Epithelial cells are a critical first line of defense against pathogens such as influenza and strategies to modulate epithelial cell signaling are therefore an appealing target for novel therapeutics. We have previously identified protein kinase D (PKD) as a key regulator of bronchial epithelial barrier integrity during viral infection and have recently identified PKD as a mediator of airway epithelial chemokine secretion. We hypothesize that stimulation with the double-stranded RNA polyI:C or Influenza A virus (IAV) infection leads to activation of PKD (specifically the PKD3 isoform) which increases epithelial chemokine release and promotes leukocyte recruitment. This project aims to determine the mechanism between PKD3 activity and epithelial human interleukin-8 (IL-8)/ murine chemokine CXCL1 secretion. To test this aim, we will assay PKD3 activity (substrate phosphorylation) following siRNA-mediated knock-down of potential receptors upstream PKD activation, and then use that information to inform identification of the adaptor molecule(s) associated with PKD (using co-immunoprecipitation). We will also assay IL-8/CXCL1 mRNA levels in human and mouse airway epithelial cell lines following knock-down of PKD isoforms to determine the isoform driving chemokine production. We will then use chromatin immunoprecipitation to identify PKD-activated transcription factors involved in IL-8/CXCL1 transcription and confirm activity with luciferase reporter assay. We further aim to investigate the therapeutic potential of PKD3 reduction/inhibition to attenuate lung injury following IAV infection. To test this aim, we will infect PKD3 deficient mice (PKD3-/-) with sub-lethal influenza A virus (IAV) and assay for leukocyte infiltration (via flow cytometry), chemokine secretion (via ELISA and multiplex array), and general pathology (histology). We will also generate bone marrow chimeric mice to assess the relative contributions of PKD3 in stromal cells vs. hematopoietic cells to lung injury and neutrophilia. We hypothesize that stimulation with the double-stranded RNA polyI:C or Influenza A virus (IAV) infection leads to activation of the PKD3 isoform which increases epithelial pro-inflammatory cytokine release and promotes leukocyte recruitment. We further hypothesize that PKD3 may play a previously unsuspected role in regulating airway epithelial anti-viral immune responses and that further study of the mechanism of PKD3-mediated cytokine release in the airway epithelium may lead to new therapeutics in combating viral respiratory infections.
This research project will increase understanding of the molecular signaling pathways involved in regulating mucosal immune responses to respiratory viruses. Specifically, we have identified protein kinase D3 (PKD3) as a potent mediator of airway epithelial chemokine production and have observed that PKD3 reduction limits airway neutrophilia, making PKD3 an attractive target for novel broad-acting therapeutics for respiratory infection.
