Allergy Asthma Immunol Res.  2013 Jul;5(4):197-206. 10.4168/aair.2013.5.4.197.

Effects of Interleukin-9 Blockade on Chronic Airway Inflammation in Murine Asthma Models

• Kim MS
• Cho KA
• Cho YJ
• Woo SY

• KMID: 2260312
• DOI: http://doi.org/10.4168/aair.2013.5.4.197

Abstract

PURPOSE
Asthma is a chronic inflammatory disease of the airways associated with structural changes and airway remodeling. Interleukin (IL)-9 has pleiotropic effects on both inflammatory cells and airway structural cells, which are involved in asthma pathogenesis. We evaluated the effects of IL-9 blockade on chronic airway inflammation.
METHODS
Acute airway inflammation was induced in Balb/c mice using aerosolized ovalbumin (OVA), whereas chronic asthma was induced by OVA exposure for 5 weeks with anti-IL-9 or isotype-matched antibody (Ab) treatment during the OVA challenge. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and lung tissues were stained to detect cellular infiltration, mucus deposition, and collagen accumulation. The levels of interferon (IFN)-gamma, IL-4, IL-5, IL-9, IL-17, and immunoglobulin E (IgE) in BALF were measured using enzyme linked immunosorbent assays, and profiles of inflammatory cells and subsets of T helper (Th) cells were analyzed using flow cytometry.
RESULTS
IL-9, IL-17, and IFN-gamma levels were significantly increased in the chronic group compared to the acute asthma group. However, the number of IL-9-positive cells was not affected, with a decrease in Th17 cells in OVA-challenged caspase-1 knockout mice. Numbers of eosinophils, neutrophils, B cells, mast cells, and Th17 cells decreased after administration of anti-IL-9 Ab. Total IgE, IL-5, IL-9, and IL-17 levels were also lower in the anti-IL-9 group.
CONCLUSIONS
Our results suggest that anti-IL-9 Ab treatment inhibits pulmonary infiltration of inflammatory cells and cytokine production, especially IL-17. These results provide a basis for the use of an anti-IL-9 Ab to combat IL-17-mediated airway inflammation.

Keyword

Interleukin-9; allergic asthma; T helper 17; anti-interleukin-9 antibody

MeSH Terms

Airway Remodeling
Animals
Asthma
B-Lymphocytes
Bronchoalveolar Lavage Fluid
Collagen
Eosinophils
Immunoglobulin E
Immunoglobulins
Inflammation
Interferons
Interleukin-17
Interleukin-4
Interleukin-5
Interleukin-9
Interleukins
Lung
Mast Cells
Mice
Mice, Knockout
Mucus
Neutrophils
Ovalbumin
Ovum
Th17 Cells
Collagen
Immunoglobulin E
Immunoglobulins
Interferons
Interleukin-17
Interleukin-4
Interleukin-5
Interleukin-9
Interleukins
Ovalbumin

Figure

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  Fig. 1 Protocol for inducing experimental acute and chronic airway inflammation models. (A) In the acute asthma model, mice were sensitized by two intraperitoneal injections of OVA on days 0 and 5, and then received aerosolized OVA challenge on days 12-15 using a nebulizer. (B) Mice were sensitized via intraperitoneal injection of OVA on days 0 and 12 to induce chronic asthma. Mice were exposed to an aerosol of 1% OVA in PBS on days 18-23 using a nebulizer. After day 26, mice were exposed to aerosolized 1% OVA three times per week for 5 weeks. OVA, ovalbumin; PBS, phosphate-buffered saline.

• 

  Fig. 2 OVA-induced airway inflammation in acute and chronic asthma groups. (A) BALF from acute (n=15) or chronic asthma (n=10) mice was collected 24 hours after the final OVA challenge, and the number of cells in the BALF was determined as described in the Materials and Methods. The data are expressed as the means±SEM (*P<0.05, groups were compared as indicated by Mann-Whitney U tests). (B) Mice were sacrificed 1 day after the last OVA challenge. Mice lungs were fixed with 4% paraformaldehyde, and tissue sections were stained with H&E. Images are presented at the original magnification of ×400. OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; SEM, standard error of the mean; H&E, hematoxylin and eosin.

• 

  Fig. 3 Comparison of cytokine profiles after OVA challenge. Concentrations of IFN-γ, IL-4, IL-5, IL-9, IL-17, and IgE in BALF from acute asthma or chronic asthma mice were measured by ELISA. Data are expressed as the means±SEM (n=5). *P<0.05 for control versus OVA-inhalation and acute-OVA versus chronic-OVA by Mann-Whitney U tests. OVA, ovalbumin; IFN, interferon; IL, interleukin; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.

• 

  Fig. 4 Caspase-1 KO mice induced airway inflammation. (A) BALF from WT C57BL/6 (n=5) or caspase-1 KO (n=5) mice was collected 24 hours after the final OVA challenge, and the number of cells in the BALF was determined. The data are expressed as the means±SEM (*P<0.05;0.05, groups were compared as indicated by Mann-Whitney U tests). (B) BAL cells were lysed, and whole-cell extracts were resolved using 12% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and probed with anti-IL-1β, anti-β-actin, and anti-PU.1 Ab. Representative blots of three independent experiments are shown. (C) Mice lungs were fixed with 4% paraformaldehyde, tissue sections were stained with H&E, and mucus was visualized by PAS staining. Images are presented at the original magnification of ×400. (D) After OVA challenge, proportions of basophils (CD49d+, FcεRI+), mast cells (c-Kit+, SSClow), eosinophils (CD49b+, SSCint), and neutrophils (Gr1+) were collected for flow cytometric analysis (n=5 for each groups). Cell numbers were calculated from the percentage of cells×BAL cell numbers. (E) The proportions of CD4+ T cells were determined according to intracellular levels of cytokines: IFN-γ+ for IFN-γ-secreting T cells, IL-4+ for IL-4-secreting T cells, IL-9+ for IL-9-secreting T cells, and IL-17+ for Th17 cells. Cell numbers were calculated from the percentage of cells subsets×BAL cell numbers. Data are expressed as the means±SEM (n=5). *P<0.05; groups were compared as indicated by unpaired t-tests. KO, knockout; BALF, bronchoalveolar lavage fluid; OVA, ovalbumin; SEM, standard error of the mean; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IL, interleukin; Ab, antibody; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff.

• 

  Fig. 5 Effects of IL-9 blockade on chronic pulmonary inflammation. (A) Anti-IL-9 or control Iso IgG Ab were administered (100 µg/mouse, intraperitoneally) once a week throughout the OVA induced development of chronic asthma. Five weeks of OVA challenge induced massive infiltration of eosinophils, and anti-IL-9 treatment significantly suppressed their infiltration. (B) BALF from each experimental mouse was collected 24 hours after the final OVA challenge, and the number of cells in the BALF was determined. (C) Differential cell counts were calculated as (total cell number of BAL cells)×(percentage of mononuclear cells, eosinophils, or neutrophils counted in the Giemsa-stained cytospin cell preparation). MNC=mononuclear cell. (D) IL-9 blockade attenuated tissue inflammation. The randomly selected histologic images were scored as the mean inflammatory index, which represents the severity of peri-bronchial inflammation. Mucus producing goblet cells were visualized by PAS staining. Sections were stained with Sirius Red solution to detect collagen deposition. Images are presented at the original magnification of ×400. PAS-positive goblet cells and Sirius Red-positive sub-epithelial areas for each bronchiole were calculated using image analysis software for quantitative analysis. Data are expressed as the mean±SEM (n=5). *P<0.05; groups were compared as indicated using the Mann-Whitney U tests. OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; IL, interleukin; PAS, periodic acid-Schiff; SEM, standard error of the mean.

• 

  Fig. 6 IL-9 blockade altered BAL cell profiles. Flow cytometric analysis shows profiles of cells from BALF. Eosinophils (CD49b+, SSCint), neutrophils (Gr1+), mast cells (c-Kit+, SSClow), basophils (CD49d+, FcεRI+), and B cells (CD19+) were assessed. Data are presented as representative results of three independent experiments (A) and a statistical graph (B). The proportions of CD4+ T cells were determined according to intracellular levels of cytokines: IFN-γ+ for IFN-γ-secreting T cells, IL-4+ for IL-4-secreting T cells, IL-9+ for IL-9-secreting T cells, IL-17+ for Th17 cells, and Foxp3+ for Treg cells. Data are presented as representative results of three independent experiments (C) and a statistical graph (D). Data are expressed as the means±SEM (n=5) *P<0.05; groups were compared as indicated by unpaired t-tests. BALF, bronchoalveolar lavage fluid; SEM, standard error of the mean.

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  Fig. 7 IL-9 blockade alters cytokine levels in BALF. Mice were sacrificed and the levels of IgE and cytokines in BALF from OVA-challenged mice with anti-IL-9 Ab or Iso IgG Ab were measured using ELISA. Data are expressed as the means±SEM (n=5). *P<0.05; groups were compared as indicated by Mann-Whitney U tests. IL, interleukin; BALF, bronchoalveolar lavage fluid; OVA, ovalbumin; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.

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