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German Cockroach Extract Proteolytic Activity Down-regulates Interleukin-13 Dependent Eotaxin-3 (CCL26) Expression in Airway Epithelial Cells

  • Author / Creator
    Alzahrani, Khadija
  • Introduction:Allergy to inhaled allergens is an important factor to develop airway inflammation. The airway epithelium is the main barrier between the host and inhaled allergens, and its activation by allergens is a key step towards the activation of innate and adaptive immunity. German cockroach (Blattella germanica) allergens can activate airway epithelial cells through multiple receptors, including protease activated receptor 2 (PAR-2), and induce primarily pro-inflammatory effects. Th2 cytokine, (interleukin-13) IL-13, is a key mediator in allergic inflammation. It activates epithelial cells and induces the production of eotaxin-3 (CCL26), a potent eosinophil chemoattractant. The interactions between the effects of IL-13 and cockroach allergens on airway epithelium have not been studied.Methods:A bronchial epithelial cell line (BEAS-2B) and normal human bronchial epithelial cells (NHBE) were cultured in pre-coated multi-well plates and stimulated with IL-13, German cockroach extract (CE) or both. CCL26 mRNA was measured by qRT-PCR and the release of CCL26 protein by ELISA. To test the role of CE proteases, heat inactivated CE (HICE), boiled CE or CE pre-incubated with protease inhibitors were used. PAR-2 activated peptides were used to examine the role of PAR-2 activation in the inhibition of IL-13 induced CCL26. Western blotting was used to assess STAT-6 phosphorylation and IL-13 protein degradation.Results:CE prevented IL-13-mediated up-regulation of CCL26 mRNA and protein from BEAS-2B and NHBE cells in a time and dose dependent manner. HICE and CE pre-incubated with aprotinin, an inhibitor of serine proteases, or soy bean trypsin inhibitor (SBTI) did not inhibit IL-13 mediated CCL26 mRNA induction. PAR-2 activation did not show inhibition of IL-13 induced CCL26 mRNA. STAT-6 activation by IL-13 was not prevented by CE. Western blot detected early onset degradation of IL-13 protein by CE even in the presence of aprotinin or with HICE but not with boiled CE.Conclusions:CE inhibited IL-13 induced CCL26 in a time and dose dependant manner. This inhibitory effect is dependent on CE proteases, particularly proteases with trypsin-like activity. Serine protease inhibitor did not inhibit IL-13 protein degradation by CE, which indicates that CE trypsin like activity prevents IL-13-induced CCL26 expression in a degradation independent manner. PAR-2 activation did not mimic the inhibitory effect of CE on IL-13 induced CCL26. This may be a mechanism for CE to decrease the detrimental effects of Th2 cytokines in allergic asthma.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3P844B7V
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.