An essential regulatory role of downstream of kinase-1 in the ovalbumin-induced murine model of asthma

Abstract

The downstream of kinase (DOK)-1 is involved in the protein tyrosine kinase (PTK) pathway in mast cells, but the role of DOK-1 in the pathogenesis of asthma has not been defined. In this study, we have demonstrated a novel regulatory role of DOK-1 in airway inflammation and physiologic responses in a murine model of asthma using lentiviral vector containing DOK-1 cDNA or DOK-1-specific ShRNA. The OVA-induced inflammatory cells, airway hyperresponsiveness, Th2 cytokine expression, and mucus response were significantly reduced in DOK-1 overexpressing mice compared to OVA-challenged control mice. The transgenic introduction of DOK-1 significantly stimulated the activation and expression of STAT-4 and T-bet, while impressively inhibiting the activation and expression of STAT-6 and GATA-3 in airway epithelial cells. On the other hand, DOK-1 knockdown mice enhanced STAT-6 expression and its nuclear translocation compared to OVA-challenged control mice. When viewed in combination, our studies demonstrate DOK-1 regulates allergen-induced Th2 immune responses by selective stimulation and inhibition of STAT-4 and STAT-6 signaling pathways, respectively. These studies provide a novel insight on the regulatory role of DOK-1 in allergen-induced Th2 inflammation and airway responses, which has therapeutic potential for asthma and other allergic diseases.

Figure 1. DOK-1 expression in the lungs after OVA sensitization and challenge.

The lentiviral vectors containing DOK-1 specific ShRNA (DOK_ShRNA) or DOK-1 cDNA (DOK) were administrated together with controls (empty vector or vector containing non-specific scrambled ShRNA) before OVA challenge. (A) Representative western blotting demonstrating DOK-1 protein expression in the lungs of the mice. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression (B) Immunofluorescent staining on the tissue sections from the lungs using Alexa Fluor 488-conjugated DOK-1 antibody and DAPI stains.

Figure 2. Effects of DOK-1 in OVA-induced inflammation and airway response.

(A) The recovery of BAL cells 24 hr after OVA challenge. NEU, Neutrophil; EOS, Eosinophil; LYM, Lymphocyte; MAC, Macrophages; TOT, total cell. (B) Eosinophil peroxidase (EPO) activity in BAL fluids of OVA-sensitized and –challenged mice. (C) Airway responsiveness to aerosolized methacholine measured by non-invasive whole body plethysmography. (D) Serum IgE and IgG2a levels detected by ELISA. The values in all the panels represent means ± S.E.M. At least 5 mice were included in each group. *P<0.05, ***P<0.001 vs. OVA-challenged mice.

Figure 3. Effect of DOK-1 in OVA-induced tissue inflammation and mucus responses.

(A) Lung sections were stained with hematoxylin and eosin, D-PAS, alcian blue for the evaluation of inflammatory cells and airway mucus responses. ×40 of original magnification. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression. At least 4 mice were included in each group. (B) Inflammatory index that scored parenchymal inflammation. At least 4 mice were included in each group. *P<0.05 (C) Mucus index evaluated by morphometric analysis representing alcian blue stained mucus cells (percentage of positive cells) in airway epithelial cells. At least 4 mice were included in each group. *P<0.05.

Figure 4. Effect of DOK-1 on T cells and cytokine/chemokine expression.

(A) FACS Histogram analysis on BAL cells from the mice after OVA sensitization and challenge. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression. Each lane indicates CD4⁺ T cell population stained with Cy5-anti-CD4 antibody. (B) BAL CD4(+)T cells gated with PE-conjugated CD4 were further evaluated by intracellular staining against Cy5-conjugated-IL-4 and FITC-conjugated-IFN-γ. (C) The levels of inflammatory Th1 and Th2 cytokines and a chemokine in BAL fluid were measured by ELISA at 24 hrs after the last OVA challenge. Data represent means ± S.E.M. Each group contains at least 7 mice. *P<0.05, ***P<0.001 vs. OVA-challenged mice.

Figure 5. Effect of DOK-1 on STAT-6 and STAT-4 signaling pathways.

Expression and/or activation (phosphorylation) of STAT6, STAT4, GATA3, and T-bet transcriptional factors were evaluated by Western blot analysis. A representative gel photo out of 6 similar independent experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.

Figure 6. DOK-1 regulation of STAT-4, STAT-6 signaling pathways (A) Expression and/or activation of DOK-1, STAT-4, GATA-3 and T-bet in STAT-6 knockout and (B) DOK-1, STAT-6, GATA-3 and T-bet in STAT-4 knockout mice were evaluated by Western blot analysis.

A representative gel photo out of five similar independent experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.

Figure 7. Effects of DOK-1 on STAT-6 and STAT-4 expression and nuclear translocation in airway epithelial cells.

(A) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-6 antibodies and DAPI stain. (B) Lung sections were stained with Alexa Fluor 488-conjugated DOK-1 and Alexa Fluor 568-conjugated STAT-4 antibodies and DAPI stains. A representative photo of 7 similar experiments. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression.