Fstl1 Promotes Asthmatic Airway Remodeling by Inducing Oncostatin M

Abstract

Chronic asthma is associated with airway remodeling and decline in lung function. In this article, we show that follistatin-like 1 (Fstl1), a mediator not previously associated with asthma, is highly expressed by macrophages in the lungs of humans with severe asthma. Chronic allergen-challenged Lys-Cre(tg) /Fstl1(Δ/Δ) mice in whom Fstl1 is inactivated in macrophages/myeloid cells had significantly reduced airway remodeling and reduced levels of oncostatin M (OSM), a cytokine previously not known to be regulated by Fstl1. The importance of the Fstl1 induction of OSM to airway remodeling was demonstrated in murine studies in which administration of Fstl1 induced airway remodeling and increased OSM, whereas administration of an anti-OSM Ab blocked the effect of Fstl1 on inducing airway remodeling, eosinophilic airway inflammation, and airway hyperresponsiveness, all cardinal features of asthma. Overall, these studies demonstrate that the Fstl1/OSM pathway may be a novel pathway to inhibit airway remodeling in severe human asthma.

Figure 1. Fstl1 is highly expressed in lungs of severe human asthma

Lungs from human asthmatics were processed for immunofluorescence microscopy to detect either Fstl1 (Fig. 1a), CD68 (Fig. 1b), or Fstl1 and CD68 (Fig. 1c). Bronchial biopsies from human subjects with severe asthma, mild asthma, or no asthma were processed for immunohistochemistry with an anti-Fstl1 Ab (n=10 subjects/group)(Fig 1d). The number of peribronchial Fstl1 positive cells were quantitated by light microscopy and image analysis in each group.

Figure 2. M2 macrophages highly express Fstl1 in response to chronic allergen

WT mice were sensitized to OVA and challenged acutely or chronically with OVA allergen (8 mice/group). Lungs were processed to detect Fstl1 mRNA by qPCR (Fig. 2a). BAL fluid levels of Fstl1 protein was measured by ELISA (Fig. 2b). Lungs from non-OVA challenged mice were processed for immunofluorescence microscopy to detect either F4/80 (Fig. 2c), Fstl1 (Fig. 2d), or arginase (Fig. 2e). Lungs from chronic OVA challenged WT mice were processed for immunofluorescence microscopy to detect either F4/80 (Fig. 2f), Fstl1 (Fig. 2g), or F4/80 and Fstl1 (Fig. 2h), as well as to detect arginase (Fig. 2i), Fstl1 (Fig. 2j), or arginase and Fstl1 (Fig. 2k). Image analysis quantitated the % of Fstl1 positive cells that co-expressed arginase (Fig. 2l). Levels of Fstl1 mRNA were quantitated by qPCR in mouse macrophages (Fig. 2m), fibroblasts (Fig. 2n), or epithelial cells (Fig. 2o) incubated with either TGFβ1 a known stimulator of Fstl1 expression, IL-4, or Fstl1.

Figure 3. Inhibition of macrophage Fstl1 expression inhibits airway remodeling

Lys-Cre^tg/Fstl1^Δ/Δ or WT mice (8 mice/group) were sensitized with OVA allergen followed by chronic exposure to OVA allergen. Levels of lung mucus were quantitated by PAS staining (Fig. 3a). Levels of peribronchial trichrome staining were quantitated by image analysis (Fig 3b). The number of peribronchial eosinophils was quantitated by MBP immunostaining and image analysis (Fig. 3c). Levels of oncostain M (OSM) were quantitated by qPCR (Fig. 3d). Levels of BAL MMP9 were quantitated by ELISA (Fig. 3e). In separate experiments, WT mouse bone marrow derived macrophages were incubated for 24 hrs with either Fstl1 (100 ng/ml) or media and levels of OSM mRNA quantitated by qPCR (Fig. 3f).

Figure 4. Chronic Fstl1 induces airway remodeling

WT mice (8 mice/group) were administered Fstl1 intranasally daily for 15 days prior to sacrifice (WT+ Fstl1 group). A control WT group did not receive Fstl1 (WT). Lungs from the different groups of WT mice were processed for PAS staining (Fig. 4a–c), Muc5ac immunostaining (Fig. 4d–e), assessment of expression of the mucus gene Muc5AC by qPCR (Fig. 4f), quantitation of peribronchial fibrosis by trichrome staining and image analysis (Fig. 4g), measurement of AHR (Fig. 4h), quantitation of lung MBP positive eosinophils (Fig. 4i), BAL inflammatory cells (Fig. 4j), and assessment of cytokine gene expression by qPCR including IL-5 (Fig. 4k), eotaxin-1 (Fig. 4l), TNFα (Fig. 4m), and oncostatin M (OSM) (Fig. 4n). Levels of serum IgE were quantitated by Elisa (Fig. 4o).

Figure 5. Blocking Oncostain M inhibits Fstl1 induced airway remodeling

WT mice (4 mice/group) were administered Fstl1 intranasally daily for 15 days, with or without pre-treatment with an anti-oncostatin M antibody (anti-OSM). A control WT group received the anti-oncostatin M antibody and no Fstl1. Levels of lung mucus were quantitated by PAS staining (Fig. 5a–d). Levels of peribronchial trichrome staining were quantitated by image analysis (Fig. 5e–h). The number of MBP+ peribronchial eosinophils were quantitated by image analysis (Fig. 5i–l). The number of Wright-Giemsa stained BAL eosinophils was quantitated by light microscopy (Fig. 5m). Levels of airway responsiveness to methacholine was assessed by flexivent (Fig. 5n). In a separate experiment, lungs from either WT mice subjected to chronic OVA challenge (WT+OVA), or WT mice not challenged with OVA (WT+ No OVA), were immunostained with an anti-OSM Ab to detect OSM positive cells in the lung.

Figure 6. Fstl1 can also directly induce remodeling in vitro

Mouse lung fibroblasts (Fig. 6a), human airway epithelial cells (Fig. 6b, 6c), or mouse lung smooth muscle cells (Fig. 6d), were incubated for 24 hrs with either Fstl1 (100 ng/ml), TGFβ1 (50 ng/ml), or media. Levels of collagen mRNA (Col1α1; Col3α1; Col5α1) expressed by fibroblasts were assessed by qPCR (Fig. 6a). Levels of mucus gene (Muc5AC)(Fig. 6b) and RANTES mRNA expression (Fig. 6c) were assessed by qPCR in human bronchial epithelial cell incubated with Fstl1, TGFβ1, or media. Levels of smooth muscle contraction (Fig. 6d, 6e) were assessed at baseline (time 0 or 0h), as well as 24 hours (24h) after incubation with either Fstl1, TGFβ1, or media (Fig. 6d, 6e). With agonist-induced smooth muscle contraction the area of the gel decreases significantly.