The effects of interleukin-33 on airways collagen deposition and matrix metalloproteinase expression in a murine surrogate of asthma

Abstract

It has been suggested that interleukin-33 (IL-33) plays an important role in the pathogenesis of asthma through a variety of pathways, but its role in airways fibrosis in asthma has not been fully elucidated. In the present study we evaluated changes in the expression of extracellular matrix proteins (ECMs) as well as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in an IL-33-induced, antigen-independent murine surrogate of asthma as well as a conventional surrogate employing per-nasal challenge of mice previously sensitized to produce an IgE response to ovalbumin (OVA). In addition, in in vitro experiments we explored the direct effects of IL-33 on the proliferation and function of murine fibroblasts. Per-nasal administration of IL-33 alone was sufficient to induce airways deposition of ECMs, including collagens I, III, V and fibronectin, to a degree comparable with that observed in the OVA-sensitized and challenged mice. These changes were associated with a local imbalance between the expression of extracellular MMPs and TIMPs. Per-nasal challenge of mice with IL-33 also induced elevated airways expression of connective tissue growth factor and fibroblast growth factor receptor 4, two key facilitators of local fibrosis, again to a degree compatible with that observed in OVA-sensitized and challenged mice. Deletion of the ST2 gene, which encodes the IL-33 receptor, abrogated these fibrotic changes in the airways in the OVA surrogate. In vitro, IL-33 significantly increased the proliferation and expression of collagen III by murine lung fibroblasts. These data suggest that direct exposure of murine airways to IL-33 is able to induce local fibrotic changes, at least partially through effects of signalling through the IL-33/ST2 axis on fibroblast function and local expression of MMPs and their inhibitors, and other fibrosis-related proteins.

Keywords: asthma; fibrosis; interleukin-33; pathogenesis; remodelling.

Figure 1

(a) Schedule of murine challenge. (b) Representative photomicrographs of collagen I immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and interleukin‐33 (IL‐33) ‐challenged mice at various time‐points as indicated (original magnification ×20). (c) Quantitative analysis of collagen I immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 2

Interleukin‐33 (IL‐33) induced fibronectin deposition in the lungs of challenged animals. (a) Representative photomicrographs of fibronectin immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of fibronectin immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 3

Interleukin‐33 (IL‐33) promotes connective tissue growth factor (CTGF) expression in the lungs of challenged animals. (a) Representative photomicrographs of CTGF immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of CTGF immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 4

Interleukin‐33 (IL‐33) promotes fibroblast growth factor receptor 4 (FGFR4) expression in the lungs of challenged animals. (a) Representative photomicrographs of FGFR4 immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of FGFR4 immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time point). *P < 0·05.

Figure 5

Interleukin‐33 (IL‐33) promotes matrix metalloproteinase 1 (MMP1) expression in the lungs of challenged animals. (a) Representative photomicrographs of MMP1 immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of MMP1 immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time point). *P < 0·05.

Figure 6

Interleukin‐33 (IL‐33) promotes matrix metalloproteinase 9 (MMP9) expression in the lungs of challenged animals. (a) Representative photomicrographs of MMP9 immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of MMP9 immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 7

Interleukin‐33 (IL‐33) fails to promote matrix metalloproteinase 2 (MMP2) expression in the lungs of challenged animals. (a) Representative photomicrographs of MMP2 immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of MMP2 immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 8

Interleukin‐33 (IL‐33) promotes tissue inhibitor of metalloproteinase 1 (TIMP1) expression in the lungs of challenged animals. (a) Representative photomicrographs of TIMP1 immunoreactivity in lung sections from saline (NS)‐, ovalbumin (OVA)‐ and IL‐33‐challenged mice at various time‐points as indicated (original magnification ×20). (b) Quantitative analysis of TIMP1 immunoreactivity. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.

Figure 9

Interleukin‐33 (IL‐33) promotes fibroblast proliferation and collagen production. (a) Representative photomicrographs of Western blot assay. (b) IL‐33 promotes fibroblast proliferation. (c, d) Quantitative analysis of fibronectin and collagen III expression. The data are expressed as the mean ± SEM (n = 5 in each group in proliferation experiment and n = 3 in each group in Western blot). *P < 0·05.

Figure 10

Effect of ST2 receptor knockdown on airways fibrosis in an ovalbumin (OVA) ‐challenged asthma model on 24 days. The data are expressed as the mean ± SEM (n = 5 in each group at each time‐point). *P < 0·05.