An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

An immune-based tool platform for in vivo cell clearance.

Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.

Impaired Myofibroblast Proliferation is a Central Feature of Pathologic Post-Natal Alveolar Simplification.

Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFß) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFß signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFß signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Macrophage Cx43 Is Necessary for Fibroblast Cytosolic Calcium and Lung Fibrosis After Injury.

Macrophages are paracrine signalers that regulate tissular responses to injury through interactions with parenchymal cells. Connexin hemichannels have recently been shown to mediate efflux of ATP by macrophages, with resulting cytosolic calcium responses in adjacent cells. Here we report that lung macrophages with deletion of connexin 43 (MacΔCx43) had decreased ATP efflux into the extracellular space and induced a decreased cytosolic calcium response in co-cultured fibroblasts compared to WT macrophages. Furthermore, MacΔCx43 mice had decreased lung fibrosis after bleomycin-induced injury. Interrogating single cell data for human and mouse, we found that P2rx4 was the most highly expressed ATP receptor and calcium channel in lung fibroblasts and that its expression was increased in the setting of fibrosis. Fibroblast-specific deletion of P2rx4 in mice decreased lung fibrosis and collagen expression in lung fibroblasts in the bleomycin model. Taken together, these studies reveal a Cx43-dependent profibrotic effect of lung macrophages and support development of fibroblast P2rx4 as a therapeutic target for lung fibrosis.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner.

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Gene silencing by EZH2 suppresses TGF-ß activity within the decidua to avert pregnancy-adverse wound healing at the maternal-fetal interface.

A little-appreciated feature of early pregnancy is that embryo implantation and placental outgrowth do not evoke wound-healing responses in the decidua, the specialized endometrial tissue that surrounds the conceptus. Here, we provide evidence that this phenomenon is partly due to an active program of gene silencing mediated by EZH2, a histone methyltransferase that generates repressive histone 3 lysine 27 trimethyl (H3K27me3) histone marks. We find that pregnancies in mice with EZH2-deficient decidual stromal cells frequently fail by mid-gestation, with the decidua showing ectopic myofibroblast formation, peri-embryonic collagen deposition, and gene expression profiles associated with transforming growth factor ß (TGF-ß)-driven fibroblast activation and fibrogenic extracellular matrix (ECM) remodeling. Analogous responses are observed when the mutant decidua is surgically wounded, while blockade of TGF-ß receptor signaling inhibits the defects and improves reproductive outcomes. Together, these results highlight a critical feature of reproductive success and have implications for the context-specific control of TGF-ß-mediated wound-healing responses elsewhere in the body.

Integrin αvß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy.

αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.

Gli1+ mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung.

Aberrant epithelial reprogramming can induce metaplastic differentiation at sites of tissue injury that culminates in transformed barriers composed of scar and metaplastic epithelium. While the plasticity of epithelial stem cells is well characterized, the identity and role of the niche has not been delineated in metaplasia. Here, we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts during scarring, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation signalling to upregulate BMP antagonism in the progenitor niche that promotes metaplasia. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation into SFTPC+ epithelium. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signalling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.

Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis.

Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1-expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1-expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis.

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking.

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.

CXCR6 regulates localization of tissue-resident memory CD8 T cells to the airways.

Resident memory T cells (TRM cells) are an important first-line defense against respiratory pathogens, but the unique contributions of lung TRM cell populations to protective immunity and the factors that govern their localization to different compartments of the lung are not well understood. Here, we show that airway and interstitial TRM cells have distinct effector functions and that CXCR6 controls the partitioning of TRM cells within the lung by recruiting CD8 TRM cells to the airways. The absence of CXCR6 significantly decreases airway CD8 TRM cells due to altered trafficking of CXCR6-/- cells within the lung, and not decreased survival in the airways. CXCL16, the ligand for CXCR6, is localized primarily at the respiratory epithelium, and mice lacking CXCL16 also had decreased CD8 TRM cells in the airways. Finally, blocking CXCL16 inhibited the steady-state maintenance of airway TRM cells. Thus, the CXCR6/CXCL16 signaling axis controls the localization of TRM cells to different compartments of the lung and maintains airway TRM cells.

Gli signaling pathway modulates fibroblast activation and facilitates scar formation in pulmonary fibrosis.

Pulmonary fibrosis is characterized by progressive and irreversible scarring of alveoli, which causes reduction of surface epithelial area and eventually respiratory failure. The precise mechanism of alveolar scarring is poorly understood. In this study, we explored transcriptional signatures of activated fibroblasts in alveolar airspaces by using intratracheal transfer in bleomycin-induced lung fibrosis. Lung fibroblasts transferred into injured alveoli upregulated genes related to translation and metabolism in the first two days, and upregulated genes related to extracellular matrix (ECM) production between day 2 and 7. Upstream analysis of these upregulated genes suggested possible contribution of hypoxia-inducible factors 1a (Hif1a) to fibroblast activation in the first two days, and possible contribution of kruppel-like factor 4 (Klf4) and glioma-associated oncogene (Gli) transcription factors to fibroblast activation in the following profibrotic phase. Fibroblasts purified based on high Acta2 expression after intratracheal transfer were also characterized by ECM production and upstream regulation by Klf4 and Gli proteins. Pharmacological inhibition of Gli proteins by GANT61 in bleomycin-induced lung fibrosis altered the pattern of scarring characterized by dilated airspaces and smaller fibroblast clusters. Activated fibroblasts isolated from GANT61-treated mice showed decreased migration capacity, suggesting that Gli signaling inhibition attenuated fibroblast activation. In conclusion, we revealed transcriptional signatures and possible upstream regulators of activated fibroblasts in injured alveolar airspaces. The altered scar formation by Gli signaling inhibition supports that activated fibroblasts in alveolar airspaces may play a critical role in scar formation.

Engraftment and proliferation potential of embryonic lung tissue cells in irradiated mice with emphysema.

Recently, there has been increasing interest in stem cell transplantation therapy, to treat chronic respiratory diseases, using lung epithelial cells or alveolospheres derived from endogenous lung progenitor cells. However, optimal transplantation strategy of these cells has not been addressed. To gain insight into the optimization of stem cell transplantation therapy, we investigated whether lung cell engraftment potential differ among different developmental stages. After preconditioning with irradiation and elastase to induce lung damage, we infused embryonic day 15.5 (E15.5) CAG-EGFP whole lung cells, and confirmed the engraftment of epithelial cells, endothelial cells, and mesenchymal cells. The number of EGFP-positive epithelial cells increased from day 7 to 28 after infusion. Among epithelial cells derived from E13.5, E15.5, E18.5, P7, P14, and P56 mice, E15.5 cells demonstrated the most efficient engraftment. In vitro, E15.5 epithelial cells showed high proliferation potential. Transcriptome analyses of sorted epithelial cells from E13.5, E15.5, E18.5, P14, and P56 mice revealed that cell cycle and cell-cell adhesion genes were highly enriched in E15.5 epithelial cells. Our findings suggest that cell therapy for lung diseases might be most effective when epithelial cells with transcriptional traits similar to those of E15.5 epithelial cells are used.

Transcriptome network analysis identifies protective role of the LXR/SREBP-1c axis in murine pulmonary fibrosis.

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Although lung fibroblasts play a central role in PF, the key regulatory molecules involved in this process remain unknown. To address this issue, we performed a time-course transcriptome analysis on lung fibroblasts of bleomycin- and silica-treated murine lungs. We found gene modules whose expression kinetics were associated with the progression of PF and human idiopathic PF (IPF). Upstream analysis of a transcriptome network helped in identifying 55 hub transcription factors that were highly connected with PF-associated gene modules. Of these hubs, the expression of Srebf1 decreased in line with progression of PF and human IPF, suggesting its suppressive role in fibroblast activation. Consistently, adoptive transfer and genetic modification studies revealed that the hub transcription factor SREBP-1c suppressed PF-associated gene expression changes in lung fibroblasts and PF pathology in vivo. Moreover, therapeutic pharmacological activation of LXR, an SREBP-1c activator, suppressed the Srebf1-dependent activation of fibroblasts and progression of PF. Thus, SREBP-1c acts as a protective hub of lung fibroblast activation in PF. Collectively, the findings of the current study may prove to be valuable in the development of effective therapeutic strategies for PF.

Lung fibroblasts express a miR-19a-19b-20a sub-cluster to suppress TGF-ß-associated fibroblast activation in murine pulmonary fibrosis.

Lung fibroblasts play a pivotal role in pulmonary fibrosis, a devastating lung disease, by producing extracellular matrix. MicroRNAs (miRNAs) suppress numerous genes post-transcriptionally; however, the roles of miRNAs in activated fibroblasts in fibrotic lungs remain poorly understood. To elucidate these roles, we performed global miRNA-expression profiling of fibroblasts from bleomycin- and silica-induced fibrotic lungs and investigated the functions of miRNAs in activated lung fibroblasts. Clustering analysis of global miRNA-expression data identified miRNA signatures exhibiting increased expression during fibrosis progression. Among these signatures, we found that a miR-19a-19b-20a sub-cluster suppressed TGF-ß-induced activation of fibroblasts in vitro. Moreover, to elucidate whether fibroblast-specific intervention against the sub-cluster modulates pathogenic activation of fibroblasts in fibrotic lungs, we intratracheally transferred the sub-cluster-overexpressing fibroblasts into bleomycin-treated lungs. Global transcriptome analysis of the intratracheally transferred fibroblasts revealed that the sub-cluster not only downregulated expression of TGF-ß-associated pro-fibrotic genes, including Acta2, Col1a1, Ctgf, and Serpine1, but also upregulated expression of the anti-fibrotic genes Dcn, Igfbp5, and Mmp3 in activated lung fibroblasts. Collectively, these findings indicated that upregulation of the miR-19a-19b-20a sub-cluster expression in lung fibroblasts counteracted TGF-ß-associated pathogenic activation of fibroblasts in murine pulmonary fibrosis.

Fra-2 negatively regulates postnatal alveolar septation by modulating myofibroblast function.

Mice that globally overexpress the transcription factor Fos-related antigen-2 (Fra-2) develop extensive pulmonary fibrosis and pulmonary vascular remodeling. To determine if these phenotypes are a consequence of ectopic Fra-2 expression in vascular smooth muscle cells and myofibroblasts, we generated mice that overexpress Fra-2 specifically in these cell types (α-SMA-rtTA;tetO-Fra-2). Surprisingly, these mice did not develop vascular remodeling or pulmonary fibrosis but did develop a spontaneous emphysema-like phenotype characterized by alveolar enlargement. Secondary septa formation is an important step in the normal development of lung alveoli, and α-smooth muscle actin (SMA)-expressing fibroblasts (myofibroblasts) play a crucial role in this process. The mutant mice showed reduced numbers of secondary septa at postnatal day 7 and enlarged alveolae starting at postnatal day 12, suggesting impairment of secondary septa formation. Lineage tracing using α-SMA-rtTA mice crossed to a floxed TdTomato reporter revealed that embryonic expression of α-SMA Cre marked a population of cells that gave rise to nearly all alveolar myofibroblasts. Comprehensive transcriptome analyses (RNA sequencing) demonstrated that the overwhelming majority of genes whose expression was significantly altered by overexpression of Fra-2 in myofibroblasts encoded secreted proteins, components of the extracellular matrix (ECM), and cell adhesion-associated genes, including coordinate upregulation of pairs of integrins and their principal ECM ligands. In addition, primary myofibroblasts isolated from the mutant mice showed reduced migration capacity. These findings suggest that Fra-2 overexpression might impair myofibroblast functions crucial for secondary septation, such as myofibroblast migration across alveoli, by perturbing interactions between integrins and locally produced components of the ECM.

Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis.

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor ß1 (TGF-ß1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-ß1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.

Intratracheal cell transfer demonstrates the profibrotic potential of resident fibroblasts in pulmonary fibrosis.

Pulmonary fibrosis is a devastating disease for which there are few effective therapies. Activated fibroblasts form subepithelial clusters known as fibroblastic foci, which are characterized by excessive collagen deposition. The origin of activated fibroblasts is controversial and needs to be clarified to understand their pathogenicity. Here, using an intratracheal adoptive cell transfer method, we show that resident fibroblasts in alveolar walls have the highest profibrotic potential. By using collagen I(α)2-green fluorescent protein and neural/glial antigen 2-DsRed fluorescent reporter mice, we identified resident fibroblasts and pericytes in the alveolar walls based on surface marker expression and ultrastructural characteristics. In the early phase of bleomycin-induced pulmonary fibrosis, activated fibroblasts migrated into epithelium-denuded alveolar airspaces. Purified resident fibroblasts delivered into injured alveoli by an intratracheal route showed similar activated signatures as activated fibroblasts and formed fibroblastic foci. Neither pericytes nor epithelial cells had the same profibrotic potential. Transferred resident fibroblasts highly up-regulated profibrotic genes including α-smooth muscle actin and were a significant source of collagen deposition. These data provide insights into the cellular mechanisms of fibrogenesis and show intratracheal cell transfer to be a useful tool for exploring novel therapeutic targets against pulmonary fibrosis.

Reduced supply of monocyte-derived macrophages leads to a transition from nodular to diffuse lesions and tissue cell activation in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses in vivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuse lesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, and Fgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.