RORgammat. Differentiation of these cells is restricted by Th1 and Th2 cytokines including IFNgamma and IL-4 which attenuate Th17 cell differentiation. The presence of Th17 cells in the airway has yet to be confirmed, yet IL-17 is expressed in both asthma and COPD. Many of the inflammatory effects of Th17 cells are attributed to the expression of this cytokine. For example, IL-17 up-regulates the expression of a number of CXCR2 chemokines including CXCL1, CXCL6 and CXCL8 together with neutrophil survival factors GM-CSF and G-CSF from the airway epithelium. This would suggest that Th17 cells are important in promoting and sustaining neutrophilic inflammation as observed in severe asthma and COPD. In addition, IL-17 can act synergistically with viral infection or other inflammatory mediators including TNF-alpha to potentiate these responses. Confirmation of the presence of Th17 cells in the airways in disease warrants further investigation since these cells would present a novel therapeutic opportunity to reduce neutrophilic inflammation in the lung.

Cigarette smoke modulates rhinovirus-induced airway epithelial cell chemokine production

Human rhinovirus (HRV) infections induce epithelial cell production of chemokines that may contribute to the pathogenesis of exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Cigarette smoking is the predominant risk factor for the development of COPD and also aggravates asthma symptoms. We examined whether cigarette smoke extract (CSE) modulates viral inflammation by altering the profile of HRV-induced epithelial chemokine production. Purified HRV-16, and CSE were used to examine the effects on CXC chemokine ligand (CXCL)8 and CXCL10 production from both primary human bronchial epithelial cells and the BEAS-2B epithelial cell line. Both CSE and HRV-16 induced CXCL8 production and, when used in combination, induced at least an additive production of CXCL8 compared with either stimulus alone. In contrast, CSE did not induce CXCL10 and markedly inhibited HRV-16-induced CXCL10 production. Inhibition of HRV-16-induced CXCL10 by CSE was mediated, at least in part, via transcriptional regulation. The increased CXCL8 production seen with the combination of CSE and HRV-16 was not due to transcriptional regulation but was associated with CXCL8 mRNA stabilisation. Thus, CSE differentially modulates HRV-16-induced chemokine production from human airway epithelial cells in a manner that might be expected to alter inflammatory cell profiles.

Expression of muscarinic receptors by human macrophages

Macrophages increase in number and are highly activated in chronic obstructive pulmonary disease (COPD). Muscarinic receptor antagonists inhibit acetylcholine-stimulated release of neutrophilic chemoattractants, suggesting that acetylcholine may regulate macrophage responses. Therefore, expression and function of components of the non-neuronal cholinergic system in monocyte-macrophage cells was investigated. RNA was isolated from monocytes, monocyte-derived macrophages (MDMs), lung and alveolar macrophages from nonsmokers, smokers and COPD patients, and expression of the high-affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter and muscarinic receptors (M1–M5) ascertained using real-time PCR. M2 and M3 receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B4 were measured by ELISA or EIA. All monocyte-macrophage cells expressed mRNA for components of the non-neuronal cholinergic system. Lung macrophages expressed significantly more M1 mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M3 mRNA. Expression of M2 and M3 protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB4 from lung macrophages (buffer 222.3±75.1 versus carbachol 1,118±622.4 pg·mL−1; n=15, p<0.05) but not IL-6 or IL-8. LTB4 release was attenuated by the M3 antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2±2.2 nM; n=9). Stimulation of macrophage M3 receptors promotes release of LTB4, suggesting that anti-muscarinic agents may be anti-inflammatory.

Effect of incremental exercise on airway and systemic inflammation in patients with COPD

Airway and systemic inflammation are features of chronic obstructive pulmonary disease (COPD), and there is growing interest in clarifying the inflammatory processes. Strenuous exercise induces an intensified systemic inflammatory response in patients with COPD, but no study has investigated the airway inflammatory and anti-inflammatory responses to exercise. Twenty steroid-naïve, ex-smokers with diagnosed COPD (forced expired volume in 1 s = 66 ± 12%) underwent baseline collection of venous blood and induced sputum followed by an incremental exercise test to symptom limitation 48 h later. Additional venous blood samples were collected following exercise at 0, 2, and 24 h, while induced sputum was collected 2 and 24 h after exercise. Sputum and blood samples were analyzed for differential cell count, CD4(+) and CD8(+) T lymphocytes (serum only), interleukin (IL)-6, IL-8, IL-10, chemokine (C-C motif) ligand 5 (CCL5), and high sensitivity C-reactive protein (serum only). There was an increase in the number of sputum eosinophils (cells/gram, P = 0.012) and a reduction in sputum IL-6 (P = 0.01) 24 h postexercise. Sputum IL-8 and CCL5 were also persistently decreased after exercise (P = 0.0098 and P = 0.0012, respectively), but sputum IL-10 did not change. There was a decrease in serum eosinophils 2 h after exercise (P = 0.0014) and a reduction in serum CCL5 immediately following and 2 h postexercise (P < 0.0001). Both serum eosinophils and CCL5 returned to baseline levels within 24 h. An acute bout of exercise resulted in a significant increase in the number of sputum eosinophils, which may be mediated by serum CCL5. However, there was also a reduction in sputum proinflammatory cytokines, suggesting some anti-inflammatory effect of exercise in the lungs of steroid-naïve patients with COPD.

Enhanced monocyte migration to CXCR3 and CCR5 chemokines in COPD.

Chronic obstructive pulmonary disease (COPD) patients exhibit chronic inflammation, both in the lung parenchyma and the airways, which is characterised by an increased infiltration of macrophages and T-lymphocytes, particularly CD8+ cells. Both cell types can express chemokine (C-X-C motif) receptor (CXCR)3 and C-C chemokine receptor 5 and the relevant chemokines for these receptors are elevated in COPD. The aim of this study was to compare chemotactic responses of lymphocytes and monocytes of nonsmokers, smokers and COPD patients towards CXCR3 ligands and chemokine (C-C motif) ligand (CCL)5. Migration of peripheral blood mononuclear cells, monocytes and lymphocytes from nonsmokers, smokers and COPD patients toward CXCR3 chemokines and CCL5 was analysed using chemotaxis assays. There was increased migration of peripheral blood mononuclear cells from COPD patients towards all chemokines studied when compared with nonsmokers and smokers. Both lymphocytes and monocytes contributed to this enhanced response, which was not explained by increased receptor expression. However, isolated lymphocytes failed to migrate and isolated monocytes from COPD patients lost their enhanced migratory capacity. Both monocytes and lymphocytes cooperate to enhance migration towards CXCR3 chemokines and CCL5. This may contribute to increased numbers of macrophages and T-cells in the lungs of COPD patients, and inhibition of recruitment using selective antagonists might be a treatment to reduce the inflammatory response in COPD. Peripheral blood mononuclear cells from COPD patients migrate in greater numbers towards CCL5 and CXCR3 chemokines http://ow.ly/X6iQw

Transcriptional and Epigenetic Modulation of Human Rhinovirus–Induced CXCL10 Production by Cigarette Smoke

Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease. Cigarette smoking is the primary risk factor for the development of chronic obstructive pulmonary disease, and 25% of individuals with asthma smoke. Smokers experience both longer and more severe colds. We previously showed that cigarette smoke extract (CSE) inhibited HRV-induced expression of a range of epithelial antiviral molecules. Here, we use CXCL10 as a model antiviral gene to examine the mechanisms by which CSE inhibits epithelial antiviral immunity. HRV-induced CXCL10 transcription depends on activation of NF-ĸB and IFN-regulatory factor-1 (IRF-1), and we now also implicate two signal transducer and activator of transcription (STAT) consensus sequences in the CXCL10 promoter in HRV-induced CXCL10 expression. CSE inhibited HRV-induced activation and nuclear translocation/binding of both NF-ĸB, and IRF-1 to their respective recognition sequences in the CXCL10 promoter. HRV also induced formation of complexes at the STAT region in the CXCL10 promoter, and HRV-induced activation of STAT-1 was inhibited by CSE. In addition, CSE inhibited HRV-induced chromatin accessibility around the transcriptional start site of the CXCL10 promoter. Although CSE inhibited HRV-induced expression of both the viral double-stranded RNA sensors, retinoic acid-inducible gene-I and melanoma differentiation-associated gene (MDA) 5, only specific short interfering RNA (siRNA) to MDA5, but not nontargeting siRNA, or siRNA to retinoic acid-inducible gene-I, inhibited HRV-induced CXCL10 induction. We conclude that CSE reduces chromatin accessibility and inhibits viral signaling via NF-ĸB, IRF-1, STAT-1, and MDA5. Thus, we show that CSE can simultaneously modulate multiple pathways linked to innate immune responses to HRV infection.

Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

Human rhinovirus (HRV) infections trigger exacerbations of lower airway diseases. HRV infects human airway epithelial cells and induces proinflammatory and antiviral molecules that regulate the response to HRV infection. Interferon (IFN)-stimulated gene of 15 kDa (ISG15) has been shown to regulate other viruses. We now show that HRV-16 infection induces both intracellular epithelial ISG15 expression and ISG15 secretion in vitro. Moreover, ISG15 protein levels increased in nasal secretions of subjects with symptomatic HRV infections. HRV-16-induced ISG15 expression is transcriptionally regulated via an IFN regulatory factor pathway. ISG15 does not directly alter HRV replication but does modulate immune signaling via the viral sensor protein RIG-I to impact production of CXCL10, which has been linked to innate immunity to viruses. Extracellular ISG15 also alters CXCL10 production. We conclude that ISG15 has a complex role in host defense against HRV infection, and that additional studies are needed to clarify the role of this molecule.

Chemokines and their Receptors as Targets for the Treatment of COPD

Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory condition, characterised by progressive, irreversible airflow obstruction. The major risk factor for development of COPD is cigarette smoking, and the disease is predicted to become the 3 rd leading cause of death by 2020. Currently, there are no pharmacological interventions that halt the progression of COPD; however one strategy is to reduce the chronic lung inflammation associated with this disease. An increased inflammatory infiltrate comprising macrophages, neutrophils and T-lymphocytes is a major hallmark of COPD. Furthermore, both macrophages and neutrophils have the ability to cause all the pathological changes associated with COPD. Chemokines that are elevated in sputum from COPD patients have the capacity to recruit neutrophils, the macrophage precursor cells, monocytes, and T-lymphocytes. Chemokines are considered predominantly chemotactic cytokines however; there is a growing body of evidence demonstrating that chemokines can also act as functional antagonists thus leading to selective recruitment of inflammatory cells. Whilst inhibition of chemokine dependent recruitment of inflammatory cells via small molecule antagonists gives rise to potential treatments for COPD, the discovery that chemokines are also natural antagonists could also be exploited in the ongoing search for treatment of this currently fatal disease.

Human β-defensin-2 production upon viral and bacterial co-infection is attenuated in COPD

Viral-bacterial co-infections are associated with severe exacerbations of COPD. Epithelial antimicrobial peptides, including human β-defensin-2 (HBD-2), are integral to innate host defenses. In this study, we examined how co-infection of airway epithelial cells with rhinovirus and Pseudomonas aeruginosa modulates HBD-2 expression, and whether these responses are attenuated by cigarette smoke and in epithelial cells obtained by bronchial brushings from smokers with normal lung function or from COPD patients. When human airway epithelial cells from normal lungs were infected with rhinovirus, Pseudomonas aeruginosa, or the combination, co-infection with rhinovirus and bacteria resulted in synergistic induction of HBD-2 (p<0.05). The combination of virus and flagellin replicated this synergistic increase (p<0.05), and synergy was not seen using a flagella-deficient mutant Pseudomonas (p<0.05). The effects of Pseudomonas aeruginosa were mediated via interactions of flagellin with TLR5. The effects of HRV-16 depended upon viral replication but did not appear to be mediated via the intracellular RNA helicases, retinoic acid-inducible gene-I or melanoma differentiation-associated gene-5. Cigarette smoke extract significantly decreased HBD-2 production in response to co-infection. Attenuated production was also observed following co-infection of cells obtained from healthy smokers or COPD patients compared to healthy controls (p<0.05). We conclude that co-exposure to HRV-16 and Pseudomonas aeruginosa induces synergistic production of HBD-2 from epithelial cells and that this synergistic induction of HBD-2 is reduced in COPD patients. This may contribute to the more severe exacerbations these patients experience in response to viral-bacterial co-infections.