Abstract
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).
Background
Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins.
Materials and Reagents
Equipment
Procedure
Data analysis
Results can be shown as a representative immunoblot (Figure 1); also see Larson-Casey et al. (2016). Figure 1. Immunoblot analysis of α-SMA in IMR-90 fibroblasts cultured in BAL fluid (BALF) from saline or bleomycin (Bleo) exposed WT mice
Recipes
Acknowledgments
This work was supported by 2R01ES015981 & VA merit review BX001135.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.