Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis.

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 µm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

Impaired PPARγ activation by cadmium exacerbates infection-induced lung injury.

Emerging data indicate an association between environmental heavy metal exposure and lung disease, including lower respiratory tract infections (LRTIs). Here, we show by single-cell RNA sequencing an increase in Pparg gene expression in lung macrophages from mice exposed to cadmium and/or infected with Streptococcus pneumoniae. However, the heavy metal cadmium or infection mediated an inhibitory posttranslational modification of peroxisome proliferator-activated receptor γ (PPARγ) to exacerbate LRTIs. Cadmium and infection increased ERK activation to regulate PPARγ degradation in monocyte-derived macrophages. Mice harboring a conditional deletion of Pparg in monocyte-derived macrophages had more severe S. pneumoniae infection after cadmium exposure, showed greater lung injury, and had increased mortality. Inhibition of ERK activation with BVD-523 protected mice from lung injury after cadmium exposure or infection. Moreover, individuals residing in areas of high air cadmium levels had increased cadmium concentration in their bronchoalveolar lavage (BAL) fluid, increased barrier dysfunction, and showed PPARγ inhibition that was mediated, at least in part, by ERK activation in isolated BAL cells. These observations suggest that impaired activation of PPARγ in monocyte-derived macrophages exacerbates lung injury and the severity of LRTIs.

Mitochondrial uncoupling protein-2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age-associated lung fibrosis.

Mitochondrial dysfunction has been associated with age-related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein-2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro-oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro-fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age-related diseases associated with impaired tissue regeneration and organ fibrosis.

Targeting Cpt1a-Bcl-2 interaction modulates apoptosis resistance and fibrotic remodeling.

The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid ß-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid ß-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.

NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression.

Pulmonary fibrosis is a progressive lung disease often occurring secondary to environmental exposure. Asbestos exposure is an important environmental mediator of lung fibrosis and remains a significant cause of disease despite strict regulations to limit exposure. Lung macrophages play an integral role in the pathogenesis of fibrosis induced by asbestos (asbestosis), in part by generating reactive oxygen species (ROS) and promoting resistance to apoptosis. However, the mechanism by which macrophages acquire apoptosis resistance is not known. Here, we confirm that macrophages isolated from asbestosis subjects are resistant to apoptosis and show they are associated with enhanced mitochondrial content of NADPH oxidase 4 (NOX4), which generates mitochondrial ROS generation. Similar results were seen in chrysotile-exposed WT mice, while macrophages from Nox4-/- mice showed increased apoptosis. NOX4 regulated apoptosis resistance by activating Akt1-mediated Bcl-2-associated death phosphorylation. Demonstrating the importance of NOX4-mediated apoptosis resistance in fibrotic remodeling, mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages exhibited increased apoptosis and were protected from pulmonary fibrosis. Moreover, resolution occurred when Nox4 was deleted in monocyte-derived macrophages in mice with established fibrosis. These observations suggest that NOX4 regulates apoptosis resistance in monocyte-derived macrophages and contributes to the pathogenesis of pulmonary fibrosis. Targeting NOX4-mediated apoptosis resistance in monocyte-derived macrophages may provide a novel therapeutic target to protect against the development and/or progression of pulmonary fibrosis.

A novel tree shrew model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapy. Animal models effectively reproducing IPF disease features are needed to study the underlying molecular mechanisms. Tree shrews are genetically, anatomically, and metabolically closer to humans than rodents or dogs; therefore, the tree shrew model presents a unique opportunity for translational research in lung fibrosis. Here we demonstrate that tree shrews have in vivo and in vitro fibrotic responses induced by bleomycin and pro-fibrotic mediators. Bleomycin exposure induced lung fibrosis evidenced by histological and biochemical fibrotic changes. In primary tree shrew lung fibroblasts, transforming growth factor beta-1 (TGF-ß1) induced myofibroblast differentiation, increased extracellular matrix (ECM) protein production, and focal adhesion kinase (FAK) activation. Tree shrew lung fibroblasts showed enhanced migration and increased matrix invasion in response to platelet derived growth factor BB (PDGF-BB). Inhibition of FAK significantly attenuated pro-fibrotic responses in lung fibroblasts. The data demonstrate that tree shrews have in vivo and in vitro fibrotic responses similar to that observed in IPF. The data, for the first time, support that the tree shrew model of lung fibrosis is a new and promising experimental animal model for studying the pathophysiology and therapeutics of lung fibrosis.

Technical advance: The use of tree shrews as a model of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with a high morbidity and mortality. Some of the mechanisms of fibrosis development have been described using rodent models; however, the relevance of findings in these animal models is difficult to assess. New innovative models are needed that closely mimic IPF disease pathology. METHODS: To overcome this unmet need of investigating IPF with a relevant model, we utilized tree shrews, which are genetically, anatomically, and metabolically similar to primates and humans. Using human antibodies and primers, we investigated the role of macrophage phenotypic switching in normal and IPF subjects and bleomycin-injured tree shrews. RESULTS: Bronchoalveolar lavage (BAL) cells from tree shrews expressed human markers, and there was recruitment of monocyte-derived macrophages (MDMs) to the lung in IPF subjects and bleomycin-injured tree shrews. MDMs were polarized to a profibrotic phenotype in IPF and in bleomycin-injured tree shrews. Resident alveolar macrophages (RAMs) expressed proinflammatory markers regardless of bleomycin exposure. Tree shrews developed bleomycin-induced pulmonary fibrosis with architectural distortion in parenchyma and widespread collagen deposition. CONCLUSION: The profibrotic polarization of macrophages has been demonstrated to be present in IPF subjects and in fibrotic mice. Although the lung macrophages have long been considered to be homogeneous, recent evidence indicates that these cells are heterogeneous during multiple chronic lung diseases. Here, we show new data that indicate a critical and essential role for macrophage-fibroblast crosstalk promoting fibroblast differentiation and collagen production. in the development and progression of fibrosis. The current data strongly suggest development of therapeutics that attenuate of the profibrotic activation of MDMs may mitigate macrophage-fibroblast interaction. These observations demonstrate that tree shrews are an ideal animal model to investigate the pathogenesis of IPF as they are genetically, anatomically, and metabolically closer to humans than the more commonly used rodent models.

Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages.

Heavy metals released into the environment have a significant effect on respiratory health. Lung macrophages are important in mounting an inflammatory response to injury, but they are also involved in repair of injury. Macrophages develop mixed phenotypes in complex pathological conditions and polarize to a predominant phenotype depending on the duration and stage of injury and/or repair. Little is known about the reprogramming required for lung macrophages to switch between these divergent functions; therefore, understanding the mechanism(s) by which macrophages promote metabolic reprogramming to regulate lung injury is essential. Here, we show that lung macrophages polarize to a pro-inflammatory, classically activated phenotype after cadmium-mediated lung injury. Because metabolic adaptation provides energy for the diverse macrophage functions, these classically activated macrophages show metabolic reprogramming to glycolysis. RNA-Seq revealed up-regulation of glycolytic enzymes and transcription factors regulating glycolytic flux in lung macrophages from cadmium-exposed mice. Moreover, cadmium exposure promoted increased macrophage glycolytic function with enhanced extracellular acidification rate, glycolytic metabolites, and lactate excretion. These observations suggest that cadmium mediates the persistence of classically activated lung macrophages to exacerbate lung injury.

Mitochondrial quality control in pulmonary fibrosis.

Mechanisms underlying the pathogenesis of pulmonary fibrosis remain incompletely understood. Emerging evidence suggests changes in mitochondrial quality control are a critical determinant in many lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary hypertension, acute lung injury, lung cancer, and in the susceptibility to pulmonary fibrosis. Once thought of as the kidney-bean shaped powerhouses of the cell, mitochondria are now known to form interconnected networks that rapidly and continuously change their size to meet cellular metabolic demands. Mitochondrial quality control modulates cell fate and homeostasis, and diminished mitochondrial quality control results in mitochondrial dysfunction, increased reactive oxygen species (ROS) production, reduced ATP production, and often induces intrinsic apoptosis. Here, we review the role of the mitochondria in alveolar epithelial cells, lung macrophages, and fibroblasts within the context of pulmonary fibrosis.

Increased flux through the mevalonate pathway mediates fibrotic repair without injury.

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to ß oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis.

Macrophage activation is implicated in the development of pulmonary fibrosis by generation of profibrotic molecules. Although NADPH oxidase 4 (NOX4) is known to contribute to pulmonary fibrosis, its effects on macrophage activation and mitochondrial redox signaling are unclear. Here, we show that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic repair after asbestos exposure. NOX4 was elevated in lung macrophages from subjects with asbestosis, and mice harboring a deletion of NOX4 in lung macrophages were protected from asbestos-induced fibrosis. NOX4 promoted lung macrophage profibrotic polarization and increased production of profibrotic molecules that induce collagen deposition. Mechanistically, NOX4 further augmented mitochondrial ROS production and induced mitochondrial biogenesis. Targeting redox signaling and mitochondrial biogenesis prevented the profibrotic polarization of lung macrophages by reducing the production of profibrotic molecules. These observations provide evidence that macrophage NOX4 is a potentially novel therapeutic target to halt the development of asbestos-induced pulmonary fibrosis.

Macrophage Rac2 Is Required to Reduce the Severity of Cigarette Smoke-induced Pneumonia.

RATIONALE: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood. OBJECTIVES: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages. METHODS: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2-/- mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae. MEASUREMENTS AND MAIN RESULTS: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice. CONCLUSIONS: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.

Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization.

Fibrosis in multiple organs, including the liver, kidney, and lung, often occurs secondary to environmental exposure. Asbestos exposure is one important environmental cause of lung fibrosis. The mechanisms that mediate fibrosis is not fully understood, although mitochondrial oxidative stress in alveolar macrophages is critical for fibrosis development. Mitochondrial Ca2+ levels can be associated with production of reactive oxygen species. Here, we show that patients with asbestosis have higher levels of mitochondrial Ca2+ compared with normal patients. The mitochondrial calcium uniporter (MCU) is a highly selective ion channel that transports Ca2+ into the mitochondrial matrix to modulate metabolism. Asbestos exposure increased mitochondrial Ca2+ influx in alveolar macrophages from wild-type, but not MCU+/-, mice. MCU expression polarized macrophages to a profibrotic phenotype after exposure to asbestos, and the profibrotic polarization was regulated by MCU-mediated ATP production. Profibrotic polarization was abrogated when MCU was absent or its activity was blocked. Of more importance, mice that were deficient in MCU were protected from pulmonary fibrosis. Regulation of mitochondrial Ca2+ suggests that MCU may play a pivotal role in the development of fibrosis and could potentially be a therapeutic target for pulmonary fibrosis.-Gu, L., Larson-Casey, J. L., Carter, A. B. Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization.

Macrophage Akt1 Kinase-Mediated Mitophagy Modulates Apoptosis Resistance and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder with increasing incidence. Mitochondrial oxidative stress in alveolar macrophages is directly linked to pulmonary fibrosis. Mitophagy, the selective engulfment of dysfunctional mitochondria by autophagasomes, is important for cellular homeostasis and can be induced by mitochondrial oxidative stress. Here, we show Akt1 induced macrophage mitochondrial reactive oxygen species (ROS) and mitophagy. Mice harboring a conditional deletion of Akt1 in macrophages (Akt1(-/-)Lyz2-cre) and Park2(-/-) mice had impaired mitophagy and reduced active transforming growth factor-ß1 (TGF-ß1). Although Akt1 increased TGF-ß1 expression, mitophagy inhibition in Akt1-overexpressing macrophages abrogated TGF-ß1 expression and fibroblast differentiation. Importantly, conditional Akt1(-/-)Lyz2-cre mice and Park2(-/-) mice had increased macrophage apoptosis and were protected from pulmonary fibrosis. Moreover, IPF alveolar macrophages had evidence of increased mitophagy and displayed apoptosis resistance. These observations suggest that Akt1-mediated mitophagy contributes to alveolar macrophage apoptosis resistance and is required for pulmonary fibrosis development.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

BACKGROUND: This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. METHODS: Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. RESULTS: It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. CONCLUSIONS: Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

Cu,Zn-Superoxide Dismutase-Mediated Redox Regulation of Jumonji Domain Containing 3 Modulates Macrophage Polarization and Pulmonary Fibrosis.

M2 macrophages are implicated in the development of pulmonary fibrosis as they generate profibrotic signals. The polarization process, at least in part, is regulated by epigenetic modulation. Because Cu,Zn-superoxide dismutase-induced H2O2 can polarize macrophages to a profibrotic M2 phenotype, we hypothesized that modulation of the redox state of the cell is involved in the epigenetic modulation of the macrophage phenotype. In this study, we show that signal transducer and activator of transcription 6 (STAT6) regulates Jumonji domain containing (Jmjd) 3, a histone H3 lysine 27 demethylase, and mutation of a redox-sensitive cysteine in STAT6 attenuates jmjd3 expression. Moreover, Jmjd3 deficiency abrogates profibrotic M2 gene expression. Treatment with leflunomide, which reduces mitochondrial reactive oxygen species production and tyrosine phosphorylation, inhibits jmjd3 expression and M2 polarization, as well as development of a fibrotic phenotype. Taken together, these observations provide evidence that the redox regulation of Jmjd3 is a unique regulatory mechanism for Cu,Zn-superoxide dismutase-mediated profibrotic M2 polarization. Furthermore, leflunomide, which reduces reactive oxygen species production and tyrosine phosphorylation, may prove to be therapeutic in the treatment of asbestos-induced pulmonary fibrosis.

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis.

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H2O2) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H2O2 attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H2O2 generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys(189)) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.

Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-ß1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention.