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Silicosis is a severe worldwide occupational hazard, characterized with lung tissue inflammation and irreversible fibrosis caused by crystalline silicon dioxide. As the most common and abundant internal modification of messenger RNAs or noncoding RNAs, N6-methyladenosine (m6A) methylation is dysregulated in the chromic period of silicosis. However, whether m6A modification is involved in the early phase of silica-induced pulmonary inflammation and fibrosis and its specific effector cells remains unknown. In this study, we established a pulmonary inflammation and fibrosis mouse model by silica particles on day 7 and day 28. Then, we examined the global m6A modification level by m6A dot blot and m6A RNA methylation quantification kits. The key m6A regulatory factors were analyzed by RTqPCR, Western blot, and immunohistochemistry (IHC) in normal and silicosis mice. The results showed that the global m6A modification level was upregulated in silicosis lung tissues with the demethylase FTO suppression after silica exposure for 7 days and 28 days. METTL3, METTL14, ALKBH5, and other m6A readers had no obvious differences between the control and silicosis groups. Then, single-cell sequencing analysis revealed that thirteen kinds of cells were recognized in silicosis lung tissues, and the mRNA expression of FTO was downregulated in epithelial cells, endothelial cells, fibroblasts, and monocytes. These results were further confirmed in mouse lung epithelial cells (MLE-12) exposed to silica and in the peripheral blood mononuclear cells of silicosis patients. In conclusion, the high level of global m6A modification in the early stage of silicosis is induced by the downregulation of the demethylase FTO, which may provide a novel target for the diagnosis and treatment of silicosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Circular RNAs (circRNAs) are functional molecules in all kinds of fibrosis diseases. The current study was performed for the exploration of circ_0007535 in pulmonary fibrosis. RNA levels for circ_0007535, miR-18a-5p, and transforming growth factor-β receptor 1 (TGFBR1) were assayed via a reverse transcription-quantitative polymerase chain reaction. Cell growth was determined by cell counting kit-8 assay for viability and ethynyl-2'-deoxyuridine assay for proliferation. Cell invasion and migration were examined by transwell assay and scratch assay. Western blot was performed for the detection of different proteins. Enzyme-linked immunosorbent assay was used to assess inflammatory response. The interaction analysis was conducted using dual-luciferase reporter assay, RNA immunoprecipitation assay, and biotin-coupled pull-down assay. Circ_0007535 was significantly upregulated by TGF-β1 in HFL1 cells. TGF-β1-induced proliferation, motility, ECM accumulation, and inflammatory reaction in HFL1 cells were alleviated by circ_0007535 knockdown. Circ_0007535 exhibited interaction with miR-18a-5p, and miR-18a-5p inhibition reversed all influences of circ_0007535 downregulation in TGF-β1-treated HFL1 cells. Circ_0007535 acted as a miR-18a-5p sponge to regulate the expression of downstream target TGFBR1. MiR-18a-5p induced TGFBR1 level inhibition to attenuate TGF-β1-mediated cell injury in HFL1 cells. This study evidenced that circ_0007535 facilitated TGF-β1-induced pulmonary fibrosis by depending on the absorption of miR-18a-5p to upregulate TGFBR1.

Background: Epithelial mesenchymal transformation (EMT) in alveolar type 2 epithelial cells (AT2) is closely associated with pulmonary fibrosis (PF). Histone deacetylase 3 (HDAC3) is an important enzyme that regulates protein stability by modulating the acetylation level of non-histones. Here, we aimed to explore the potential role and regulatory mechanisms associated with HDAC3 in PF., Methods: We quantified HDAC3 expression both in lung tissues from patients with PF and from bleomycin (BLM)-treated mice. HDAC3 was also detected in TGF-β1-treated AT2. The mechanistic activity of HDAC3 in pulmonary fibrosis and EMT was also explored., Results: HDAC3 was highly expressed in lung tissues from patients with PF and bleomycin (BLM)-treated mice, especially in AT2. Lung tissues from AT2-specific HDAC3-deficient mice stimulated with BLM showed alleviative fibrosis and EMT. Upstream of HDAC3, TGF-β1/SMAD3 directly promoted HDAC3 transcription. Downstream of HDAC3, we also found that genetic or pharmacologic inhibition of HDAC3 inhibited GATA3 expression at the protein level rather than mRNA. Finally, we found that intraperitoneal administration of RGFP966, a selective inhibitor of HDAC3, could prevent mice from BLM-induced pulmonary fibrosis and EMT., Conclusion: TGF-β1/SMAD3 directly promoted the transcription of HDAC3, which aggravated EMT in AT2 and pulmonary fibrosis in mice via deacetylation of GATA3 and inhibition of its degradation. Our results suggest that targeting HDAC3 in AT2 may provide a new therapeutic target for the prevention of PF., (© 2023. The Author(s).)

The global COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 virus has resulted in a significant number of patients experiencing persistent symptoms, including post-COVID pulmonary fibrosis (PCPF). This study aimed to identify novel therapeutic targets for PCPF using single-cell RNA-sequencing data from lung tissues of COVID-19 patients, idiopathic pulmonary fibrosis (IPF) patients, and a rat transforming growth factor beta-1-induced fibrosis model treated with antifibrotic drugs. Patients with COVID-19 had lower alveolar macrophage counts than healthy controls, whereas patients with COVID-19 and IPF presented with elevated monocyte-derived macrophage counts. A comparative transcriptome analysis showed that macrophages play a crucial role in IPF and COVID-19 development and progression, and fibrosis- and inflammation-associated genes were upregulated in both conditions. Functional enrichment analysis revealed the upregulation of inflammation and proteolysis and the downregulation of ribosome biogenesis. Cholesterol efflux and glycolysis were augmented in both macrophage types. The study suggests that antifibrotic drugs may reverse critical lung fibrosis mediators in COVID-19. The results help clarify the molecular mechanisms underlying pulmonary fibrosis in patients with severe COVID-19 and IPF and highlight the potential efficacy of antifibrotic drugs in COVID-19 therapy. Collectively, all these findings may have significant implications for the development of new treatment strategies for PCPF., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Pulmonary fibrosis is a chronic and progressive fibrotic disease that results in impaired gas exchange, ventilation, and eventual death. The pro-fibrotic environment is instigated by various factors, leading to the transformation of epithelial cells into myofibroblasts and/or fibroblasts that trigger fibrosis. Epithelial mesenchymal transition (EMT) is a biological process that plays a critical role in the pathogenesis of pulmonary fibrosis. Epigenetic regulation of tissue-stromal crosstalk involving DNA methylation, histone modifications, non-coding RNA, and chromatin remodeling plays a key role in the control of EMT. The review investigates the epigenetic regulation of EMT and its significance in pulmonary fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pulmonary fibrosis is a progressive lung disorder. Evidence has shown that hsa_circular (circ)RNA_0001861 is dysregulated in pulmonary fibrosis. However, the detailed function of hsa_circRNA_0001861 in pulmonary fibrosis remains unexplored. To investigate the function of hsa_circRNA_0001861 in pulmonary fibrosis, human pulmonary fibroblasts in vitro were used, and cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining were performed to assess cell viability and proliferation, respectively. Western blot analysis and reverse transcription-quantitative PCR (RT-qPCR) were used to evaluate protein and mRNA levels. Meanwhile, the relationship among hsa_circRNA_0001861, miR-296-5p and BCL-2 binding component 3 (BBC3) was investigated by RNA pull-down assays. Furthermore, an in vivo model of lung fibrosis was constructed to assess the function of hsa_circRNA_0001861 in lung fibrosis. The data revealed that TGF‑β1 significantly increased the proliferation of pulmonary fibroblasts, while this phenomenon was markedly abolished by hsa_circRNA_0001861 overexpression. hsa_circRNA_0001861 overexpression markedly inhibited TGF‑β1‑induced fibrosis in pulmonary fibroblasts through the mediation of α-smooth muscle actin, E-cadherin, collagen III and fibronectin 1. Meanwhile, hsa_circRNA_0001861 could bind with miR-296-5p, and BBC3 was identified to be the downstream mRNA of miR-296-5p. In addition, the upregulation of hsa_circRNA_0001861 clearly reversed TGF‑β1‑induced fibrosis and proliferation in pulmonary fibroblasts through the upregulation of BBC3. Furthermore, hsa_circRNA_0001861 upregulation markedly alleviated pulmonary fibrosis in vivo. Hsa_circRNA_0001861 upregulation attenuated pulmonary fibrosis by modulating the miR-296-5p/BBC3 axis. Hence, the present study may provide some insights for the discovery of new methods against pulmonary fibrosis.

Ferroptosis, a newly discovered type of regulated cell death, has been implicated in numerous human diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease with poor prognosis and limited treatment options. Emerging evidence has linked ferroptosis and glutamate-determined cell fate which is considered a new light on the etiology of pulmonary fibrosis. Here, we observed that N-methyl d-aspartate receptor (NMDAR) activation promoted cell damage and iron deposition in MLE-12 cells in a dose-, time-, and receptor-dependent manner. This mediated substantial Ca 2+ influx, upregulated the expression levels of nNOS and IRP1, and affected intracellular iron homeostasis by regulating the expression of iron transport-related proteins (i.e., TFR1, DMT1, and FPN). Excessive iron load promoted the continuous accumulation of total intracellular and mitochondrial reactive oxygen species, which ultimately led to ferroptosis. NMDAR inhibition reduced lung injury and pulmonary fibrosis in bleomycin-induced mice. Bleomycin stimulation upregulated the expression of NMDAR1, nNOS, and IRP1 in mouse lung tissues, which ultimately led to iron deposition via regulation of the expression of various iron metabolism-related genes. NMDAR activation initiated the pulmonary fibrosis process by inducing iron deposition in lung tissues and ferroptosis of alveolar type II cells. Our data suggest that NMDAR activation regulates the expression of iron metabolism-related genes by promoting calcium influx, increasing nNOS and IRP1 expression, and increasing iron deposition by affecting cellular iron homeostasis, ultimately leading to mitochondrial damage, mitochondrial dysfunction, and ferroptosis. NMDAR activation-induced ferroptosis of alveolar type II cells might be a key event to the initiation of pulmonary fibrosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The incidence of pulmonary fibrosis is on the rise, and existing treatments have limited efficacy in improving patient survival. The purpose of this study was to reveal the potential of Krüppel-like factor (KLF)15 activation in alleviating pulmonary fibrosis. Transforming growth factor beta (TGF-β) was utilized to induce lung fibroblasts to establish an in vitro model of pulmonary fibrosis. The impacts of TGF-β and KLF15 level on cell proliferation, migration, extracellular matrix (ECM) accumulation, and endoplasmic reticulum stress (ERS) were assessed. Additionally, tunicamycin, an ERS agonist, was used to investigate the role of ERS in KLF15 regulation. The results showed that KLF15 was dropped in response to TGF-β treatment. However, KLF15 overexpression reduced cell proliferation, migration, ECM accumulation, and ERS, alleviating the effects of TGF-β stimulation. Subsequent treatment with tunicamycin diminished the effects of KLF15 overexpression, demonstrating that ERS mediated the modulation of KLF15. KLF15 acts against ERS and suppresses excessive proliferation and ECM accumulation in lung fibroblast. These findings suggest that activating KLF15 is a promising strategy for alleviating pulmonary fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Fibroblast growth factor (FGF) signaling plays a crucial role in lung development and repair. Fibroblast growth factor 2 (FGF2) can inhibit fibrotic gene expression and suppress the differentiation of pulmonary fibroblasts (PFs) into myofibroblasts in vitro, suggesting that FGF2 is a potential target for inhibiting pulmonary fibrosis. To gain deeper insights into the molecular mechanism underlying FGF2-mediated regulation of PFs, we performed mRNA sequencing analysis to systematically and globally uncover the regulated genes and biological functions of FGF2 in PFs. Gene Ontology analysis revealed that the differentially expressed genes regulated by FGF2 were enriched in multiple cellular functions including extracellular matrix (ECM) organization, cytoskeleton formation, β-catenin-independent Wnt signaling pathway, supramolecular fiber organization, epithelial cell proliferation, and cell adhesion. Gene Set Enrichment Analysis and cellular experiments confirmed that FGF2 can suppress ECM and actin filament organization and increase PFs proliferation. Taken together, these findings indicate that FGF2 acts as an upstream regulator of the inhibition of PFs activation and may play a regulatory role in pulmonary fibrosis., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Epigenetics indicates that certain phenotypes of an organism can undergo heritable changes in the absence of changes in the genetic DNA sequence. Many studies have shown that epigenetic patterns play an important role in the lung and lung diseases. Pulmonary fibrosis (PF) is also a type of lung disease. PF is an end-stage change of a large group of lung diseases, characterized by fibroblast proliferation and massive accumulation of extracellular matrix, accompanied by inflammatory injury and histological destruction, that is, structural abnormalities caused by abnormal repair of normal alveolar tissue. It causes loss of lung function in patients with multiple complex diseases, leading to respiratory failure and subsequent death. However, current treatment options for IPF are very limited and no drugs have been shown to significantly prolong the survival of patients. Therefore, based on a systematic understanding of the disease mechanisms of PF, this review integrates the role of epigenetics in the development and course of PF, describes preventive and potential therapeutic targets for PF, and provides a theoretical basis for further exploration of the mechanisms of PF., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Silicosis is a fatal occupational respiratory disease caused by the prolonged inhalation of respirable silica. The core event of silicosis is the heightened activity of fibroblasts, which excessively synthesize extracellular matrix (ECM) proteins. Our previous studies have highlighted that human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hucMSC-EVs) hold promise in mitigating silicosis and the significant role played by microRNAs (miRNAs) in this process. Delving deeper into this mechanism, we found that miR-148a-3p was the most abundant miRNA of the differential miRNAs in hucMSC-EVs, with the gene heat shock protein 90 beta family member 1 (Hsp90b1) as a potential target. Notably, miR-148a-3p's expression was downregulated during the progression of silica-induced pulmonary fibrosis both in vitro and in vivo, but was restored after hucMSC-EVs treatment ( p < 0.05). Introducing miR-148a-3p mimics effectively hindered the collagen synthesis and secretion of fibroblasts induced by transforming growth factor-β1 (TGF-β1) ( p < 0.05). Confirming our hypothesis, Hsp90b1 was indeed targeted by miR-148a-3p, with significantly reduced collagen activity in TGF-β1-treated fibroblasts upon Hsp90b1 inhibition ( p < 0.05). Collectively, our findings provide compelling evidence that links miR-148a-3p present in hucMSC-EVs with the amelioration of silicosis, suggesting its therapeutic potential by specifically targeting Hsp90b1, thereby inhibiting fibroblast collagen activities. This study sheds light on the role of miR-148a-3p in hucMSC-EVs, opening avenues for innovative therapeutic interventions targeting molecular pathways in pulmonary fibrosis.

Background: Pulmonary fibrosis (PF), the most common clinical type of irreversible interstitial lung disease with one of the worse prognoses, has a largely unknown molecular mechanisms that underlies its progression. CD5 molecule-like (CD5L) functions in an indispensable role during inflammatory responses; however, whether CD5L functions in regulating bleomycin (BLM)-induced lung fibrosis is less clear., Methods: Herein, we describe the engineering of Cd5l knockout mice using CRISPR/Cas9 gene editing technology. The BLM-induced model of acute lung injury represents the most widely used experimental rodent model for PF., Results: Taking advantage of this model, we demonstrated that both CD5L mRNA and protein were enriched in the lungs of mice following BLM-induced pulmonary fibrosis. Inhibition of CD5L prevented mice from BLM-induced lung fibrosis and injury. In particular, a lack of CD5L significantly attenuated inflammatory response and promoted M2 polarization in the lung of this pulmonary fibrosis model as well as suppressing macrophage apoptosis., Conclusions: Collectively, our data support that CD5L deficiency can suppress the development of pulmonary fibrosis, and also provides new molecular targets for the use of immunotherapy to treat lung fibrosis., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Silicosis is a progressive pulmonary fibrosis disease caused by long-term inhalation of a large amount of free crystalline silica, which seriously threatens the health of relevant workers and causes a huge amount of disease burden. The pathogenesis of silicosis is complex and unclear, it has been reported that long non coding RNA (lncRNA) plays an important role in the pathogenesis of silicosis. In order to improve the understanding of the disease and provide directions for the prevention and treatment of silicosis, this article reviewed the mechanism of lncRNA in the pathogenesis and disease progression of silicosis.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) have common pathophysiological features, such as the significant remodeling of pulmonary parenchyma and vascular wall. There is no effective specific drug in clinical treatment for these two diseases, resulting in a worse prognosis and higher mortality. This study aimed to screen the common key genes and immune characteristics of PF and PH by means of bioinformatics to find new common therapeutic targets. Expression profiles are downloaded from the Gene Expression Database. Weighted gene co-expression network analysis is used to identify the co-expression modules related to PF and PH. We used the ClueGO software to enrich and analyze the common genes in PF and PH and obtained the protein-protein interaction (PPI) network. Then, the differential genes were screened out in another cohort of PF and PH, and the shared genes were crossed. Finally, RT-PCR verification and immune infiltration analysis were performed on the intersection genes. In the result, the positive correlation module with the highest correlation between PF and PH was determined, and it was found that lymphocyte activation is a common feature of the pathophysiology of PF and PH. Eight common characteristic genes ( ACTR2, COL5A2, COL6A3, CYSLTR1, IGF1, RSPO3, SCARNA17 and SEL1L ) were gained. Immune infiltration showed that compared with the control group, resting CD4 memory T cells were upregulated in PF and PH. Combining the results of crossing characteristic genes in ImmPort database and RT-PCR, the important gene IGF1 was obtained. Knocking down IGF1 could significantly reduce the proliferation and apoptosis resistance in pulmonary microvascular endothelial cells, pulmonary smooth muscle cells, and fibroblasts induced by hypoxia, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β1 (TGF-β1), respectively. Our work identified the common biomarkers of PF and PH and provided a new candidate gene for the potential therapeutic targets of PF and PH in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Wang, Yan, Zhao, He, Jiang, Luo, Qiu, Miao, Gong, Yuan and Wu.)

Biallelic pathogenic variants in the surfactant protein (SP)-B gene (SFTPB) have been associated with fatal forms of interstitial lung diseases (ILD) in newborns and exceptional survival in young children. We herein report the cases of two related adults with pulmonary fibrosis due to a new homozygous SFTPB pathogenic variant, c.582G>A p.(Gln194=). In vitro transcript studies showed that this SFTPB synonymous pathogenic variant induces aberrant splicing leading to three abnormal transcripts with the preservation of the expression of a small proportion of normal SFTPB transcripts. Immunostainings on lung biopsies of the proband showed an almost complete loss of SP-B expression. This hypomorphic splice variant has thus probably allowed the patients' survival to adulthood while inducing an epithelial cell dysfunction leading to ILD. Altogether, this report shows that SFTPB pathogenic variants should be considered in atypical presentations and/or early-onset forms of ILD particularly when a family history is identified., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Silicosis is a global occupational pulmonary disease due to the accumulation of silica dust in the lung. Lacking effective clinical drugs makes the treatment of this disease quite challenging in clinics largely because the pathogenic mechanisms remain obscure. Interleukin 33 (IL33), a pleiotropic cytokine, could promote wound healing and tissue repair via the receptor ST2. However, the mechanisms governing the involvement of IL33 in silicosis progression remain to be further explored. Here, we demonstrated that the IL33 levels in the lung sections were significantly overexpressed after bleomycin and silica treatment. Chromatin immunoprecipitation assay, knockdown, and reverse experiments were performed in lung fibroblasts to prove gene interaction following exogenous IL33 treatment or cocultured with silica-treated lung epithelial cells. Mechanistically, we illustrated that silica-stimulated lung epithelial cells secreted IL33 and further promoted the activation, proliferation, and migration of pulmonary fibroblasts by activating the ERK/AP-1/NPM1 signaling pathway in vitro. And more, treatment with NPM1 siRNA-loaded liposomes markedly protected mice from silica-induced pulmonary fibrosis in vivo. In conclusion, the involvement of NPM1 in the progression of silicosis is regulated by the IL33/ERK/AP-1 signaling axis, which is the potential therapeutic target candidate in developing novel antifibrotic strategies for pulmonary fibrosis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pulmonary fibrosis is a severe lung interstitial disease characterized by the destruction of lung tissue structure, excessive activation and proliferation of fibroblasts, secretion and accumulation of a large amount of extracellular matrix (ECM), and impaired lung function. Due to the complexity of the disease, a suitable animal model to mimic human pulmonary fibrosis has not yet been established. Precision-cut lung slice (PCLS) has been a widely used in vitro method to study lung physiology and pathogenesis in recent years. This method is an in vitro culture technology at the level between organs and cells, because it can preserve the lung tissue structure and various types of airway cells in the lung tissue, simulate the in vivo lung environment, and conduct the observation of various interactions between cells and ECM. Therefore, PCLS can compensate for the limitations of other models such as cell culture. In order to explore the role of discoidin domain receptor 2 (DDR2) in pulmonary fibrosis, Ddr2 flox/flox mice were successfully constructed. The Cre-LoxP system and PCLS technology were used to verify the deletion or knockdown of DDR2 in mouse PCLS. Transforming growth factor β1 (TGF-β1) can induce fibrosis of mouse PCLS in vitro, which can simulate the in vivo environment of pulmonary fibrosis. In the DDR2 knock down-PCLS in vitro model, the expression of various fibrosis-related factors induced by TGF-β1 was significantly reduced, suggesting that knocking down DDR2 can inhibit the formation of pulmonary fibrosis. The results provide a new perspective for the clinical study of DDR2 as a therapeutic target in pulmonary fibrosis.

Pro-proliferative, M2-like polarization of macrophages is a critical step in the development of fibrosis and remodeling in chronic lung diseases such as pulmonary fibrosis and pulmonary hypertension. Macrophages in healthy and diseased lungs express gremlin 1 (Grem1), a secreted glycoprotein that acts in both paracrine and autocrine manners to modulate cellular function. Increased Grem1 expression plays a central role in pulmonary fibrosis and remodeling, however, the role of Grem1 in M2-like polarization of macrophages has not previously been explored. The results reported here show that recombinant Grem1 potentiated M2-like polarization of mouse macrophages and bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 could partially rescue this effect. Taken together, these findings reveal that gremlin 1 is required for M2-like polarization of macrophages. NEW & NOTEWORTHY We show here that gremlin 1 potentiated M2 polarization of mouse bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 partially rescued this effect. Taken together, these findings reveal a previously unknown requirement for gremlin 1 in M2 polarization of macrophages and suggest a novel cellular mechanism promoting fibrosis and remodeling in lung diseases.

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.

Silicosis is a refractory pneumoconiosis of unknown etiology that is characterized by diffuse lung fibrosis, and microRNA (miRNA) dysregulation is connected to silicosis. Emerging evidence suggests that miRNAs modulate pulmonary fibrosis through autophagy; however, its underlying molecular mechanism remains unclear. In agreement with miRNA microarray analysis, the qRT-PCR results showed that miR-29a-3p was significantly decreased in the pulmonary fibrosis model both in vitro and in vivo. Increased autophagosome was observed via transmission electron microscopy in lung epithelial cell models and lung tissue of silicosis mice. The expression of autophagy-related proteins LC3α/β and Beclin1 were upregulated. The results from using 3-methyladenine, an autophagy inhibitor, or rapamycin, an autophagy inducer, together with TGF-β1, indicated that autophagy attenuates fibrosis by protecting lung epithelial cells. In TGF-β1-treated TC-1 cells, transfection with miR-29a-3p mimics activated protective autophagy and reduced alpha-smooth muscle actin and collagen I expression. miRNA TargetScan predicted, and dual-luciferase reporter experiments identified Akt3 as a direct target of miR-29a-3p. Furthermore, Akt3 expression was significantly elevated in the silicosis mouse model and TGF-β1-treated TC-1 cells. The mammalian target of rapamycin (mTOR) is a central regulator of the autophagy process. Silencing Akt3 inhibited the transduction of the mTOR signaling pathway and activated autophagy in TGF-β1-treated TC-1 cells. These results show that miR-29a-3p overexpression can partially reverse the fibrotic effects by activating autophagy of the pulmonary epithelial cells regulated by the Akt3/mTOR pathway. Therefore, targeting miR-29a-3p may provide a new therapeutic strategy for silica-induced pulmonary fibrosis.

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To date, pulmonary fibrosis remains an unmet medical need. In this study, we evaluated the potency of mesenchymal stromal cell (MSC) secretome components to prevent pulmonary fibrosis development and facilitate fibrosis resolution. Surprisingly, the intratracheal application of extracellular vesicles (MSC-EVs) or the vesicle-depleted secretome fraction (MSC-SF) was not able to prevent lung fibrosis when applied immediately after the injury caused by bleomycin instillation in mice. However, MSC-EV administration induced the resolution of established pulmonary fibrosis, whereas the vesicle-depleted fraction did not. The application of MSC-EVs caused a decrease in the numbers of myofibroblasts and FAPa + progenitors without affecting their apoptosis. Such a decrease likely occurred due to their dedifferentiation caused by microRNA (miR) transfer by MSC-EVs. Using a murine model of bleomycin-induced pulmonary fibrosis, we confirmed the contribution of specific miRs (miR-29c and miR-129) to the antifibrotic effect of MSC-EVs. Our study provides novel insights into possible antifibrotic therapy based on the use of the vesicle-enriched fraction of the MSC secretome., (© 2023. The Author(s).)

This study investigated the effect of Gualou Xiebai Decoction on rats with bleomycin-induced pulmonary fibrosis. The rats were randomly divided into a control group, a model group, a low-dose Gualou Xiebai Decoction group(2.4 g·kg~(-1)), a high-dose Gualou Xiebai Decoction group(4.8 g·kg~(-1)), and pirfenidone group(150 mg·kg~(-1)). The model of pulmonary fibrosis was established by intratracheal instillation of bleomycin in all groups, except the control group. Since the second day of modeling, the corresponding drugs were given to rats by intragastric administration, once a day for 14 d and 28 d. The hematoxylin-eosin(HE) staining was used to evaluate the degree of inflammatory injury in lung tissues. The immunofluorescence staining was used to detect the expression of CD68 and CD163 in lung tissues of rats. The levels of tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10) in serum and brochoalveolar lavage fluid(BALF) were detected by enzyme-linked immunosorbent assay(ELISA). The expression of pyroptosis-related genes in lung tissues of rats was detected by qRT-PCR. The results of HE staining and immunofluorescence staining showed that the lung tissue structure was normal in the control group. In addition, there were alveolar collapse or even closure in lung tissues of rats in the model group, with obvious inflammatory cell infiltration, and the expression of CD68 and CD163 was significantly up-regulated. As compared with the model group, the lung tissue structure of rats in the Gualou Xiebai Decoction groups was significantly improved, with alleviated inflammation, and the expression of CD68 and CD163 was decreased. As compared with the control group, the level of TNF-α in serum and BALF of rats in the model group was significantly increased(P&lt;0.01), the mRNA expression levels of alpha smooth muscle actin(α-SMA), collagen type Ⅰ alpha 1 chain(Col1a1), caspase-1, IL-1β, IL-18, gasdermin D(Gsdmd), and NOD-like receptor thermal protein domain associated protein 3(NLRP3) in lung tissues were significantly increased(P&lt;0.05, P&lt;0.01), and the mRNA expression level of E-cadherin was significantly decreased(P&lt;0.01). As compared with the model group, the level of TNF-α in serum and BALF was significantly down-regulated in the high-dose Gualou Xiebai Decoction group(P&lt;0.05, P&lt;0.01), and that of IL-10 was up-regulated(P&lt;0.05, P&lt;0.01). The mRNA expression levels of α-SMA, Col1a1, caspase-1, IL-18, Gsdmd, NLRP3 and IL-1β in lung tissues were significantly decreased(P&lt;0.05, P&lt;0.01) in the high-dose Gualou Xiebai Decoction group, and the mRNA expression level of E-cadherin was significantly increased(P&lt;0.05, P&lt;0.01). In conclusion, Gualou Xiebai Decoction can down-regulate the levels of inflammatory factors and related genes and effectively mitigate pulmonary fibrosis by regulating the pyroptosis pathways.

An adverse outcome pathway (AOP) framework can be applied as an efficient tool for the rapid screening of environmental chemicals. For the development of an AOP, a database mining approach can support an expert derivation approach by gathering a wider range of evidence than in a literature review. In this study, data from various databases were integrated and analyzed to supplement the AOP leading to pulmonary fibrosis by analyzing additional evidence using a data mining approach and establishing an application domain for chemicals. First, we collected chemicals, genes, and phenotypes that were studied and related to pulmonary fibrosis through the Comparative Toxicogenomics Database (CTD). CGPD-tetramers constructed by linking each related chemical, gene, phenotype, and disease can provide the basic components for the assembly of putative AOPs. Next, an AOP network was established by connecting eight existing AOPs for pulmonary fibrosis developed by expert derivation from the AOP Wiki. Finally, the pulmonary fibrosis AOP network was proposed by integrating the AOP network from AOP Wiki and the CGPD-tetramers from the CTD. To prioritize potential chemical stressors in the AOP network, 61 chemicals were ranked using the relevance of the chemical to the AOP and chemical exposure information from the CompTox Chemicals Dashboard. The approach proposed in this study can guide the utilization of available evidence from various databases as well as the literature in constructing AOP networks related to specific diseases.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused an ongoing pandemic of a pathology termed Coronavirus Disease 19 (COVID-19). Several studies reported that both COVID-19 and RTEL1 variants are associated with shorter telomere length, but a direct association between the two is not generally acknowledged. Here we demonstrate that up to 8.6% of severe COVID-19 patients bear RTEL1 ultra-rare variants, and show how this subgroup can be recognized., Methods: A cohort of 2246 SARS-CoV-2-positive subjects, collected within the GEN-COVID Multicenter study, was used in this work. Whole exome sequencing analysis was performed using the NovaSeq6000 System, and machine learning methods were used for candidate gene selection of severity. A nested study, comparing severely affected patients bearing or not variants in the selected gene, was used for the characterisation of specific clinical features connected to variants in both acute and post-acute phases., Results: Our GEN-COVID cohort revealed a total of 151 patients carrying at least one RTEL1 ultra-rare variant, which was selected as a specific acute severity feature. From a clinical point of view, these patients showed higher liver function indices, as well as increased CRP and inflammatory markers, such as IL-6. Moreover, compared to control subjects, they present autoimmune disorders more frequently. Finally, their decreased diffusion lung capacity for carbon monoxide after six months of COVID-19 suggests that RTEL1 variants can contribute to the development of SARS-CoV-2-elicited lung fibrosis., Conclusion: RTEL1 ultra-rare variants can be considered as a predictive marker of COVID-19 severity, as well as a marker of pathological evolution in pulmonary fibrosis in the post-COVID phase. This notion can be used for a rapid screening in hospitalized infected people, for vaccine prioritization, and appropriate follow-up assessment for subjects at risk. Trial Registration NCT04549831 ( www., Clinicaltrial: org )., (© 2023. The Author(s).)

Background: Reduction of histone deacetylase (HDAC) 2 expression and activity may contribute to amplified inflammation in patients with severe asthma. Connective tissue growth factor (CTGF) is a key mediator of airway fibrosis in severe asthma. However, the role of the HDAC2/Sin3A/methyl-CpG-binding protein (MeCP) 2 corepressor complex in the regulation of CTGF expression in lung fibroblasts remains unclear., Methods: The role of the HDAC2/Sin3A/MeCP2 corepressor complex in endothelin (ET)-1-stimulated CTGF production in human lung fibroblasts (WI-38) was investigated. We also evaluated the expression of HDAC2, Sin3A and MeCP2 in the lung of ovalbumin-induced airway fibrosis model., Results: HDAC2 suppressed ET-1-induced CTGF expression in WI-38 cells. ET-1 treatment reduced HDAC2 activity and increased H3 acetylation in a time-dependent manner. Furthermore, overexpression of HDAC2 inhibited ET-1-induced H3 acetylation. Inhibition of c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 attenuated ET-1-induced H3 acetylation by suppressing HDAC2 phosphorylation and reducing HDAC2 activity. Overexpression of both Sin3A and MeCP2 attenuated ET-1-induced CTGF expression and H3 acetylation. ET-1 induced the disruption of the HDAC2/Sin3A/MeCP2 corepressor complex and then prompted the dissociation of HDAC2, Sin3A, and MeCP2 from the CTGF promoter region. Overexpression of HDAC2, Sin3A, or MeCP2 attenuated ET-1-stimulated AP-1-luciferase activity. Moreover, Sin3A- or MeCP2-suppressed ET-1-induced H3 acetylation and AP-1-luciferase activity were reversed by transfection of HDAC2 siRNA. In an ovalbumin-induced airway fibrosis model, the protein levels of HDAC2 and Sin3A were lower than in the control group; however, no significant difference in MeCP2 expression was observed. The ratio of phospho-HDAC2/HDAC2 and H3 acetylation in the lung tissue were higher in this model than in the control group. Overall, without stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex inhibits CTGF expression by regulating H3 deacetylation in the CTGF promoter region in human lung fibroblasts. With ET-1 stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex is disrupted and dissociated from the CTGF promoter region; this is followed by AP-1 activation and the eventual initiation of CTGF production., Conclusions: The HDAC2/Sin3A/MeCP2 corepressor complex is an endogenous inhibitor of CTGF in lung fibroblasts. Additionally, HDAC2 and Sin3A may be of greater importance than MeCP2 in the pathogenesis of airway fibrosis., (© 2023. The Author(s).)

Pulmonary fibrosis is a common and severe fibrotic lung disease with high morbidity and mortality. Recent studies have reported a large number of unwanted myofibroblasts appearing in pulmonary fibrosis, and shown that the sustained activation of myofibroblasts is essential for unremitting interstitial fibrogenesis. However, the origin of these myofibroblasts remains poorly understood. Here, we create new mouse models of pulmonary fibrosis and identify a previously unknown population of endothelial cell (EC)-like myofibroblasts in normal lung tissue. We show that these EC-like myofibroblasts significantly contribute myofibroblasts to pulmonary fibrosis, which is confirmed by single-cell RNA sequencing of human pulmonary fibrosis. Using the transcriptional profiles, we identified a small molecule that redirects the differentiation of EC-like myofibroblasts and reduces pulmonary fibrosis in our mouse models. Our study reveals the mechanistic underpinnings of the differentiation of EC-like myofibroblasts in pulmonary fibrosis and may provide new strategies for therapeutic interventions., Competing Interests: Conflict of interest: The authors have no potential conflicts of interest to disclose., (Copyright ©The authors 2023.)

Silicosis is one of several potentially fatal occupational pathologies caused by the prolonged inhalation of respirable crystalline silica. Previous studies have shown that lung epithelial-mesenchymal transition (EMT) plays a significant role in the fibrosis effect of silicosis. Human umbilical cord mesenchymal stem cells-derived Extracellular vesicles (hucMSC-EVs) have attracted great interest as a potential therapy of EMT and fibrosis-related diseases. However, the potential effects of hucMSC-EVs in inhibiting EMT in silica-induced fibrosis, as well as its underlying mechanisms, remain largely unknown. In this study, we used the EMT model in MLE-12 cells and observed the effects and mechanism of hucMSC-EVs inhibition of EMT. The results revealed that hucMSC-EVs can indeed inhibit EMT. MiR-26a-5p was highly enriched in hucMSC-EVs but was down-regulated in silicosis mice. We found that miR-26a-5p in hucMSC-EVs was over-expressed after transfecting miR-26a-5p expressing lentivirus vectors into hucMSCs. Subsequently, we explored if miR-26a-5p, attained from hucMSC-EVs, was involved in inhibiting EMT in silica-induced lung fibrosis. Our findings suggested that hucMSC-EVs could deliver miR-26a-5p into MLE-12 cells and cause the inhibition of the Adam17/Notch signalling pathway to ameliorate EMT in silica-induced pulmonary fibrosis. These findings might represent a novel insight into treating silicosis fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Numerous studies have proved that epithelial-mesenchymal transition (EMT) of lung epithelial cells is one of the important causes of radiation-induced pulmonary fibrosis (RIPF). Aldose reductase (AR) is a monomer enzyme in the polyglycolic metabolic pathway and belongs to the aldo-keno reductase protein superfamily. Our previous studies have found that AR as one of the most significantly up-regulated genes was associated with the development of bleomycin-induced PF in rats. It is not clear whether aldose reductase is related to the regulation of radiation-induced EMT and mediates RIPF. AR-knockout mice, wild-type mice and lung epithelial cells were induced by radiation to establish a RIPF animal model and EMT system, to explore whether AR is mediation to RIPF through the EMT pathway. In vivo, AR deficiency significantly alleviated radiation-induced histopathological changes, reduced collagen deposition and inhibited collagen I, matrix metalloproteinase 2 (MMP2) and Twist1 expression. In addition, AR knockout up-regulated E-cadherin expression and up-regulated α-SMA and Vimentin expression. In vitro, AR, collagen I and MMP2 expression were increased in lung epithelial cells after radiation, which was accompanied by Twist1 expression up-regulation and EMT changes evidenced by decreased E-cadherin expression and increased α-SMA and Vimentin expression. Knockdown or inhibition of AR inhibited the expressions of Twist1, MMP2 and collagen I, and reduced cell migration and reversed radiation-induced EMT. These results indicated that aldose reductase may be related to radiation-induced lung epithelial cells EMT, and that inhibition of aldose reductase might be a promising treatment for RIPF.

Epigenetic has been implicated in pulmonary fibrosis. However, there is limited information regarding the biological role of the epigenetic reader MeCP2 in pulmonary fibrosis. The aim of this study was to investigate the role of MeCP2 and its target WIF1 in pulmonary fibrosis. The pathological changes and collagen depositions was analyzed by H&E, Masson's Trichrome Staining and Sirius Red staining. MeCP2, WIF1, α-SMA, Wnt1, β-catenin, and collagen I expression were analyzed by western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, respectively. The effects of MeCP2 on pulmonary fibrosis involve epigenetic mechanisms, using cultured cells, animal models, and clinical samples. Herein, our results indicated that MeCP2 level was up-regulated, while WIF1 was decreased in Bleomycin (BLM)-induced mice pulmonary fibrosis tissues, patients pulmonary fibrosis tissues and TGF-β1-induced lung fibroblast. Knockdown of MeCP2 by siRNA can rescue WIF1 downregulation in TGF-β1-induced lung fibroblast, inhibited lung fibroblast activation. The DNA methylation inhibitor 5-azadC-treated lung fibroblasts have increased WIF1 expression with reduced MeCP2 association. In addition, we found that reduced expression of WIF1 caused by TGF-β1 is associated with the promoter methylation status of WIF1. Moreover, in vivo studies revealed that knockdown of MeCP2 mice exhibited significantly ameliorated pulmonary fibrosis, decreased interstitial collagen deposition, and increased WIF1 expression. Taken together, our study showed that epigenetic reader MeCP2 repressed WIF1 facilitates lung fibroblast proliferation, migration and pulmonary fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rationale and Objectives: Up to 20% of idiopathic interstitial lung disease is familial, referred to as familial pulmonary fibrosis (FPF). An integrated analysis of FPF genetic risk was performed by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds. Methods: Whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds was performed, followed by cosegregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell-type enrichment analysis, and coexpression network construction. Measurements and Main Results: It was found that 14.9-23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomere-related genes. New candidate genes were identified in a small number of families-including SYDE1 , SERPINB8 , GPR87 , and NETO1 -and tools were developed for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single-cell transcriptomics with prioritized candidate genes, expression of RV-containing genes was discovered to be enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells. Conclusions: In the most comprehensive FPF genetic study to date, the prevalence of RVs in known FPF-related genes was defined, and new candidate genes and pathways relevant to FPF were identified. However, new RV-containing genes shared across multiple kindreds were not identified, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Objective: Acute lung injury (ALI) is a common complication of critical diseases with high morbidity and mortality. This study explored the regulatory role and mechanism of high mobility histone box 1 protein (HMGB1) on pulmonary fibrosis (PF) after ALI in rats through nucleotide oligomerization domain-like receptor protein-3 (NLRP3) inflammasome., Methods: PF rat models after ALI were established by induction of bleomycin. Degree of fibrosis was assessed by Masson staining and Ashcroft scoring. Hydroxyproline (Hyp) contents in lung tissues and rat lung tissue morphology were detected by enzyme-linked-immunosorbent serologic assay (ELISA) and hematoxylin and eosin staining. The levels of NLRP3, major proteins of NLRP3 inflammasome (NLRP3/ASC/caspase-1), and downstream inflammatory cytokines interleukin (IL)-1 and IL-18 were determined using immunohistochemistry, Western blotting analysis, and ELISA. The nuclear/cytoplasmic nuclear factor erythroid 2-related factor 2 (Nrf2) levels and HO-1 levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis. Rats was injected with lentivirus carrying short hairpin (sh)-HMGB1 and zinc protoporphyria (ZNPP) (HO-1 inhibitor) to assess the effects of HMGB1 and HO-1 on PF and NLRP3 inflammasome activation., Results: Bleomycin induced PF after ALI in rats, manifested as patchy fibrosis, atelectasis, and excessive expansion, and increased Aschcroft score and Hyp content. Bleomycin treatment enhanced levels of NLRP3, ASC, caspase-1, IL-1, and IL-18 in rat lung tissues, which promoted activation of NLRP3 inflammasome. HMGB1 was up-regulated in bleomycin-induced rats. HMGB1 knockdown partially reversed NLRP3 inflammasome activation and PF progression. HMGB1 knockdown promoted Nrf2 nuclear translocation and up-regulated HO-1. Suppression of HO-1 partially reversed inhibition of HMGB1 knockdown on NLRP3 inflammasome activation and PF., Conclusion: HMGB1 can activate NLRP3 inflammasomes and promote PF by inhibiting the Nrf2/HO-1 pathway., Competing Interests: The authors declared that they had no competing interests to assert.

Increased apoptosis of alveolar epithelial cells is a prominent feature of pulmonary fibrosis. Macrophage efferocytosis, phagocytosis of apoptotic cells by macrophages, is crucial for maintaining tissue homeostasis. Expression of Mer tyrosine kinase (MERTK, an important recognition receptor in efferocytosis) in macrophages is thought to be associated with fibrosis. However, how macrophage MERTK affects pulmonary fibrosis and whether it depends on efferocytosis are not yet clear. Here, we found elevated MERTK expression in lung macrophages from IPF patients and mice with bleomycin-induced pulmonary fibrosis. In vitro experiments showed that macrophages overexpressing MERTK exhibit profibrotic effects and that macrophage efferocytosis abrogates the profibrotic effect of MERTK by downregulating MERTK, forming a negative regulatory loop. In pulmonary fibrosis, this negative regulation is defective, and MERTK mainly exhibits profibrotic effects. Our study reveals a previously unsuspected profibrotic effect of elevated macrophage MERTK in pulmonary fibrosis and defective regulation of efferocytosis function as a result of that elevation, suggesting that targeting MERTK in macrophages may help to attenuate pulmonary fibrosis., (© 2023. The Author(s).)

Background: Radiotherapy is an essential treatment for chest cancer. Radiation-induced pulmonary fibrosis (RIPF) is an almost irreversible interstitial lung disease; however, its pathogenesis remains unclear., Methods: We analyzed specific changes in cell populations and potential markers by using single-cell sequencing datasets from the Sequence Read Archive database, PERFORMED from control (0 Gy) and thoracic irradiated (20 Gy) mouse lungs at day 150 post-radiation. We performed IHC and ELISA on lung tissue and cells to validate the potential marker cytokines identified by the analysis on rat thoracic irradiated molds (30 Gy)., Results: Single-cell sequencing analysis showed changes in abundance across cell types and at the single-cell level, with B and T cells showing the most significant changes in abundance. And four cytokines, CCL5, ICAM1, PF4, and TNF, were significantly upregulated in lung tissues of RIPF rats and cell supernatants after ionizing radiation., Conclusion: Cytokines CCL5, ICAM1, PF4, and TNF may play essential roles in radiation pulmonary fibrosis. They are potential targets for the treatment of radiation pulmonary fibrosis., (© 2023. The Author(s).)

Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme neuraminidase 3 (NEU3) causing release of active TGF-β1 and TGF-β1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-β1 upregulation of the translation mechanism. In human lung fibroblasts, TGF-β1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. A total of 180 of these mRNAs shared a common 20-nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-β1 upregulation of NEU3 translation, while insertion of this motif in 2 mRNAs insensitive to TGF-β1 caused TGF-β1 to upregulate their translation. RNA-binding proteins including DEAD box helicase 3, X-linked (DDX3), bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. We found that DDX3 was upregulated in the fibrotic lesions in patients with pulmonary fibrosis, and inhibiting DDX3 in fibroblasts reduced TGF-β1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and tissue levels of DDX3, TGF-β1, and NEU3. These results suggest that inhibiting an mRNA-binding protein that mediates TGF-β1 upregulation of translation can reduce pulmonary fibrosis.

It has been reported that particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) could induce epithelial-mesenchymal transition (EMT)- and extracellular matrix (ECM)-related pulmonary fibrosis (PF). The transcription factor Nrf2 alleviated PM2.5-induced PF by antagonizing oxidative stress. The N 6 -methyladenosine (m 6 A) modification plays a significant role in the stress response. However, the effect of m 6 A modification on the mechanisms of Nrf2-mediated defense against PM2.5-induced PF remained unknown. Here, we explored the role and the underlying molecular mechanisms of m 6 A methylation of Nrf2 mRNA in PM2.5-induced PF. We established filtered air (FA), unfiltered air (UA), and concentrated PM2.5 air (CA) group mice model and 0, 50, and 100 μg/mL PM2.5-treated 16HBE cell models. The extent of lung fibrosis in mice and fibrosis indicators were detected by histopathological analysis, immunohistochemical staining and western blotting. The molecular mechanism of m 6 A-modified Nrf2 was demonstrated by m 6 A-methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), qRT-PCR and T3 ligase-based PCR. Our data showed that PM2.5 exposure for 16 weeks could induce pulmonary fibrosis and activate Nrf2 signaling pathway. m 6 A methyltransferase METTL3 was upregulated after PM2.5 treatment in vivo and in vitro. Moreover, METTL3 mediated m 6 A modification of Nrf2 mRNA and promoted Nrf2 translation in mice and 16HBE cells after PM2.5 exposure. Mechanistically, three m 6 A-modified sites (1317, 1376 and 935; numbered relative to the first nucleotide of 3'UTR) of Nrf2 mRNA were identified in PM2.5-treatment 16HBE cells. Furthermore, the m 6 A binding proteins YTHDF1/IGF2BP1 promoted Nrf2 translation by binding to m 6 A residues of Nrf2 mRNA. Our results revealed the mechanism of m 6 A mediated Nrf2 signaling pathway against oxidative stress, which affected the development of PM2.5-induced PF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pulmonary fibrosis (PF) is an interstitial lung disease characterized by the destruction of the pulmonary parenchyma caused by excessive extracellular matrix deposition. Despite the well-known etiological factors such as senescence, aberrant epithelial cell and fibroblast activation, and chronic inflammation, PF has recently been recognized as a metabolic disease and abnormal lipid signature was observed both in serum and bronchoalveolar lavage fluid (BALF) of PF patients and mice PF model. Clinically, observational studies suggest a significant link between high-fat diet (HFD) and PF as manifested by high intake of saturated fatty acids (SFAs) and meat increases the risk of PF and mice lung fibrosis. However, the possible mechanisms between HFD and PF remain unclear. In the current review we emphasize the diversity effects of the epigenetic dysregulation induced by HFD on the fibrotic factors such as epithelial cell injury, abnormal fibroblast activation and chronic inflammation. Finally, we discuss the potential ways for patients to improve their conditions and emphasize the prospect of targeted therapy based on epigenetic regulation for scientific researchers or drug developers.

Genetic predisposition to pulmonary fibrosis has been confirmed by the discovery of several gene mutations that cause pulmonary fibrosis. Although genetic sequencing of familial pulmonary fibrosis (FPF) cases is embedded in routine clinical practice in several countries, many centres have yet to incorporate genetic sequencing within interstitial lung disease (ILD) services and proper international consensus has not yet been established. An international and multidisciplinary expert Task Force (pulmonologists, geneticists, paediatrician, pathologist, genetic counsellor, patient representative and librarian) reviewed the literature between 1945 and 2022, and reached consensus for all of the following questions: 1) Which patients may benefit from genetic sequencing and clinical counselling? 2) What is known of the natural history of FPF? 3) Which genes are usually tested? 4) What is the evidence for telomere length measurement? 5) What is the role of common genetic variants (polymorphisms) in the diagnostic workup? 6) What are the optimal treatment options for FPF? 7) Which family members are eligible for genetic sequencing? 8) Which clinical screening and follow-up parameters may be considered in family members? Through a robust review of the literature, the Task Force offers a statement on genetic sequencing, clinical management and screening of patients with FPF and their relatives. This proposal may serve as a basis for a prospective evaluation and future international recommendations., Competing Interests: Conflict of interest: R. Borie reports grants from Boehringer Ingelheim, consulting fees from Sanofi, lecture honoraria from Boehringer Ingelheim, Roche and Sanofi, and travel support from Boehringer Ingelheim, outside the submitted work. K. Antoniou reports consulting fees from Boehringer Ingelheim, Roche and GlaxoSmithKline, lecture honoraria from Boehringer Ingelheim, Roche, Chiesi, Menarini, GlaxoSmithKline and AstraZeneca, outside the submitted work. F. Bonella reports lecture honoraria from Boehringer Ingelheim, Fujirebio, Galapagos NV and Roche, travel support from Boehringer Ingelheim and Roche, participation on advisory boards with Boehringer Ingelheim, BMS, Fujirebio, Galapagos NV, GlaxoSmithKline, Roche and Takeda, outside the submitted work. B. Crestani reports grants from BMS, Boehringer Ingelheim and Roche, consulting fees from Apellis, lecture honoraria from Boehringer Ingelheim, BMS, Roche, Sanofi, Novartis, AstraZeneca and Chiesi, and is in receipt of equipment from Translate Bio, outside the submitted work. A. Froidure reports grants and consulting fees from Boehringer Ingelheim, lecture honoraria from Boehringer Ingelheim and GlaxoSmithKline, and travel support from Sanofi, outside the submitted work. L. Galvin reports travel support from the European Lung Foundation, European Respiratory Society and European Pulmonary Fibrosis Federation, leadership roles with the European Pulmonary Fibrosis Federation, Irish Lung Fibrosis Association, European Lung Foundation and European Reference Network On Rare Respiratory Diseases, outside the submitted work. M. Griese reports grants and travel support from Boehringer Ingelheim, outside the submitted work. M. Molina-Molina reports grants from Roche and Boehringer Ingelheim, consulting fees from Esteve-Teijin, Ferrer, Roche and Boehringer Ingelheim, and lecture honoraria from Chiesi, Roche and Boehringer Ingelheim, outside the submitted work. V. Poletti reports lecture honoraria from AMBU, Boehringer Ingelheim, ERBE and Roche, outside the submitted work. E. Renzoni reports grants from Boehringer Ingelheim, and lecture honoraria from Boehringer Ingelheim, Roche and Novartis, outside the submitted work. C.H.M. van Moorsel reports grants from the European Respiratory Society supporting the present manuscript, grants and lecture honoraria from Boehringer Ingelheim, and a leadership role as co-chair of the European Respiratory Society Task Force for genetics in pulmonary fibrosis, outside the submitted work. All other authors have nothing to disclose., (Copyright ©The authors 2023. For reproduction rights and permissions contact permissions@ersnet.org.)

Objective: For respiratory failure patients, mechanical ventilation (MV) is a life-saving therapy to maintain respiratory function. However, MV could also cause damage to pulmonary structures, result in ventilator-induced lung injury (VILI) and eventually progress to mechanical ventilation-induced pulmonary fibrosis (MVPF). Mechanically ventilated patients with MVPF are closely related to increased mortality and poor quality of life in long-term survival. Thus, a thorough understanding of the involved mechanism is necessary., Methods: We used next-generation sequencing to identify differentially expressed non-coding RNAs (ncRNAs) in BALF EVs which were isolated from Sham and MV mice. Bioinformatics analysis was conducted to identify the engaged ncRNAs and related signaling pathways in the process of MVPF., Results: We found 1801 messenger RNAs (mRNA), 53 micro RNAs (miRNA), 273 circular RNAs (circRNA) and 552 long non-coding RNAs (lncRNA) in mice BALF EVs of two groups, which showed significant differential expression. TargetScan predicted that 53 differentially expressed miRNAs targeted 3105 mRNAs. MiRanda revealed that 273 differentially expressed circRNAs were associated with 241 mRNAs while 552 differentially expressed lncRNAs were predicated to target 20528 mRNAs. GO, KEGG pathway analysis and KOG classification showed that these differentially expressed ncRNA-targeted mRNAs were enriched in fibrosis related signaling pathways and biological processes. By taking the intersection of miRNAs target genes, circRNAs target genes and lncRNAs target genes, we found 24 common key genes and 6 downregulated genes were confirmed by qRT-PCR., Conclusions: Changes in BALF-EV ncRNAs may contribute to MVPF. Identification of key target genes involved in the pathogenesis of MVPF could lead to interventions that slow or reverse fibrosis progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tang, Hu, Mei, Zhou, Feng, Jin, Dai, Xing, Gao, Xu and He.)

Molecular clock REV-ERBα is central to regulating lung injuries, and decreased REV-ERBα abundance mediates sensitivity to pro-fibrotic insults and exacerbates fibrotic progression. In this study, we determine the role of REV-ERBα in fibrogenesis induced by bleomycin and Influenza A virus (IAV). Bleomycin exposure decreases the abundance of REV-ERBα, and mice dosed with bleomycin at night display exacerbated lung fibrogenesis. Rev-erbα agonist (SR9009) treatment prevents bleomycin induced collagen overexpression in mice. Rev-erbα global heterozygous (Rev-erbα Het) mice infected with IAV showed augmented levels of collagens and lysyl oxidases compared with WT-infected mice. Furthermore, Rev-erbα agonist (GSK4112) prevents collagen and lysyl oxidase overexpression induced by TGFβ in human lung fibroblasts, whereas the Rev-erbα antagonist exacerbates it. Overall, these results indicate that loss of REV-ERBα exacerbates the fibrotic responses by promoting collagen and lysyl oxidase expression, whereas Rev-erbα agonist prevents it. This study provides the potential of Rev-erbα agonists in the treatment of pulmonary fibrosis., (© 2023. The Author(s).)

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast-to-myofibroblast activation. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)-induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild-type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF-β-driven endothelial-to-mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition-related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA-treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM-induced pulmonary fibrosis, partly through the reduction in TGF-β expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Introduction: The genetic risk factors for Coronavirus disease-2019 (COVID19)-associated pulmonary fibrosis (CAPF) are not clearly defined. Mutations in the genes encoding telomerase reverse transcriptase (TERT) and mucin 5B (MUC5B) are well-known genetic risk factors for pulmonary fibrosis. In this study, we aimed to show whether the most common proven mutations of pulmonary fibrosis affect the development of CAPF., Materials and Methods: Forty-eight patients who were matched for age, gender, COVID-19 disease severity, and respiratory support type and needed high flow nasal cannula, non-invasive mechanical ventilator, or invasive mechanical ventilator due to COVID-19 were followed up prospectively. Eighteen patients were excluded from the follow-up due to known structural lung disease, collagen tissue disease, and occupational exposure to fibrosis. The patients were called for follow-up three months after discharge, and CT was performed. Those with fibrosis (n= 15) in the third-month follow-up CT were included in the CAPF group, and those with complete resolution (n= 15) were included in the control group. Blood samples were taken for genetic analysis., Result: TERT gene study revealed that six (40%) of the fibrosis group was normal, while five were heterozygous (33.3%). MUC5B polymorphism was not detected in 10 (66.7%) of the fibrosis group., Conclusions: Individuals with TERT mutations may be at a higher risk for CAPF. Further studies are needed to clarify the genetic risk factors for CAPF.

Objective: This study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms., Methods: The mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃β1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis., Results: The mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-β1/Smad signaling pathway., Conclusion: Inhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃β1 and bleomycin, and then inhibit the fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pulmonary fibrosis (PF) is a form of progressive lung disease characterized by chronic inflammation and excessive extracellular matrix (ECM) deposition. However, the protein changes in fibrotic ECM during PF and their contribution to fibrosis progression are unclear. Here we show that changes in expression of ECM components and ECM remodeling had occurred in silica-instilled mice. The macrophage-derived glycoprotein nonmetastatic melanoma protein B (GPNMB) captured by fibrotic ECM may activate resident normal fibroblasts around the fibrotic foci. Functional experiments demonstrated the activation of fibroblasts in fibrotic ECM, which was alleviated by GPNMB-neutralizing antibodies or macrophage deletion in the ECM of silica-instilled mice. Moreover, the Serpinb2 expression level was increased in fibroblasts in fibrotic ECM, and the expression of CD44 was increased in silica-instilled mice. In conclusion, macrophage-derived GPNMB is trapped by fibrotic ECM during transport and may activate fibroblasts via the CD44/Serpinb2 pathway, thus leading to the further development of fibrosis., (© 2023. The Author(s).)