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. 2011 Jul 29;286(30):26667-79.
doi: 10.1074/jbc.M110.193987. Epub 2011 May 25.

The protease allergen Pen c 13 induces allergic airway inflammation and changes in epithelial barrier integrity and function in a murine model

Jui-Chieh Chen  1 , Jiing-Guang Chuang, Yu-Yi Su, Bor-Luen Chiang, You-Shuei Lin, Lu-Ping Chow
Affiliations

The protease allergen Pen c 13 induces allergic airway inflammation and changes in epithelial barrier integrity and function in a murine model

Jui-Chieh Chen et al. J Biol Chem. .

Abstract

Fungal allergens are associated with the development of asthma, and some have been characterized as proteases. Here, we established an animal model of allergic airway inflammation in response to continuous exposure to proteolytically active Pen c 13, a major allergen secreted by Penicillium citrinum. In functional analyses, Pen c 13 exposure led to increased airway hyperresponsiveness, significant inflammatory cell infiltration, mucus overproduction, and collagen deposition in the lung, dramatically elevated serum levels of total IgE and Pen c 13-specific IgE and IgG1, and increased production of the Th2 cytokines IL-4, IL-5, and IL-13 by splenocytes stimulated in vitro with Pen c 13. To examine the mechanisms involved in the regulation of allergenicity by Pen c 13, we performed two-dimensional fluorescence difference gel electrophoresis analysis combined with nano-LC-MS/MS, followed by bioinformatics analysis to identify potential targets that associated with allergic inflammation, which suggested that galectin-3 and laminin might be involved in novel pathogenic mechanisms. Finally, we focused on junctional proteins between cells, because, in addition to opening of the epithelial barrier by environmental proteases possibly being the initial step in the development of asthma, these proteins are also associated with actin rearrangement. Taken together, our findings indicate that Pen c 13 exposure causes junctional structure alterations and actin cytoskeletal rearrangements, resulting in increased permeability and airway structural changes. These effects probably change the lung microenvironment and foster the development of allergic sensitization.

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Figures

FIGURE 1.
FIGURE 1.
Functional analyses of the ability of Pen c 13 to facilitate allergic sensitization and airway inflammation. A, schematic representation of the mouse model for inducing allergic inflammation by consecutive intratracheal administration of allergen. B, effect of n-Pen c 13 on methacholine-induced airway resistance in mice. AHR against aerosolized methacholine was determined by evaluation of percentage changes from the base-line level of airway resistance (n = 6–9/group). †, significant difference from control (naïve, PBS, and d-Pen c 13), p < 0.05. C, total cell count (left) and differential cell count (right) in the bronchoalveolar lavage. D, serum levels of total IgE (top left), specific IgE (top right), specific IgG1 (bottom left), and specific IgG2a (bottom right). Sera were collected from naïve mice (untreated) and mice intratracheally treated with PBS, d-Pen c 13, or n-Pen c 13. n = 4–10/group, p < 0.001 compared with naïve (*), PBS-treated (**), or d-Pen c 13-sensitized mice (***), respectively. The data are expressed as the mean ± S.E. Differences between experimental groups were assessed by one-way analysis of variance followed by the Newman-Keuls multiple comparison test. The levels of the specific IgE, IgG1, and IgG2a were expressed as ELISA unit (E.U.). The E.U. is calculated as the absorbance of sample subtracted by the blank (Asample − Ablank) divided by the absorbance of positive control subtracted by the blank (Apositive − Ablank).
FIGURE 2.
FIGURE 2.
Th2-associated cytokine production after in vitro stimulation of splenocytes with d-Pen c 13. A, production of IL-4, IL-5, IL-13, and IFN-γ by splenocytes after in vitro stimulation with d-Pen c 13 or medium alone. n = 4–11/group; data are expressed as means ± S.E. B, the increased levels of cytokine secretion were determined by subtracting background activity of cells incubated with medium only without antigen. The data are expressed as the mean ± S.E. (error bars). p < 0.05 compared with PBS-treated (*) or d-Pen c 13-sensitized mice (**), respectively. Statistical analysis was performed using one-way analysis of variance followed by the Newman-Keuls multiple comparison test.
FIGURE 3.
FIGURE 3.
Histological evidence of induced inflammatory cell recruitment, mucus hypersecretion, and collagen deposition. Histopathologic analysis of a lung from a naïve mouse (A, B, I, and M) or a PBS-treated (C, D, J, and N), d-Pen c 13-sensitized (E, F, K, and O), or n-Pen c 13-sensitized (G, H, L, and P) mouse is shown. Lung sections were stained with H&E staining (magnification: ×100 (left) and ×400 (right)) for assessment of inflammatory changes, with PAS staining (magnification: ×200) for visualization of mucus-secreting cells in the intrapulmonary conducting airways (purple-red staining), and with Masson's trichrome staining (magnification: ×200) for showing collagen deposition (blue staining). The results shown are representative of those from at least four experiments. Q, the left lobe of the mice was harvested and assayed for hydroxyproline analysis. Values are expressed as the means ± S.E. (error bars) (n = 6) for each group. p < 0.001 compared with naïve (*), PBS-treated (**), or d-Pen c 13-sensitized mice (***), respectively. Differences between experimental groups were assessed by one-way analysis of variance followed by the Newman-Keuls multiple comparison test.
FIGURE 4.
FIGURE 4.
2-D DIGE analysis of lung proteome alterations in a mouse model. A, overlaid two-dimensional gel images of tissue lysates from n-Pen c 13-sensitized mice labeled with Cy3 (green), PBS-treated mice labeled with Cy5 (red), protein spot patterns. B, representative two-dimensional electrophoresis profiles of lung samples using the pooled standard proteins showing altered expression after n-Pen c 13 treatment and the spots identified by nano-LC-MS/MS spectrometry, which are numbered and shown in supplemental Table 3. C, classification of identified proteins based on their functional annotations using gene ontology of the UniProt Knowledgebase and Rat Genome Database (available on the World Wide Web).
FIGURE 5.
FIGURE 5.
Protein validation by Western blotting. A, confirmation for selected proteins that were identified as differentially expressed by 2-D DIGE. B, confirmation of the two proteins, laminin γ-1 and galectin-3, hypothetically identified by bioinformatics analysis. Left panels, representative immunoblots of lung lysates obtained from n-Pen c 13-sensitized mice (+) and PBS-treated mice (−). β-Actin was used as the loading control. Right panels, quantitative analysis of results of four Western blots performed using ImageQuantTM TL software. The data are shown as the mean ± S.E. (error bars).
FIGURE 6.
FIGURE 6.
Disruption of junctional proteins by Pen c 13 increases epithelial paracellular permeability. A, immunoblots of junctional proteins from the lung tissues of PBS-treated or n-Pen c 13-sensitized mice. The results shown are representative of those from four separate experiments. B, immunoblots of junctional proteins from NCI-H441 cell monolayers under control conditions and after treatment for 0.5, 1, or 2 h with n-Pen c 13 (30 nm). The results shown are representative of those from at least four separate experiments. C, representative photomicrographs obtained by confocal microscopy after immunofluorescence staining for occludin, ZO-1, and E-cadherin in NCI-H441 cells exposed to PBS or n-Pen c 13 (30 nm) for 0.5 h. The results shown are representative of those from at least four separate experiments. Bar, 25 μm. D, time course of the decrease in the TEER of NCI-H441 cell monolayers after applying n-Pen c 13 (10 nm). Values are expressed as the means ± S.E. (error bars) (n = 3) for each measurement time point. p < 0.05 versus control.
FIGURE 7.
FIGURE 7.
Schematic overview of possible signaling pathways in Pen c 13 allergen-induced allergic airway inflammation. Potential mechanisms by Pen c 13 might concomitantly disrupt the epithelial barrier and release IL-8. The epithelium damage might bring active Pen c 13 into contact with target cells and influence Th2 and IgE regulation via several signaling pathways.
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References

    1. Lai C. K., De Guia T. S., Kim Y. Y., Kuo S. H., Mukhopadhyay A., Soriano J. B., Trung P. L., Zhong N. S., Zainudin N., Zainudin B. M. (2003) J. Allergy Clin. Immunol. 111, 263–268 - PubMed
    1. Rabe K. F., Adachi M., Lai C. K., Soriano J. B., Vermeire P. A., Weiss K. B., Weiss S. T. (2004) J. Allergy Clin. Immunol. 114, 40–47 - PubMed
    1. Gould H. J., Sutton B. J. (2008) Nat. Rev. Immunol. 8, 205–217 - PubMed
    1. Elias J. A., Zhu Z., Chupp G., Homer R. J. (1999) J. Clin. Invest. 104, 1001–1006 - PMC - PubMed
    1. Folli C., Descalzi D., Scordamaglia F., Riccio A. M., Gamalero C., Canonica G. W. (2008) Curr. Opin. Allergy Clin. Immunol. 8, 367–375 - PubMed

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