Macrophage-derived GPNMB Trapped by Fibrotic Extracellular Matrix Promotes Pulmonary Fibrosis

doi:10.21203/rs.3.rs-1360030/v1

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Macrophage-derived GPNMB Trapped by Fibrotic Extracellular Matrix Promotes Pulmonary Fibrosis

https://doi.org/10.21203/rs.3.rs-1360030/v1

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posted 24 Mar, 2022

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Pulmonary fibrosis (PF) is a form of progressive lung disease characterized by chronic inflammation and excessive extracellular matrix (ECM) deposition. The pulmonary ECM undergoes profound changes during fibrosis. However, the protein changes that occur in fibrotic ECM during silica-induced progressive PF and their contribution to fibrosis progression are unclear. Here, we established a model of progressive PF induced by silica, observed the effect of fibrotic ECM on normal fibroblast activation and explored the possible mechanism. Proteomic analysis of the ECM revealed that obvious changes in the expression of ECM components and ECM remodeling occurred. Single-cell RNA sequencing (scRNA-seq) combined with spatial transcriptomics revealed that during transport, macrophage-derived glycoprotein nonmetastatic melanoma protein B (GPNMB) captured by fibrotic ECM may activate resident normal fibroblasts around fibrotic foci. Functional experiments showed activation of normal fibroblasts in fibrotic ECM, which was alleviated by GPNMB-neutralizing antibodies or macrophage deletion in the ECM of silica-instilled mice, implying that macrophage-derived GPNMB may contribute to progressive PF. Transcriptome analysis showed that the Serpinb2 expression level was increased in fibroblasts in fibrotic ECM, and the expression of CD44, a cell receptor for GPNMB, was increased in mice after silica instillation according to scRNA-seq combined with spatial transcriptomics. Therefore, macrophage-derived GPNMB is trapped by fibrotic ECM during transport and may activate fibroblasts via the CD44/Serpinb2 pathway, leading to further development of fibrosis.

Extracellular matrix

pulmonary fibrosis

fibroblast

macrophage

proteomics

Pulmonary fibrosis (PF) is a type of chronic lung disease that leads to a progressive and irreversible decline in lung function (King et al., 2011; Schafer et al., 2017). The incidence of PF is increasing year by year, and the prognosis of PF is poor. A variety of lung diseases eventually lead to PF, and the mechanism of PF has not been fully elucidated. PF is characterized by chronic inflammation, excessive deposition of extracellular matrix (ECM) components and lung tissue scarring resulting from aberrant wound healing in susceptible individuals (Chanda et al., 2019; Richeldi et al., 2017; Wijsenbeek and Cottin, 2020). Fiber proliferation is an integral part of host defense, and it ceases or regresses after mitigation or termination of the harmful stimulus (Herrera et al., 2018). However, a large amount of data have proven that PF progresses once it is established, suggesting that the initiation and sustained development of fibrosis can be independent of each other (Parker et al., 2014).

The ECM, which is present in all tissues, is a highly dynamic noncellular structure that serves as a physical scaffold for tissues and organs (Theocharis et al., 2016). The lung ECM plays important roles in lung health from the earliest stages of development and throughout adulthood and is involved in the regulation of developmental organogenesis, homeostasis and injury repair responses. In addition, the ECM is essential for cell functions such as survival, proliferation, migration, and differentiation via biochemical or biomechanical cues (Hynes, 2009). The ECM is composed of hundreds of proteins, including fiber-forming proteins such as collagens, laminins, fibronectin, elastin, glycoproteins, proteoglycans (PGs) and glycosaminoglycans, which form a complex three-dimensional matrix network. All cell types, especially fibroblasts, can synthesize and secrete matrix macromolecules, such as collagens and fibronectin, to participate in the construction of the ECM under the control of multiple signals (Bonnans et al., 2014; Humphrey et al., 2014). Data show that components of the ECM can serve as ligands for cell receptors to interact with cells and then transmit signals that orchestrate cell behaviors (Humphries et al., 2019). Studies have found that ECM components interact with cells via their surface receptors, such as integrins, CD44, and cell surface PGs. The ECM can also bind and locally release growth factors and cytokines; for example, fibroblast growth factor (FGF) binds avidly to heparin and heparin sulfate, a component of many ECM PGs, and can be released from the ECM by protein degradation (Hynes, 2009; Wijelath et al., 2006). Therefore, the ECM serves as a localized reservoir for soluble growth factors, and many growth factors may utilize heparan sulfate as a cofactor when they bind to their signal receptors. The ECM plays key roles in cell fates.

A large amount of data have proven that aberrant proliferation of activated fibroblasts and abnormally excessive collagen deposition and ECM remodeling occur during PF progression. Once activated, fibroblasts could synthesize and secrete excessive matrix proteins, resulting in abnormal remodeling of the ECM (Klingberg et al., 2013; Liu et al., 2021; Wollin et al., 2015). Recently, it was reported that in idiopathic pulmonary fibrosis (IPF), a common type of clinical PF, abnormal fibrotic ECM (that is, the ECM after lung fibrosis is established) can promote the activation of normal fibroblasts through a positive feedback loop by downregulating the expression of antifibrotic miR-29 family members in normal fibroblasts and further aggravate PF (Parker et al., 2014), which may explain why fibrosis can be self-sustaining once it is established. However, the possible mechanism by which fibrotic ECM affects cell fate and leads to exacerbation of PF has not been well defined.

To explore the contribution of fibrotic ECM to pathological gene expression in normal fibroblasts, we established a mouse model of chronic PF via intratracheal instillation of silica. Unlike the bleomycin-induced lung injury model, which exhibits transient physiological fibrosis (Schiller et al., 2015), the silica-induced model present progressive irreversible PF, allowing us to explore the true mechanism of the disease. We observed changes in ECM in mice 56 days after silica instillation and analyzed the changes in the protein levels of ECM components by proteomics. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were used to analyze the possible contributors to these changes. Normal fibroblasts were transplanted into decellularized mouse lung ECM, and the effects of normal ECM and fibrotic ECM on cell behaviors and the possible mechanism were evaluated.

Fibrotic ECM affects normal lung fibroblast function in silicosis mice

To explore the contribution of the fibrotic ECM to normal fibroblast function, chronic PF was first established in mice via intratracheal instillation of silica. The CT imaging of the mouse chest and hematoxylin and eosin (H&E) staining showed obvious collagen deposition and PF in mice 56 days after silica instillation (Fig. 1A), which is consistent with previous reports (Cheng et al., 2021b; Jiang et al., 2021). Furthermore, a pulmonary function test was performed, and it was found that several pulmonary function indicators, including inspiratory capacity (IC, the volume inspired during slow inspiration), the expiratory reserve volume (ERV), forced vital capacity (FVC, the volume expired during fast expiration) and functional residual capacity (FRC), were significantly decreased in the mice (Fig. 1B). These data showed that PF was well established 56 days after silica instillation, suggesting that the model of progressive PF was successfully constructed and could be used for further research.

To better study the changes in lung ECM after PF and explore the influences of lung ECM on fibroblast behaviors in vitro, mouse lung ECM was decellularized via treatment with detergents, salts and DNase following the steps shown in Fig. 1C to avoid the effects of endogenous cellular components on the results. We first confirmed that all cellular and nuclear material was eliminated via the decellularization process. H&E staining, Masson trichrome staining and DAPI staining showed that decellularized ECM reserved a similar architecture, albeit without evidence of cells (Figure S1A-C). Western blotting and qPCR data showed that there were hardly any cellular proteins or mRNAs in the acellular ECM (Figure S1D-F). Then, we cultured fibroblasts in the decellularized ECM, and the data showed that the cells survived (Figure S1). These results indicated that cells and nuclear material were cleared effectively by decellularization and that cells repopulated in the decellularized ECM, suggesting that the in vitro model could be used for our subsequent experiments.

What happened to the lung ECM 56 days after silica instillation? H&E staining and Masson trichrome staining showed excessive collagen deposition and obvious structural changes in the fibrotic ECM (Fig. 1D). Moreover, collagen I, fibronectin, collagen III and α-SMA presented obvious increases in expression as well as abnormal deposition (Figs. 1D,S2A and B). In addition, analysis of Young’s modulus showed that the stiffness of the fibrotic ECM was increased (Fig. 1E). These results indicated that the lung ECM was altered after fibrosis was established. To explore whether fibrotic ECM influences normal fibroblast function, we assessed the viability, proliferation, migration and activation of normal fibroblasts cultured in normal ECM and fibrotic ECM. The Cell Counting Kit-8 (CCK-8) assay showed that fibrotic ECM induced a clear increase in cell viability (Fig. 1F), and more cells were harvested from fibrotic ECM than from normal ECM (Fig. 1G). Fibroblasts can synthesize and secrete interstitial-associated proteins when activated (Karsdal et al., 2017), so we also studied mRNA expression levels in fibroblasts cultured in different types of ECM. The data showed that compared with normal ECM, fibrotic ECM increased α-SMA, collagen I and fibronectin mRNA expression levels in fibroblasts (Fig. 1H and J). In addition, fibrotic ECM promoted fibroblast migration (Figs. 1K, S2Cand D). All the above experimental results proved that the fibrotic ECM influences normal fibroblast function, leading to PF aggravation.

Protein expression in the fibrotic ECM changes significantly

The changes in the fibrotic ECM toned to be elucidated to determine the mechanism of fibroblast activation. The ECM is difficult to analyze biochemically because of its complexity and insolubility, and we tried to obtain detailed information about ECM components by mass spectrometry-based proteomics to. Analysis of the quality of the proteomics results showed that the results were reliable (Figures S3A-G). The data from all specimens were similar, and samples from the same group were most similar to each other (Fig. 2A). A total of 143 proteins with upregulated expression and 127 proteins with downregulated expression were identified in fibrotic ECM compared with normal ECM (Fig. 2B). Glycoprotein nonmetastatic melanoma protein B (GPNMB) was identified as the most highly upregulated protein in PF (Figs. 2C and S3H-J). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that GPNMB is related to pathways such as ECM-receptor interaction, lysosome, focal adhesion, protein digestion and absorption, complement and coagulation cascades, PI3K-Akt signaling pathway, apoptosis and phagosome (Fig. 2D), and Gene Ontology (GO) enrichment analysis revealed that GPNMB is enriched is identical protein binding, collagen-containing ECM, ECM, and tissue development (Fig. 2E and F). Western blotting analysis and immunofluorescence staining confirmed that GPNMB expression was substantially upregulated in the fibrotic ECM (Fig. 2G and H). Proteomics analysis showed that the increase GPNMB expression in the fibrotic ECM might be related to fibroblast activation.

GPNMB protein trapped by fibrotic ECM is involved in the effect of ECM on lung fibroblast function

Experiments were designed to assess the effect of GPNMB on fibroblast function. The viability of fibroblasts, as assessed by the CCK-8 assay, increased after GPNMB treatment (Fig. 3A). We harvested many more fibroblasts from the GPNMB-treated group (Fig. 3B), and the number of Ki67-positive cells was increased after treatment with GPNMB (Fig. 3C and D). Fibroblasts exposed to GPNMB migrated much more quickly than those exposed to vehicle (Fig. 3E and F). Moreover, α-SMA, collagen I and fibronectin mRNA expression levels were increased in fibroblasts of the GPNMB-treated group. These data proved that GPNMB can regulate normal fibroblast behaviors.

Then, we asked whether increased GPNMB expression in the fibrotic ECM affects normal fibroblast behaviors. Normal fibroblasts were cultured in different types of ECM with a GPNMB-neutralizing antibody, and then fibroblast migration was evaluated. The fibrotic ECM promoted fibroblast migration, and this effect was ameliorated by the GPNMB-neutralizing antibody (Fig. 3G). Therefore, increased GPNMB expression in the fibrotic ECM indeed influences normal fibroblast behaviors.

GPNMB captured by fibrotic ECM might be derived from macrophages

While the increase in GPNMB expression in the fibrotic ECM plays such an important role, it is unclear where this GPNMB is derived from. To identify the cellular origin of GPNMB, we obtained whole lungs from four groups of mice (the NS-7 day group, SiO₂-7 day group, NS-56 day group, and SiO₂-56 day group) and performed scRNA-seq. The transcriptomic data for the four groups were normalized, and graph-based clustering was applied to analyze and compare the samples, which yielded 20 cell clusters (Fig. 4A). The cell type for each cluster was then annotated according to canonical cell markers from CellMarker (Figure S4A). The data showed that the relative percentage of macrophages was obviously increased in the SiO₂-7 day group mice and even more in the SiO₂-56 day group mice (Figs. 4A and S4B), implying that macrophages participated in the inflammatory stage as well as the fibrotic stage. GPNMB was mainly derived from macrophages according to scRNA-seq analysis (Fig. 4B), and its expression was increased obviously in SiO₂-treated mice, especially in SiO₂-56 day group mice (Fig. 4A). Moreover, scRNA-seq combined with spatial transcriptomics showed that more GPNMB-positive macrophages accumulated in the focus area in SiO₂-7 day group mice and SiO₂-56 day group mice (Fig. 4C). Immunofluorescence staining revealed that macrophage infiltration and localization of GPNMB in macrophages were increased in the lungs of SiO₂-56 day group mice compared with those of NS-56 day group mice (Fig. 4D). As a result, we speculated that GPNMB protein in fibrotic ECM, which showed an increase in expression, may be derived from macrophages.

The effect of fibrotic ECM on lung fibroblast activation is alleviated by macrophage depletion

To prove that the cellular origin of GPNMB is macrophages, we administered clodronate liposomes to mice every 7 days to remove macrophages because macrophages are constantly produced (shown in Fig. 5A). The data indicated that administration of macrophage scavengers resulted in a marked increase in the number of macrophages in mice (Figure S5A and B). The lungs were obviously damaged and became solidified after exposure to the macrophage scavengers (Figure S5C). CT imaging of the chest showed that macrophage deletion by clodronate liposomes relieved PF induced by silica instillation (Fig. 5B). H&E staining and Masson trichrome staining showed that silica instillation caused obvious destruction of the alveolar structure and collagen deposition, which were mitigated by macrophage deletion (Figs. 5B and S5D). H&E staining and Masson trichrome staining also revealed that macrophage deletion inhibited the obvious structural changes in fibrotic ECM and excessive collagen deposition (Fig. 5C).

To assess the effect of fibrotic ECM on normal fibroblast behaviors after macrophage deletion in mice, we cultured fibroblasts in ECM harvested from mice in the four groups. Cell viability and fibroblast number were decreased in the fibrotic ECM of silicosis mice subjected to macrophage deletion (Fig. 5D and E). Macrophage deletion also relieved fibroblast migration induced by fibrotic ECM (Fig. 5F and G). These data indicated that the effect of fibrotic ECM on lung fibroblast function is relieved by macrophage depletion.

GPNMB protein expression is decreased in fibrotic ECM after macrophage depletion

Next, we evaluated GPNMB protein levels in fibrotic ECM after macrophage depletion to confirm the cellular origin of GPNMB in fibrotic ECM. As shown in Fig. 7A, a large number of macrophages infiltrated and colocalized with GPNMB in the lungs of mice 7 days and 56 days after silica instillation, and macrophage depletion decreased the GPNMB level, suggesting that macrophages increased in quantity and synthesized and secreted GPNMB during the progression of PF. Concomitantly, macrophage depletion decreased GPNMB protein levels in the lung ECM of mice subjected to silica instillation, further confirming the macrophage origin of GPNMB (Fig. 6A).

To further confirm our results, we treated RAW264.7 and THP-1 macrophages with silica and then observed the change in GPNMB protein levels. Western blot analysis showed that GPNMB protein levels increased after silica treatment (Figs. 6B and C, S6A and B). However, whether GPNMB produced by macrophages following stimulation with silica could bind to the ECM was unclear. Thus, we cultured RAW264.7 cells in normal ECM in the presence of silica. Surprisingly, we found that GPNMB levels in the ECM increased when RAW264.7 cells were cultured with silica (Fig. 6D); however, this result does not fully confirm that GPNMB produced by macrophages indeed bound to the ECM since the effects of cellular components could not be excluded. Therefore, we decellularized ECM with which RAW264.7 cells had been cultured. Immunofluorescence staining showed that GPNMB produced by macrophages could reliably bind to the ECM (Fig. 6D), indicating that the GPNMB in the fibrotic ECM of silicosis mice was derived from macrophages.

The CD44/Serpinb2 pathway is associated with changes in fibroblast function driven by fibrotic ECM

The increase in GPNMB expression plays a nonnegligible role in the effect of the fibrotic ECM on fibroblast activation confirmed above; however, the related mechanism remains unclear. To explore the possible mechanism, we cultured normal fibroblasts in normal ECM and fibrotic ECM, and then gene expression in fibroblasts was assessed via transcriptome analysis. The data from all samples were similar, and the data from samples in the same group were most similar to each other (Fig. 7A). Sixteen mRNAs with upregulated expression and 54 proteins with downregulated expression were identified in fibroblasts cultured in fibrotic ECM compared with those cultured in normal ECM (Fig. 7B). KEGG pathway analysis revealed that Serpinb2 was enriched in pathways such as regulation of actin cytoskeleton, tight junction, cell adhesion molecules (CAMs), calcium signaling pathway, focal adhesion, PI3K-Akt signaling pathway (Fig. 7C), and GO enrichment analysis showed that mRNAs that showed upregulated expression in fibroblasts cultured in fibrotic ECM were enriched in the terms external side of plasma membrane, sequence-specific DNA binding, DNA binding transcription factor activity, regulation of transcription and DNA-templated (Fig. 7D). Serpinb2, an inhibitor of extracellular urokinase plasminogen activator (uPA), was identified as the most highly upregulated mRNA possibly related to fibroblast activation. Western blot analysis confirmed that Serpinb2 levels were substantially increased in fibroblasts treated with GPNMB (Figs. 7E and S6C). scRNA-seq showed that Serpinb2 expression was increased in SiO₂-56 day group mice (Fig. 7F); however, whether GPNMB was contributed to this increase in Serpinb2 expression was unclear. Protein interaction network analysis showed that Serpinb2 could be regulated via CD44 (Fig. 7G), a receptor that mediates cell–cell and cell-matrix interactions through its affinity for HA and other ligands, such as collagens and matrix metalloproteinases (MMPs). The data in Fig. 7H show that CD44 expression was obviously increased in SiO₂-treated mice, especially in SiO₂-56 day group mice. Moreover, scRNA-seq combined with spatial transcriptomics revealed that CD44 levels were increased in the focus area in SiO₂-7 day group mice and SiO₂-56 day group mice (Fig. 7I). Figure 7J shows that CD44 was abundantly expressed in fibroblasts, implying that GPNMB might truly regulate Serpinb2 expression via CD44.

To further prove our hypothesis, fibroblasts were treated with GPNMB protein. To our surprise, Western blotting suggested that the CD44 protein level did not increase after GPNMB treatment (Figure S6D and E). Then, fibroblasts were incubated with transforming growth factor-β (TGF-β), which is a significant inflammatory factor that promotes fibrosis formation, and Western blotting showed that CD44 expression increased markedly (Figs. 7K and S6F), indicating that increased CD44 expression may be a result of the inflammatory environment in PF. These results suggested that CD44/Serpinb2 is related to changes in fibroblast function driven by increased GPNMB expression in the fibrotic ECM.

Considering the importance of GPNMB, we analyzed the protein levels of GPNMB in patients with PF. The data showed that GPNMB levels were increased in PF patients (Figures S7A-C), further suggesting that GPNMB protein levels are related to progressive PF.

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.

These data indicated that the additional GPNMB protein derived from macrophages in fibrotic ECM increased the Serpinb2 level in normal fibroblasts via CD44, leading to changes in cell behaviors that promoted the progression of PF. Drugs that can inhibit the abnormal increase in GPNMB levels in the ECM may have the potential to treat PF.

Detailed methods can be found in the online supplement.

Mice

C57BL/6 mice (male, 20 ± 2 g) purchased from Hangzhou Ziyuan Experimental Animal Co., Ltd. (Hangzhou, China), were housed at 23 °C and 50% humidity on a 12-h light/dark cycle with free access to food and water. All animal procedures were approved by the Laboratory Animal Care and Use Committee of Southeast University and were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Establishment of a mouse model of PF

Silica (Sigma) was sterilized and suspended (50 mg/ml) in sterile normal saline (NS). The mice were intratracheally administered the silica suspension (100 μL) after anesthesia with pentobarbital sodium (1%, 50 mg/kg). Mice in the NS group were given an equivalent volume of NS in the same way.

Decellularization of lung matrices

Decellularized ECM was harvested as previously reported with some modifications (Booth et al., 2012).

Proteomic analysis of the ECM (PXD028194)

ECM proteins were analyzed by liquid chromatography–mass spectrometry (LC–MS). The raw LC–MS/MS data were processed using Proteome Discover 2.4 (Thermo, USA).

Cell culture and treatment

Mouse lung fibroblast cells (MLG, ScienCell) were cultured in DMEM containing fetal bovine serum (FBS, 10%) and penicillin–streptomycin (PS). After they reached 80–90% confluency, the cells were exposed to GPNMB protein (R&D Systems) or TGF-β1 (Kingsley Biotechnology) and then used for subsequent experiments.

Reseeding acellular matrices

Fibroblasts were seeded dropwise on acellular slices and cultured in DMEM containing FBS (10%) and PS The medium was changed every other day.

Western blotting

RIPA buffer (Beyotime) containing a protease inhibitor cocktail (Roche) was used to isolate protein from samples. Primary antibodies against collagen I (1:1000, BioWorld), α-SMA (1:1000, Proteintech), FN1 (1:1000, Proteintech), GPNMB (1:1000, Abcam), Serpinb2 (1:1000, Affinity) and CD44 (1:1000, Proteintech) were used. GAPDH (1:1000, BioWorld) was used as an internal reference protein.

Cell migration assay

A 2D scratch assay was performed to assess cell migration capacity after treatment. Images of the scratch were captured at 0 h, 6 h and 12 h after addition of fresh medium. A 3-dimensional (3D) migration assay was used to evaluate cell migration as described previously with some modifications (Yang et al., 2018).

Histopathological analysis

Frozen slices were subjected to H&E (Biyun Tian) and Masson's trichrome (Biyun Tian) staining according to the manufacturer’s instructions.

Immunofluorescence staining

Frozen lung sections or cells were incubated with primary antibodies against collagen I (1:200, BioWorld), α-SMA (1:200, Proteintech), FN1 (1:200, Proteintech), collagen III (1:200, BioWorld), GPNMB (1:300, Abcam), or Adgre1 (1:300, Abcam). Cy3-conjugated (1:500, Invitrogen) and/or Alexa Fluor 488-conjugated (1:300, Invitrogen) secondary antibodies were added to the sections or cells. The cell nuclei were labeled with Hoechst 33342 or DAPI if needed.

Statistics

All data are presented as the mean ± standard error of the mean (SEM). Comparisons between two groups were analyzed using a two-tailed Student’s t test. Differences were considered significant at p<0.05. All analyses were conducted using GraphPad Prism version 8.0.

Author contributions

J.W., X.Z. and M.Y. performed the experiments, interpreted the data, prepared the figures, and wrote the manuscript. J.Y., M.L., S.W and Y.C. performed the experiments and interpreted the data. W.J. designed the experiments, interpreted the data, and wrote the manuscript. J.C. provided laboratory space and funding, designed the experiments, interpreted the data, wrote the manuscript, and directed the project. All the authors read, discussed, and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.

Acknowledgments

This study is the result of work that was partially supported by the resources and facilities at the Core Laboratory at the Medical School of Southeast University. This study is the result of work that was partially supported by resources and facilities at the Core Laboratory at the Medical School of Southeast University. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81972987, 81773796, 81901809 and 81700068), the National Key R&D Program of China (No. 2017YFA0104303) and Natural Science Foundation of Jiangsu Province (BK20190355).

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Macrophage-derived GPNMB Trapped by Fibrotic Extracellular Matrix Promotes Pulmonary Fibrosis

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Abstract

Figures

Introduction

Results

Fibrotic ECM affects normal lung fibroblast function in silicosis mice

Protein expression in the fibrotic ECM changes significantly

GPNMB captured by fibrotic ECM might be derived from macrophages

The effect of fibrotic ECM on lung fibroblast activation is alleviated by macrophage depletion

GPNMB protein expression is decreased in fibrotic ECM after macrophage depletion

The CD44/Serpinb2 pathway is associated with changes in fibroblast function driven by fibrotic ECM

Discussion

Conclusion

Star Methods

Declarations

References

Competing Interests

Supplementary Files

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