Avian Influenza Viruses Infect Primary Human Bronchial Epithelial Cells Unconstrained by Sialic Acid α2,3 Residues

Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

<p>Abstract</p> <p>Background</p> <p>Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂is also produced endogenously in the lung during acute inflammatory responses. NO₂can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺cells in NO₂-promoted allergic sensitization.</p> <p>Methods</p> <p>We systemically depleted CD11c⁺cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺cells from wildtype mice were studied after exposure to NO₂and ovalbumin for cellular phenotype by flow cytometry and <it>in vitro </it>cytokine production.</p> <p>Results</p> <p>Transient depletion of CD11c⁺cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂exposure. By 48 hours, CD11c⁺MHCII⁺DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^-and CD11c⁺CD11b⁺pulmonary cells exposed to NO₂<it>in vivo </it>increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺CD11c⁺MHCII⁺DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺T cells from naïve mice and CD11c⁺pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 <it>in vitro </it>and augmented antigen-induced IL-5 production.</p> <p>Conclusions</p> <p>CD11c⁺cells are critical for NO₂-promoted allergic sensitization. NO₂exposure causes pulmonary CD11c⁺cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.</p

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Abstract Background Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis. Methods Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31. Results IL-13 induced increasing levels of MUC5AC protein, and TGF-β2, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β2, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β2 neutralizing antibody reversed the increase in collagen content and SHG signal. Conclusion Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β2 at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β2 secretion from the airway epithelium.</p

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background: Inflammatory cytokines (e. g. IL-13) and mechanical perturbations (e. g. scrape injury) to the epithelium release profibrotic factors such as TGF-beta(2), which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis. Methods: Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were cocultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22-25 or days 28-31. Results: IL-13 induced increasing levels of MUC5AC protein, and TGF-beta(2), while decreasing beta-Tubulin IV at day 22 and 28 in the NHBE. TGF-beta(2), soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-beta(2) neutralizing antibody reversed the increase in collagen content and SHG signal. Conclusion: Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-beta(2) at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-beta(2) secretion from the airway epithelium

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background: Inflammatory cytokines (e. g. IL-13) and mechanical perturbations (e. g. scrape injury) to the epithelium release profibrotic factors such as TGF-beta(2), which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis. Methods: Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were cocultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22-25 or days 28-31. Results: IL-13 induced increasing levels of MUC5AC protein, and TGF-beta(2), while decreasing beta-Tubulin IV at day 22 and 28 in the NHBE. TGF-beta(2), soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-beta(2) neutralizing antibody reversed the increase in collagen content and SHG signal. Conclusion: Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-beta(2) at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-beta(2) secretion from the airway epithelium

(A) Sircol soluble collagen assay was performed as described in the , which quantifies the amount of soluble collagen in the cell culture supernatant and newly synthesized salt soluble collagen in the matrix

The amount of soluble collagen secreted in the media at day 25 in the IL-13 pre-treated NHBE at 1 and 10 ng/ml co-cultured with NHLF is augmented as compared to the untreated NHBE co-culture; * p < 0.01 and addition of TGFβneutralizing antibody (10 μg/ml) abolishes this increase (p < 0.01 compared to respective condition without TGFβneutralizing antibody). (B) At day 25 there is an increase in newly synthesized salt soluble collagen content in the matrix in the IL-13 pre-treated NHBE at 1 and 10 ng/ml followed by co-culture with NHLF as compared to the untreated NHBE co-culture; * p < 0.01 and the IL-13 pre-treated NHBE at 10 ng/ml co-culture collagen levels are elevated as compared to the IL-13 pretreated NHBE at 1 ng/ml co-culture; # p < 0.01. Also, addition of the TGFβneutralizing antibody abolishes this increase (p < 0.01 compared to respective condition without TGFβantibody). The media and matrix collagen levels are normalized to respective levels obtained from NHLF embedded in collagen gels ("NHLF only"). (C, D) Representative Second harmonic generated (SHG) images (scale bar = 50 μm) of collagen fibrils at day 25 are shown along with the quantification of signal intensities. The SHG signals from the collagen secreted by NHLF embedded in rat tail collagen gels which were co-cultured with the IL-13 pre-treated NHBE at 10 ng/ml are elevated compared to the untreated NHBE co-culture; * p < 0.01 and this increase is inhibited on incubation with TGFβneutralizing antibody in the 3 day co-culture period (p < 0.01 compared to respective condition without TGFβantibody). Addition of goat IgG did not alter the increased levels of collagen in the matrix and media in the pre-treated NHBE-NHLF co-culture. (E) Exogenous active TGF-βat 0.05, 0.1, 0.5, 1 and 10 ng/ml is added in 50:50 epithelial media to NHLF embedded in collagen gels for a period of 3 days. There is a significant increase in the newly synthesized salt soluble collagen content in the matrix with addition of increasing concentration of active TGF-β(* p < 0.01 compared to only NHLF condition). All values are normalized to those obtained from "NHLF only" condition. All experiments were performed using 3 donors, grown in duplicate, with 3–6 wells for each condition.<p><b>Copyright information:</b></p><p>Taken from "IL-13 induces a bronchial epithelial phenotype that is profibrotic"</p><p>http://respiratory-research.com/content/9/1/27</p><p>Respiratory Research 2008;9(1):27-27.</p><p>Published online 18 Mar 2008</p><p>PMCID:PMC2292179.</p><p></p

A new look at the pathogenesis of asthma

Asthma is an inflammatory disorder of the conducting airways that has strong association with allergic sensitization. The disease is characterized by a polarized Th-2 (T-helper-2)-type T-cell response, but in general targeting this component of the disease with selective therapies has been disappointing and most therapy still relies on bronchodilators and corticosteroids rather than treating underlying disease mechanisms. With the disappointing outcomes of targeting individual Th-2 cytokines or manipulating T-cells, the time has come to re-evaluate the direction of research in this disease. A case is made that asthma has its origins in the airways themselves involving defective structural and functional behaviour of the epithelium in relation to environmental insults. Specifically, a defect in barrier function and an impaired innate immune response to viral infection may provide the substrate upon which allergic sensitization takes place. Once sensitized, the repeated allergen exposure will lead to disease persistence. These mechanisms could also be used to explain airway wall remodelling and the susceptibility of the asthmatic lung to exacerbations provoked by respiratory viruses, air pollution episodes and exposure to biologically active allergens. Variable activation of this epithelial-mesenchymal trophic unit could also lead to the emergence of different asthma phenotypes and a more targeted approach to the treatment of these. It also raises the possibility of developing treatments that increase the lung's resistance to the inhaled environment rather than concentrating all efforts on trying to suppress inflammation once it has become established.<br/