Asthma is exacerbated by viral infection and viral pathogenesis is altered in asthmatics due to a defective innate response in asthma. Is the defective innate response in epithelium follow through to an enhanced activation of DCs (via TSLP) leading to a switch to Th2 driven inflammation (Allergy)?
The hypothesis is that defective response of the asthmatic epithelium to viral infection results in alterations in the functionality of dendritic cells.
This project will investigate the crosstalk between epithelial cells, the dendritic cells in response to viruses and aeroallergens, using an in vitro co-culture model.
Asthma is a chronic immune-mediated inflammatory disease, characterised by structural and functional changes of airway follow by non-specific airway hyperreactivity.
The conducting airway and the lung parenchyma are the two major anatomically compartments of the respiratory tract. At the surface of the conducting airway lines the respiratory epithelium, which includes a variety of cells: ciliated cells, goblet cells and basal cells. Cells of the immune systems are found as well in the airway, such as: dendritic cells, mast cells, T cells, B cells .
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Airway epithelial cells represent a mechanical barrier and immune defence against harmful inhaled substances and pathogens [3, 4]. Pattern-recognition receptors (PRRs) which recognize compounds of allergens and pathogens (the so-called pathogens-associated molecular-pattern, PAMP), have been found to be expressed in airway epithelial cells, including toll like receptors (TLRs), NOD-like receptors (NLRs), retinoic acid inducible gene I RIG-like receptors (RLRs)[5, 6]. Following the TLR signalling in response to different antigens, several transcription factors are activated, including NF-kB and interferon (INF) regulatory factors (IRFs) leading to production of a multitude of cytokines and chemokines able to regulate the function of immune cells . Th2 cytokine IL-4 and the rhinovirus synergistically enhanced thymic stromal lymphopoietin (TSLP) production from epithelial cells . Epithelial cell-derived thymic stromal lymphopoietin (TSLP) activates DCs and polarize naive T cells into Th2 cells .
including thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferons Î± and Î², tumor necrosis factor -Î± (TNF-Î±), RANTES, MIP3Î± (CCL20), IL1Î², IL-25, IL-33 and osteopontin [8-11]. This array of cytokines and chemokines induces the regulation of immune and inflammatory cells.
The immune cells can react to intracellular molecules released by damaged cells. These molecules are called damage associated molecular patterns (DAMPs). Adenosine triphosphate (ATP), heat shock proteins (HSP), high mobility group box 1 (HMGB-1) protein, uric acid, heparin sulphate are a few examples of DAMPs[13-15].
Immunology of asthma and allergy (APC>Tcell+Bcell>IgE(sensitsation)>(effector)>MC>bronchoconstriction>inflammation including eosinophils>mucus)
Viral exacerbations of asthma
It has been previously reported that close to 80% of asthma exacerbations are due to viral infections, with rhinovirus being the most common [16-18]. Part of the antiviral response of the epithelium is expression of type I interferon (IFN), interferon Î± and Î², and type III INFs, interferon Î». It has been previously shown that there is a deficiency of interferon production by bronchial epithelial cells in response to rhinovirus infection [20, 21]. The expression of costimulatory molecule B7-H1 (an inhibitor of T-cell activation) is up-regulated by interferon Î² on monocytes and dendritic cells. Therefore, by producing interferon Î², healthy epithelial cells are effective inhibitors of T cell activation, and this immunosuppressive effect might be reduced in asthmatic epithelial cells. Rate et al. 2009 demonstrated in a epithelial-monocytes cells co-culture model that type I interferons influence as well the way of monocytes differentiate into DC phenotypically and functionally, resulting in a significant attenuation of TLR3, TLR4, CD80 and HLA-DR expression . Human rhinovirus inhibits T cells activation, by inducing sialoadhesin and B7-H1 expression on dendritic cells.
Background on dendritic cells
Dendritic cells, which are professional antigen presenting cells, play an important role in the pathogenesis of asthma . In the airway they are found within the epithelium, and they are able to extend their protrusions into the lumen of the conducting airway to sample foreign antigens [26, 27]. Activation of DCs by an antigen, involves the maturation of DC, reached by up regulation of the costimulatory molecules (CD40, CD80, CD86); and the migration of DCs to the lymph node. Once activated, they cause an adaptive immune response, by inducing proliferation and polarization of T lymphocytes .
Interaction between dendritic cells and epithelial cells in asthma, TSLP
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The recruitment of DC precursors or immature DC from the bloodstream into the airway, is control by the chemokines . The degree of DCs differentiation can influence the responsiveness of the cells to different chemokines. Monocyte chemotactic proteins (MCP)-1, -2, -3 and -4, RANTES, macrophage inflammatory protein 1Î± (MIP1 Î±), MIP3Î± can induce DCs migration.
Differentation of monocytes into dendritic cells
It has been previously shown that airway epithelial cells can induce the differentiation and maturation of monocytes into functional dendritic cells through secretion of different cytokines, like: IL15, .
In vitro coculture systems have been developed to investigate the interaction between immune cells and epithelial cells, but all of them used cell lines rather than primary cells derived from patients with asthma [23, 31, 32]. Most of these model used transwell inserts that separate the two types of cells, but allow the diffusion of soluble molecules.
The complete mechanism through which epithelial cells regulate the function of the DCs is still unknown. An in vitro model will be used to characterise the activation of DCs in the presence of primary bronchial epithelial cells and/or epithelial cell lines.
Using in situ hybridization it has been previously found that bronchial epithelial cells express higher level of TSLP in asthmatic patients compared with healthy patients.
To investigate the hypothesis the following experimental plan will be performed (condensed literature review)
A stock of rhinovirus from major group (RV16) and minor group (RV1B) will be generated using Ohio Hela cells (Papi et al 1999).
Primary epithelial cells from normal and asthmatic patients will be infected at different multiplicity of infections and the immune responses is assessed by measuring using quantitative real time PCR the expression of selected cytokines and chemokines genes that have an effect on DCs. Their expression pattern will be confirmed by examining their protein level by ELISA.
UV inactivated supernatants from infected primary epithelial cells will be used to characterise the effect on DCs of soluble cytokines and chemokines released by the epithelial cells into the media in response to viral infections.
experimental chapter 1 - adult epithelial cells response to virus (RV1b) and differences between asthmatics and normal patients in terms of cytokines, chemokines that have effect on DCs (InfB, GMCSF, TSLP mRNA other DC relevant activating mediators). Detected by mRNA, confirm protein. Then to confirm responses with RV16 (time v dose response for effect on DCs). ALL be with supernatants (UV inactivated)
experimental chapter 2 - co-culture of epithelial cells with DCs. Activate epithelium with IL4 (TSLP), IL13 to see effect on DCs
paediatric cells similar to adult? Dendritic cells response to allergen and virus
experimental chapter 3 - inducing TSLP other things from epithelium with allergen (Derp1), virus, IL13
experimental chapter 4 - epithelial DC communication in response to allergen, virus, IL-13
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