Asthma and obesity are frequently associated, and obesity has been considered a factor contributing to both an increase in severity of asthma and to its development. The present document summarizes the proceedings of a symposium held in Montreal, Quebec, on November 2, 2006, under the auspices of the Réseau en santé respiratoire du Fonds de la recherche en santé du Québec in collaboration with the McGill University – Strauss Severe Asthma Program, Université Laval (Quebec City) and Université de Montréal. It includes an overview of the various aspects of the relationships between asthma and obesity with regard to animal models; genetic, hormonal and physiological determinants; influence of comorbidities (eg, sleep apnea syndrome); epidemiology; clinical and psychological features; and management of asthma in the obese population.
Airway inflammation; Asthma; Body mass index; Lung function; Obesity; Sleep apnea syndrome
IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD.
Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. The IL-17 superfamily, which mediates cross-talk between the adaptive and innate immune systems, has been associated with diminished responses to steroids. Increasing evidence supports elevated IL-17 expression in the lung of COPD subjects. However, whether cells of the immune system (systemic) and/or local lung cells are contributing to the elevated IL-17 remains unclear. To address this issue, we utilized a human parenchymal lung tissue explant culture system with cigarette smoke exposure to investigate the expression of IL-17 and the mechanisms involved.
Parenchymal lung tissue removed from 10 non-COPD and 8 COPD patients was sectioned and cultured with different concentrations of cigarette smoke extract (CSE) for 3 or 6 hours. Tissue viability was evaluated by LDH (lactate dehydrogenase) in culture supernatants. Western blot and real-time PCR were performed to evaluate IL-17A/F expression. To investigate the mechanisms, pharmacological inhibitors for MAPK p38, ERK1/2, NF-κB and PI3K pathways were added into the culture media.
No tissue damage was observed after the cigarette smoke exposure for 3 h or 6 h compared with the control media. At the protein level, the expression of both IL-17A (2.4 ± 0.6 fold) and IL-17 F (3.7 ± 0.7 fold) in the tissue from non-COPD subjects was significantly increased by 5% of CSE at 3 h. For COPD subjects, IL-17A/F expression were significantly increased only at 6 h with 10% of CSE (IL-17A: 4.2 ± 0.8 fold; IL-17 F: 3.3 ± 0.8 fold). The increased expression of IL-17A/F is also regulated at the mRNA level. The inhibitors for NF-κB and PI3K pathways significantly inhibited CSE-induced IL-17A/F expression from lung tissue of non-COPD subjects.
We found the evidence that the expression of both IL-17A and IL-17 F is increased by the cigarette smoke exposure in explants from both non-COPD and COPD subjects, supporting that local lung cells contribute IL-17 production. The elevated IL-17A/F expression is dependent on NF-κB and PI3K pathways. These observations add to the growing evidence which suggests that Th17 cytokines play a significant role in COPD.
COPD; IL-17; Cigarette smoke; Tissue explants
Vitamin D deficiency has been declared a public health problem for both adults and children worldwide. Asthma and related allergic diseases are the leading causes of morbidity in children. The objective of this study was to investigate the potential role of Vitamin D deficiency in childhood asthma and other allergic diseases such as allergic rhinitis and wheezing.
Materials and Methods:
This cross-sectional study was conducted in Primary Health Care Centers (PHCs), from March 2012 to October 2013. A total of 2350 Qatari children below the age of 16 were selected from PHCs, and 1833 agreed to participate in this study giving a response rate of (78%). Face-to-face interviews with parents of all the children were based on a questionnaire that included variables such as socio-demographic information, assessment of nondietary covariates, Vitamin D intake, type of feeding, and laboratory investigations. Their health status was assessed by serum Vitamin D (25-hydoxyvitamin D), family history and body mass index.
Most of the children who had asthma (38.5%), allergic rhinitis (34.8%) and wheezing (35.7%) were below 5 years. Consanguinity was significantly higher in parents of children with allergic rhinitis (48.6%), followed by those with asthma (46.4%) and wheezing (40.8%) than in healthy children (35.9%) (P < 0.001). The proportion of severe Vitamin D deficiency was significantly higher in children with wheezing (23.4%), allergic rhinitis (18.5%), and asthma (17%) than in healthy children (10.5%). Exposure to the sun was significantly less in Vitamin D deficient children with asthma (60.3%), allergic rhinitis (62.5%) and wheezing (64.4%) than in controls (47.1%) (P = 0.008). It was found that Vitamin D deficiency was a significant correlate for asthma (odds ratio [OR] =2.31; P < 0.001), allergic rhinitis (OR = 1.59; P < 0.001) and wheezing (relative risk = 1.29; P = 0.05).
The study findings revealed a high prevalence of Vitamin D deficiency in children with asthma and allergic diseases. Vitamin D deficiency was a strong correlate for asthma, allergic rhinitis and wheezing.
Allergic rhinitis; asthma; children; predictors; Qatar; Vitamin D; wheezing
In the past, asthma was considered mainly as a childhood disease. However, asthma is an important cause of morbidity and mortality in the elderly nowadays. In addition, the burden of asthma is more significant in the elderly than in their younger counterparts, particularly with regard to mortality, hospitalization, medical costs or health-related quality of life. Nevertheless, asthma in the elderly is still been underdiagnosed and undertreated. Therefore, it is an imperative task to recognize our current challenges and to set future directions. This project aims to review the current literature and identify unmet needs in the fields of research and practice for asthma in the elderly. This will enable us to find new research directions, propose new therapeutic strategies, and ultimately improve outcomes for elderly people with asthma. There are data to suggest that asthma in older adults is phenotypically different from young patients, with potential impact on the diagnosis, assessment and management in this population. The diagnosis of AIE in older populations relies on the same clinical findings and diagnostic tests used in younger populations, but the interpretation of the clinical data is more difficult. The challenge today is to encourage new research in AIE but to use the existing knowledge we have to make the diagnosis of AIE, educate the patient, develop a therapeutic approach to control the disease, and ultimately provide a better quality of life to our elderly patients.
Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects.
Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22.
Primary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics.
The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.
drug response; genetic variants; pharmacogenomics; vitamin D receptor
The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR−/−) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR−/− mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR−/− mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.
Eosinophilic airway inflammation is heterogeneous in asthmatic patients. We recently described a distinct subtype of asthma defined by the expression of genes inducible by TH2 cytokines in bronchial epithelium. This gene signature, which includes periostin, is present in approximately half of asthmatic patients and correlates with eosinophilic airway inflammation. However, identification of this subtype depends on invasive airway sampling, and hence noninvasive biomarkers of this phenotype are desirable.
We sought to identify systemic biomarkers of eosinophilic airway inflammation in asthmatic patients.
We measured fraction of exhaled nitric oxide (Feno), peripheral blood eosinophil, periostin, YKL-40, and IgE levels and compared these biomarkers with airway eosinophilia in asthmatic patients.
We collected sputum, performed bronchoscopy, and matched peripheral blood samples from 67 asthmatic patients who remained symptomatic despite maximal inhaled corticosteroid treatment (mean FEV1, 60% of predicted value; mean Asthma Control Questionnaire [ACQ] score, 2.7). Serum periostin levels are significantly increased in asthmatic patients with evidence of eosinophilic airway inflammation relative to those with minimal eosinophilic airway inflammation. A logistic regression model, including sex, age, body mass index, IgE levels, blood eosinophil numbers, Feno levels, and serum periostin levels, in 59 patients with severe asthma showed that, of these indices, the serum periostin level was the single best predictor of airway eosinophilia (P = .007).
Periostin is a systemic biomarker of airway eosinophilia in asthmatic patients and has potential utility in patient selection for emerging asthma therapeutics targeting TH2 inflammation.
Asthma; biomarker; sputum; bronchoscopy; eosinophil; TH2; IL-13; periostin; IgE; Feno
Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17–induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.
Glucocorticoid-induced TNF receptor-related protein ligand (GITRL), a ligand for the T cell co-stimulatory molecule GITR, is expressed by keratinocytes and involved in chemokine production. The expression of GITRL in skin inflammation is unknown.
This study investigated cytokine regulation of keratinocyte GITRL expression.
GITRL expression was evaluated in cytokine treated human epidermal keratinocytes (HEK)s, murine PAM 212 cell line, murine and human skin explants by real time PCR, flow cytometry and immunostaining. Functional responses to GITR fusion protein were examined by real time PCR and ELISA. GITRL expression in AD and psoriasis was studied by immunohistochemistry.
Skin biopsies from STAT6VT transgenic mice, which develop spontaneous atopic skin inflammation, were found by immunofluoresence, to have increased keratinocyte GITRL expression. Exposure to Th2 cytokines augmented GITRL mRNA expression in the murine PAM 212 keratinocytic cell line and murine skin explants. In contrast, GITRL mRNA and protein expression was only increased in HEKs and human skin explants in the presence of the combination of TNFα and Th2 cytokines. A synergistic effect of Th2 cytokines and GITR fusion protein on production of CCL17, the Th2 chemokine, by murine keratinocytes was demonstrated. Immunohistochemical staining showed that acute AD lesions have increased expression of GITRL compared with normal skin, chronic AD lesions and psoriatic plaques.
Conclusions and Clinical Relevance
Our studies demonstrate that GITRL expression is augmented by Th2 cytokines and TNFα in keratinocytes. Increased GITRL expession in acute AD skin lesions is shown. This data suggests a link between cytokine regulated keratinocyte GITRL expression and its role in inflammatory responses in AD.
Skin; Th2 cytokines; atopic dermatitis
Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.
In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.
Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.
The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.
Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.
Asthma; Eosinophils; Th17 cytokines; Pro-fibrotic cytokines; TGF-β; IL-11
asthma pathogenesis; autophagy; lung function; polymorphism; SNP; ATG5; autophagosome
Asthma is a common disease of children with a complex genetic origin. Understanding the genetic basis of asthma susceptibility will allow disease prediction and risk stratification.
We sought to identify asthma susceptibility genes in children.
A nested case-control genetic association study of children of Caucasian European ancestry from a birth cohort was conducted. Single nucleotide polymorphisms (SNPs, n=116,024) were genotyped in pools of DNA samples from cohort children with physician-diagnosed asthma (n=112) and normal controls (n=165). A genomic region containing the ATPAF1 gene was significantly associated with asthma. Additional SNPs within this region were genotyped in individual samples from the same children and in eight independent study populations consisting of Caucasian, African American, Hispanic, or other ancestries. SNPs were also genotyped or imputed in two consortia control populations. ATPAF1 expression was measured in bronchial biopsies from asthmatics and controls.
Asthma was associated with a cluster of SNPs and SNP haplotypes containing the ATPAF1 gene with two SNPs achieving significance at a genome-wide level (p=2.26×10−5 to 2.2×10−8). Asthma severity was also associated with SNPs and haplotypes in the primary population. SNP and/or gene-level associations were confirmed in the four non-Hispanic populations. Haplotype associations were confirmed in the non-Hispanic populations (p=0.045 to 0.0009). ATPAF1 total RNA expression was significantly (p<0.01) higher in bronchial biopsies from asthmatics than controls.
Genetic variation in the ATPAF1 gene predisposes children of different ancestry to asthma.
asthma; ATPAF1; children; gene; genetic; genome-wide association; purinergic; respiratory; single nucleotide polymorphism; SNP
Inhaled corticosteroids represent the most common treatment for asthma. Although most asthmatic patients respond well, a significant proportion of severe asthmatics require higher doses or even fail to respond to oral or inhaled corticosteroids. We previously reported that glucocorticoid receptor-beta is associated with corticosteroid resistance in airway epithelial cells from asthmatic patients and that Th-17 cytokines increase steroid insensitivity via a mechanism involving GR-beta upregulation. We aim to investigate whether IL-17A and F cytokines enhance steroid unresponsiveness in PBMCs from normal subjects and severe asthmatics via the upregulation of GR-beta isoform.
PBMCs were cultured for 48 hours in the presence or absence of IL-2, IL-4, IL-17A, IL-17F or IL-23 cytokines. Expression of GR-alpha, GR-beta, GILZ and IL-6 was determined using Q-RT-PCR and/or Western blotting. Response to Dexamethasone was determined on the inhibition of PHA-induced proliferation by Dexamethasone (IC50) using either 3H-thymidine or CFSE-labelled cells. Response of the cells to Dexamethasone-induced apoptosis was determined by Annexin-V staining.
Treatment of PBMCs with IL-17A+IL-17F combined significantly decreased the mRNA expression of GR-alpha while that of GR-beta was significantly upregulated. IL-2+IL-4 in combination significantly decreased GR-alpha expression but had no effect on GR-beta receptor expression. IL17A+IL17F+IL23 combined induced the highest ratio of GR-beta/GR-alpha in PBMC from normal subjects. Either IL-17A+F or IL-2+IL-4 combinations significantly decreased the inhibitory effect of Dexamethasone on PBMC proliferation (IL-17A+F IC50 = 190 nM Dex; IL-2+4 IC50 = 1060 nM Dex), when compared to the control without cytokine stimulation. In the presence of Dexamethasone, IL-2+IL-4 but not IL-17A+IL-17F, inhibited the expression of the glucocorticoid-inducible leucine zipper gene (GILZ) in PBMCs from both normal (60%) and asthmatics (45–50%), which was correlated with significantly higher apoptosis in cells stimulated with IL-2+IL-4.
IL-17A, IL-17F, IL-2, and IL-4, which are known to be upregulated in the blood and lung tissue of asthmatics, contribute to steroid insensitivity of severe asthmatic patients by modulating the expression of GR-alpha and GR-beta receptors on peripheral blood PBMCs. GR-beta could protect PBMCs from Dex-induced apoptosis. Furthermore, the increased GR-beta/GR-alpha ratios by both IL-17A+F and IL-2+4 cytokines correlates with the decreased inhibitory effect of Dexamethasone on PHA-induced PBMC proliferation.
B lymphocytes are known to be important cytokine sources in inflammation and play a pathogenic role by producing autoantibodies in a number of chronic immunological diseases. However, B cell depletion therapy induced an exacerbation of symptoms in some patients with autoimmune disorders, revealing that B cells play a critical anti-inflammatory role mediated by IL-10 release. We therefore investigated the human B cell regulatory subset producing IL-10 in response to stimulation.
Highly purified B cells were obtained from tonsils by using a multiple-step separation procedure which included rosette depletion, adherence depletion, CD3+ cell magnetic-activated depletion and CD19+ magnetic-activated positive cell selection. CD20+ purity was verified by flow cytometry. The CD19+CD20+ B cells were stimulated with CpG oligonucleotide, IL-4, IFN-gamma, anti-CD40, IL-17A and IL-17F, either alone or in combination. The expression of both IL-6 and IL-10 mRNA was analyzed by quantitative RT-PCR and by ELISA. B regulatory cell subsets expressing IL-10 and the markers CD5 and CD1d were quantified by FACS analysis. B cell proliferation was determined by 3H thymidine incorporation or CFSE labeling.
Expression of IL-10 mRNA and protein in purified B cells from tonsils was weakly stimulated by anti-CD40 antibody, CpG oligonucleotide or with IL-17. When B cells were simultaneously stimulated with IL-17, anti-CD40 antibody and CpG oligonucleotide, the mRNA and protein expression of IL-10 was strongly increased (n = 3; P ≤ 0.001). B cells proliferation was also significantly increased. In contrast, stimulation with IL-4 alone or in combination with anti-CD40 antibody, decreased the expression of IL-10 (n = 3; P ≤ 0.001).
TLR9 receptor stimulation synergizes with CD40 and IL-17 receptors stimulation in the induced proliferation and potent release of IL-10 cytokine while decreasing IL-6 production in B cells. These novel findings provide evidence that B lymphocytes might be an important source of the anti-inflammatory IL-10 cytokine, and provide novel evidence that stimulation of B lymphocytes with IL-17 cytokine could be an important regulatory mechanism in immune responses.
Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. Its is well documented that the smooth muscle mass in asthmatic airways is increased due to hypertrophy and hyperplasia of the ASM cells. Moreover, eosinophils have been proposed in different studies to play a major role in airway remodeling. Here, we hypothesized that eosinophils modulate the airways through enhancing ASM cell proliferation. The aim of this study is to examine the effect of eosinophils on ASM cell proliferation using eosinophils isolated from asthmatic and normal control subjects.
Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. ASM cells were incubated with eosinophils or eosinophil membranes and ASM proliferation was estimated using thymidine incorporation. The mRNA expression of extracellular matrix (ECM) in ASM cells was measured using quantitative real-time PCR. The effect of eosinophil-derived proliferative cytokines on ASM cells was determined using neutralizing antibodies. The role of eosinophil derived Cysteinyl Leukotrienes in enhancing ASM was also investigated.
Co-culture with eosinophils significantly increased ASM cell proliferation. However, there was no significant difference in ASM proliferation following incubation with eosinophils from asthmatic versus normal control subjects. Co-culture with eosinophil membranes had no effect on ASM proliferation. Moreover, there was no significant change in the mRNA expression of ECM proteins in ASM cells following co-culture with eosinophils when compared with medium alone. Interestingly, blocking the activity of cysteinyl Leukotries using antagonists inhibited eosinophil-derived ASM proliferation.
Eosinophils enhances the proliferation of ASM cells. This role of eosinophil does not seem to depend on ASM derived ECM proteins nor on Eosinophil derived TGF-β or TNF-α. Eosinophil seems to induce ASM proliferation via the secretion of Cysteinyl Leukotrienes.
Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8+ T cells within the central and peripheral airways. We hypothesized that the CD8+ T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways.
Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8+ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology.
No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8+ T cells. Exposure of CD8+ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8+ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10.
Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8+ T cells in COPD. CD8+ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8+ T cells in COPD.
COPD; Toll-like receptors; CD8+ T cell; cigarette smoke; cytokine
Airway remodelling refers to the structural changes that occur in both large and small airways relevant to miscellaneous diseases including asthma. In asthma, airway structural changes include subepithelial fibrosis, increased smooth muscle mass, gland enlargement, neovascularization and epithelial alterations. Although controversial, airway remodelling is commonly attributed to an underlying chronic inflammatory process. These remodelling changes contribute to thickening of airway walls and, consequently, lead to airway narrowing, bronchial hyper-responsiveness, airway edema and mucous hypersecretion. Airway remodelling is associated with poor clinical outcomes among asthmatic patients. Early diagnosis and prevention of airway remodelling has the potential to decrease disease severity, improve control and prevent disease expression. The relationship between structural changes and clinical and functional abnormalities clearly deserves further investigation. The present review briefly describes the characteristic features of airway remodelling observed in asthma, its clinical consequences and relevance for physicians, and its modulation by therapeutic approaches used in the treatment of asthmatic patients.
Allergy; Asthma; Remodelling; Rhinitis
Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease of the lung. The nature of the immune reaction in COPD raises the possibility that IL-17 and related cytokines may contribute to this disorder. This study analyzed the expression of IL-17A and IL-17F as well as the phenotype of cells producing them in bronchial biopsies from COPD patients.
Bronchoscopic biopsies of the airway were obtained from 16 COPD subjects (GOLD stage 1-4) and 15 control subjects. Paraffin sections were used for the investigation of IL-17A and IL-17F expression in the airways by immunohistochemistry, and frozen sections were used for the immunofluorescence double staining of IL-17A or IL-17F paired with CD4 or CD8. In order to confirm the expression of IL-17A and IL-17F at the mRNA level, a quantitative RT-PCR was performed on the total mRNA extracted from entire section or CD8 positive cells selected by laser capture microdissection.
IL-17F immunoreactivity was significantly higher in the bronchial biopsies of COPD patients compared to control subjects (P < 0.0001). In the submucosa, the absolute number of both IL-17A and IL-17F positive cells was higher in COPD patients (P < 0.0001). After adjusting for the total number of cells in the submucosa, we still found that more cells were positive for both IL-17A (P < 0.0001) and IL-17F (P < 0.0001) in COPD patients compared to controls. The mRNA expression of IL-17A and IL-17F in airways of COPD patients was confirmed by RT-PCR. The expression of IL-17A and IL-17F was co-localized with not only CD4 but also CD8, which was further confirmed by RT-PCR on laser capture microdissection selected CD8 positive cells.
These findings support the notion that Th17 cytokines could play important roles in the pathogenesis of COPD, raising the possibility of using this mechanism as the basis for novel therapeutic approaches.
Chronic Obstructive Pulmonary Disease; IL-17; Tc17 cells
Airway inflammation in chronic obstructive pulmonary disease (COPD) is characterised by infiltration of CD8+ T cells and CD68+ macrophages and an increased number of neutrophils, whereas few studies have described the presence of eosinophils. Although the anti‐inflammatory effects of corticosteroids in stable COPD are unclear, recent studies suggest that combination therapy could be beneficial. A study was therefore undertaken to evaluate combined salmeterol/fluticasone propionate (SFC) and fluticasone propionate (FP) alone on inflammatory cells in the airways of patients with COPD.
Patients were treated in a randomised, double blind, parallel group, placebo‐controlled trial with either a combination of 50 µg salmeterol and 500 µg FP twice daily (SFC, n = 19, 19 men, mean age 62 years), 500 µg FP twice daily (n = 20, 15 men, mean age 64 years) or placebo (n = 21, 17 men, mean age 66 years) for 3 months. At the start and end of treatment bronchoscopy with bronchial biopsies was performed and the numbers of CD8+ T lymphocytes, CD68+ macrophages, neutrophils and eosinophils were measured.
CD8+ cells were significantly reduced by SFC compared with placebo (difference −98.05 cells/mm2; 95% CI −143.14 to −52.9; p<0.001). Such a marked effect was not seen with FP alone (−44.67 cells/mm2; 95% CI −90.92 to 1.57; p = 0.06). CD68+ macrophages were also reduced by SFC compared with placebo (difference −31.68 cells/mm2; 95% CI −61.07 to −2.29; p = 0.03) but not by FP. SFC did not significantly change neutrophils and eosinophils compared with placebo.
SFC has airway anti‐inflammatory effects not seen with inhaled corticosteroids alone.
Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma.
Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax.
Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay.
Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion.
Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.
asthma; airway hyperresponsiveness; gene expression; smooth muscle; myosin