Tgf-Beta isoform specific regulation of airway inflammation and remodelling in a murine model of asthma

Abstract

The TGF-beta family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-beta, TGF-beta(1-3), are expressed in the airways and TGF-beta(1) and -beta(2) are increased in asthma. However, there is little information on the specific roles of individual TGF-beta isoforms. In this study we assess the roles of TGF-beta(1) and TGF-beta(2) in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-beta neutralising antibodies. Antibodies to both isoforms inhibited TGF-beta mediated Smad signalling. Anti-TGF-beta(1) and anti-TGF-beta(2) inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-beta(1) also specifically regulated airway and fibroblast decorin deposition by TGF-beta(1). Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-beta(1) also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-beta(1) and TGF-beta(2) were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-beta(1) and TGF-beta(2) exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases.

Figure 1. Localisation of TGF-β₁, -β₂ and -β₃ in normal and OVA challenged mouse airways.

Representative images from control lungs (A, E and I) and OVA sensitised and challenged mice 3–7 d (B, C, F, G, J and K) and 12 d (D, H and L) after final OVA challenge, immunostained (brown) for TGF-β₁ (A-D), TGF-β₂ (E-H) and TGF-β₃ (I-L). Different cell populations are indicated by arrows; BE - bronchial epithelial cell, F - fibroblast-like cell, G - goblet cell, M - macrophage, Pmn - polymorphonuclear cell, ATII - type II alveolar epithelial cell. Representative images are shown from n = 6–8 animals per experimental group. Scale bar represents 50 µm.

Figure 2. Treatment with isoform specific TGF-β antibodies inhibits signalling via the Smad pathway.

(A-C) Representative images of airways stained for phosphorylated Smad 2/3 (brown) from saline sensitised and challenged mice treated with irrelevant IgG (A), anti-TGF-β₁ (B) or anti-TGF-β₂ (C) 12 days after final challenge. Phosphorylated Smad 2/3 localises to the nucleus. Note the reduction in intensity of staining and number of positive cells in the airways of animals treated with TGF-β antibodies (B-C) compared with controls (A). Scale bar represents 25 µm. (D) Quantification of the proportion of bronchial epithelial cells negative for phosphorylated Smad 2/3 in IgG controls and saline (Sal) or ovalbumin (Ova) sensitised and challenged animals treated with TGF-β antibodies. Each value represents the mean ± SEM of measurements from 9–10 animals per group. * P<0.05 or ** P<0.001 compared with control.

Figure 3. TGF-β signalling in remodelling mouse airways.

Phosphorylated Smad 2/3 staining of control (A) and remodelling (B) mouse airway 3 days following final challenge. Cells showing active TGF-β signalling via Smad 2/3 indicated by brown nuclear staining include bronchial epithelial cells (BE), fibroblast-like cells (F), macrophages (M) and type II alveolar epithelial cell (ATII). Scale bar represent 25 µm.

Figure 4. TGF-β isoform shared regulation of allergen-induced sub-epithelial collagen deposition.

Control (A) and OVA sensitised and challenged (B) mouse airways 12 days following final challenge demonstrating allergen-induced increase in sub-epithelial collagen deposition depicted by blue staining of the thickened airway wall using a modified Martius Scarlet Blue stain. Scale bar represents 50 µm. (C) Quantification of the area of collagen staining demonstrating OVA-induced increase in sub-epithelial collagen deposition and its inhibition by treatment with antibodies to TGF-β₁ or -β₂. Each value represents the mean ± SEM of measurements from 7–10 animals per group. **, P<0.001 compared with relative control. ⁺, P<0.02 and ⁺⁺, P<0.01 compared with OVA control groups.

Figure 5. TGF-β isoform specific regulation of allergen-induced sub-epithelial decorin deposition.

Control (A) and OVA sensitised and challenged (B) mouse airways 12 days following final challenge demonstrating allergen-induced increase in sub-epithelial decorin deposition depicted by brown staining of the thickened airway wall by immunohistochemistry. Scale bar represents 50 µm. (C) Quantification of the area of decorin staining demonstrating OVA-induced increase in sub-epithelial decorin deposition and its inhibition by treatment with antibodies to TGF-β₁ but not TGF-β₂. Each value represents the mean ± SEM of measurements from 9–10 animals per group. *, P<0.001 compared with relative control. ⁺, P<0.01 compared with OVA control and not significantly different to the saline anti-TGF-β₁ group.

Figure 6. Effect of TGF-β isoforms on murine lung fibroblast decorin production.

Mouse lung fibroblasts were incubated with or without TGF-β isoforms for 48 h and decorin production assessed by immunocytochemistry. Control cells and cells incubated with TGF-β₂ showed little or no staining for decorin. In contrast cells incubated with TGF-β₁ showed increased decorin staining (brown) localised to the peri-nuclear cytoplasm (arrows). Scale bar represents 50 µm.

Figure 7. TGF-β isoform shared and selective effects on allergen-induced inflammation.

Graphs show total BAL cell numbers and the relative contributions of monocytes/macrophages, eosinophils, lymphocytes and neutrophils isolated from animals 12 days following final challenge. Each bar represents the mean ± SEM of values from 9–14 animals per group. *, P<0.05. **, P<0.01. ***, P<0.001. Photomicrographs show staining of lung sections for the macrophage and macrophage progenitor surface antigen, F4/80, demonstrating inhibition of macrophage accumulation (arrows) around the airways of animals treated with antibodies to TGF-β₁ compared with sections from animals treated with control IgG or anti-TGF-β₂. Scale bar represents 50 µm.

Figure 8. Schematic representation of the source and roles of TGF-β₁ and TGF-β₂ in OVA-induced airway inflammation and remodelling.

Following OVA sensitisation and challenge there appears to be a shift in the localisation/production of TGF-β₁ (A) and TGF-β₂ (B) away from bronchial epithelial cells with increased localisation/production of TGF-β₁ to macrophages and TGF-β₂ to macrophages and eosinophils. TGF-β₁ and TGF-β₂ are potent inhibitors of bronchial epithelial proliferation, the reduced localisation of TGF-βs to these cells may be directed towards epithelial proliferation and repair. However, greater levels of inflammatory cell derived TGF-β may counteract this. TGF-β₁ is capable of inducing epithelial-mesenchymal transition of bronchial epithelial cells in vitro but whether this occurs in this model or asthmatic airways is uncertain. TGF-β₁ plays a critical role in the recruitment/maintenance of macrophages and the synthesis/deposition of decorin as well as contributing to the recruitment/maintenance of eosinophils, lymphocytes and synthesis/deposition of collagen. TGF-β₂ appears to have a more restricted role, contributing to the recruitment/maintenance of eosinophils, lymphocytes and the synthesis/deposition of collagen. Red arrows denote synthesis/localisation of TGF-β isoforms, blue illustrates functions of the isoforms based on this study and the literature. The width of the arrows symbolise the apparent relative importance of the source of that isoform or its function.