Hypoxia Promotes Invadosome Formation by Lung Fibroblasts.

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

The role of quercetin in ameliorating bleomycin-induced pulmonary fibrosis: insights into autophagy and the SIRT1/AMPK signaling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by a constant incidence rate. Unfortunately, effective pharmacological treatments for this condition are lacking and the identification of novel therapeutic approaches and underlying pathological mechanisms are required. This study investigated the potential of quercetin in alleviating pulmonary fibrosis by promoting autophagy and activation of the SIRT1/AMPK pathway. METHODS: Mouse models of IPF were divided into four treatment groups: control, bleomycin (BLM), quercetin (Q), and quercetin + EX-527 (Q + E) treatment. Pulmonary fibrosis was induced in the mouse models through intratracheal instillation of BLM. Various indexes were identified through histological staining, Western blotting analysis, enzyme-linked immunosorbent assay, immunohistochemistry, and transmission electron microscopy. RESULTS: Quercetin treatment ameliorated the pathology of BLM-induced pulmonary fibrosis of mice by reducing α-smooth muscle actin (α-SMA), collagen I (Col I), and collagen III (Col III) levels, and also improved the level of E-cadherin in lung tissue. Furthermore, Quercetin significantly enhanced LC3II/LC3I levels, decreased P62 expression, and increased the number of autophagosomes in lung tissue. These effects were accompanied by the activation of the SIRT1/AMPK pathway. Treatment with EX-527, an inhibitor for SIRT1, reversed all effects induced by quercetin. CONCLUSION: This study showed that quercetin could alleviate pulmonary fibrosis and improve epithelial-mesenchymal transition by acting on the SIRT1/AMPK signaling pathway, which may be achieved by regulating the level of autophagy.

Highlighting fibroblast plasticity in lung fibrosis: the WI-38 cell line as a model for investigating the myofibroblast and lipofibroblast switch.

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-ß1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-ß1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-ß1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-ß1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-ß1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.

Single-cell RNA sequencing reveals special basal cells and fibroblasts in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most predominant type of idiopathic interstitial pneumonia and has an increasing incidence, poor prognosis, and unclear pathogenesis. In order to investigate the molecular mechanisms underlying IPF further, we performed single-cell RNA sequencing analysis on three healthy controls and five IPF lung tissue samples. The results revealed a significant shift in epithelial cells (ECs) phenotypes in IPF, which may be attributed to the differentiation of alveolar type 2 cells to basal cells. In addition, several previously unrecognized basal cell subtypes were preliminarily identified, including extracellular matrix basal cells, which were increased in the IPF group. We identified a special population of fibroblasts that highly expressed extracellular matrix-related genes, POSTN, CTHRC1, COL3A1, COL5A2, and COL12A1. We propose that the close interaction between ECs and fibroblasts through ligand-receptor pairs may have a critical function in IPF development. Collectively, these outcomes provide innovative perspectives on the complexity and diversity of basal cells and fibroblasts in IPF and contribute to the understanding of possible mechanisms in pathological lung fibrosis.

Usefulness of Transbronchial Lung Cryobiopsy When Starting Antifibrotic Treatment and Predicting Progressive Fibrosing Interstitial Lung Disease: Descriptive Research.

BACKGROUND: Although transbronchial lung cryobiopsy (TBLC) is widely used in diagnostic algorithms for various interstitial lung diseases (ILDs), its real-world utility in the therapeutic decision-making strategy for ILD patients remains unclear, in particular, when judging the time to start antifibrotic agents. METHODS: We analyzed medical records of 40 consecutive patients with idiopathic or fibrotic hypersensitivity pneumonitis who underwent TBLC. A TBLC-based usual interstitial pneumonia (UIP) score was used to assess three morphologic descriptors: patchy fibrosis, fibroblastic foci, and honeycombing. RESULTS: In our 40 patients with ILD, the most frequent radiological feature was indeterminate for UIP (45.0%). Final diagnosis included idiopathic pulmonary fibrosis (22.5%), fibrotic nonspecific interstitial pneumonia (5.0%), fibrotic hypersensitivity pneumonitis (35.0%), and unclassifiable ILD (37.5%). Linear mixed-effects analysis showed that declines in the slopes of %FVC and %DLCO in patients with TBLC-based UIP "Score ≥ 2" were significantly steeper than those of patients with "Score ≤ 1." During follow-up of patients with Score ≥ 2 (n = 24), more than half of them (n = 17) received an antifibrotic agent, with most patients (n = 13) receiving early administration of the antifibrotic agent within 6 months after the TBLC procedure. CONCLUSIONS: TBLC-based UIP Score ≥ 2 indicated the increased possibility of a progressive fibrosis course that may prove helpful in predicting progressive pulmonary fibrosis/progressive fibrosing ILD even if disease is temporarily stabilized due to anti-inflammatory agents. Patients may benefit from early introduction of antifibrotic agents by treating clinicians.

Pulmonary matrix-derived hydrogels from patients with idiopathic pulmonary fibrosis induce a proinflammatory state in lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF), one of the most common forms of interstitial lung disease, is a poorly understood, chronic, and often fatal fibroproliferative condition with only two FDA-approved medications. Understanding the pathobiology of the fibroblast in IPF is critical to evaluating and discovering novel therapeutics. Using a decellularized lung matrix derived from patients with IPF, we generate three-dimensional hydrogels as in vitro models of lung physiology and characterize the phenotype of fibroblasts seeded into the hydrogels. When cultured in IPF extracellular matrix hydrogels, IPF fibroblasts display differential contractility compared with their normal counterparts, lose the classical myofibroblast marker α-smooth muscle actin, and increase expression of proinflammatory cytokines compared with fibroblasts seeded two-dimensionally on tissue culture dishes. We validate this proinflammatory state in fibroblast-conditioned media studies with monocytes and monocyte-derived macrophages. These findings add to a growing understanding of the lung microenvironment effect on fibroblast phenotypes, shed light on the potential role of fibroblasts as immune signaling hubs during lung fibrosis, and suggest intervention in fibroblast-immune cell cross-talk as a possible novel therapeutic avenue.

Stimuli-Specific Senescence of Primary Human Lung Fibroblasts Modulates Alveolar Stem Cell Function.

Aging is the main risk factor for chronic lung diseases (CLDs) including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Accordingly, hallmarks of aging like cellular senescence are increased in these patients in different lung cell types including fibroblasts. However, little is known about the different triggers that induce a senescence phenotype in different disease backgrounds and its role in CLD pathogenesis. Therefore, we characterized senescence in primary human lung fibroblasts (phLF) from control, IPF, or COPD patients at baseline and after exposure to disease-relevant insults (H2O2, bleomycin, TGF-ß1) and studied their capacity to support progenitor cell potential in a lung organoid model. Bulk-RNA sequencing revealed that phLF from IPF and COPD activate different transcriptional programs but share a similar senescence phenotype at baseline. Moreover, H2O2 and bleomycin but not TGF-ß1 induced senescence in phLF from different disease origins. Exposure to different triggers resulted in distinct senescence programs in phLF characterized by different SASP profiles. Finally, co-culture with bleomycin- and H2O2-treated phLF reduced the progenitor cell potential of alveolar epithelial progenitor cells. In conclusion, phLF from COPD and IPF share a conserved senescence response that varies depending on the insult and impairs alveolar epithelial progenitor capacity ex vivo.

Regulation of myofibroblast dedifferentiation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a lethal, progressive, and irreversible condition that has become a significant focus of medical research due to its increasing incidence. This rising trend presents substantial challenges for patients, healthcare providers, and researchers. Despite the escalating burden of pulmonary fibrosis, the available therapeutic options remain limited. Currently, the United States Food and Drug Administration has approved two drugs for the treatment of pulmonary fibrosis-nintedanib and pirfenidone. However, their therapeutic effectiveness is limited, and they cannot reverse the fibrosis process. Additionally, these drugs are associated with significant side effects. Myofibroblasts play a central role in the pathophysiology of pulmonary fibrosis, significantly contributing to its progression. Consequently, strategies aimed at inhibiting myofibroblast differentiation or promoting their dedifferentiation hold promise as effective treatments. This review examines the regulation of myofibroblast dedifferentiation, exploring various signaling pathways, regulatory targets, and potential pharmaceutical interventions that could provide new directions for therapeutic development.

Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

Clinical, radiological and histopathological features of patients with familial pulmonary fibrosis.

BACKGROUND: In familial pulmonary fibrosis (FPF) at least two biological relatives are affected. Patients with FPF have diverse clinical features. RESEARCH QUESTION: We aimed to characterize demographic and clinical features, re-evaluate high-resolution computed tomography (HRCT) scans and histopathology of surgical lung biopsies, assess survival and investigate the suitability of risk prediction models for FPF patients. STUDY DESIGN: A retrospective cohort study. METHODS: FPF data (n = 68) were collected from the medical records of Oulu University Hospital (OUH) and Oulaskangas District Hospital between 1 Jan 2000 and 11 Jan 2023. The inclusion criterion was pulmonary fibrosis (PF) (ICD 10-code J84.X) and at least one self-reported relative with PF. Clinical information was gathered from hospital medical records. HRCT scans and histology were re-evaluated. RESULTS: Thirty-seven (54.4%) of the patients were men, and 31 (45.6%) were women. The mean ages of the women and men were 68.6 and 61.7 years, respectively (p = 0.003). Thirty-seven (54.4%) patients were nonsmokers. The most common radiological patterns were usual interstitial pneumonia (UIP) (51/75.0%), unclassifiable (8/11.8%) and nonspecific interstitial pneumonia (NSIP) (3/4.4%). Pleuroparenchymal fibroelastosis (PPFE) was observed as a single or combined pattern in 13.2% of the patients. According to the 2022 guidelines for idiopathic pulmonary fibrosis (IPF), the patients were categorized as UIP (31/45.6%), probable UIP (20/29.4%), indeterminate for UIP (7/10.3%) or alternative diagnosis (10/14.7%). The histopathological patterns were UIP (7/41.2%), probable UIP (1/5.9%), indeterminate for UIP (8/47.2%) and alternative diagnosis (1/5.9%). Rare genetic variants were found in 9 patients; these included telomerase reverse transcriptase (TERT, n = 6), telomerase RNA component (TERC, n = 2) and regulator of telomere elongation helicase 1 (RTEL1, n = 1). Half of the patients died (n = 29) or underwent lung transplantation (n = 5), with a median survival of 39.9 months. The risk prediction models composite physiology index (CPI), hazard ratio (HR) 1.07 (95.0% CI 1.04-1.10), and gender-age-physiology index (GAP) stage I predicted survival statistically significantly (p<0.001) compared to combined stages II and III. CONCLUSIONS: This study confirmed the results of earlier studies showing that FPF patients' radiological and histopathological patterns are diverse. Moreover, radiological and histological features revealed unusual patterns and their combinations.

ATP-induced fibrogenic pathway in circulating cells obtained by idiopathic pulmonary fibrotic (IPF) patients is not blocked by nintedanib and pirfenidone.

Idiopathic pulmonary fibrosis (IPF) is a severe disability due to progressive lung dysfunction. IPF has long been viewed as a non-immune form of pulmonary fibrosis, but nowadays it is accepted that a chronic inflammatory response can exacerbate fibrotic patterns. IL-1-like cytokines and ATP are highly detected in the lung and broncho-alveolar lavage fluid of IPF patients. Because ATP binds the purinergic receptor P2RX7 involved in the release of IL-1-like cytokines, we aimed to understand the role of P2RX7 in IPF. PBMCs from IPF patients were treated with nintedanib or pirfenidone in the presence of ATP. Under these conditions, PBMCs still released IL-1-like cytokines and the pro-fibrotic TGFß. Bulk and scRNAseq demonstrated that lung tissues of IPF patients had higher levels of P2RX7, especially on macrophages, which were correlated to T cell activity and inflammatory response with a TGFBI and IL-10 signature. A subcluster of macrophages in IPF lung tissues had 2055 genes that were not in common with the other subclusters, and that were involved in metabolic and PDGF, FGF and VEGF associated pathways. These data confirmed what observed on circulating cells that, although treated with anti-fibrotic agents, nintedanib or pirfenidone, they were still able to release IL-1 cytokines and the fibrogenic TGFß. In conclusion, these data imply that because nintedanib and pirfenidone do not block ATP-induced IL-1-like cytokines and TGFß induced during P2RX7 activation, it is plausible to consider P2RX7 on circulating cells and/or tissue biopsies as potential pharmacological tool for IPF patients.

Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Discovery of novel pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione (PBD) derivatives as selective HDAC6 inhibitors for the efficient treatment of idiopathic pulmonary fibrosis (IPF) in vitro and in vivo.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive fibrotic phenotype. Immunohistochemical studies on HDAC6 overexpression in IPF lung tissues confirmed that IPF is associated with aberrant HDAC6 activity. We herein developed a series of novel HDAC6 inhibitors that can be used as potential pharmacological tools for IPF treatment. The best-performing derivative H10 showed good selectivity for multiple isoforms of the HDAC family. The structural analysis and structure-activity relationship studies of H10 will contribute to optimizing the binding mode of the new molecules. The pharmacological mechanism of H10 to inhibit pulmonary fibrosis was validated, and its ability to inhibit the IPF phenotype was also demonstrated. Moreover, H10 showed satisfactory metabolic stability. The efficacy of H10 was also determined in a mouse model of bleomycin-induced pulmonary fibrosis. The results highlighted in this paper may provide a reference for the identification of new drug molecules for the treatment of IPF.

Micro-CT-assisted identification of the optimal time-window for antifibrotic treatment in a bleomycin mouse model of long-lasting pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating and fatal lung disease characterized by the excessive formation of scar tissue and decline of lung function. Despite extensive research, only two FDA-approved drugs exist for IPF, with limited efficacy and relevant side effects. Thus, there is an urgent need for new effective therapies, whose discovery strongly relies on IPF animal models. Despite some limitations, the Bleomycin (BLM)-induced lung fibrosis mouse model is widely used for antifibrotic drug discovery and for investigating disease pathogenesis. The initial acute inflammation triggered by BLM instillation and the spontaneous fibrosis resolution that occurs after 3 weeks are the major drawbacks of this system. In the present study, we applied micro-CT technology to a longer-lasting, triple BLM administration fibrosis mouse model to define the best time-window for Nintedanib (NINT) treatment. Two different treatment regimens were examined, with a daily NINT administration from day 7 to 28 (NINT 7-28), and from day 14 to 28 (NINT 14-28). For the first time, we automatically derived both morphological and functional readouts from longitudinal micro-CT. NINT 14-28 showed significant effects on morphological parameters after just 1 week of treatment, while no modulations of these biomarkers were observed during the preceding 7-14-days period, likely due to persistent inflammation. Micro-CT morphological data evaluated on day 28 were confirmed by lung histology and bronchoalveolar lavage fluid (BALF) cells; Once again, the NINT 7-21 regimen did not provide substantial benefits over the NINT 14-28. Interestingly, both NINT treatments failed to improve micro-CT-derived functional parameters. Altogether, our findings support the need for optimized protocols in preclinical studies to expedite the drug discovery process for antifibrotic agents. This study represents a significant advancement in pulmonary fibrosis animal modeling and antifibrotic treatment understanding, with the potential for improved translatability through the concurrent structural-functional analysis offered by longitudinal micro-CT.

Bayesian-frequentist hybrid inference framework for single cell RNA-seq analyses.

BACKGROUND: Single cell RNA sequencing technology (scRNA-seq) has been proven useful in understanding cell-specific disease mechanisms. However, identifying genes of interest remains a key challenge. Pseudo-bulk methods that pool scRNA-seq counts in the same biological replicates have been commonly used to identify differentially expressed genes. However, such methods may lack power due to the limited sample size of scRNA-seq datasets, which can be prohibitively expensive. RESULTS: Motivated by this, we proposed to use the Bayesian-frequentist hybrid (BFH) framework to increase the power and we showed in simulated scenario, the proposed BFH would be an optimal method when compared with other popular single cell differential expression methods if both FDR and power were considered. As an example, the method was applied to an idiopathic pulmonary fibrosis (IPF) case study. CONCLUSION: In our IPF example, we demonstrated that with a proper informative prior, the BFH approach identified more genes of interest. Furthermore, these genes were reasonable based on the current knowledge of IPF. Thus, the BFH offers a unique and flexible framework for future scRNA-seq analyses.

Macrophages as determinants and regulators of systemic sclerosis-related interstitial lung disease.

BACKGROUND: Interstitial lung disease (ILD) is the primary cause of mortality in systemic sclerosis (SSc), an autoimmune disease characterized by tissue fibrosis. SSc-related ILD (SSc-ILD) occurs more frequently in females aged 30-55 years, whereas idiopathic pulmonary fibrosis (IPF) is more prevalent in males aged 60-75 years. SSc-ILD occurs earlier than IPF and progresses rapidly. FCN1, FABP4, and SPP1 macrophages are involved in the pathogenesis of lung fibrosis; SPP1 macrophages demonstrate upregulated expression in both SSc-ILD and IPF. To identify the differences between SSc-ILD and IPF using single-cell analysis, clarify their distinct pathogeneses, and propose directions for prevention and treatment. METHODS: We performed single-cell RNA sequencing on NCBI Gene Expression Omnibus (GEO) databases GSE159354 and GSE212109, and analyzed lung tissue samples across healthy controls, IPF, and SSc-ILD. The primary measures were the filtered genes integrated with batch correction and annotated cell types for distinguishing patients with SSc-ILD from healthy controls. We proposed an SSc-ILD pathogenesis using cell-cell interaction inferences, and predicted transcription factors regulating target genes using SCENIC. Drug target prediction of the TF gene was performed using Drug Bank Online. RESULTS: A subset of macrophages activates the MAPK signaling pathway under oxidative stress. Owing to the lack of inhibitory feedback from ANNEXIN and the autoimmune characteristics, this leads to an earlier onset of lung fibrosis compared to IPF. During initial lung injury, fibroblasts begin to activate the IL6 pathway under the influence of SPP1 alveolar macrophages, but IL6 appears unrelated to other inflammatory and immune cells. This may explain why tocilizumab (an anti-IL6-receptor antibody) only preserves lung function in patients with early SSc-ILD. Finally, we identified BCLAF1 and NFE2L2 as influencers of MAPK activation in macrophages. Metformin downregulates NFE2L2 and could serve as a repurposed drug candidate. CONCLUSIONS: SPP1 alveolar macrophages play a role in the profibrotic activity of IPF and SSc-ILD. However, SSc-ILD is influenced by autoimmunity and oxidative stress, leading to the continuous activation of MAPK in macrophages. This may result in an earlier onset of lung fibrosis than in IPF. Such differences could serve as potential research directions for early prevention and treatment.

Multiomic analysis of monocyte-derived alveolar macrophages in idiopathic pulmonary fibrosis.

BACKGROUND: Monocyte-derived alveolar macrophages (Mo_AMs) are increasingly recognised as potential pathogenic factors for idiopathic pulmonary fibrosis (IPF). While scRNAseq analysis has proven valuable in the transcriptome profiling of Mo_AMs, the integration analysis of multi-omics may provide additional dimensions of understanding of these cellular populations. METHODS: We performed multi-omics analysis on 116 scRNAseq, 119 bulkseq and five scATACseq lung tissue samples from IPF. We built a large-scale IPF scRNAseq atlas and conducted the Monocle 2/3 as well as the Cellchat to explore the developmental path and intercellular communication on Mo_AMs. We also reported the difference in metabolisms, tissue repair and phagocytosis between Mo_AMs and tissue-resident alveolar macrophages (TRMs). To determine whether Mo_AMs affected pulmonary function, we projected clinical phenotypes (FVC%pred) from the bulkseq dataset onto the scRNAseq atlas. Finally, we used scATATCseq to uncover the upstream regulatory mechanisms and determine key drivers in Mo_AMs. RESULTS: We identified three Mo_AMs clusters and the trajectory analysis further validated the origin of these clusters. Moreover, via the Cellchat analysis, the CXCL12/CXCR4 axis was found to be involved in the molecular basis of reciprocal interactions between Mo_AMs and fibroblasts through the activation of the ERK pathway in Mo_AMs. SPP1_RecMacs (RecMacs, recruited macrophages) were higher in the low-FVC group than in the high-FVC group. Specifically, compared with TRMs, the functions of lipid and energetic metabolism as well as tissue repair were higher in Mo_AMs than TRMs. But, TRMs may have higher level of phagocytosis than TRMs. SPIB (PU.1), JUNB, JUND, BACH2, FOSL2, and SMARCC1 showed stronger association with open chromatin of Mo_AMs than TRMs. Significant upregulated expression and deep chromatin accessibility of APOE were observed in both SPP1_RecMacs and TRMs. CONCLUSION: Through trajectory analysis, it was confirmed that SPP1_RecMacs derived from Monocytes. Besides, Mo_AMs may influence FVC% pred and aggravate pulmonary fibrosis through the communication with fibroblasts. Furthermore, distinctive transcriptional regulators between Mo_AMs and TRMs implied that they may depend on different upstream regulatory mechanisms. Overall, this work provides a global overview of how Mo_AMs govern IPF and also helps determine better approaches and intervention therapies.

Succinate promotes pulmonary fibrosis through GPR91 and predicts death in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is believed to be associated with a notable disruption of cellular energy metabolism. By detecting the changes of energy metabolites in the serum of patients with pulmonary fibrosis, we aimed to investigate the diagnostic and prognostic value of energy metabolites in IPF, and further elucidated the mechanism of their involvement in pulmonary fibrosis. Through metabolomics research, it was discovered that the TCA cycle intermediates changed dramatically in IPF patients. In another validation cohort of 55 patients with IPF compared to 19 healthy controls, it was found that succinate, an intermediate product of TCA cycle, has diagnostic and prognostic value in IPF. The cut-off levels of serum succinate were 98.36 µM for distinguishing IPF from healthy controls (sensitivity, 83.64%; specificity, 63.16%; likelihood ratio, 2.27, respectively). Moreover, a high serum succinate level was independently associated with higher rates of disease progression (OR 13.087, 95%CI (2.819-60.761)) and mortality (HR 3.418, 95% CI (1.308-8.927)). In addition, accumulation of succinate and increased expression of the succinate receptor GPR91 were found in both IPF patients and BLM mouse models of pulmonary fibrosis. Reducing succinate accumulation in BLM mice alleviated pulmonary fibrosis and 21d mortality, while exogenous administration of succinate can aggravate pulmonary fibrosis in BLM mice. Furthermore, GPR91 deficiency protected against lung fibrosis caused by BLM. In vitro, succinate promoted the activation of lung fibroblasts by activating ERK pathway through GPR91. In summary, succinate is a promising biomarker for diagnosis and prognosis of IPF. The accumulation of succinate may promote fibroblast activation through GPR91 and pulmonary fibrosis.

Fibroblast growth factor 21 alleviates idiopathic pulmonary fibrosis by inhibiting PI3K-AKT-mTOR signaling and stimulating autophagy.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease with an unclear pathogenesis and no available specific drug treatment. The principal etiological factors are lung inflammation caused by environmental factors, damage to alveolar epithelial cells, leading to epithelial-mesenchymal transition (EMT), and the abnormal proliferation of fibroblasts. Here, we have demonstrated that fibroblast growth factor 21 (FGF21) ameliorates IPF via the autophagy pathway. We administered FGF21 to bleomycin (BLM)-treated mice, which ameliorated their defects in lung function, reduced the accumulation of collagen, restored tissue structure, reduced the deposition of hydroxyproline, reduced the expression of collagen I and α-SMA and increased the expression of E-cadherin. The expression of LC3BII and the number of autophagosomes were significantly higher in the lungs. The expression of AKT and mTOR was significantly reduced by FGF21 treatment. We also determined the effects of FGF21 in A549 cells treated with TGF-ß, and found that FGF21 significantly inhibits activation of the AKT signaling pathway, thereby reducing TGF-ß-induced EMT and preventing the uncontrolled proliferation of fibroblasts. We conclude that FGF21 ameliorates IPF by inhibiting the PI3K-AKT-mTOR signaling pathway and activating autophagy, which provides a theoretical basis for FGF21 to be used for the treatment of IPF.

Realveolarization with inhalable mucus-penetrating lipid nanoparticles for the treatment of pulmonary fibrosis in mice.

The stemness loss-associated dysregeneration of impaired alveolar type 2 epithelial (AT2) cells abolishes the reversible therapy of idiopathic pulmonary fibrosis (IPF). We here report an inhalable mucus-penetrating lipid nanoparticle (LNP) for codelivering dual mRNAs, promoting realveolarization via restoring AT2 stemness for IPF treatment. Inhalable LNPs were first formulated with dipalmitoylphosphatidylcholine and our in-house-made ionizable lipids for high-efficiency pulmonary mucus penetration and codelivery of dual messenger RNAs (mRNAs), encoding cytochrome b5 reductase 3 and bone morphogenetic protein 4, respectively. After being inhaled in a bleomycin model, LNPs reverses the mitochondrial dysfunction through ameliorating nicotinamide adenine dinucleotide biosynthesis, which inhibits the accelerated senescence of AT2 cells. Concurrently, pathological epithelial remodeling and fibroblast activation induced by impaired AT2 cells are terminated, ultimately prompting alveolar regeneration. Our data demonstrated that the mRNA-LNP system exhibited high protein expression in lung epithelial cells, which markedly extricated the alveolar collapse and prolonged the survival of fibrosis mice, providing a clinically viable strategy against IPF.