Interleukin-17A and vascular remodelling in severe asthma; lack of evidence for a direct role.

BACKGROUND: Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. OBJECTIVE: To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. METHODS: Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. RESULTS: Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. CONCLUSIONS & CLINICAL RELEVANCE: Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets.

Human ß-defensin-3 induces IL-8 release and apoptosis in airway smooth muscle cells.

BACKGROUND: Human airway smooth muscle cells (ASMCs) may have a pro-inflammatory role through the release of inflammatory mediators. Increasing evidence indicates that human ß-defensins (HBDs) are related to pathogenesis of asthma. OBJECTIVES: To examine the plasma level of HBD-1, HBD-2 and HBD-3 in asthmatic patients and the expression of their mouse orthologues in the lung tissue of a mouse model of chronic severe asthma. Further to investigate the effect of HBD-3 on the release of the pro-inflammatory cytokine IL-8 and to explore the mechanisms. METHODS: The plasma levels of HBD-1, HBD-2 and HBD-3 from 34 healthy controls and 25 asthmatic patients were determined by ELISA. The expression of mouse ß-defensins MBD-1, MBD-3 and MBD-14 in the lung tissue of asthmatic mice was detected by Western blot. The ASMCs were cultured with HBD-3 for 24 hour, and then the supernatant level of IL-8 was evaluated by ELISA and the cell viability was examined by WST-1 assay. The signalling pathway was investigated with blocking antibodies or pharmacological inhibitors. RESULTS: The plasma levels of HBD-1 and HBD-3 were elevated in asthmatic patients, and the expression of MBD-14, the mouse orthologue for HBD-3, was increased in asthmatic mice. HBD-3-induced IL-8 production in a CCR6 receptor-specific manner and was dependent on multiple signalling pathways. Moreover, HBD-3-induced cell apoptosis concurrently, which was dependent on the ERK1/2 MAPK pathway. Mitochondrial ROS regulated both HBD-3-induced IL-8 production and cell apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE: These observations provide clear evidence of an important new mechanism for the promotion of airway inflammation and tissue remodelling with potential relevance for the treatment of asthma.

Phenotyping of difficult asthma using longitudinal physiological and biomarker measurements reveals significant differences in stability between clusters.

BACKGROUND: Although the heterogeneous nature of asthma has prompted asthma phenotyping with physiological or biomarker data, these studies have been mostly cross-sectional. Longitudinal studies that assess the stability of phenotypes based on a combination of physiological, clinical and biomarker data are currently lacking. Our objective was to assess the longitudinal stability of clusters derived from repeated measures of airway and physiological data over a 1-year period in moderate and severe asthmatics. METHODS: A total of 125 subjects, 48 with moderate asthma (MA) and 77 with severe asthma (SA) were evaluated every 3 months and monthly, respectively, over a 1-year period. At each 3-month time point, subjects were grouped into 4 asthma clusters (A, B, C, D) based on a combination of clinical (duration of asthma), physiological (FEV1 and BMI) and biomarker (sputum eosinophil count) variables, using k-means clustering. RESULTS: Majority of subjects in clusters A and C had severe asthma (93 % of subjects in cluster A and 79.5 % of subjects in cluster C at baseline). Overall, a total of 59 subjects (47 %) had stable cluster membership, remaining in clusters with the same subjects at each evaluation time. Cluster A was the least stable (21 % stability) and cluster B was the most stable cluster (71 % stability). Cluster stability was not influenced by changes in the dosage of inhaled corticosteroids. CONCLUSION: Asthma phenotyping based on clinical, physiologic and biomarker data identified clusters with significant differences in longitudinal stability over a 1-year period. This finding indicates that the majority of patients within stable clusters can be phenotyped with reasonable accuracy after a single measurement of lung function and sputum eosinophilia, while patients in unstable clusters will require more frequent evaluation of these variables to be properly characterized.

Exacerbation risk in severe asthma is stratified by inflammatory phenotype using longitudinal measures of sputum eosinophils.

BACKGROUND: Airway inflammatory phenotyping is increasingly applied to subjects with asthma. However, its relationship to clinical outcomes in difficult asthma is incompletely elucidated. OBJECTIVE: The goal of our study was to determine the relationship between exacerbation rates and phenotypes of difficult asthma based on the longitudinal measures of sputum eosinophils and neutrophils. METHODS: Subjects in the longitudinal observational study from two tertiary care centres that completed 1 year of observation and provided at least three sputum samples were classified by inflammatory phenotypes using previously established thresholds. Kaplan-Meier curves and univariable and multivariable Cox proportional hazard models were used to determine the association between inflammatory phenotypes and exacerbation rate. RESULTS: During the study, 115 exacerbations occurred in 73 severe asthmatic subjects. Subjects with the persistently eosinophilic phenotype had a significantly shorter time to first exacerbation and greater risk of exacerbation over a 1-year period than those with the non-eosinophilic phenotype based on the univariable and multivariable Cox proportional hazard model (hazard ratio [HR], 3.24; 95% confidence interval [CI], 1.35-7.72; adjusted HR, 3.90; 95% CI, 1.34-11.36). No significant differences in time to first exacerbation or exacerbation risk over a 1-year period were observed among the neutrophilic phenotypes. CONCLUSIONS: The persistent eosinophilic phenotype is associated with increased exacerbation risk compared with the non-eosinophilic phenotype in severe asthma. No differences in time to first exacerbation or exacerbation risk over a 1-year period were detected among neutrophilic phenotypes.

Human bronchial and parenchymal fibroblasts display differences in basal inflammatory phenotype and response to IL-17A.

BACKGROUND: Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodelling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. OBJECTIVE: To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodelling or the ability of these fibroblasts to respond to IL-17A. METHODS: Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. RESULTS: At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. CONCLUSIONS AND CLINICAL RELEVANCE: These findings provide insight into the inflammatory and remodelling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A.

Monocyte-derived fibrocytes induce an inflammatory phenotype in airway smooth muscle cells.

BACKGROUND: Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma, but the pathological significance is unknown. OBJECTIVE: We sought to explore whether FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects. METHODS: Fibrocytes were isolated from CD14+ monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects was analysed by (3) H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-ß1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC + FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC + FC, where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). RESULTS: Fibrocytes did not affect ASMC proliferation and expression of TGF-ß1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8/IL-6 and produced more IL-8/IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production. CONCLUSIONS AND CLINICAL RELEVANCE: Fibrocytes promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.

IL-21 receptor signalling partially mediates Th2-mediated allergic airway responses.

BACKGROUND: Interleukin-21 (IL-21) has been implicated in the development of Th2-mediated immune responses; however, the exact role it plays in allergic diseases is not well understood. OBJECTIVE: To elucidate the contribution of IL-21 receptor signalling to Th2-dependent immune responses in the lung. METHODS: We compared allergic airway responses in wild-type BALB/c and Il21r-deficient mice exposed to local airway challenge with house dust mite (HDM). RESULTS: We demonstrate that IL-21R-deficiency reduces HDM-driven airway hyperresponsiveness (AHR) with only partial effects on airway inflammation. Concomitant with the reduction in AHR in Il21r-deficient mice, significant suppression was observed in protein levels of the Th2 cytokines IL-4, and IL-13. In contrast, IL-21R-deficiency was associated with an increase in PBS- and allergen-driven IgE levels, while IgG1 and IgG2a levels were decreased. Moreover, our results suggest that IL-21 may contribute to AHR through its ability to both directly induce Th2 cell survival and to impair regulatory T-cell suppression of Th2 cytokine production. Importantly, we show that IL-21-positive cells are increased in the bronchial mucosa of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that IL-21 plays an important role in the allergic diathesis by enhancing Th2 cytokine production through multiple mechanisms including the suppression of Treg inhibitory effects on Th2 cell cytokine production.

Effect of topical corticosteroids on allergic airway inflammation and disease severity in obstructive sleep apnoea.

BACKGROUND: The incidence of sleep-related breathing disorders is correlated with lower and upper airway inflammatory diseases, such as asthma and allergic rhinitis. We hypothesized that corticosteroids treatment would lead to a greater reduction in disease severity in obstructive sleep apnoea syndrome (OSAS) patients with concomitant allergic rhinitis vs. non-allergic OSAS patients by reducing the level of inflammation in upper airway tissues. OBJECTIVE: This study was performed to determine whether treatment with intranasal corticosteroids could reduce upper airway inflammation and improve sleep parameters in obstructive sleep apnoea syndrome patients with or without concomitant allergic rhinitis. METHODS: Obstructive sleep apnoea syndrome patients with (n = 34) or without (n = 21) documented allergic rhinitis voluntarily enrolled in the study and were assessed at baseline and after corticosteroids treatment for 10-12 weeks. Sleep studies were performed and biopsies were obtained from the inferior turbinate, nasopharynx, and uvula. The apnoea-hypopnoea index, sleep quality, and level of daytime alertness were determined, and immunocytochemistry was used to phenotype tissue inflammation. RESULTS: Standard sleep indices improved following treatment in the entire cohort of obstructive sleep apnoea syndrome patients, with greater improvement seen in the allergic rhinitis group. Allergic rhinitis patients demonstrated significantly improved O2 saturation and a lower supine apnoea-hypopnoea index score after corticosteroid treatment; similar improvements were not seen in the non-allergic rhinitis group. Eosinophilia was detected at all three sites in the allergic rhinitis group, but not in the non-allergic rhinitis group. Following treatment, fewer eosinophils and CD4 lymphocytes were documented at all three biopsy sites in the allergic group; the reduction in inflammation was less apparent in the non-allergic rhinitis group. CONCLUSION: This study has provided important molecular and clinical evidence regarding the ability of corticosteroids to reduce upper airway inflammation and improve obstructive sleep apnoea syndrome morbidity patients with concomitant allergic rhinitis.

PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production.

Studies examining the role of programmed death 1 (PD-1) ligand 2 (PD-L2)/PD-1 in asthma have yielded conflicting results. To clarify its role, we examined the PD-L2 expression in biopsies from human asthmatics and the lungs of aeroallergen-treated mice. PD-L2 expression in bronchial biopsies correlated with the severity of asthma. In mice, allergen exposure increased PD-L2 expression on pulmonary myeloid dendritic cells (DCs), and PD-L2 blockade diminished allergen-induced airway hyperresponsiveness (AHR). By contrast, PD-1 blockade had no impact, suggesting that PD-L2 promotes AHR in a PD-1-independent manner. Decreased AHR was associated with enhanced serum interleukin (IL)-12 p40, and in vitro stimulation of DCs with allergen and PD-L2-Fc reduced IL-12 p70 production, suggesting that PD-L2 inhibits allergen-driven IL-12 production. In our model, IL-12 did not diminish T helper type 2 responses but rather directly antagonized IL-13-inducible gene expression, highlighting a novel role for IL-12 in regulation of IL-13 signaling. Thus, allergen-driven enhancement of PD-L2 signaling through a PD-1-independent mechanism limits IL-12 secretion, exacerbating AHR.

Do small airway abnormalities characterize asthma phenotypes? In search of proof.

The role of small airway abnormalities in asthma pathogenesis has been extensively studied and debated for several decades. However, whether or not small airway abnormalities play a relevant role in specific phenotypes of asthmatic patients and contribute to clinical presentation is largely unknown. In the present review, we evaluated available data on the role of small airways in severe asthma, with a further focus on asthma in smokers and asthma in the elderly. These phenotypes are characterized by a poor response to treatment and they can represent a model of greater small airway impairment. In severe asthmatics, small airway involvement has been shown through evidence of both distal inflammation and of increased air trapping. The few available data on asthmatics who smoke, and elderly asthmatics, similarly suggests that small airway abnormalities contribute to the pathogenesis of the disease. In this perspective, there could be a rationale for specifically assessing small airway impairment in these patients and for clinical studies evaluating whether pharmacological approaches targeting the more peripheral airways result in clinical benefits beyond conventional therapy.

Pathogenesis of severe asthma.

Patients with severe asthma have asthma symptoms which are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction. Epidemiological and clinical evidences point to the fact that severe asthma is not a single phenotype. Cluster analyses have identified subclasses of severe asthma using parameters such as patient characteristics, and cytokine profiles have also been useful in classifying moderate and severe asthma. The IL-4/IL-13 signalling pathway accounts for the symptoms experienced by a subset of severe asthmatics with allergen-associated symptoms and high serum immunoglobulin E (IgE) levels, and these patients are generally responsive to anti-IgE treatment. The IL-5/IL-33 signalling pathway is likely to play a key role in the disease pathogenesis of those who are resistant to high doses of inhaled corticosteroid but responsive to systemic corticosteroids and anti-IL5 therapy. The IL-17 signalling pathway is thought to contribute to 'neutrophilic asthma'. Although traditionally viewed as players in the defence mechanism against viral and intracellular bacterial infection, mounting evidence supports a role for Th1 cytokines such as IL-18 and IFN-γ in severe asthma pathogenesis. Furthermore, these cytokine signalling pathways interact to contribute to the spectrum of clinical pathological outcomes in severe asthma. To date, glucocorticoids are the most effective anti-asthma drugs available, yet severe asthma patients are typically resistant to the effects of glucocorticoids. Glucocorticoid receptor dysfunction and histone deacetylase activity reduction are likely to contribute to glucocorticoid resistance in severe asthma patients. This review discusses recent development in different cytokine signalling pathways, their interactions and steroid resistance, in the context of severe asthma pathogenesis.

Induction of GITRL expression in human keratinocytes by Th2 cytokines and TNF-α: implications for atopic dermatitis.

BACKGROUND: 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 remains unknown. OBJECTIVES: This study investigated cytokine regulation of keratinocyte GITRL expression. METHODS: Glucocorticoid-induced TNF receptor 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. RESULTS: 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 TNF-α 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 TNF-α in keratinocytes. Increased GITRL expression in acute AD skin lesions is shown. This observation suggests a link between cytokine-regulated keratinocyte GITRL expression and its role in inflammatory responses in AD.

Expression and regulation of CCL15 by human airway smooth muscle cells.

BACKGROUND: Structural cells are an important reservoir of chemokines that coordinate the influx of various immune cells to the lungs of asthmatics. Airway smooth muscle cells (ASMC) are an important source of these chemokines. CCL15 is a recently described chemo-attractant for neutrophils, eosinophils, monocytes and lymphocytes. OBJECTIVE: To determine the production and the regulation of CCL15 by ASMC and to investigate its production in asthmatic airways. METHODS: Human ASMC were obtained from main bronchial airway segments of patients with mild, moderate and severe asthma. To induce chemokine production, cells were incubated with IL-4, IL-13, TNF-α or IFN-γ in presence or absence of dexamethasone, mithramycin A (SP-1 inhibitor) or the IKK-2 inhibitor, AS602868. CCL15 mRNA expression was evaluated by real-time PCR. Immunoreactive CCL15 was detected by immuno-fluorescence and CCL15 protein concentration in the supernatant was measured using ELISA. RESULTS: CCL15 is constitutively expressed in human ASMC and is strongly up-regulated by TNF-α. This up-regulation is inhibited by dexamethasone, mithramycin A and AS602868. TNF-α-induced CCL15 levels can be synergistically enhanced by the presence of IFN-γ, at both the transcriptional and translation level. This synergism is NF-κB-dependent. Asthmatic biopsies demonstrated higher expression of CCL15 compared with non-asthmatic controls. CONCLUSION AND CLINICAL RELEVANCE: Our results show that ASMC are a potent source of CCL15 in the airways and may directly participate in the recruitment of inflammatory cells to asthmatic airways. Targeting the production of CCL15 by ASMC might reduce the inflammatory response within the airways of asthmatic patients.

Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip.

The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

Fabrication of three-dimensional scaffolds using precision extrusion deposition with an assisted cooling device.

In the field of biofabrication, tissue engineering and regenerative medicine, there are many methodologies to fabricate a building block (scaffold) which is unique to the target tissue or organ that facilitates cell growth, attachment, proliferation and/or differentiation. Currently, there are many techniques that fabricate three-dimensional scaffolds; however, there are advantages, limitations and specific tissue focuses of each fabrication technique. The focus of this initiative is to utilize an existing technique and expand the library of biomaterials which can be utilized to fabricate three-dimensional scaffolds rather than focusing on a new fabrication technique. An expanded library of biomaterials will enable the precision extrusion deposition (PED) device to construct three-dimensional scaffolds with enhanced biological, chemical and mechanical cues that will benefit tissue generation. Computer-aided motion and extrusion drive the PED to precisely fabricate micro-scaled scaffolds with biologically inspired, porosity, interconnectivity and internal and external architectures. The high printing resolution, precision and controllability of the PED allow for closer mimicry of tissues and organs. The PED expands its library of biopolymers by introducing an assisting cooling (AC) device which increases the working extrusion temperature from 120 to 250 °C. This paper investigates the PED with the integrated AC's capabilities to fabricate three-dimensional scaffolds that support cell growth, attachment and proliferation. Studies carried out in this paper utilized a biopolymer whose melting point is established to be 200 °C. This polymer was selected to illustrate the newly developed device's ability to fabricate three-dimensional scaffolds from a new library of biopolymers. Three-dimensional scaffolds fabricated with the integrated AC device should illustrate structural integrity and ability to support cell attachment and proliferation.

Increased upper airway cytokines and oxidative stress in severe obstructive sleep apnoea.

Inflammation may contribute to upper airway pathophysiology in obstructive sleep apnoea (OSA). Our objective was to compare upper airway pro-inflammatory cytokine expression, oxidative stress and connective tissue deposition in severe (n = 25) versus mild (n = 17) OSA patients. Upper airway surgical specimens were separated by predominance of either mucosal or muscle tissue. Expression levels of interleukin (IL)-1α, IL-6, interferon-γ, RANTES (regulated on activation, normal T-cell expressed and secreted), transforming growth factor (TGF)-ß and l-selectin were measured by ribonuclease protection assay. Oxidative stress was assessed via protein carbonyl group detection by immunoblotting. Histochemistry was employed for immunolocalisation of selected cytokines and connective tissue morphometry. In the severe OSA group, expression of IL-1α, IL-6 and TGF-ß was significantly higher in mucosa-predominant tissues, whereas in muscle-predominant specimens, RANTES expression was greater in severe OSA. Increased protein carbonylation was observed in severe OSA within both mucosal and muscle compartments. Immunohistochemistry localised TGF-ß to submucosal and perimuscular inflammatory cells, while IL-6 was primarily localised to myocytes. Consistent with the pro-fibrotic cytokine profile observed in mucosa-predominant tissue, morphometric analysis revealed greater submucosal and perimuscular connective tissue in severe OSA subjects. There is increased pro-inflammatory and pro-fibrotic cytokine expression, oxidative stress, and connective tissue deposition in upper airway tissues from severe versus mild OSA patients.

Children with atopic histories exhibit impaired lipopolysaccharide-induced Toll-like receptor-4 signalling in peripheral monocytes.

BACKGROUND: The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll-like receptor (TLR)-4 on CD4(+) cells in nasal mucosa. OBJECTIVE: To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4(+) leucocytes. METHODS: Peripheral blood mononuclear cells from children (aged 2-18) and adults with or without a history of atopic conditions were cultured with/without IL-4 or LPS for up to 24 h. Expression of surface TLR-4, CD14, CD4, CD3, as well as of intracellular phosphorylated (p42/p44) ERK and p38 mitogen-activated protein kinase (MAPK) were assessed by flow cytometry. RESULTS: A history of atopy in children was associated with impaired LPS-induced TLR-4-dependent phosphorylation of (p42/44) ERK and p38 MAPK by CD4(+) monocytes. Decreased LPS signalling was reproduced by pre-incubation of control cells with recombinant IL-4. LPS stimulation also decreased TLR-4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4(+) T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non-atopic children, TLR-4 expression on monocytes of children with atopic histories decreased as a function of age. CONCLUSIONS: This study provides evidence for defective LPS recognition on circulating CD4(+) leucocytes of subjects with atopic histories compared with those from non-atopic children. CD4(+) TLR4(+) monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR-4-mediated innate immune function compared with non-atopic children.