Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation.

The epithelial-mesenchymal transition (EMT) and fibroblast activation are major events in idiopathic pulmonary fibrosis pathogenesis. Here, we investigated whether growth arrest-specific protein 6 (Gas6) plays a protective role in lung fibrosis via suppression of the EMT and fibroblast activation. rGas6 administration inhibited the EMT in isolated mouse ATII cells 14 days post-BLM treatment based on morphologic cellular alterations, changes in mRNA and protein expression profiles of EMT markers, and induction of EMT-activating transcription factors. BLM-induced increases in gene expression of fibroblast activation-related markers and the invasive capacity of primary lung fibroblasts in primary lung fibroblasts were reversed by rGas6 administration. Furthermore, the hydroxyproline content and collagen accumulation in interstitial areas with damaged alveolar structures in lung tissue were reduced by rGas6 administration. Targeting Gas6/Axl signaling events with specific inhibitors of Axl (BGB324), COX-2 (NS-398), EP1/EP2 receptor (AH-6809), or PGD2 DP2 receptor (BAY-u3405) reversed the inhibitory effects of rGas6 on EMT and fibroblast activation. Finally, we confirmed the antifibrotic effects of Gas6 using Gas6-/- mice. Therefore, Gas6/Axl signaling events play a potential role in inhibition of EMT process and fibroblast activation via COX-2-derived PGE2 and PGD2 production, ultimately preventing the development of pulmonary fibrosis.

A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model.

BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.

[Research progress of PD-L1 in inflammatory lung diseases].

Programmed death-ligand 1 (PD-L1) is important in maintaining central and peripheral immune tolerance in normal tissues, mediating tumor immune escape and keeping the balance between anti- and pro-inflammatory responses. Inflammation plays an important role in inflammatory lung diseases. This article reviews the research progress and potential clinical value of PD-L1 in inflammatory lung diseases, including acute lung injury, chronic obstructive pulmonary disease, asthma and idiopathic pulmonary fibrosis.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts.

BACKGROUND: Abnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined. METHODS: Oil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs. RESULTS: Injured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα. CONCLUSIONS: These data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

Exploring therapeutic targets for molecular therapy of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic and progressive interstitial lung disease with a poor prognosis. Idiopathic pulmonary fibrosis is characterized by repeated alveolar epithelial damage leading to abnormal repair. The intercellular microenvironment is disturbed, leading to continuous activation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and ultimately fibrosis. Moreover, pulmonary fibrosis was also found as a COVID-19 complication. Currently, two drugs, pirfenidone and nintedanib, are approved for clinical therapy worldwide. However, they can merely slow the disease's progression rather than rescue it. These two drugs have other limitations, such as lack of efficacy, adverse effects, and poor pharmacokinetics. Consequently, a growing number of molecular therapies have been actively developed. Treatment options for IPF are becoming increasingly available. This article reviews the research platform, including cell and animal models involved in molecular therapy studies of idiopathic pulmonary fibrosis as well as the promising therapeutic targets and their development progress during clinical trials. The former includes patient case/control studies, cell models, and animal models. The latter includes transforming growth factor-beta, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid, interleukin-13, Rho-associated coiled-coil forming protein kinase family, and Janus kinases/signal transducers and activators of transcription pathway. We mainly focused on the therapeutic targets that have not only entered clinical trials but were publicly published with their clinical outcomes. Moreover, this work provides an outlook on some promising targets for further validation of their possibilities to cure the disease.

[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via µPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

Identifying a survival-associated cell type based on multi-level transcriptome analysis in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a five-year survival rate of less than 40%. There is significant variability in survival time among IPF patients, but the underlying mechanisms for this are not clear yet. METHODS AND RESULTS: We collected single-cell RNA sequence data of 13,223 epithelial cells taken from 32 IPF patients and bulk RNA sequence data from 456 IPF patients in GEO. Based on unsupervised clustering analysis at the single-cell level and deconvolution algorithm at bulk RNA sequence data, we discovered a special alveolar type 2 cell subtype characterized by high expression of CCL20 (referred to as ATII-CCL20), and found that IPF patients with a higher proportion of ATII-CCL20 had worse prognoses. Furthermore, we uncovered the upregulation of immune cell infiltration and metabolic functions in IPF patients with a higher proportion of ATII-CCL20. Finally, the comprehensive decision tree and nomogram were constructed to optimize the risk stratification of IPF patients and provide a reference for accurate prognosis evaluation. CONCLUSIONS: Our study by integrating single-cell and bulk RNA sequence data from IPF patients identified a special subtype of ATII cells, ATII-CCL20, which was found to be a risk cell subtype associated with poor prognosis in IPF patients. More importantly, the ATII-CCL20 cell subtype was linked with metabolic functions and immune infiltration.

Astragaloside IV inhibits idiopathic pulmonary fibrosis through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway.

As the main active ingredient of Astragalus, Astragaloside IV (AS-IV) can ameliorate pulmonary fibrosis. In this experiment, we studied how AS-IV reduces idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) or TGF-ß1 was treated in mice or alveolar epithelial cells to mimic IPF in vivo as well as in vitro. ASV-IV alleviated levels of inflammatory cytokines and fibrosis markers in IPF model. Through detection of autophagy-related genes, ASV-IV was observed to induce autophagy in IPF. Besides, ASV-IV inhibited miR-21 expression in IPF models, and overexpression of miR-21 could reverse the protective potential of ASV-IV on IPF. PTEN was targeted by miR-21 and was up-regulated by ASV-IV in IPF models. In addition, levels of inflammatory cytokines and fibrosis markers, autophagy, as well as the PI3K/AKT/mTOR pathway regulated by ASV-IV could be neutralized after treatment with autophagy inhibitors, miR-21 mimics, or si-PTEN. Our study demonstrates that ASV-IV inhibits IPF through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway, suggesting that ASV-IV could be acted to be a promising therapeutic method for IPF.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Discoidin domain receptor 2 signaling through PIK3C2α in fibroblasts promotes lung fibrosis.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFß and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The gene expression of CALD1, CDH2, and POSTN in fibroblast are related to idiopathic pulmonary fibrosis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung dysfunction due to excessive collagen production and tissue scarring. Despite recent advancements, the molecular mechanisms remain unclear. Methods: RNA sequencing identified 475 differentially expressed genes (DEGs) in the TGF-ß1-induced primary lung fibrosis model. Gene expression chips GSE101286 and GSE110147 from NCBI gene expression omnibus (GEO) database were analyzed using GEO2R, revealing 94 DEGs in IPF lung tissue samples. The gene ontology (GO) and pathway enrichment, Protein-protein interaction (PPI) network construction, and Maximal Clique Centrality (MCC) scoring were performed. Experimental validation included RT-qPCR, Immunohistochemistry (IHC), and Western Blot, with siRNA used for gene knockdown. A co-expression network was constructed by GeneMANIA. Results: GO enrichment highlighted significant enrichment of DEGs in TGF-ß cellular response, connective tissue development, extracellular matrix components, and signaling pathways such as the AGE-RAGE signaling pathway and ECM-receptor interaction. PPI network analysis identified hub genes, including FN1, COL1A1, POSTN, KIF11, and ECT2. CALD1 (Caldesmon 1), CDH2 (Cadherin 2), and POSTN (Periostin) were identified as dysregulated hub genes in both the RNA sequencing and GEO datasets. Validation experiments confirmed the upregulation of CALD1, CDH2, and POSTN in TGF-ß1-treated fibroblasts and IPF lung tissue samples. IHC experiments probed tissue-level expression patterns of these three molecules. Knockdown of CALD1, CDH2, and POSTN attenuated the expression of fibrotic markers (collagen I and α-SMA) in response to TGF-ß1 stimulation in primary fibroblasts. Co-expression analysis revealed interactions between hub genes and predicted genes involved in actin cytoskeleton regulation and cell-cell junction organization. Conclusions: CALD1, CDH2, and POSTN, identified as potential contributors to pulmonary fibrosis, present promising therapeutic targets for IPF patients.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. METHODS: We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. RESULTS: We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. CONCLUSION: Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

SLC15A3 plays a crucial role in pulmonary fibrosis by regulating macrophage oxidative stress.

Idiopathic pulmonary fibrosis (IPF) is a fatal and irreversible disease with few effective treatments. Alveolar macrophages (AMs) are involved in the development of IPF from the initial stages due to direct exposure to air and respond to external oxidative damage (a major inducement of pulmonary fibrosis). Oxidative stress in AMs plays an indispensable role in promoting fibrosis development. The oligopeptide histidine transporter SLC15A3, mainly expressed on the lysosomal membrane of macrophages and highly expressed in the lung, has proved to be involved in innate immune and antiviral signaling pathways. In this study, we demonstrated that during bleomycin (BLM)- or radiation-induced pulmonary fibrosis, the recruitment of macrophages induced an increase of SLC15A3 in the lung, and the deficiency of SLC15A3 protected mice from pulmonary fibrosis and maintained the homeostasis of the pulmonary microenvironment. Mechanistically, deficiency of SLC15A3 resisted oxidative stress in macrophages, and SLC15A3 interacted with the scaffold protein p62 to regulate its expression and phosphorylation activation, thereby regulating p62-nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant stress pathway protein, which is related to the production of reactive oxygen species (ROS). Overall, our data provided a novel mechanism for targeting SLC15A3 to regulate oxidative stress in macrophages, supporting the therapeutic potential of inhibiting or silencing SLC15A3 for the precautions and treatment of pulmonary fibrosis.

Antifibrotic effects of vitamin D3 on human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Up to now, no treatment can stop the progression of IPF. Vitamin D3 (VD) reduces experimental lung fibrosis in murine models and depletion of vitamin D3 might be associated with the reduced survival of patients with IPF. In this context, we determined if VD can prevent the pro-fibrotic functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and control HLFs were derived from surgical lung biopsies collected from patients with IPF or with primary lung cancer, respectively. VD (3-100 nM) markedly reduced the basal and PDGF-induced proliferation of HLFs. VD also altered cell cycle by increasing the percentage of IPF HLFs arrested in the G0/G1 phase, and by downregulating the expression of various cell cycle regulatory proteins. In addition, VD barely prevented the TGF-ß1-induced differentiation in HLFs. At 100 nM, VD slightly reduced the expression of the pro-fibrotic marker α-smooth muscle actin, and had no effect on fibronectin and collagen-1 expression. In contrast, 100 nM VD strongly inhibited the aerobic glycolytic metabolism induced by TGF- ß1. Finally, VD reduced both the secretion of lactate, the levels of lactate deshydrogenase mRNA and the activity of intracellular LDH in IPF HLFs. In conclusion, our study shows that VD reduced pro-fibrotic functions of HLFs. These findings suggest that it might be interesting to assess the potential clinical benefits of vitamin D supplementation in patients with IPF, especially on lung function decline.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Traditional Chinese medicine inspired dual-drugs loaded inhalable nano-therapeutics alleviated idiopathic pulmonary fibrosis by targeting early inflammation and late fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a highly debilitating and fatal chronic lung disease that is difficult to cure clinically. IPF is characterized by a gradual decline in lung function, which leads to respiratory failure and severely affects patient quality of life and survival. Oxidative stress and chronic inflammation are believed to be important pathological mechanisms underlying the onset and progression of IPF, and the vicious cycle of NOX4-derived ROS, NLRP3 inflammasome activation, and p38 MAPK in pulmonary fibrogenesis explains the ineffectiveness of single-target or single-drug interventions. In this study, we combined astragaloside IV (AS-IV) and ligustrazine (LIG) based on the fundamental theory of traditional Chinese medicine (TCM) of "tonifying qi and activating blood" and loaded these drugs onto nanoparticles (AS_LIG@PPGC NPs) that were inhalable and could penetrate the mucosal barrier. Our results suggested that inhalation of AS_LIG@PPGC NPs significantly improved bleomycin-induced lung injury and fibrosis by regulating the NOX4-ROS-p38 MAPK and NOX4-NLRP3 pathways to treat and prevent IPF. This study not only demonstrated the superiority, feasibility, and safety of inhalation therapy for IPF intervention but also confirmed that breaking the vicious cycle of ROS and the NLRP3 inflammasome is a promising strategy for the successful treatment of IPF. Moreover, this successful nanoplatform is a good example of the integration of TCM and modern medicine.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture.

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3-4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air-liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFß-2, ZO-1 and DN12. When stimulated with exogenous TGFß, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFß-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1ß, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFß isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFß signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Circulating metabolic profile in idiopathic pulmonary fibrosis: data from the IPF-PRO Registry.

BACKGROUND: The circulating metabolome, reflecting underlying cellular processes and disease biology, has not been fully characterized in patients with idiopathic pulmonary fibrosis (IPF). We evaluated whether circulating levels of metabolites correlate with the presence of IPF, with the severity of IPF, or with the risk of clinically relevant outcomes among patients with IPF. METHODS: We analyzed enrollment plasma samples from 300 patients with IPF in the IPF-PRO Registry and 100 individuals without known lung disease using a set of targeted metabolomics and clinical analyte modules. Linear regression was used to compare metabolite and clinical analyte levels between patients with IPF and controls and to determine associations between metabolite levels and measures of disease severity in patients with IPF. Unadjusted and adjusted univariable Cox regression models were used to evaluate associations between circulating metabolites and the risk of mortality or disease progression among patients with IPF. RESULTS: Levels of 64 metabolites and 5 clinical analytes were significantly different between patients with IPF and controls. Among analytes with greatest differences were non-esterified fatty acids, multiple long-chain acylcarnitines, and select ceramides, levels of which were higher among patients with IPF versus controls. Levels of the branched-chain amino acids valine and leucine/isoleucine were inversely correlated with measures of disease severity. After adjusting for clinical factors known to influence outcomes, higher levels of the acylcarnitine C:16-OH/C:14-DC were associated with all-cause mortality, lower levels of the acylcarnitine C16:1-OH/C14:1DC were associated with all-cause mortality, respiratory death, and respiratory death or lung transplant, and higher levels of the sphingomyelin d43:2 were associated with the risk of respiratory death or lung transplantation. CONCLUSIONS: IPF has a distinct circulating metabolic profile characterized by increased levels of non-esterified fatty acids, long-chain acylcarnitines, and ceramides, which may suggest a more catabolic environment that enhances lipid mobilization and metabolism. We identified select metabolites that were highly correlated with measures of disease severity or the risk of disease progression and that may be developed further as biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov; No: NCT01915511; URL: www. CLINICALTRIALS: gov .