Lower airway bacterial colonisation (LABC) in COPD patients is associated with increased exacerbation frequency and faster lung function decline. Defective macrophage phagocytosis in COPD drives ...inflammation, but how defective macrophage function contributes to exacerbations is not clear. This study investigated the association between macrophage phagocytosis and exacerbation frequency, LABC and clinical parameters.
Monocyte-derived macrophages (MDM) were generated from 92 stable COPD patients, and at the onset of exacerbation in 39 patients. Macrophages were exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4 h, then phagocytosis measured by fluorimetry and cytokine release by ELISA. Sputum bacterial colonisation was measured by PCR.
Phagocytosis of H. influenzae was negatively correlated with exacerbation frequency (r = 0.440, p < 0.01), and was significantly reduced in frequent vs. infrequent exacerbators (1.9 × 10
RFU vs. 2.5 × 10
RFU, p < 0.01). There was no correlation for S. pneumoniae. There was no association between phagocytosis of either bacteria with age, lung function, smoking history or treatment with inhaled corticosteroids, or long-acting bronchodilators. Phagocytosis was not altered during an exacerbation, or in the 2 weeks post-exacerbation. In response to phagocytosis, MDM from exacerbating patients showed increased release of CXCL-8 (p < 0.001) and TNFα (p < 0.01) compared to stable state.
Impaired COPD macrophage phagocytosis of H. influenzae, but not S. pneumoniae is associated with exacerbation frequency, resulting in pro-inflammatory macrophages that may contribute to disease progression. Targeting these frequent exacerbators with drugs that improve macrophage phagocytosis may prove beneficial.
Sirtuin-1 (SIRT1) and SIRT6, NAD
-dependent Class III protein deacetylases, are putative anti-aging enzymes, down-regulated in patients with chronic obstructive pulmonary disease (COPD), which is ...characterized by the accelerated ageing of the lung and associated with increased oxidative stress. Here, we show that oxidative stress (hydrogen peroxide) selectively elevates microRNA-34a (miR-34a) but not the related miR-34b/c, with concomitant reduction of SIRT1/-6 in bronchial epithelial cells (BEAS2B), which was also observed in peripheral lung samples from patients with COPD. Over-expression of a miR-34a mimic caused a significant reduction in both mRNA and protein of SIRT1/-6, whereas inhibition of miR-34a (antagomir) increased these sirtuins. Induction of miR-34a expression with H
O
was phosphoinositide-3-kinase (PI3K) dependent as it was associated with PI3Kα activation as well as phosphatase and tensin homolog (PTEN) reduction. Importantly, miR-34a antagomirs increased SIRT1/-6 mRNA levels, whilst decreasing markers of cellular senescence in airway epithelial cells from COPD patients, suggesting that this process is reversible. Other sirtuin isoforms were not affected by miR-34a. Our data indicate that miR-34a is induced by oxidative stress via PI3K signaling, and orchestrates ageing responses under oxidative stress, therefore highlighting miR-34a as a new therapeutic target and biomarker in COPD and other oxidative stress-driven aging diseases.
Background: The pathophysiology of chronic obstructive pulmonary disease (COPD) features pulmonary inflammation with a predominant alveolar macrophage involvement. Bronchoalveolar macrophages from ...patients with COPD release increased amounts of inflammatory cytokines in vitro, an effect that is not inhibited by the glucocorticosteroid dexamethasone. Resveratrol (3,5,4′-trihydroxystilbene) is a component of red wine extract that has anti-inflammatory and antioxidant properties. A study was undertaken to determine whether or not resveratrol would inhibit cytokine release in vitro by alveolar macrophages from patients with COPD. Methods: Alveolar macrophages were isolated from bronchoalveolar lavage (BAL) fluid from cigarette smokers and from patients with COPD (n=15 per group). The macrophages were stimulated with either interleukin (IL)-1β or cigarette smoke media (CSM) to release IL-8 and granulocyte macrophage-colony stimulating factor (GM-CSF). The effect of resveratrol was examined on both basal and stimulated cytokine release. Results: Resveratrol inhibited basal release of IL-8 in smokers and patients with COPD by 94% and 88% respectively, and inhibited GM-CSF release by 79% and 76% respectively. Resveratrol also inhibited stimulated cytokine release. Resveratrol reduced IL-1β stimulated IL-8 and GM-CSF release in both smokers and COPD patients to below basal levels. In addition, resveratrol inhibited CSM stimulated IL-8 release by 61% and 51% respectively in smokers and COPD patients, and inhibited GM-CSF release by 49% for both subject groups. Conclusions: Resveratrol inhibits inflammatory cytokine release from alveolar macrophages in COPD. Resveratrol or similar compounds may be effective pharmacotherapy for macrophage pathophysiology in COPD.
The contingent negative variation (CNV) is a long-latency electroencephalography (EEG) surface negative potential with cognitive and motor components, observed during response anticipation. CNV is an ...index of cortical arousal during orienting and attention, yet its functional neuroanatomical basis is poorly understood. We used functional magnetic resonance imaging (fMRI) with simultaneous EEG and recording of galvanic skin response (GSR) to investigate CNV-related central neural activity and its relationship to peripheral autonomic arousal. In a group analysis, blood oxygenation level dependent (BOLD) activity during the period of CNV generation was enhanced in thalamus, somatomotor cortex, bilateral midcingulate, supplementary motor, and insular cortices. Enhancement of CNV-related activity in anterior and midcingulate, SMA, and insular cortices was associated with decreases in peripheral sympathetic arousal. In a subset of subjects in whom we acquired simultaneous EEG and fMRI data, we observed activity in bilateral thalamus, anterior cingulate, and supplementary motor cortex that was modulated by trial-by-trial amplitude of CNV. These findings provide a likely functional neuroanatomical substrate for the CNV and demonstrate modulation of components of this neural circuitry by peripheral autonomic arousal. Moreover, these data suggest a mechanistic model whereby thalamocortical interactions regulate CNV amplitude.
BackgroundCOPD is associated with cellular senescence and fibrosis. Extracellular vesicles (EVs) are membrane-derived vesicles involved in intercellular communication. EVs contain miRNAs, mRNA and ...proteins and have been implicated in COPD to induce senescence and the transition of fibroblast to myofibroblasts. This study examined whether EVs derived from COPD fibroblasts drive senescence in healthy recipient fibroblasts. Changes in expression of p21CIP1 and alpha-smooth muscle actin (αSMA) were chosen as markers of senescence and transition of fibroblasts to myofibroblasts respectively.MethodsLarge EVs, and small EVs were isolated from media from non-smoker (NS) and COPD fibroblasts cultured with or without H2O2. EVs were labelled with phk67 and uptake measured by flow cytometry. Healthy recipient fibroblasts were cultured with EVs or EV-free media for 24h and 48h and protein expression of p21CIP1 and αSMA measured using western blots and CXCL8 release by ELISA.ResultsThere was a time-dependent uptake of EVs into recipient cells with no difference between EVs from control or COPD fibroblasts with 91.8 ± 3.8% of recipient cells phk67 positive by 48h (n=4). Incubation of recipient fibroblasts (n=2–5) with large EVs from either non-smokers or COPD subjects did not alter the expression of p21CIP1 or αSMA at 24h. Similarly large EVs from fibroblasts exposed to H2O2 had no effect on these markers in recipient cells. By contrast, at 48h (figure 1), small EVs from COPD cells showed a trend to increased expression of p21CIP1 and EVs from both non-smokers and COPD subjects increased expression of αSMA. Incubation of recipient cells with large EVs from non-smoker fibroblasts that had been cultured with or without H2O2 increased release of CXCL8 (0.36±0.15ng/ml to 5.43±3.92ng/ml and 5.44±5.23ng/ml respectively) and small EVs from COPD fibroblasts induced CXCL8 release at 48h (0.36±0.15ng/ml to 3.75±3.16ng/ml).ConclusionsLarge and small EVs tend to increase the expression of p21CIP1 and αSMA in recipient fibroblasts. These results are confirmed by the uptake analysis showing that maximum uptake of EVs from both NS and COPD fibroblasts is reached after 48h. Altogether, these data suggest that EVs participate in COPD pathophysiology by spreading senescence in recipient fibroblasts.Abstract P208 Figure 1Effect of Large and Small EVs on p21CIP1 and αSMA Expression in NS Fibroblasts Stimulated for 48h. Healthy fibroblasts from non-smoker (NS) subjects were incubated with large and small EVs derived from healthy NS or COPD fibroblasts, derived from cells that had been cultured in the absence or presence of 100µM H2O2. Cells were also stimulated with media only (NT) and media containing H2O2 as controls. Cells were lysed after 48h (a, d) and the expression of p21CIP1 (b, e) and αSMA (c, f) was measured relative to β-actin expression and data presented as mean±SEM. Representative blots are presented in panels a and d.
Pulmonary macrophages are a target for inhaled therapies. Combinations of long-acting beta(2)-agonists (LABA) and glucocorticosteroids have been developed for asthma and chronic obstructive pulmonary ...disease (COPD). This study examined two LABA, salmeterol and formoterol, and the glucocorticosteroid, budesonide, on cytokine release from monocyte-derived macrophages (MDM) to determine whether anti-inflammatory effects observed in patients are due to inhibition of macrophages. MDM were incubated in the absence or presence of LABA or budesonide prior to stimulation with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-alpha, granulocyte macrophage-colony stimulating factor (GM-CSF) and CXC chemokine ligand (CXCL)8 were measured by ELISA. Formoterol and salmeterol inhibited LPS-stimulated release of TNF-alpha (mean effective concentration (EC(50)) 2.4+/-1.8 and 3.5+/-2.7 nM, respectively; n = 11-16), GM-CSF (EC(50) 24.6+/-2.1 and 52.4+/-40.8 nM, respectively, n = 11-12) but not CXCL8 from LPS-stimulated MDM. Budesonide inhibited release of all three cytokines (EC(50) TNF-alpha: 1.2+/-0.4 nM; GM-CSF: 0.4+/-0.2 nM; CXCL8: 0.4+/-0.1 nM; n = 3-4). Formoterol but not salmeterol elevated cAMP in these cells. These effects were attenuated by beta-adrenoceptor antagonists, propranolol and ICI118551. Salmeterol (10(-7) M) also inhibited formoterol-induced cAMP and formoterol-mediated attenuation of cytokine release. Combining budesonide (0.3 nM) with formoterol, inhibited TNF-alpha release additively. LABA may inhibit inflammatory cytokine release from macrophages in a cAMP-independent manner and act additively with budesonide.
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 (M(1)-M(5)) ascertained using real-time PCR. M(2) and M(3) receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B(4) 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 M(1) mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M(3) mRNA. Expression of M(2) and M(3) protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB(4) 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. LTB(4) release was attenuated by the M(3) antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2 ± 2.2 nM; n = 9). Stimulation of macrophage M(3) receptors promotes release of LTB(4), suggesting that anti-muscarinic agents may be anti-inflammatory.
Background and purpose:
Macrophages release cytokines that may contribute to pulmonary inflammation in conditions such as chronic obstructive pulmonary disease. Thus, inhibition of macrophage ...cytokine production may have therapeutic benefit. p38 MAPK may regulate cytokine production, therefore, the effect of two p38 MAPK inhibitors, SB239063 and SD‐282, on the release of TNF‐α, GM‐CSF and IL‐8 from human macrophages was investigated.
Experimental approach:
Cytokine release was measured by ELISA. Immunoblots and mRNA expression studies were performed to confirm p38 MAPK isoform expression and activity. Macrophages were isolated from lung tissue of current smokers, ex‐smokers and emphysema patients and exposed to lipopolysaccharide. These cells then released cytokines in a concentration‐dependent manner.
Key results:
SB239063 only inhibited TNF‐α release (EC50 0.3±0.1 μM). Disease status had no effect on the efficacy of SB239063. SD‐282 inhibited both TNF‐α and GM‐CSF release from macrophages (EC50 6.1±1.4 nM and 1.8±0.6 μM respectively) but had no effect on IL‐8 release. In contrast, both inhibitors suppressed cytokine production in monocytes.
Conclusions and Implications:
The differential effects of p38 MAPK inhibitors between macrophages and monocytes could not be explained by differences in p38 MAPK isoform expression or activity. However, the stability of TNF‐α mRNA was significantly increased in macrophages compared to monocytes. These data suggest a differential involvement for p38 MAPK in macrophage cytokine production compared with monocytes. These effects are not due to lack of p38 activation or p38α expression in macrophages but may reflect differential effects on the stability of cytokine mRNA.
British Journal of Pharmacology (2006) 149, 393–404. doi:10.1038/sj.bjp.0706885
Millennial and multi-millennial tree-ring chronologies can provide useful proxy records of past climate, giving insight into a more complete range of natural climate variability prior to the 20th ...century. Since the 1980s a multi-millennial tree-ring chronology has been developed from kauri (Agathis australis) from the upper North Island, New Zealand. Previous work has demonstrated the sensitivity of kauri to the El Niño-Southern Oscillation (ENSO). Here we present recent additions and extensions to the late Holocene kauri chronology (LHKC), and assess the potential of a composite master chronology, AGAUc13, for palaeoclimate reconstruction. The updated composite kauri chronology now spans 4491 years (2488 BCE–2002 CE) and includes data from 18 modern sites, 25 archaeological sites, and 18 sub-fossil (swamp) kauri sites. Consideration of the composition and statistical quality of AGAUc13 suggests the LHKC has utility for palaeoclimate reconstruction but there are caveats. These include: (a) differences in character between the three assemblages including growth rate and sensitivity; (b) low sample depth and low statistical quality in the 10th–13th century CE, when the record transitions from modern and archaeological material to the swamp kauri; (c) a potential difference in amplitude of the signal in the swamp kauri; (d) a westerly bias in site distribution prior to 911 CE; (e) variable statistical quality across the entire record associated with variable replication; and (f) complex changes in sample depth and tree age and size which may influence centennial scale trends in the data. Further tree ring data are required to improve statistical quality, particularly in the first half of the second millennium CE.
•The updated and revised late Holocene kauri chronology spans 4491 years (2488 BCE–2002 CE).•The chronology includes data from modern, archaeological, and sub-fossil sites.•The character of the three data sets is different.•There is a potential difference in amplitude of the signal in sub-fossil kauri.•Complex changes in chronology composition may influence centennial scale trends.
The contributions of disease escape and disease resistance to the responses of wheat to septoria tritici leaf blotch (STB) were analysed in a set of 226 lines, including modern cultivars, breeding ...lines and their progenitors dating back to the origin of scientific wheat breeding. Field trials were located in the important wheat-growing region of eastern England and were subject to natural infection by Mycosphaerella graminicola. STB scores were related to disease-escape traits, notably height, leaf spacing, leaf morphology and heading date, and to the presence of known Stb resistance genes and isolate-specific resistances. The Stb6 resistance gene was associated with a reduction of 19% in the level of STB in the complete set of 226 lines and with a 33% reduction in a subset of 139 lines of semidwarf stature. Greater plant height was strongly associated with reduced STB in the full set of lines, but only weakly in the semidwarf lines. Shorter leaf length was also associated with reduced STB, but, in contrast to earlier reports, lines with more prostrate leaves had more STB on average, probably because they tended to have longer leaves. Several lines, notably cvs Pastiche and Exsept, had low mean levels of STB which could not be explained by either escape traits or specific resistance genes, implying that they have unknown genes for partial resistance to STB.
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