SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

YTHDC1 delays cellular senescence and pulmonary fibrosis by activating ATR in an m6A-independent manner.

Accumulation of DNA damage in the lung induces cellular senescence and promotes age-related diseases such as idiopathic pulmonary fibrosis (IPF). Hence, understanding the mechanistic regulation of DNA damage repair is important for anti-aging therapies and disease control. Here, we identified an m6A-independent role of the RNA-binding protein YTHDC1 in counteracting stress-induced pulmonary senescence and fibrosis. YTHDC1 is primarily expressed in pulmonary alveolar epithelial type 2 (AECII) cells and its AECII expression is significantly decreased in AECIIs during fibrosis. Exogenous overexpression of YTHDC1 alleviates pulmonary senescence and fibrosis independent of its m6A-binding ability, while YTHDC1 deletion enhances disease progression in mice. Mechanistically, YTHDC1 promotes the interaction between TopBP1 and MRE11, thereby activating ATR and facilitating DNA damage repair. These findings reveal a noncanonical function of YTHDC1 in delaying cellular senescence, and suggest that enhancing YTHDC1 expression in the lung could be an effective treatment strategy for pulmonary fibrosis.

Pharmacological expansion of type 2 alveolar epithelial cells promotes regenerative lower airway repair.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

Deep Learning-based Segmentation of CT Scans Predicts Disease Progression and Mortality in IPF.

RATIONALE: Despite evidence demonstrating a prognostic role for CT scans in IPF, image-based biomarkers are not routinely used in clinical practice or trials. OBJECTIVES: Develop automated imaging biomarkers using deep learning based segmentation of CT scans. METHODS: We developed segmentation processes for four anatomical biomarkers which were applied to a unique cohort of treatment-naive IPF patients enrolled in the PROFILE study and tested against a further UK cohort. The relationship between CT biomarkers, lung function, disease progression and mortality were assessed. MEASUREMENTS AND MAIN RESULTS: Data was analysed from 446 PROFILE patients. Median follow-up was 39.1 months (IQR 18.1-66.4) with cumulative incidence of death of 277 over 5 years (62.1%). Segmentation was successful on 97.8% of all scans, across multiple imaging vendors at slice thicknesses 0.5-5mm. Of 4 segmentations, lung volume showed strongest correlation with FVC (r=0.82, p<0.001). Lung, vascular and fibrosis volumes were consistently associated across cohorts with differential five-year survival, which persisted after adjustment for baseline GAP score. Lower lung volume (HR 0.98, CI 0.96-0.99, p=0.001), increased vascular volume (HR 1.30, CI 1.12-1.51, p=0.001) and increased fibrosis volume (HR 1.17, CI 1.12-1.22, p=<0.001) were associated with reduced two-year progression-free survival in the pooled PROFILE cohort. Longitudinally, decreasing lung volume (HR 3.41; 95% CI 1.36-8.54; p=0.009) and increasing fibrosis volume (HR 2.23; 95% CI 1.22-4.08; p=0.009) were associated with differential survival. CONCLUSIONS: Automated models can rapidly segment IPF CT scans, providing prognostic near and long-term information, which could be used in routine clinical practice or as key trial endpoints. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Bexotegrast in Patients with Idiopathic Pulmonary Fibrosis: The INTEGRIS-IPF Study.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a rare and progressive disease, which causes progressive cough, exertional dyspnea, impaired quality of life and death. OBJECTIVES: Bexotegrast (PLN 74809) is an oral, once-daily, investigational drug in development for the treatment of IPF. METHODS: This Phase 2a, multicenter, clinical trial, randomized participants with IPF to receive oral, once daily bexotegrast 40 mg, 80 mg, 160 mg, 320 mg, or placebo, with or without background IPF therapy (pirfenidone or nintedanib), in an approximately 3:1 ratio in each bexotegrast dose cohort, for at least 12 weeks. The primary endpoint was incidence of treatment-emergent adverse events (TEAEs). Exploratory efficacy endpoints included change from baseline in forced vital capacity (FVC); quantitative lung fibrosis (QLF) extent (%) and changes from baseline in fibrosis-related biomarkers. MEASUREMENTS AND MAIN RESULTS: Bexotegrast was well tolerated with similar rates of TEAEs in the pooled bexotegrast and placebo groups (62/89 [69.7%] and 21/31 [67.7%], respectively). Diarrhea was the most common TEAE; most participants with diarrhea also received nintedanib. Bexotegrast treated participants experienced a reduction in FVC decline over 12 weeks vs. placebo, with or without background therapy. A dose-dependent antifibrotic effect of bexotegrast was observed with QLF imaging and a decrease in fibrosis-associated biomarkers was observed with bexotegrast vs. placebo. CONCLUSIONS: Bexotegrast demonstrated a favorable safety and tolerability profile, up to 12 weeks for the doses studied. Exploratory analyses suggest an antifibrotic effect according to FVC, QLF imaging, and circulating levels of fibrosis biomarkers. Clinical trial registration available at www. CLINICALTRIALS: gov, ID: NCT04396756. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Meaningful Endpoints for Idiopathic Pulmonary Fibrosis (IPF) Clinical Trials: Emphasis on 'Feels, Functions, Survives'. Report of a Collaborative Discussion in a Symposium with Direct Engagement from Representatives of Patients, Investigators, the National Institutes of Health, a Patient Advocacy Organization, and a Regulatory Agency.

Background: Idiopathic pulmonary fibrosis (IPF) carries significant mortality and unpredictable progression, with limited therapeutic options. Designing trials with patient-meaningful endpoints, enhancing the reliability and interpretability of results, and streamlining the regulatory approval process are of critical importance to advancing clinical care in IPF. Methods: A landmark in-person symposium in June 2023 assembled 43 participants from the US and internationally, including patients with IPF, investigators, and regulatory representatives, to discuss the immediate future of IPF clinical trial endpoints. Patient advocates were central to discussions, which evaluated endpoints according to regulatory standards and the FDA's 'feels, functions, survives' criteria. Results: Three themes emerged: 1) consensus on endpoints mirroring the lived experiences of patients with IPF; 2) consideration of replacing forced vital capacity (FVC) as the primary endpoint, potentially by composite endpoints that include 'feels, functions, survives' measures or FVC as components; 3) support for simplified, user-friendly patient-reported outcomes (PROs) as either components of primary composite endpoints or key secondary endpoints, supplemented by functional tests as secondary endpoints and novel biomarkers as supportive measures (FDA Guidance for Industry (Multiple Endpoints in Clinical Trials) available at: https://www.fda.gov/media/162416/download). Conclusions: This report, detailing the proceedings of this pivotal symposium, suggests a potential turning point in designing future IPF clinical trials more attuned to outcomes meaningful to patients, and documents the collective agreement across multidisciplinary stakeholders on the importance of anchoring IPF trial endpoints on real patient experiences-namely, how they feel, function, and survive. There is considerable optimism that clinical care in IPF will progress through trials focused on patient-centric insights, ultimately guiding transformative treatment strategies to enhance patients' quality of life and survival.

The Regulation of Fatty Acid Synthase by Exosomal miR-143-5p and miR-342-5p in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease caused by an aberrant repair of injured alveolar epithelial cells. The maintenance of the alveolar epithelium and its regeneration after the damage is fueled by alveolar type II (ATII) cells. Injured cells release exosomes containing microRNAs (miRNAs), which can alter the recipient cells' function. Lung tissue, ATII cells, fibroblasts, plasma, and exosomes were obtained from naive patients with IPF, patients with IPF taking pirfenidone or nintedanib, and control organ donors. miRNA expression was analyzed to study their impact on exosome-mediated effects in IPF. High miR-143-5p and miR-342-5p levels were detected in ATII cells, lung tissue, plasma, and exosomes in naive patients with IPF. Decreased FASN (fatty acid synthase) and ACSL-4 (acyl-CoA-synthetase long-chain family member 4) expression was found in ATII cells. miR-143-5p and miR-342-5p overexpression or ATII cell treatment with IPF-derived exosomes containing these miRNAs lowered FASN and ACSL-4 levels. Also, this contributed to ATII cell injury and senescence. However, exosomes isolated from patients with IPF taking nintedanib or pirfenidone increased FASN expression in ATII cells compared with naive patients with IPF. Furthermore, fibroblast treatment with exosomes obtained from naive patients with IPF increased SMAD3, CTGF, COL3A1, and TGFß1 expression. Our results suggest that IPF-derived exosomes containing miR-143-5p and miR-342-5p inhibited the de novo fatty acid synthesis pathway in ATII cells. They also induced the profibrotic response in fibroblasts. Pirfenidone and nintedanib improved ATII cell function and inhibited fibrogenesis. This study highlights the importance of exosomes in IPF pathophysiology.

Single-cell combined with transcriptome sequencing to explore the molecular mechanism of cell communication in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a common, chronic, and progressive lung disease that severely impacts human health and survival. However, the intricate molecular underpinnings of IPF remains elusive. This study aims to delve into the nuanced molecular interplay of cellular interactions in IPF, thereby laying the groundwork for innovative therapeutic approaches in the clinical field of IPF. Sophisticated bioinformatics methods were employed to identify crucial biomarkers essential for the progression of IPF. The GSE122960 single-cell dataset was obtained from the Gene Expression Omnibus (GEO) compendium, and intercellular communication potentialities were scrutinized via CellChat. The random survival forest paradigm was established using the GSE70866 dataset. Quintessential genes were selected through Kaplan-Meier (KM) curves, while immune infiltration examinations, functional enrichment critiques and nomogram paradigms were inaugurated. Analysis of intercellular communication revealed an intimate potential connections between macrophages and various cell types, pinpointing five cardinal genes influencing the trajectory and prognosis of IPF. The nomogram paradigm, sculpted from these seminal genes, exhibits superior predictive prowess. Our research meticulously identified five critical genes, confirming their intimate association with the prognosis, immune infiltration and transcriptional governance of IPF. Interestingly, we discerned these genes' engagement with the EPITHELIAL_MESENCHYMAL_TRANSITION signalling pathway, which may enhance our understanding of the molecular complexity of IPF.

Unveiling the role of copper metabolism and STEAP2 in idiopathic pulmonary fibrosis molecular landscape.

Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disease characterized by progressive fibrosis and poor prognosis. Despite advancements in treatment, the pathophysiological mechanisms of IPF remain elusive. Herein, we conducted an integrated bioinformatics analysis combining clinical data and carried out experimental validations to unveil the intricate molecular mechanism of IPF. Leveraging three IPF datasets, we identified 817 upregulated and 560 downregulated differentially expressed genes (DEGs). Of these, 14 DEGs associated with copper metabolism were identified, shedding light on the potential involvement of disrupted copper metabolism in IPF progression. Immune infiltration analysis revealed dysregulated immune cell infiltration in IPF, with a notable correlation between copper metabolism-related genes and immune cells. Weighted gene co-expression network analysis (WGCNA) identified a central module correlated with IPF-associated genes, among which STEAP2 emerged as a key hub gene. Subsequent in vivo and in vitro studies confirmed the upregulation of STEAP2 in IPF model. Knockdown of STEAP2 using siRNA alleviated fibrosis in vitro, suggesting potential pathway related to copper metabolism in the pathophysiological progression of IPF. Our study established a novel link between immune cell infiltration and dysregulated copper metabolism. The revelation of intracellular copper overload and upregulated STEAP2 unravelled a potential therapeutic option. These findings offer valuable insights for future research and therapeutic interventions targeting STEAP2 and associated pathways in IPF.

Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5-/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Presence of focal usual interstitial pneumonia is a key prognostic factor in progressive pulmonary fibrosis.

AIMS: Progressive pulmonary fibrosis (PPF) is a newly recognised clinical phenotype of interstitial lung diseases in the 2022 interstitial pulmonary fibrosis (IPF) guidelines. This category is based entirely on clinical and radiological factors, and the background histopathology is unknown. Our objective was to investigate the histopathological characteristics of PPF and to examine the correlation between usual interstitial pneumonia (UIP) and prognosis in this new disease type. We hypothesised that the presence of UIP-like fibrosis predicts patients' survival in PPF cases. METHODS AND RESULTS: We selected 201 cases fulfilling the clinical criteria of PPF from case archives. Cases diagnosed as IPF by a multidisciplinary team were excluded. Whole slide images were evaluated by three pathologists who were blinded to clinical and radiological data. We measured areas of UIP-like fibrosis and calculated what percentage of the total lesion area they occupied. The presence of focal UIP-like fibrosis amounting to 10% or more of the lesion area was seen in 148 (73.6%), 168 (83.6%) and 165 (82.1%) cases for each pathologist, respectively. Agreement of the recognition of UIP-like fibrosis in PPF cases was above κ = 0.6 between all pairs. Survival analysis showed that the presence of focal UIP-like fibrosis correlated with worsened survival under all parameters tested (P < 0.001). CONCLUSIONS: The presence of UIP-like fibrosis is a core pathological feature of clinical PPF, and its presence within diseased areas is associated with poorer prognosis. This study highlights the importance of considering the presence of focal UIP-like fibrosis in the evaluation and management of PPF.

Hospitalisation patterns in interstitial lung diseases: data from the EXCITING-ILD registry.

BACKGROUND: Interstitial lung diseases (ILD) comprise a heterogeneous group of mainly chronic lung diseases with more than 200 entities and relevant differences in disease course and prognosis. Little data is available on hospitalisation patterns in ILD. METHODS: The EXCITING-ILD (Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases) registry was analysed for hospitalisations. Reasons for hospitalisation were classified as all cause, ILD-related and respiratory hospitalisations, and patients were analysed for frequency of hospitalisations, time to first non-elective hospitalisation, mortality and progression-free survival. Additionally, the risk for hospitalisation according to GAP index and ILD subtype was calculated by Cox proportional-hazard models as well as influencing factors on prediction of hospitalisation by logistic regression with forward selection. RESULTS: In total, 601 patients were included. 1210 hospitalisations were recorded during the 6 months prior to registry inclusion until the last study visit. 800 (66.1%) were ILD-related, 59.3% of admissions were registered in the first year after inclusion. Mortality was associated with all cause, ILD-related and respiratory-related hospitalisation. Risk factors for hospitalisation were advanced disease (GAP Index stages II and III) and CTD (connective tissue disease)-ILDs. All cause hospitalisations were associated with pulmonary hypertension (OR 2.53, p = 0.005). ILD-related hospitalisations were associated with unclassifiable ILD and concomitant emphysema (OR = 2.133, p = 0.001) as well as with other granulomatous ILDs and a positive smoking status (OR = 3.082, p = 0.005). CONCLUSION: Our results represent a crucial contribution in understanding predisposing factors for hospitalisation in ILD and its major impact on mortality. Further studies to characterize the most vulnerable patient group as well as approaches to prevent hospitalisations are warranted.

Disease trajectories in interstitial lung diseases - data from the EXCITING-ILD registry.

BACKGROUND: Interstitial lung diseases (ILD) comprise a heterogeneous group of mainly chronic lung diseases with different disease trajectories. Progression (PF-ILD) occurs in up to 50% of patients and is associated with increased mortality. METHODS: The EXCITING-ILD (Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases) registry was analysed for disease trajectories in different ILD. The course of disease was classified as significant (absolute forced vital capacity FVC decline > 10%) or moderate progression (FVC decline 5-10%), stable disease (FVC decline or increase < 5%) or improvement (FVC increase ≥ 5%) during time in registry. A second definition for PF-ILD included absolute decline in FVC % predicted ≥ 10% within 24 months or ≥ 1 respiratory-related hospitalisation. Risk factors for progression were determined by Cox proportional-hazard models and by logistic regression with forward selection. Kaplan-Meier curves were utilised to estimate survival time and time to progression. RESULTS: Within the EXCITING-ILD registry 28.5% of the patients died (n = 171), mainly due to ILD (n = 71, 41.5%). Median survival time from date of diagnosis on was 15.5 years (range 0.1 to 34.4 years). From 601 included patients, progression was detected in 50.6% of the patients (n = 304) with shortest median time to progression in idiopathic NSIP (iNSIP; median 14.6 months) and idiopathic pulmonary fibrosis (IPF; median 18.9 months). Reasons for the determination as PF-ILD were mainly deterioration in lung function (PFT; 57.8%) and respiratory hospitalisations (40.6%). In multivariate analyses reduced baseline FVC together with age were significant predictors for progression (OR = 1.00, p < 0.001). Higher GAP indices were a significant risk factor for a shorter survival time (GAP stage III vs. I HR = 9.06, p < 0.001). A significant shorter survival time was found in IPF compared to sarcoidosis (HR = 0.04, p < 0.001), CTD-ILD (HR = 0.33, p < 0.001), and HP (HR = 0.30, p < 0.001). Patients with at least one reported ILD exacerbation as a reason for hospitalisation had a median survival time of 7.3 years (range 0.1 to 34.4 years) compared to 19.6 years (range 0.3 to 19.6 years) in patients without exacerbations (HR = 0.39, p < 0.001). CONCLUSION: Disease progression is common in all ILD and associated with increased mortality. Most important risk factors for progression are impaired baseline forced vital capacity and higher age, as well as acute exacerbations and respiratory hospitalisations for mortality. Early detection of progression remains challenging, further clinical criteria in addition to PFT might be helpful.

The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis.

BACKGROUND: Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice. METHODS: Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann-Whitney tests were performed using GraphPad Prism software. RESULTS: Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma. CONCLUSION: IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

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.

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-ß1/Smad signaling pathway.

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⁃ß1 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-ß1/Smad signaling pathway. CONCLUSION: Inhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃ß1 and bleomycin, and then inhibit the fibrosis.

Design, synthesis, and evaluation of pirfenidone-NSAIDs conjugates for the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal, chronic and progressive lung disease that threaten public health like many cancers. In this study, targeting the significant driving factor, inflammatory response, of the IPF, several conjugates of pirfenidone (PFD) with non-steroidal anti-inflammatory drugs (NSAIDs), along with their derivatives, were designed and synthesized to enhance the anti-IPF potency of PFD. Among these compounds, the (S)-ibuprofen-PFD conjugate 5b exhibited the most potent anti-proliferation activity against NIH3T3 cells, demonstrating up to a 343-fold improvement compared to PFD (IC50 = 0.04 mM vs IC50 = 13.72 mM). Notably, 5b exhibited superior activity in inhibiting the migration of macrophages induced by TGF-ß compared to PFD. Additionally, 5b demonstrated significant suppression of TGF-ß-induced migration of NIH3T3 cells and induction of apoptosis in NIH3T3 cells. Mechanistic studies revealed that 5b reduced the expression of collagen I and α-SMA by inhibiting the TGF-ß/SMAD3 pathway. In a bleomycin-induced pulmonary fibrosis model, treatment with 5b (40 mg/kg/day, orally) exhibited a more pronounced effect on reducing the degree of histopathological changes in lung tissue and alleviating collagen deposition compared to PFD (100 mg/kg/day, orally). Moreover, 5b could block the expression of collagen I, α-SMA, fibronectin, and pro-inflammatory factors (IL-6, IFN-γ, and TNF-α) compared to PFD, while demonstrating low toxicity in vivo. These preliminary results indicated that the hybridization of PFD with NSAIDs represented an effective modification approach to improve the anti-IPF potency of PFD. Consequently, 5b emerged as a promising candidate for the further development of new anti-IPF agents.

Inhibition of TGF-ß1/Smad3 signaling by compound 5aa: A potential treatment for idiopathic pulmonary fibrosis.

The incidence of idiopathic pulmonary fibrosis (IPF) has been steadily increasing each year, posing significant challenges in its treatment. In this study, we conducted the design and synthesis of 23 new inhibitors that specifically target the TGF-ß1/Smad3 pathway. Initially, we employed a cell model of TGF-ß-induced pulmonary fibrosis, using cell survival rate and HYP expression as indicators to identify the potent ingredient 5aa, which demonstrated significant anti-pulmonary fibrosis activity. Subsequently, we induced mice with bleomycin (BLM) to establish an experimental animal model of pulmonary fibrosis, and evaluated the pharmacodynamics of 5aa in vivo against pulmonary fibrosis. The alterations in HYP and collagen levels in BLM-induced pulmonary fibrosis mice were analyzed using ELISA and immunohistochemistry techniques. The results indicated that compound 5aa effectively suppressed the fibrotic response induced by TGF-ß1, inhibited the expression of the fibrotic marker α-SMA, and hindered the EMT process in NIH3T3 cells. Additionally, oral administration of 5aa demonstrated significant therapeutic effects in a mouse model of IPF, comparable to the established drug Nintedanib. Moreover, compound 5aa exhibited higher bioavailability in vivo compared to Nintedanib. These collective outcomes suggest that 5aa holds promise as a potential inhibitor of TGF-ß1/Smad3 signaling for the treatment of IPF.

Serum C-reactive protein is associated with earlier mortality across different interstitial lung diseases.

BACKGROUND AND OBJECTIVE: The acute-phase protein C-reactive protein (CRP) is known to be associated with poor outcomes in cancer and cardiovascular disease, but there is limited evidence of its prognostic implications in interstitial lung diseases (ILDs). We therefore set out to test whether baseline serum CRP levels are associated with mortality in four different ILDs. METHODS: In this retrospective study, clinically measured CRP levels, as well as baseline demographics and lung function measures, were collected for ILD patients first presenting to the Royal Brompton Hospital between January 2010 and December 2019. Cox regression analysis was used to determine the relationship with 5-year mortality. RESULTS: Patients included in the study were: idiopathic pulmonary fibrosis (IPF) n = 422, fibrotic hypersensitivity pneumonitis (fHP) n = 233, rheumatoid arthritis associated ILD (RA-ILD) n = 111 and Systemic Sclerosis associated ILD (SSc-ILD) n = 86. Patients with a recent history of infection were excluded. Higher CRP levels were associated with shorter 5-year survival in all four disease groups on both univariable analyses, and after adjusting for age, gender, smoking history, immunosuppressive therapy and baseline disease severity (IPF: HR (95% CI): 1.3 (1.1-1.5), p = 0.003, fHP: 1.5 (1.2-1.9), p = 0.001, RA-ILD: 1.4 (1.1-1.84), p = 0.01 and SSc-ILD: 2.7 (1.6-4.5), p < 0.001). CONCLUSION: Higher CRP levels are independently associated with reduced 5-year survival in IPF, fHP, RA-ILD and SSc-ILD.