Despite the profound changes that the ECM undergoes during PF, there have been insufficient studies on the changes in the expression of ECM proteins during progressive PF, and the mechanism by which the fibrotic ECM affects cell fates and then leads to exacerbation of PF has not been well defined. Here, we found that an increase in the protein expression of GPNMB, which was mainly derived from macrophages, in the fibrotic ECM promoted normal fibroblast activation via the CD44/Serpinb2 pathway, possibly accounting for exacerbation of PF after its establishment.
Here, we used silica instead of bleomycin, which is commonly used to induce PF formation, to establish a progressive PF model in mice. Bleomycin can induce transient physiological fibrosis that gradually resolves within four to eight weeks (Rock et al., 2011; Schiller et al., 2015). Considering the purpose of our exploration, silica was used to construct a PF model for further study. Our data showed that the pulmonary function of mice was significantly decreased 56 days after silica instillation, and CT imaging of the mouse chest and H&E staining showed extensive alveolar structure collapse and excessive collagen deposition, indicating well-established PF. Previous reports have also pointed out that PF develops 56 days after silica intratracheal instillation (Cheng et al., 2021a; Dinh et al., 2020; Liu et al., 2017). Therefore, we collected data and samples 56 days after silica treatment in our study.
As an important component of tissues and organs, the ECM can not only provide physical support for cells but also regulate cell behaviors. A large amount of data has proven that excessive collagen deposition and obvious ECM remodeling occur after PF and that these processes affect the cell functions, such as activation, proliferation, migration and adhesion (Haak et al., 2018; Herrera et al., 2018). Fibrotic ECM downregulates the expression of antifibrotic miR-29 family members and activates a profibrotic positive feedback loop, leading to exacerbation of PF (Herrera et al., 2018; Parker et al., 2014). Therefore, considering the contribution of the ECM to cell behaviors, the initial damage might lead to the development of only a small fibrotic region in the lung and fibrosis of the ECM, which impacts and activates nearby fibroblasts, leading to surrounding lung tissue reconstruction and the spread of fibrosis. To explore the possibility of this phenomenon, we harvested pulmonary ECM from normal mice and silica-treated mice via decellularization and then cultured fibroblasts with the ECM. The structure of the harvested ECM and the successful removal of the cellular components were experimentally verified. The ECM we obtained retained a relatively complete and orderly structure, and intracellular components were undetectable, indicating that the ECM could be used for further study. In fact, the data indeed showed changes in fibrotic ECM, and the proliferation, migration and activation of fibroblasts cultured in the silica-induced fibrotic ECM changed obviously. This aroused our curiosity. What alterations occurred in the ECM that led to changes in cell function?
Since the structure of the ECM is complex and most ECM components are insoluble macromolecular proteins, it is difficult to explore this structure. We used mass spectrometry-based proteomics to assess and analyze the changes in protein expression in lung ECM from normal mice and silica-treated mice. Fortunately, we were able to map the changes in ECM protein expression between the two groups. A total of 143 proteins with upregulated expression and 127 proteins with downregulated expression were identified in fibrotic ECM compared with normal ECM. Through protein interaction network analysis, we confirmed that GPNMB trapped by fibrotic ECM might play a nonnegligible role in the activation of fibroblasts. GPNMB, a transmembrane glycoprotein that exists in a soluble form, is widely expressed in many cell types and participates in the regulation of multiple functions in various tissues. GPNMB is also known as osteoactivin because of its role in osteoblast differentiation and increasing bone mineral deposition (Tsou and Sawalha, 2020). GPNMB indeed affected cell proliferation, migration and activation, but these findings were not enough to confirm that GPNMB trapped by fibrotic ECM contributed to these changes. To explore the role of GPNMB captured by fibrotic ECM, we used neutralizing antibodies to assess the effect of GPNMB on the fibrotic ECM. The effect of the fibrotic ECM on normal fibroblasts was alleviated after treatment with a GPNMB-neutralizing antibody, confirming the contribution of GPNMB trapped in the fibrotic ECM to these changes.
Since the increase in GPNMB expression plays an important role in the effect of fibrotic ECM, where does the excess GPNMB come from? By combining mass spectrometry-based proteomics, scRNA-seq and spatial transcriptomics, we confirmed that GPNMB might be derived from macrophages. Macrophages are activated in response to silica, leading to changes in cell morphology and cell secretion patterns, which play roles in the progression of PF (Du et al., 2019; Fang et al., 2018; Li et al., 2017). We employed clodronate liposomes to remove macrophages in mice (Rivera et al., 2007; Winkler et al., 2010), and H&E staining and Masson trichrome staining showed that PF was significantly alleviated after clodronate liposome administration. Moreover, ECM from silica-treated mice subjected to macrophage deletion had a less marked effect on the viability, proliferation, and migration of normal fibroblasts and exhibited a significant decrease in GPNMB expression. These data suggested that GPNMB in the fibrotic ECM might be derived from macrophages and involved in regulating cell behaviors.
ECM components can serve as ligands to allow cell receptors to interact with cells and then transmit signals that orchestrate cell behaviors (Chaudhuri et al., 2020; Walraven and Hinz, 2018). How does GPNMB in fibrotic ECM regulate cell function? Transcriptome analysis showed that Serpinb2 mRNA expression was upregulated in fibroblasts in fibrotic ECM compared with those in normal ECM. Serpinb2, a regulator of inflammatory processes, is involved in aging, injury, and repair (Sen et al., 2020). Studies have shown that the activity of uPA is associated with lung fibrosis and that uPA overexpression enhances the resolution of established lung fibrosis (Horowitz et al., 2019). Serpinb2 is an inhibitor of extracellular uPA and might be related to lung fibrosis development. Our data showed that Serpinb2 levels were increased in GPNMB-treated fibroblasts. Studies have found that ECM components interact with cells via their surface receptors, and in both forms, GPNMB has been shown to interact with many different partners, such as integrins, heparan sulfate PG, tyrosine kinase receptors, and transporters. CD44 is a transmembrane glycoprotein that is widely expressed on various cell types and is a receptor for GPNMB (Neal et al., 2018; Tsou and Sawalha, 2020; Wynn, 2011). CD44 plays an important role in various cellular events, including activation, proliferation, migration, and adhesion, via cell-matrix or cell–cell interactions. scRNA-seq and spatial transcriptomics showed that CD44 expression was increased in the lungs of silica-treated mice. However, the CD44 level did not increase after exposure to GPNMB. All kinds of cells take part in tissue repair and reconstruction. They play diverse roles, including releasing many inflammatory factors, such as TGF-β1, to form an inflammatory environment at the site of lung injury (Horowitz et al., 2019). TGF-β1 is an important fibrogenic factor, and its expression has been found to increase during the fibrosis process (Wynn and Ramalingam, 2012). We wondered whether the inflammatory environment during fibrosis leads to upregulation of CD44 expression. Therefore, we treated fibroblasts with TGF-β1, and the CD44 expression level indeed increased, consistent with our hypothesis. Our data showed that fibrotic ECM captured GPNMB derived from macrophages, leading to a local increase in the protein level of GPNMB and that some inflammatory factors released by cells during fibrosis increased CD44 levels in normal fibroblasts around the fibrotic ECM. Therefore, GPNMB trapped by fibrotic ECM encountered CD44 in normal fibroblasts and Serpinb2 levels increased, leading to cell activation and further promotion of PF development.