Modulation of airway inflammation by immunostimulatory CpG oligodeoxynucleotides in a murine model of allergic aspergillosis

Abstract

Allergic aspergillosis is a Th2 T-lymphocyte-mediated pulmonary complication in patients with atopic asthma and cystic fibrosis. Therefore, any therapeutic strategy that selectively inhibits Th2 T-cell activation may be useful in downregulating allergic lung inflammation in asthma. In the present study, we developed a CpG oligodeoxynucleotide (ODN)-based immune intervention of allergic inflammation in a mouse model of allergic aspergillosis. Four different groups of mice were used in a short-term immunization protocol. Three experimental groups of animals (groups 1 to 3) were sensitized with Aspergillus fumigatus antigens. Animals in group 1 were immunized with A. fumigatus antigen alone, while those in group 2 were treated with CpG-ODN 1 day before the first antigen immunization, and the animals in group 3 received the first CpG-ODN administration between the antigen treatments. The animals in group 4 served as controls and were given phosphate-buffered saline. Allergen-specific serum immunoglobulins and total immunoglobulin E in different groups of animals were measured by enzyme-linked immunosorbent assay, while airway remodeling and cytokine production were studied by immunohistochemistry. The results demonstrated that CpG-ODN administration either before (group 2) or between (group 3) antigen treatments resulted in reduced total immunoglobulin E levels and peripheral blood eosinophil numbers compared to A. fumigatus allergen-sensitized group 1 animals. Similarly, treatment with CpG-ODN also downregulated inflammatory cell infiltration, goblet cell hyperplasia, and basement membrane thickening compared to A. fumigatus-sensitized mice. The distinct reduction in peripheral blood eosinophilia and airway remodeling in CpG-ODN-treated mice emphasized its usefulness as an immunomodulating agent for allergic fungal diseases.

FIG. 1.

Effect of CpG-ODN on A. fumigatus-specific IgG1, IgG 2a, and IgG2b in the sera of different groups of animals. Bars represent mean optical density values (mean + standard error) from five mice in each group studied by enzyme-linked immunosorbent assay. The immunoglobulin levels in pre- and postimmunization serum samples are presented for group 1, group 2, group 3, and group 4 from left to right within each grouping. The asterisk (*) indicates a statistically significant difference in immunoglobulin levels between A. fumigatus-sensitized group 1 and CpG-treated group 2 and group 3 mice (P < 0.05).

FIG. 2.

(A) Total serum IgE in A. fumigatus-sensitized and CpG-ODN-treated mice. Bars represent mean optical values (mean + standard error). The asterisk (*) indicates a statistically significant difference in total IgE levels among A. fumigatus-sensitized group 1 and CpG-treated group 2 and group 3 mice (P < 0.05). (B) Eosinophils in the peripheral blood of A. fumigatus-sensitized and CpG-ODN-treated mice. The bars represent mean cell counts (mean + standard error) in samples from five mice in each group. The asterisk (*) represents a statistically significant difference between A. fumigatus-sensitized group 1 and CpG-treated group 2 and group 3 (P < 0.001).

FIG. 3.

Immunomodulatory effects of CpG-ODN treatment in lung sections of A. fumigatus-sensitized and CpG-ODN-treated mice (magnifications, ×60). (A) IL-4-expressing cells in the lung section of a CpG-treated (group 2) mouse. (B) IL-4-expressing cells in the lung section of an A. fumigatus-sensitized mouse with no CpG treatment (group 1). The arrow indicates the cytokine-expressing cells. (C) IL-5-expressing cells in the lung section of a group 2 animal. (D) IL-5-expressing cells in the lung section of a group 1 animal. The arrow indicates IL-5-expressing cells. (E) IFN-γ-expressing cells on the lung section of a group 2 animal. The arrow indicates IFN-γ-producing cells. (F) IFN-γ staining of the lung section from a group 1 mouse. (G) Eosinophil peroxidase staining of the lung section from a CpG-treated group 2 animal. (H) Eosinophil peroxidase staining of the lung section from a group 1 animal. (I) The number of IL-4-, IL-5-, IFN-γ-, and EPO-positive cells in lung sections enumerated quantitatively. Results are represented as mean + standard error (for details, see Materials and Methods). The asterisk (*) represents a statistically significant difference in cytokine- and EPO-positive cells in lung sections from A. fumigatus-sensitized and CpG-treated mice (P < 0.001).

FIG. 3.

Immunomodulatory effects of CpG-ODN treatment in lung sections of A. fumigatus-sensitized and CpG-ODN-treated mice (magnifications, ×60). (A) IL-4-expressing cells in the lung section of a CpG-treated (group 2) mouse. (B) IL-4-expressing cells in the lung section of an A. fumigatus-sensitized mouse with no CpG treatment (group 1). The arrow indicates the cytokine-expressing cells. (C) IL-5-expressing cells in the lung section of a group 2 animal. (D) IL-5-expressing cells in the lung section of a group 1 animal. The arrow indicates IL-5-expressing cells. (E) IFN-γ-expressing cells on the lung section of a group 2 animal. The arrow indicates IFN-γ-producing cells. (F) IFN-γ staining of the lung section from a group 1 mouse. (G) Eosinophil peroxidase staining of the lung section from a CpG-treated group 2 animal. (H) Eosinophil peroxidase staining of the lung section from a group 1 animal. (I) The number of IL-4-, IL-5-, IFN-γ-, and EPO-positive cells in lung sections enumerated quantitatively. Results are represented as mean + standard error (for details, see Materials and Methods). The asterisk (*) represents a statistically significant difference in cytokine- and EPO-positive cells in lung sections from A. fumigatus-sensitized and CpG-treated mice (P < 0.001).

FIG. 4.

Lungs from A. fumigatus-sensitized (group 1) and CpG-ODN -treated (group 2) mice were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, periodic acid-Schiff base, and Trichrome. (A) Hematoxylin and eosin staining of group 2 lung sections, showing moderate inflammatory infiltrates adjacent to bronchioles and small blood vessels without eosinophil infiltrations (magnifications, ×60), (B) Hematoxylin and eosin staining of a lung section from a group 1 animal showing extensive peribronchaeolar and perivascular inflammation. The bronchial epithelium is hyperplastic (×60). (C) Higher magnification of A (×400). (D) Higher magnification of B (×400), showing the large number of eosinophils and distinct epithelial hyperplasia. (E) Periodic acid-Schiff staining of the lung section from a group 2 mouse, showing very few goblet cells around bronchial mucosa. (F) Periodic acid-Schiff staining of the lung section from a group 1 mouse, showing the large number of mucous glycoconjugate-producing goblet cells in bronchial mucosa. (G and H) Higher magnifications (×400) of E and F, respectively. (I) Trichrome staining of a section from a group 2 mouse exhibiting less peribronchial collagen deposition (×60). (J) There was significantly more collagen deposition around the airways of group 1 compared to CpG-ODN-treated animals.