A Comparison of the Inflammatory and Proteolytic Effects of Dung Biomass and Cigarette Smoke Exposure in the Lung

Rationale

Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.

Methods

We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.

Results

The in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.

Conclusion

Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.     

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Background

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Methods

Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.

Results

We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.

Conclusions

CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.     

Cigarette smoke enhances Th-2 driven airway inflammation and delays inhalational tolerance

Background

Active smoking increases asthma severity and is related to diminished treatment efficacy. Animal models in which inhalation of both allergen and mainstream cigarette smoke are combined can help us to understand the complex interaction between both agents. We have recently shown that, in allergic mice, the airway inflammation can be cleared by repeated allergen challenge, resulting in the establishment of a state of inhalational tolerance.

Methods

In this study, we assessed in vivo the impact of cigarette smoke on the efficacy and time course of this form of tolerance induction. We exposed sensitized mice to concurrent mainstream cigarette smoke and allergen (Ovalbumin- OVA) and measured the airway inflammation at different time points.

Results

We first confirmed that aerosolized OVA administered for a prolonged time period (4–8 weeks) resulted in the establishment of tolerance. Concurrent OVA and smoke exposure for 2 weeks showed that tobacco smoke enhanced the Th-2 driven airway inflammation in the acute phase. In addition, the induction of the tolerance by repeated inhalational OVA challenge was delayed significantly by the tobacco smoke, since 4 weeks of concurrent exposure resulted in a more persistent eosinophilic airway inflammation, paralleled by a more mature dendritic cell phenotype. However, smoke exposure could not prevent the establishment of tolerance after 8 weeks of antigen exposure as shown by both histopathology (disappearance of the Th-2 driven inflammation) and by in vivo functional experiments. In these tolerized mice, some of the inflammatory responses to the smoke were even attenuated.

Conclusion

Cigarette smoke enhances acute allergic inflammation and delays, but does not abrogate the development of tolerance due to prolonged challenge with inhaled antigen in experimental asthma.     

Smoking accelerates aging of the small airway epithelium

Background

Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking.

Methods

Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function.

Results

Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers.

Conclusion

These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0094-1) contains supplementary material, which is available to authorized users.     

Flavonoid ingredients of Ginkgo biloba leaf extract regulate lipid metabolism through Sp1-mediated carnitine palmitoyltranferase 1A up-regulation

Background

Lipid accumulation is the primary evidence of non-alcoholic fatty liver disease (NAFLD). Ginkgo biloba extract (GBE) and its flavonoid ingredients (quercetin, kaempferol, and isorhamnetin) could lessen the lipid accumulation associated with up-regulation of the rate-limiting enzyme, carnitine palmitoyltransferase 1A (CPT1A), in the β-oxidation of long-chain fatty acids. In this study, we investigated the mechanisms by which GBE and its flavonoids induced expression of CPT1A.

Results

CPT1A inhibition with RNAi resulted in triglyceride accumulation in HepG2 cells. Through deletion and mutation analysis of CPT1A’s promoter combined with electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments, the CPT1A promoter region (−50 to −5 nt) was determined to contain two putative Sp1 binding sites, namely Sp1a and Sp1b, which might act as the GBE regulation response DNA element. Sp1 might be induced to transfer from cytoplasma to nucleus to bind the promoter region of −50 to −5 nt by GBE. The regulatory effects of GBE on CPT1A were also verified on the flavonoid ingredients quercetin, kaempferol, and isorhamnetin.

Conclusion

Sp1 was crucial in regulating CPT1A expression with GBE and its flavonoid ingredients, and the −50 to −5 nt region of CPT1A promoter played important roles in Sp1 binding.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0087-x) contains supplementary material, which is available to authorized users.     

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background

Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.

Methods

The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).

Results

In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.

Conclusions

This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.     

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

Background

Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.

Methods

A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.

Results

Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.

Conclusions

Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.     

A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation

Introduction

Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.

Methods

Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.

Results

RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.

Conclusions

RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.     

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

Upregulation of Gelatinases and Epithelial–Mesenchymal Transition in Small Airway Remodeling Associated with Chronic Exposure to Wood Smoke

Background

Peribronchiolar fibrosis is an important feature of small airway remodeling (SAR) in cigarette smoke-induced COPD. The aim of this study was to investigate the role of gelatinases (MMP9, MMP2) and epithelial-mesenchymal transition (EMT) in SAR related to wood smoke (WS) exposure in a rat model.

Methods

Forty-eight female Sprague-Dawley rats were randomly divided into the WS group, the cigarette smoke (CS) group and the clean air control group. After 4 to 7 months of smoke exposure, lung tissues were examined with morphometric measurements, immunohistochemistry and Western blotting. Serum MMP9 and TIMP1 concentrations were detected by ELISA. In vitro, primary rat tracheal epithelial cells were stimulated with wood smoke condensate for 7 days.

Results

The COPD-like pathological alterations in rats exposed chronically to WS were similar to those exposed to CS; the area of collagen deposition was significantly increased in the small airway walls of those exposed to WS or CS for 7 months. The expression of gelatinases in rats induced by WS or CS exposure was markedly increased in whole lung tissue, and immunohistochemistry showed that MMP9, MMP2 and TIMP1 were primarily expressed in the airway epithelium. The serum levels of MMP9 and TIMP1 were significantly higher in rats secondary to WS or CS exposure. Few cells that double immunostained for E-cadherin and vimentin were observed in the airway subepithelium of rats exposed to WS for 7 months (only 3 of these 8 rats). In vitro, the expression of MMP9 and MMP2 proteins was upregulated in primary rat tracheal epithelial cells following exposure to wood smoke condensate for 7 days by Western blotting; positive immunofluorescent staining for vimentin and type I collagen was also observed.

Conclusions

These findings suggest that the upregulation of gelatinases and EMT might play a role in SAR in COPD associated with chronic exposure to wood smoke.     

Maternal smoking and the retinoid pathway in the developing lung

Background

Maternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model.

Methods

Female C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro.

Results

Compared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3–5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells.

Conclusions

A murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.     

The fire ephemeral Tersonia cyathiflora (Gyrostemonaceae) germinates in response to smoke but not the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one

Background and Aims

Tersonia cyathiflora (Gyrostemonaceae) is a fire ephemeral with an obligate requirement for smoke to germinate. Whether it is stimulated to germinate by 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR₁), the butenolide isolated from smoke that stimulates the germination of many other smoke-responsive species, is tested.

Methods

Seeds of T. cyathiflora were buried in autumn following collection and were exhumed 1 year later, as this alleviates dormancy and enables seeds to germinate in response to smoke-water. Exhumed seeds were tested with smoke-water and KAR₁. Fresh preparations of these solutions were again tested on seeds exhumed 2 months later under a broader range of conditions. They were also tested on Grevillea eriostachya (Proteaceae) and Stylidium affine (Stylidiaceae) to confirm the activity of KAR₁.

Key Results

T. cyathiflora seeds germinated in response to smoke-water but not to KAR₁. In contrast, G. eriostachya and S. affine germinated in response to both smoke-water and KAR₁.

Conclusions

Although many smoke-responsive seeds germinate in the presence of KAR₁, this does not apply universally. This suggests that other chemical(s) in smoke-water may play an important role in stimulating the germination of certain species.     

Icariin Ameliorates Cigarette Smoke Induced Inflammatory Responses via Suppression of NF-κB and Modulation of GR In Vivo and In Vitro

Purpose

To investigate the effects of icariin, a major constituent of flavonoids isolated from the herb Epimedium, on cigarette smoke (CS) induced inflammatory responses in vivo and in vitro.

Methods

In vivo, BALB/c mice were exposed to smoke of 15 cigarettes for 1 h/day, 6 days/week for 3 months and dosed with icariin (25, 50 and 100 mg/kg) or dexamethasone (1 mg/kg). In vitro, A549 cells were incubated with icariin (10, 50 and 100 µM) followed by treatments with CSE (2.5%).

Results

We found that icariin significantly protected pulmonary function and attenuated CS-induced inflammatory response by decreasing inflammatory cells and production of TNF-α, IL-8 and MMP-9 in both the serum and BALF of CS-exposed mice and decreasing production of TNF-α and IL-8 in the supernatant of CSE-exposed A549 cells. Icariin also showed properties in inhibiting the phosphorylation of NF-κB p65 protein and blocking the degradation of IΚB-α protein. Further studies revealed that icariin administration markedly restore CS-reduced GR protein and mRNA expression, which might subsequently contribute to the attenuation of CS-induced respiratory inflammatory response.

Conclusion

Together these results suggest that icariin has anti-inflammatory effects in cigarette smoke induced inflammatory models in vivo and in vitro, possibly achieved by suppressing NF-κB activation and modulating GR protein expression.     

Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction

Background

Proteases have been shown to degrade airway mucin proteins and to damage the epithelium impairing mucociliary clearance. There are increased proteases in the COPD airway but changes in protease-antiprotease balance and mucin degradation have not been investigated during the course of a COPD exacerbation. We hypothesized that increased protease levels would lead to mucin degradation in acute COPD exacerbations.

Methods

We measured neutrophil elastase (NE) and alpha 1 protease inhibitor (A1-PI) levels using immunoblotting, and conducted protease inhibitor studies, zymograms, elastin substrate assays and cigarette smoke condensate experiments to evaluate the stability of the gel-forming mucins, MUC5AC and MUC5B, before and 5–6 weeks after an acute pulmonary exacerbation of COPD (n = 9 subjects).

Results

Unexpectedly, mucin concentration and mucin stability were highest at the start of the exacerbation and restored to baseline after 6 weeks. Consistent with these data, immunoblots and zymograms confirmed decreased NE concentration and activity and increased A1-PI at the start of the exacerbation. After recovery there was an increase in NE activity and a decrease in A1-PI levels. In vitro, protease inhibitor studies demonstrated that serine proteases played a key role in mucin degradation. Mucin stability was further enhanced upon treating with cigarette smoke condensate (CSC).

Conclusion

There appears to be rapid consumption of secreted proteases due to an increase in antiproteases, at the start of a COPD exacerbation. This leads to increased mucin gel stability which may be important in trapping and clearing infectious and inflammatory mediators, but this may also contribute acutely to mucus retention.     

Cigarette smoking,cadmium exposure,and zinc intake on obstructive lung disorder

Background and objective

This study examined whether zinc intake was associated with lower risk of smoking-induced obstructive lung disorder through interplay with cadmium, one of major toxicants in cigarette smoke.

Methods

Data were obtained from a sample of 6,726 subjects aged 40+ from the Third National Health and Nutrition Examination Survey. The forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were measured using spirometry. Gender-, ethnicity-, and age-specific equations were used to calculate the lower limit of normal (LLN) to define obstructive lung disorder as: observed FEV1/FVC ratio and FEV1 below respective LLN. Zinc intake was assessed by questionnaire. Logistic regression analysis was applied to investigate the associations of interest.

Results

The analyses showed that an increased prevalence of obstructive lung disorder was observed among individuals with low zinc intake regardless of smoking status. The adjusted odds of lung disorder are approximately 1.9 times greater for subjects in the lowest zinc-intake tertile than those in the highest tertile (odds ratio = 1.89, 95% confidence interval = 1.22-2.93). The effect of smoking on lung function decreased considerably after adjusting for urinary cadmium. Protective association between the zinc-to-cadmium ratio (log-transformed) and respiratory risk suggests that zinc may play a role in smoking-associated lung disorder by modifying the influence of cadmium.

Conclusions

While zinc intake is associated with lower risk of obstructive lung disorder, the role of smoking cession and/or prevention are likely to be more important given their far greater effect on respiratory risk. Future research is warranted to explore the mechanisms by which zinc could modify smoking-associated lung disease.