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Summary Background Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. Methods Human lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. Findings RAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. Interpretation The inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in context Evidence before this study Pulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this study We demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGFβ-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidence Our findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.

Abstract Overgrowth of the fungus Wallemia mellicola in the intestines of mice enhances the severity of asthma. Wallemia mellicola interacts with the immune system through Dectin-2 expressed on the surface of myeloid and intestinal epithelial cells. Using Dectin-2-deficient mice, we show that the interaction of W. mellicola with Dectin-2 is essential for the gut-lung pathways, enhancing the severity of asthma in mice with W. mellicola intestinal dysbiosis. These findings offer better insight into dysbiosis-associated inflammation and highlight the role pattern recognition receptors have in immune recognition of commensal fungi in the gut, leading to alterations in immune function in the lungs.

Abstract The neutral amino acid glutamine plays a central role in TGF-β (transforming growth factor-β)–induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5 (solute carrier transporter 1A5). In the present work, we demonstrated that profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and that targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat patients with fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from idiopathic pulmonary fibrosis lungs. The expression of profibrotic targets, cell migration, and anchorage-independent growth by TGF-β required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy; suppressed mTOR, HIF (hypoxia-inducible factor), and Myc signaling; and impaired mitochondrial function, ATP production, and glycolysis. Pharmacological inhibition of SLC1A5 by the small-molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving and attenuated fibrosis in a bleomycin-treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, providing a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

Idiopathic pulmonary fibrosis (IPF) remains an intractably fatal disorder, despite the recent advent of anti-fibrotic medication. Successful treatment of IPF, like many chronic diseases, may benefit from the concurrent use of multiple agents that exhibit synergistic benefit. In this light, phosphodiesterase type 5 inhibitors (PDE5-Is), have been studied in IPF primarily for their established pulmonary vascular effects. However, recent data suggest certain PDE5-Is, particularly vardenafil, may also reduce transforming growth factor beta 1 (TGF-β1) activation and extracellular matrix (ECM) accumulation, making them a potential target for therapy for IPF. We evaluated fibroblast TGF-β1-driven extracellular matrix (ECM) generation and signaling as well as epithelial mesenchymal transformation (EMT) with pretreatment using the PDE5-I vardenafil. In addition, combinations of vardenafil and nintedanib were evaluated for synergistic suppression of EMC using a fibronectin enzyme-linked immunosorbent assay (ELISA). Finally, the effects of vardenafil on fibrosis were investigated in a bleomycin mouse model. Our findings demonstrate that vardenafil suppresses ECM generation alone and also exhibits significant synergistic suppression of ECM in combination with nintedanib in vitro. Interestingly, vardenafil was shown to improve fibrosis markers and increase survival in bleomycin-treated mice. Vardenafil may represent a potential treatment for IPF alone or in combination with nintedanib. However, additional studies will be required.

Transforming growth factor beta (TGF‐β) plays a significant role in diverse biologic processes, including the regulation of cell growth, survival, differentiation, development, inflammation, immunity, hematopoiesis, and tissue remodeling. TGF‐β is essential for wound healing, stimulates matrix molecule deposition, and has been implicated in the pathogenesis of a variety of fibrotic disorders, including idiopathic pulmonary fibrosis, scleroderma, radiation‐induced pulmonary fibrosis, and asthma. Furthermore, activation of profibrotic TGF‐β signaling has a central role in glucose‐induced cell hypertrophy, induces glucose transporter 1, and regulates mitochondrial energetics. However, the mechanisms by which TGF‐β induces specific metabolic alterations in the fibrogenic process have not been fully defined. In this study, we demonstrate that TGF‐β stimulates type I TGF β receptor (TβRI) translocation to the mitochondria and induction of hexokinase 2 (HK2) via both Smad‐dependent and –independent pathways. Also, we showed that TbRI association with HK2 and the mitochondrial voltage‐dependent ion channel (VDAC). These findings suggest that TβRI plays a novel role in controlling mitochondrial function and regulation of glucose metabolism. Support or Funding Information This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from the National Institutes of General Medical Sciences. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract In the current work we show that the profibrotic actions of TGF‐β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF‐β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF‐β. We further demonstrated that the NAD‐dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF‐β. Lastly, administration of the GLS1 inhibitor CB‐839 attenuated bleomycin‐induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF‐β‐dependent manner.

Abstract Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two‐thirds of IPF patients die within 2‐5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFβ) and insulin‐like growth factor‐1 (IGF‐1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF‐1 in the initiation and progression of TGFβ‐induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF‐1 via TGFβ in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF‐1's C1 promoter as mediating the increase in IGF‐1 transcription by TGFβ in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFβ‐dependent Igf‐1 expression in myofibroblasts; (4) demonstrates IGF‐1R activation is essential to support TGFβ‐driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF‐1R inhibitor OSI‐906. These findings expand our knowledge of IGF‐1's role as a novel fibrotic‐switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

TGF-β–dependent metabolic dysregulation contributes to lung fibrosis.

Abstract Transforming growth factor‐beta (TGFβ) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFβ is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFβ induces the expression of the immunoinhibitory molecule Programed death‐ligand 1 (PD‐L1) in human and murine fibroblasts in a Smad2/3‐ and YAP/TAZ‐dependent manner. Furthermore, PD‐L1 knockdown decreased the TGFβ‐dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha‐smooth muscle actin (α‐SMA), and cell migration/wound healing. In addition to an endogenous role for PD‐L1 in profibrotic TGFβ signaling, TGFβ stimulated‐human lung fibroblast‐derived PD‐L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

Abstract Fibroproliferative diseases are a leading cause of morbidity and mortality featuring localized and systematic tissue/organ fibrosis, which account for nearly 45% of all deaths in the developed world, with an unknown etiology and no effective treatment. Transforming growth factor beta (TGF-β) is a ubiquitous cytokine that contributes to fibroblast activation, myofibroblast differentiation, collagen overproduction and pathological tissue fibrosis. In that mounting evidence indicate that altered glutamine metabolism has critical roles in regulating signaling pathways, maintaining redox balance and macromolecular biosynthesis, we wished to test the general hypothesis that intervention of these metabolic processes could provide novel approaches to developing anti-fibrotic therapies. To that end, in the current work we provide a framework integrating glutamine dysregulation with profibrotic TGF-β signaling. We found that Glutaminase 1 (GLS1), which converts glutamine to glutamate, is induced by TGF-β and is also elevated in (i) fibroblasts isolated from Idiopathic Pulmonary Fibrosis (IPF) patients; and (ii) the murine bleomycin (i.e., TGF-β driven) model of lung fibrosis. While phosphorylation of Smad2 and Smad3 occurs independently of GLS1, TGF-β stimulated expression of profibrotic targets (i.e., Collagen I, PAI-1, CTGF, α-SMA and fibronectin) as well as biological actions (i.e., cell migration and anchorage independent growth) require GLS1 activity. Moreover, knockdown of Smad2 or Smad3 as well as inhibition of MAPK, PI3K, Akt or mTOR abrogates the induction of GLS1 by TGF-β; documenting that TGF-β regulates GLS1 expression via both Smad and non-Smad signaling. As GLS1 represents a critical component mediating the fibroproliferative actions of TGF-β, in order to identify the operative molecular mechanism(s) we focused on (i) a group of histone deacetylases called sirtuins (SIRT) which are known to elicit antifibrotic effects; and (ii) the Forkhead box protein O4 (FOXO4) transcription factor which has been shown to bind the GLS1 promoter. We found that there is not only a decrease in SIRT7 and FOXO4 coincident with the induction of GLS1 by TGF-β, but TGF-β inhibits SIRT7 deacetylation activity which in turn prevents FOXO4 deacetylation and inhibition of GLS1 expression. Last, consistent with SIRT7 and FOXO4 being negative regulators of profibrotic TGF-β signaling, their levels were also decreased in IPF fibroblasts and bleomycin-induced lung fibrosis. These studies point to an exciting, novel and unexplored connection between sirtuins, FOXOs and glutamine metabolism by elucidating the mechanism(s) by which epigenetic and transcriptional processes cooperate to regulate glutamine metabolism and fibrotic development in a TGF-β dependent manner. Citation Format: Malay Choudhury, Xueqian Yin, Jeong-Han Kang, Mahefatiana Andrianifahanana, Edward B. Leof. Cooperativity between glutaminase 1 and histone deacetylase SIRT7 regulates TGF-β mediated lung fibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1847.