Soo Jung Cho

Early Growth Response Gene 1–mediated Apoptosis Is Essential for Transforming Growth Factor β1–induced Pulmonary Fibrosis

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-β1 in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-β1–induced responses have not been defined. To address these issues, we targeted bioactive TGF-β1 to the murine lung using a novel externally regulatable, triple transgenic system. TGF-β1 produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-β1–induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-β1–induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-β1 in vivo and define the critical role of Egr-1 in the TGF-β1 phenotype. They also demonstrate that Egr-1–mediated apoptosis is a prerequisite for TGF-β1–induced fibrosis and remodeling.

Transforming Growth Factor (TGF)-β1 Stimulates Pulmonary Fibrosis and Inflammation via a Bax-dependent, Bid-activated Pathway That Involves Matrix Metalloproteinase-12

Fibrosis, apoptosis, and the exaggerated production of transforming growth factor (TGF)-β1 are juxtaposed in a variety of pulmonary diseases including the interstitial lung diseases and asthma. In these disorders, the relationships between these responses are not well defined. In addition, the apoptosis pathways that contribute to these responses and the mechanism(s) of their contribution have not been described. We hypothesized that BH3 domain-only protein-induced apoptosis plays an important role in the pathogenesis of TGF-β1-induced pulmonary responses. To test this hypothesis, we characterized the effects of transgenic TGF-β1 in mice with wild type (WT) and null Bax loci. To investigate the mechanisms of Bax activation and its effector functions, we also compared the effects of TGF-β1 in mice with WT and null Bid and matrix metalloproteinase (MMP)-12 loci, respectively. These studies demonstrate that TGF-β1 is a potent stimulator of Bax, Bid, and MMP-12. The studies also demonstrate that Bax and Bid play key roles in the pathogenesis of TGF-β1-induced inflammation, fibrosis, and apoptosis; that TGF-β1 stimulates MMP-12, TIMP-1, and cathepsins and inhibits MMP-9 and p21 via Bax- and Bid-dependent mechanisms; and that TGF-β1-stimulated pulmonary fibrosis is ameliorated in MMP-12-deficient animals. Finally, they demonstrate that Bax, Bid, and MMP-12 play similar roles in bleomycin-induced fibrosis, thereby highlighting the importance of this Bid-activated, Bax-mediated pathway and downstream MMP-12 in a variety of fibrogenic settings.

Age-Dependent Susceptibility to Pulmonary Fibrosis Is Associated with NLRP3 Inflammasome Activation

Aging has been implicated in the development of pulmonary fibrosis, which has seen a sharp increase in incidence in those older than 50 years. Recent studies demonstrate a role for the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome and its regulated cytokines in experimental lung fibrosis. In this study, we tested the hypothesis that age-related NLRP3 inflammasome activation is an important predisposing factor in the development of pulmonary fibrosis. Briefly, young and aged wild-type and NLRP3(-/-) mice were subjected to bleomycin-induced lung injury. Pulmonary fibrosis was determined by histology and hydroxyproline accumulation. Bone marrow and alveolar macrophages were isolated from these mice. NLRP3 inflammasome activation was assessed by co-immunoprecipitation experiments. IL-1β and IL-18 production was measured by ELISA. The current study demonstrated that aged wild-type mice developed more lung fibrosis and exhibited increased morbidity and mortality after bleomycin-induced lung injury, when compared with young mice. Bleomycin-exposed aged NLRP3(-/-) mice had reduced fibrosis compared with their wild-type age-matched counterparts. Bone marrow-derived and alveolar macrophages from aged mice displayed higher levels of NLRP3 inflammasome activation and caspase-1-dependent IL-1β and IL-18 production, which was associated with altered mitochondrial function and increased production of reactive oxygen species. Our study demonstrated that age-dependent increases in alveolar macrophage mitochondrial reactive oxygen species production and NLRP3 inflammasome activation contribute to the development of experimental fibrosis.

Glucose Transporter 1–Dependent Glycolysis Is Increased during Aging-Related Lung Fibrosis, and Phloretin Inhibits Lung Fibrosis

&NA; Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer, and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage, and reactive oxygen species generation. Although glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in the lung remain unclear. The objective of this study was to determine if glucose transporter 1 (GLUT1)‐induced glycolysis regulates age‐dependent fibrogenesis of the lung. Mouse and human lung tissues were analyzed for GLUT1 and glycolytic markers using immunoblotting. Glycolytic function was measured using a Seahorse apparatus. To study the effect of GLUT1, genetic inhibition of GLUT1 was performed by short hairpin RNA transduction, and phloretin was used for pharmacologic inhibition of GLUT1. GLUT1‐dependent glycolysis is activated in aged lung. Genetic and pharmacologic inhibition of GLUT1 suppressed the protein expression of &agr;‐smooth muscle actin, a key cytoskeletal component of activated fibroblasts, in mouse primary lung fibroblast cells. Moreover, we demonstrated that the activation of AMP‐activated protein kinase, which is regulated by GLUT1‐dependent glycolysis, represents a critical metabolic pathway for fibroblast activation. Furthermore, we demonstrated that phloretin, a potent inhibitor of GLUT1, significantly inhibited bleomycin‐induced lung fibrosis in vivo. These results suggest that GLUT1‐dependent glycolysis regulates fibrogenesis in aged lung and that inhibition of GLUT1 provides a potential target of therapy of age‐related lung fibrosis.

NLRP3 inflammasome activation in aged macrophages is diminished during Streptococcus pneumoniae infection

Pneumococcal infections are the eigth leading cause of death in the United States, and it is estimated that older patients (≥65 yr of age) account for the most serious cases. The goal of our current study is to understand the impact of biological aging on innate immune responses to Streptococcus pneumoniae, a causative agent of bacterial pneumonia. With the use of in vitro and in vivo aged murine models, our findings demonstrate that age-enhanced unfolded protein responses (UPRs) contribute to diminished inflammasome assembly and activation during S. pneumoniae infection. Pretreatment of aged mice with endoplasmic reticulum chaperone and the stress-reducing agent tauroursodeoxycholic acid (TUDCA) decreased mortality in aged hosts that was associated with increased NLRP3 inflammasome activation, improved pathogen clearance, and decreased pneumonitis during infection. Taken together, our data provide new evidence as to why older persons are more susceptible to S. pneumoniae and provide a possible therapeutic target to decrease morbidity and mortality in this population.

Laminin α1 is a genetic modifier of TGF-β1–stimulated pulmonary fibrosis

The pathogenetic mechanisms underlying the pathologic fibrosis in diseases such as idiopathic pulmonary fibrosis (IPF) are poorly understood. To identify genetic factors affecting susceptibility to IPF, we analyzed a murine genetic model of IPF in which a profibrotic cytokine (TGF-β1) was expressed in the lungs of 10 different inbred mouse strains. Surprisingly, the extent of TGF-β1-induced lung fibrosis was highly strain dependent. Haplotype-based computational genetic analysis and gene expression profiling of lung tissue obtained from fibrosis-susceptible and -resistant strains identified laminin α1 (Lama1) as a genetic modifier for susceptibility to IPF. Subsequent studies demonstrated that Lama1 plays an important role in multiple processes that affect the pulmonary response to lung injury and susceptibility to fibrosis, which include: macrophage activation, fibroblast proliferation, myofibroblast transformation, and the production of extracellular matrix. Also, Lama1 mRNA expression was significantly increased in lung tissue obtained from IPF patients. These studies identify Lama1 as the genetic modifier of TGF-β1 effector responses that significantly affects the development of pulmonary fibrosis.

Decreased NLRP3 inflammasome expression in aged lung may contribute to increased susceptibility to secondary Streptococcus pneumoniae infection

&NA; Post‐viral pneumococcal pneumonia is a leading morbidity and mortality in older patients (≥65 years of age). The goal of our current study is to understand the impact of chronological aging on innate immune responses to a secondary, post viral infection with Streptococcus pneumoniae, a causative agent of bacterial pneumonia. Using aged murine models of infection, our findings demonstrate increased morbidity and mortality in aged mice within 48 h post‐secondary S. pneumoniae infection. Increased susceptibility of aged mice was associated with decreased TLR1, TLR6, and TLR9 mRNA expression and diminished IL1&bgr; mRNA expression. Examination of NLRP3 inflammasome expression illustrated decreased NLRP3 mRNA expression and decreased IL1&bgr; production in aged lung in response to secondary S. pneumoniae infection. HighlightsOur study was designed to understand the impact of chronological aging on innate immune responses to a secondary, post influenza infection.Increased susceptibility of aged mice to secondary S. pneumoniae infection was associated with decreased IL1&bgr; mRNA expression.Examination of NLRP3 inflammasome expression illustrated decreased IL1&bgr; production in aged lung in response to secondary infection.