Chronic obstructive pulmonary disease (COPD) is a highly heterogeneous disease with complex pathogenesis. Identifying high-risk populations and implementing timely prevention strategies are critical to reducing the disease burden. Single-cell RNA sequencing of lung tissue from control never-smokers, patients with pre-COPD, and COPD patients revealed a novel T cell subset characterized by high expression of metallothionein (MT) genes, designated MT-high T cells. These cells were progressively depleted in the lungs with disease progression. A similar decline was observed in the peripheral blood using flow cytometry, highlighting the potential of these cells to serve as an accessible biomarker of disease progression. Functional analysis indicated that MT-high T cells suppress CD8+ T cell cytotoxic activity, suggesting a key immunoregulatory role in disease pathogenesis. Receiver operating characteristic curve analysis demonstrated the excellent potential of MT-high T cell frequency to predict susceptibility to COPD. These findings establish MT-high T cells as promising biomarkers for identifying individuals at risk for COPD and as novel targets for future therapeutic and prophylactic strategies.

Pulmonary fibrosis (PF) is a group of debilitating chronic lung diseases characterized by progressive scarring of the lung parenchyma. Despite advances in understanding its multifactorial pathogenesis, these insights have yet to translate into broadly effective treatment options. Emerging evidence underscores the critical roles of essential trace metals in driving fibrotic progression. In this review, we introduce the Metallic Trinity framework, which conceptualizes copper, zinc, and iron as an interdependent network within the pulmonary metallome. Notably, perturbations in any single metallic component disrupt the homeostasis of the others, initiating a cascade of molecular and cellular events that promote fibrogenesis. Drawing on data from both preclinical models and clinical studies, we delineate the individual roles and dynamic crosstalk among these metals in shaping the fibrotic lung microenvironment. This integrated perspective provides novel insights into the mechanisms by which trace metal imbalances contribute to aberrant tissue remodeling, inflammation, and cellular dysfunction. Furthermore, we explore therapeutic strategies aimed at restoring metal equilibrium and highlight the potential of these elements as biomarkers. By reframing PF through the lens of trace metal interdependence, the Metallic Trinity paradigm provides a rationale for exploring new strategies for precision diagnostics and targeted interventions, with the potential to improve therapeutic approaches for this devastating condition.

Pulmonary fibrosis represents one of the most challenging frontiers in respiratory medicine, characterized by progressive scarring that ultimately leads to respiratory failure and death. Despite decades of research, therapeutic options remain frustratingly limited, with only three Food and Drug Administration-approved agents that merely slow disease progression without reversing established fibrosis. This therapeutic impasse stems largely from the translational disconnect between traditional preclinical models and human disease complexity. Animal models fail to recapitulate the chronic, progressive nature of human fibrosis, while conventional cell culture systems cannot capture the intricate three-dimensional architecture and multicellular interactions that drive fibrotic remodeling. The emergence of new approach methodologies has catalyzed a paradigm shift in pulmonary fibrosis research, offering unprecedented opportunities to model human disease with greater fidelity. This review examines three revolutionary platforms that are reshaping our understanding of fibrotic mechanisms: precision-cut lung slices that preserve native tissue architecture, self-organizing lung organoids that enable patient-specific disease modeling, and microfluidic lung-on-chip systems that recreate physiological breathing mechanics. We critically evaluate the unique capabilities and inherent limitations of each technology, explore their applications in mechanistic studies and drug discovery, and discuss emerging hybrid approaches that promise to accelerate therapeutic development. By synthesizing current evidence and identifying future directions, this review provides a roadmap for leveraging these innovative technologies to finally break through the therapeutic ceiling in pulmonary fibrosis.

Abstract Background This study aimed to elucidate B cell subset pathology in COPD, a poorly characterized area, with a focus on its similarities to and differences from classical autoimmune disorders. Methods The single-cell RNA-sequencing (scRNA-seq) data from COPD and autoimmune diseases were obtained from Gene Expression Omnibus (GEO) for comparative analyses of B cell subsets and functions via differentially expressed genes (DEGs), KEGG, protein-protein interaction (PPI), and cell–cell communication analyses. Serum IgG4 was measured by ELISA and correlated with clinical parameters. The peripheral blood B cells were sorted by flow cytometry for single-cell B cell receptor (BCR) sequencing. A v-Abl/Bcl2 pro-B cell line was stimulated with cigarette smoke extract (CSE) to assess abnormal development in vitro . Results In lung tissue, IgG4 + plasma cells were enriched and expressed BCR activation/inflammatory genes and TNF/NF-κB/MAPK pathways. Serum IgG4 concentrations correlated negatively with pre-and post-bronchodilator FEV 1 /FVC. B cell interacted with monocytes, macrophages, fibroblasts and endothelial cells via IL-1β/IL-6, integrin and chemokine signalling, contributing to chronic inflammation and remodelling. In peripheral blood, transitional T1 B cells were increased, accompanied by λ-chain enrichment and increased IGLV1-47 usage, as well as enrichment of autoimmune pathways. In the bone marrow, the numbers of pre-B I cells were increased while those of small pre-B III cells were reduced, with altered expression of BCR development genes. CSE stimulation of the pro-B cell line reduced λ5 expression in a concentration-dependent manner. Conclusions The autoimmune abnormalities in COPD appear more restricted, although IgG4 antibody generation may contribute to immune-mediated lung damage.

BackgroundAnti-synthetase syndrome (ASS) associated interstitial lung disease (ILD) usually responds to immunosuppressive therapy, but recurrence is common. We report a 58-year-old man with ASS-ILD who developed recurrent ILD within one year after bilateral lung transplantation (LTx). Despite triple immunosuppression (glucocorticoids, tacrolimus, mycophenolate mofetil), systemic inflammation persisted. This case represents an in vivo model of ASS-ILD recurrence, warranting further investigation into underlying mechanisms and novel therapeutic strategies.MethodsPeripheral blood mononuclear cells (PBMCs) were collected at 56 and 84 weeks post-transplant for single-cell RNA sequencing (scRNA-seq), while lung tissue was analyzed via spatial transcriptomics. Control data came from five naïve ASS-ILD patients and two clinically stable connective tissue disease associated ILD (CTD-ILD) patients post-LTx. Differential gene expression and pathway enrichment analyses were performed to identify therapeutic targets.ResultsExploratory PBMC scRNA-seq analysis suggested enrichment of interferon-, interleukin- and JAK-STAT-related signaling programs in circulating monocytes and neutrophils. Based on this immune activation profile, a multidrug treatment adjustment was implemented, including short-term glucocorticoid augmentation, replacement of mycophenolate mofetil with Janus kinase inhibitor tofacitinib, and replacement of tacrolimus with cyclosporine A. Following treatment adjustment, systemic inflammatory markers declined and interstitial lesions in both lungs were markedly alleviated on imaging. Subsequent spatial transcriptomic analysis of lung tissue revealed persistent interferon-related signaling and identified pro-fibrotic transcriptional programs in alveolar macrophages and transitional type II alveolar cells. Functional enrichment suggested a potential association between systemic inflammatory activation and localized fibrotic remodeling within the lung microenvironment.ConclusionsThis case illustrates the potential utility of scRNA-seq and spatial transcriptomics for characterizing immune-related transcriptional programs in recurrent ASS-ILD after LTx. Transcriptomic profiling informed therapeutic decision-making in this complex clinical setting, and clinical improvement was observed following treatment adjustment. These findings generate exploratory insights into potential therapeutic targets in recurrent ASS-ILD.

Pulmonary hypertension (PH) is a severe disease characterized by pulmonary vascular remodeling in which various immune cells play a critical role in vascular remodeling, although the details are still vague. Furthermore, current clinical treatments primarily focus on pulmonary vasodilation, but do not fundamentally address vascular remodeling itself. Here, first significant changes in neutrophils during the development of PH are demonstrated and show that neutrophil depletion can effectively attenuate disease progression. Moreover, the data show that neutrophil-derived S100A9 is the key mediator to promote vascular remodeling, while both knockout and inhibition of S100A9 can prevent PH. In a co-culture system of neutrophils and endothelial cells (ECs), hypoxic stimulation leads to increased S100A9 secretion by neutrophils, which activates the RAGE/PI3K/AKT pathway and causes dysfunction of ECs. These findings suggest that neutrophil-derived S100A9 mediated neutrophil-EC crosstalk plays an important role in pulmonary vascular remodeling, providing a promising strategy for treatment of PH.

Tolerance to dietary antigens is critical for avoiding deleterious type 2 immune responses resulting in food allergy (FA) and anaphylaxis1,2. However, the mechanisms resulting in both the maintenance and failure of tolerance to food antigens are poorly understood. Here we demonstrate that the goblet-cell-derived resistin-like molecule β (RELMβ)3,4 is a critical regulator of oral tolerance. RELMβ is abundant in the sera of both patients with FA and mouse models of FA. Deletion of RELMβ protects mice from FA and the development of food-antigen-specific IgE and anaphylaxis. RELMβ disrupts food tolerance through the modulation of the gut microbiome and depletion of indole-metabolite-producing Lactobacilli and Alistipes. Tolerance is maintained by the local production of indole derivatives driving FA protective RORγt+ regulatory T (Treg) cells5 through activation of the aryl hydrocarbon receptor. RELMβ antagonism in the peri-weaning period restores oral tolerance and protects genetically prone offspring from developing FA later in life. Together, we show that RELMβ mediates a gut immune-epithelial circuit regulating tolerance to food antigens-a novel mode of innate control of adaptive immunity through microbiome editing-and identify targetable candidates in this circuit for prevention and treatment of FA.

To the Editor: Common respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma induce significant health burdens worldwide.[1,2] Airway/lung microbiome and microbial metabolites have been proven to be closely related to multiple respiratory diseases.[3] However, few relevant studies show characteristics and horizontal comparisons of metabolites of dominant bacteria in respiratory diseases. Additionally, most results are based on association analyses of microbiome sequences, especially those of the gut microbiome, and untargeted metabolomics, without evidence that the metabolites are directly produced and secreted by certain bacteria.[4] In this context, our study aimed to analyze and compare the key metabolites and metabolic pathways of common bacteria involved in respiratory diseases. Based on the culture results of bacteria in clinical samples of patients with respiratory diseases and the reports of cohort studies,[5] we analyzed metabolic changes of five common bacteria, namely, Streptococcus pneumoniae (Sp), Staphylococcus aureus (Sa), Moraxella catarrhal (Mc), Pseudomonas aeruginosa (Pa), and Haemophilus influenzae (Hin). Firstly, we cultivated type strains of the five bacteria until the optical density at 600 nm (OD600) reached 1.0, collected their supernatants [Supplementary Materials, https://links.lww.com/CM9/C341], and then conducted untargeted metabolomics analysis of supernatants to investigate the metabolic profile under the same conditions, attempting to minimize the influence of variabilities and unknown factors. The metabolites with variable influence in projection (VIP) >1 and P <0.05 and fold change (FC) ≥2 or FC ≤0.5 were considered differentially abundant metabolites. Then, we compared the similarity and specificity of differentially abundant metabolites among these bacteria to identify possible key metabolites and related pathways, which might provide a shortcut for further exploration of the role of bacterial metabolites in respiratory diseases. Metabolites annotated by each secondary classification under the primary classification of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were shown in the positive and negative ion modes. In the positive ion mode, most metabolites were annotated to biological pathways such as metabolism. The top 5 secondary classifications included global and overview maps (100 metabolites), amino acid metabolism (47 metabolites), metabolism of cofactors and vitamins (20 metabolites), nucleotide metabolism (17 metabolites), and metabolism of other amino acids (16 metabolites), accounting for 81.97% (200/244) of the metabolites annotated to the metabolism pathway. In the negative ion mode, most metabolites were annotated to biological pathways such as metabolism again. The top 5 secondary classifications included global and overview maps (107 metabolites), amino acid metabolism (38 metabolites), carbohydrate metabolism (29 metabolites), nucleotide metabolism (27 metabolites), and lipid metabolism (22 metabolites), accounting for 76.90% (223/290) of the metabolites annotated to the metabolism pathway. The metabolites annotated by superclass in the human metabolome database (HMDB) in the positive ion mode were as follows: hydrocarbon derivatives (1 metabolite), alkaloids and derivatives (6 metabolites), organic nitrogen compounds (11 metabolites), phenylpropanoids and polyketides (14 metabolites), nucleosides, nucleotides, and analogues (20 metabolites), organic oxygen compounds (20 metabolites), benzenoids (34 metabolites), lipids and lipid-like molecules (58 metabolites), organoheterocyclic compounds (65 metabolites), and organic acids and derivatives (98 metabolites). In the negative ion mode, the metabolites annotated by the superclass in the HMDB were as follows: alkaloids and derivatives (3 metabolites), organic nitrogen compounds (5 metabolites), phenylpropanoids and polyketides (10 metabolites), benzenoids (19 metabolites), organic oxygen compounds (26 metabolites), nucleosides, nucleotides, and analogues (29 metabolites), organoheterocyclic compounds (32 metabolites), organic acids and derivatives (64 metabolites), lipids and lipid-like molecules (68 metabolites). The results of screening of differentially abundant metabolites revealed that between the supernatants of the Sp group and the control medium group, 432 differentially abundant metabolites were identified in the positive ion mode, including 176 upregulated and 256 downregulated metabolites. In the negative ion mode, 249 differentially abundant metabolites were identified, including 137 upregulated and 112 downregulated metabolites. Among the supernatants of the Sa group and the control medium group, 103 differentially abundant metabolites were identified in the positive ion mode, including 38 upregulated and 65 downregulated metabolites. In the negative ion mode, 56 differentially abundant metabolites were identified, including 16 upregulated and 40 downregulated metabolites. Between the supernatants of Mc group and the control medium group, 137 differentially abundant metabolites were identified in the positive ion mode, including 71 upregulated and 66 downregulated metabolites. In the negative ion mode, 76 differentially abundant metabolites were identified, including 33 upregulated and 43 downregulated metabolites. Among the supernatants of the Pa group and the control medium group, 407 differentially abundant metabolites were identified in the positive ion mode, including 178 upregulated and 229 downregulated metabolites. In the negative ion mode, 253 differentially abundant metabolites were identified, including 123 upregulated and 130 downregulated metabolites. Among the supernatants of the Hin group and the control medium group, 133 differentially abundant metabolites were identified in the positive ion mode, including 73 upregulated and 60 downregulated metabolites. In the negative ion mode, 81 differentially abundant metabolites were identified, including 31 upregulated and 50 downregulated metabolites. We then compared overlapping and unique differentially abundant metabolites among different bacteria [Figure 1]. In the positive ion mode, there were no common metabolites among the five kinds of bacteria. However, 114, 18, 33, 76, and 46 metabolites were found to be specific for Sp, Sa, Mc, Pa, and Hin, respectively. The top 5 upregulated metabolites in the supernatant of Sp were thioguanine, bicyclo prostaglandin E2, triacanthine, 4-hydroxyretinoic acid, and xanthurenic acid, whereas the top 5 downregulated metabolites were alanine-leucine (ala-leu), 3′-hydroxy-stanozolol, maltotetrose, 6-hydroxymelatonin, and S-adenosylhomocysteine. The top 5 upregulated metabolites in the supernatant of Sa were dihydroceramide (d16:0/16:0), 8-hydroxy-eicosapentaenoic acid, digalactosyl monoacylglycerol (18:2), tetramethylpyrazine, and norfentanyl, whereas the top 5 downregulated metabolites were ergothioneine, cafestol, trans-2-butene-1,4-dicarboxylic acid, 4-hydroxy estrone 1-N3-adenine, and prostaglandin A3. The top 5 upregulated metabolites in the supernatant of Mc were L-tyrosine methyl ester, urocanic acid, 6-aminonicotinamide, T-2 triol, and haloperidol-d4, whereas the top 5 downregulated metabolites were pantethine, ingenol-3-angelate, UR-144 N-(2-hydroxypentyl) metabolite, all-trans-retinal and gatifloxacin. The top 5 upregulated metabolites in the supernatant of the Pa were sterigmatocystin, astragaloside IV, 6β-naltrexol-d3, norbutorphanol, and milbemycin A4 oxime, whereas the top 5 downregulated metabolites were stearamide, N-acetyl-L-tyrosine, L-asparagine, L-adrenaline, and S-adenosyl-L-methionine. The top 5 upregulated metabolites in the supernatant of Hin belonged to phosphatidyl ethanolamine, including PE (12:0/12:0), PE (8:0/8:0), PE (9:0/9:0), PE (11:0/11:0) and PE (11:0/13:1), whereas the top 5 downregulated metabolites were spermidine, cytosine, cytidine 5′-monophosphate (hydrate), N4-acetylcytidine, and gedunin.Figure 1: Common and specific differentially abundant metabolites among supernatants of different bacteria in the positive (A) and negative (B) ion modes. BHI: Brain-heart infusion medium; Hin: Haemophilus influenzae; Mc: Moraxella catarrhal; Pa: Pseudomonas aeruginosa; Sa: Staphylococcus aureus; Sp: Streptococcus pneumoniae.In the negative ion mode, docosapentaenoic acid and 8,11,14-eicosatrienoic acid were two common metabolites among the five kinds of bacteria. In addition, there were 75, 5, 14, 53, and 37 metabolites specific for Sp, Sa, Mc, Pa, and Hin, respectively. The top 5 upregulated metabolites in the supernatant of Sp were deoxyribose 5-phosphate, d-mannose 6-phosphate, d-glucose 6-phosphate, chaetocin, and piceatannol, whereas the top 5 downregulated metabolites were stachyose, methylmalonate, pantothenic acid, serotonin, and xanthosine. The upregulated metabolites in the supernatant of Sa were N-lauroylsarcosine, thromboxane B1, inosine-5′-monophosphate and N-acetyl-L-histidine, whereas the only one downregulated metabolite was 5′-adenylic acid. The top 5 upregulated metabolites in the supernatant of Mc were guanosine 5′-diphosphate (GDP), 18-hydroxy-eicosapentaenoic acid, nicotinamide N-oxide, 19-nortestosterone and geranylgeranyl pyrophosphate, whereas the top downregulated metabolites were oleoyl ethanolamide, bilirubin, stearic acid and cymarin. The top 5 upregulated metabolites in the supernatant of Pa were 3-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 3-hydroxypicolinic acid, cholic acid, and protoporphyrin IX, whereas the top 5 downregulated metabolites were adenylosuccinic acid, adenine, quinic acid, 3-(3-methoxyphenyl) propionic acid and 7-hydroxy-3,4-dihydrocarbostyril. The top 5 upregulated metabolites in the supernatant of Hin belonged to phosphatidyl ethanolamine class, including PE (12:0/12:0), PE (8:0/8:0), PE (9:0/9:0), PE (11:0/11:0), and PE (11:0/13:1), and top 5 downregulated metabolites were uridine monophosphate (UMP), guanosine-3′,5′-cyclic monophosphate, adenosine-3′,5′-cyclic monophosphate, biopterin and ribulose-5-phosphate. Metabolic pathway enrichment analysis revealed that in the positive ion mode, there were no significantly enriched metabolic pathways of differentially abundant metabolites in supernatants of Sp and Pa. The most significantly enriched pathways were purine metabolism in the supernatant of Sa and arachidonic acid metabolism in the supernatant of Mc. The significantly enriched pathways of the differentially abundant metabolites in the supernatant of Hin were purine metabolism and biosynthesis of unsaturated fatty acids, respectively. In the negative ion mode, there were no significantly enriched metabolic pathways of differentially abundant metabolites in the supernatants of Sp and Pa. The most enriched significant pathway was purine metabolism in the supernatant of Sa. The most enriched pathways in the supernatant of Mc were biosynthesis of unsaturated fatty acids, nicotinate and nicotinamide metabolism, fatty acid biosynthesis, and carbon fixation in photosynthetic organisms, respectively. The significantly enriched pathways of differentially abundant metabolites in the supernatant of Hin were also identified, including pathways such as alanine, aspartate and glutamate metabolism, the citrate cycle, carbon fixation pathways in prokaryotes, β-alanine metabolism, furfural degradation, nicotinate and nicotinamide metabolism, histidine metabolism, amino sugar and nucleotide sugar metabolism, glyoxylate and dicarboxylate metabolism, and the biosynthesis of unsaturated fatty acids. In summary, we conducted untargeted metabolomics on common bacteria detected clinically in respiratory diseases and described the features of their metabolic profiles. We also screened a series of common and specific differentially abundant metabolites of each bacterium. We hope these data will provide the basis for further exploring the role of microbiome metabolites in the pathogenesis, diagnosis, and prevention of respiratory diseases. Acknowledgments We thank Robin James Storer, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript. Funding This study was supported by grants from the National Natural Science Foundation of China (Nos. 82090013, 82071805, and 82241011) and Beijing Natural Science Foundation (Nos. 7212001, 7242010, and 7242002). Conflicts of interest None.

B-1 cells are derived from a subpopulation of B lymphocytes which have a specific developmental process, unique phenotype and location, and distinct functions in comparison with conventional B-2 cells. The origin of B-1 cells is not completely clear, with two existing hypotheses concerning their lineage and differentiation pathways. B-1 cells are located principally in the peritoneal and pleural cavities, but are also distributed in secondary lymphoid tissues, at mucosal sites and in the blood and bone marrow. B-1 cells regulate immune responses and maintain homeostasis by secretion of natural antibodies (nAbs), and participate in the adaptive immune response through phagocytosis and presentation of antigens to T cells. B-1 cells are associated with many diseases including autoimmune, infectious and inflammatory diseases. This review focuses on the origin and biological functions of B-1 cells as well as their involvement in human disease, and discusses advances in the understanding of the heterogeneity of B-1 cells under specific pathophysiological features, as partly clarified by single-cell sequencing analysis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and degenerative interstitial lung disease characterized by complex etiology, unclear pathogenesis, and high mortality. Long noncoding RNAs (lncRNAs) have been identified as key regulators in modulating the initiation, maintenance, and progression of pulmonary fibrosis. However, the precise pathological mechanisms through which lncRNAs are involved in IPF remain limited and require further elucidation. A novel lncABCE1-5 was identified as significantly decreased by an ncRNA microarray analysis in our eight IPF lung samples compared with three donor tissues and validated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in clinical lung samples. To investigate the biological function of ABCE1-5, we performed loss- and gain-of-function experiments in vitro and in vivo. LncABCE1-5 silencing promoted A549 cell migration and A549 and bronchial epithelial cell line (BEAS-2B) cell apoptosis while enhancing the expression of proteins associated with extracellular matrix deposition, whereas overexpression of ABCE1-5 partially attenuated transforming growth factor-beta (TGF-β)-induced fibrogenesis. Forced ABCE1-5 expression by intratracheal injection of adeno-associated virus 6 revealing the antifibrotic effect of ABCE1-5 in bleomycin (BLM)-treated mice. Mechanistically, RNA pull-down (RPD)-mass spectrometry and RNA immunoprecipitation assay demonstrated that ABCE1-5 directly binds to keratin14 (krt14) sequences, potentially impeding its expression by perturbing mRNA stability. Furthermore, decreased ABCE1-5 levels can promote krt14 expression and enhance the phosphorylation of both mTOR and Akt; overexpression of ABCE1-5 in BLM mouse lung tissue significantly attenuated the elevated levels of p-mTOR and p-AKT. Knockdown of krt14 reversed the activation of mTOR signaling mediated by ABCE1-5 silencing. Collectively, the downregulation of ABCE1-5 mediated krt14 activation, thereby activating mTOR/AKT signaling, to facilitate pulmonary fibrosis progression in IPF.NEW & NOTEWORTHY In the present study, our data first reveal that a novel lncRNA ABCE1-5 could inhibit pulmonary fibrosis through interacting with krt14 and negative regulation of its expression, and indicated ABCE1-5 also regulates the phosphorylation of mTOR and Akt, thus acting on extracellular matrix remodeling in lung fibrosis procession. These results suggest that novel molecules within the ABCE1-5-krt14-mTOR axis may serve as potential candidates for clinical application in IPF.

Respiratory syncytial virus (RSV) infection has been associated with disruption of the airway epithelial barrier, potentially increasing the risk of asthma development. However, whether and how RSV and RSV-induced IL-33 contribute to this process are still unclear. In vivo, 7-day-old C57BL/6 mice were infected perinasally with RSV, then viral replication, lung inflammation and barrier integrity were evaluated at various time points postinfection. In vitro, human epithelial cells were infected with RSV in the presence or absence of IL-33, and the expression and localization of apical junction complex proteins (AJC) were assessed by western blot analysis and immunofluorescence staining. The involvement of components of the IL-33/ST2/MyD88 axis was further verified through blockade of endogenous IL-33 signaling and pharmacological inhibition of MyD88. Exposure to RSV infection resulted in impairment of the airway epithelial barrier, as indicated by reduced expression of tight junction proteins (ZO-1, Occludin) and adherents junction protein (E-cadherin) in the lung tissues. These effects on epithelial barrier disruption were significantly attenuated in St2-/- mice compared with wild-type controls. In vitro, the RSV-induced epithelial barrier disruption was exacerbated by topical application of exogenous IL-33, partially through activation of MyD88-mediated NF-κB signaling. Notably, knockdown of St2 by siRNA transfection or pharmacological inhibition of MyD88 partially restored the expression of E-cadherin, ZO-1 and Occludin in RSV-infected epithelial cells. RSV infection triggers robust IL-33 release from airway epithelial cells, leading to disrupted expression of AJC protein via activation of the MyD88-dependent NF-κB signaling pathway. These findings highlight the IL-33/ST2/MyD88 axis as a critical mediator of epithelial barrier dysfunction, which may represent a potential target for therapeutic intervention in RSV-mediated lung diseases.

Pulmonary fibrosis, a life-threatening respiratory condition affecting millions globally, is characterized by progressive lung scarring that severely compromises respiratory function. With few effective treatment options available, it carries a poor prognosis for those affected. Disrupted iron homeostasis is increasingly implicated in its pathogenesis, yet the precise mechanisms linking iron overload to fibrotic progression remain elusive. This study unveils a novel pathway by which iron accumulation orchestrates fibrotic remodeling via secreted phosphoprotein 1 (SPP1)-mediated reprogramming of alveolar type 2 (AT2) cells. Using an integrated approach combining analysis of public single-cell and single-nucleus RNA sequencing datasets with functional validation across multiple murine models of pulmonary fibrosis (iron-induced, bleomycin-induced, and silica-induced), we demonstrate that iron overload within AT2 cells triggers a coordinated transcriptional cascade affecting iron handling, immune cell recruitment, and cellular differentiation. Mechanistically, SPP1 emerges as a key mediator, functioning both externally as a paracrine signal for macrophage recruitment following iron-induced secretion from AT2 cells and internally as a driver of pathological epithelial transitions, specifically fostering the development of a Krt8+ alveolar intermediate phenotype. The clinical relevance of these findings is substantiated by analysis of human idiopathic pulmonary fibrosis specimens using publicly available single-cell and spatial transcriptomic datasets. These analyses reveal conserved pathway activation and a distinctive spatial organization of SPP1-expressing AT2 cells within remodeled tissue microenvironments, notably in close proximity to macrophages. By establishing SPP1 as a critical nexus between iron dysregulation and fibrotic progression, our work identifies the SPP1 signaling axis as a compelling therapeutic target for this devastating condition.NEW & NOTEWORTHY This study reveals a novel mechanism linking iron dysregulation to pulmonary fibrosis through SPP1-mediated reprogramming of alveolar type 2 cells. We demonstrate SPP1's dual role: externally coordinating macrophage recruitment and internally directing pathological epithelial transitions toward a Krt8+ intermediate state. These findings, validated across multiple mouse models and human specimens, identify the SPP1 signaling axis as a promising therapeutic target, offering new hope for treating this devastating condition where treatment options have historically been limited.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Figures S1–S2. Table S1. Video S1. Video S2. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Objective: In a previous study we have shown that, in the presence of interleukin (IL)-33, repeated, per-nasal challenge of murine airways with Streptococcus pneumoniae (S. pneumoniae) organisms induces human asthma-like airways inflammation. It is not clear, however, whether this effect is unique or manifest in response to other common respiratory pathogens.Methods: To explore this, airways of BALB/c mice were repeatedly challenged per-nasally with formaldehyde-inactivated bacterial bodies in the presence or absence of murine recombinant IL-33. Serum concentrations of S.pneumoniae, Moraxella catarrhalis (M.catarrhalis) and Haemophilus influenzae (H.influenzae) lysates-specific IgE were measured in patients with asthma and control subjects.Results: We showed that in the presence of IL-33, repeated, per-nasal airways exposure to the bodies of these bacteria induced airways hyperresponsiveness (AHR) in the experimental mice. This was accompanied by cellular infiltration into bronchoalveolar lavage fluid (BALF), eosinophilic infiltration and mucous hypertrophy of the lung tissue, with elevated local expression of some type 2 cytokines and elevated, specific IgG and IgE in the serum. The precise characteristics of the inflammation evoked by exposure to each bacterial species were distinguishable.Conclusions: These results suggest that in the certain circumstances, inhaled or commensal bacterial body antigens of both Gram-positive (S. pneumoniae) and Gram-negative (M. catarrhalis and H. influenzae) respiratory tract bacteria may initiate type 2 inflammation typical of asthma in the airways. In addition, we demonstrated that human asthmatic patients manifest elevated serum concentrations of M.catarrhalis- and H.influenzae-specific IgE.

The pulmonary lymphatic system has emerged as a critical regulator of lung homeostasis and a key contributor to the pathogenesis of respiratory diseases. As the primary conduit responsible for maintaining fluid balance and facilitating immune cell trafficking, the integrity of lymphatic vessels is essential for preserving normal pulmonary structure and function. Lymphatic abnormalities manifest across a broad spectrum of pulmonary disorders, underscoring their significance in respiratory health and disease. This review provides an overview of pulmonary lymphatic biology and delves into the involvement of lymphatics in four major lung diseases: chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, and lung transplant rejection. We examine how lymphatic abnormalities manifest in each of these conditions and investigate the mechanisms through which lymphatic remodeling and dysfunction contribute to disease progression. Furthermore, we explore the therapeutic potential of targeting the lymphatic system to ameliorate these debilitating respiratory conditions. Despite the current knowledge, several crucial questions remain unanswered, such as the spatial and temporal dynamics of lymphatic changes, the molecular crosstalk between lymphatics and the lung microenvironment, and the distinction between protective versus detrimental lymphatic phenotypes. Unraveling these mysteries holds the promise of identifying novel molecular regulators, characterizing lymphatic endothelial phenotypes, and uncovering bioactive mediators. By harnessing this knowledge, we can pave the way for the development of innovative disease-modifying therapies targeting the lymphatic highway in lung disorders.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent chronic respiratory disease characterised by irreversible airways obstruction associated with chronic airways inflammation and remodelling, while the pathogenesis and the mechanistic differences between patients remain to be fully elucidated. We previously reported that alarmin cytokine IL-33 may contribute to the production of autoantibodies against respiratory epithelial cells. Here we expand the hypothesis that pulmonary autoimmune responses induced by airway microbiota also contribute to the progression of COPD. We focused on Edwardsiella tarda which we detected uniquely in the induced sputum of patients with acute exacerbations of COPD. Pernasal challenge of the airways of WT mice with supernatants of cultured E. tarda induced marked, elevated expression of IL-33 in the lung tissues. Immunisation of animals with supernatants of cultured E. tarda resulted in significantly elevated airways inflammation, the formation of tertiary lymphatic structures and significantly elevated proportions of T follicular helper T cells in the lung tissue and mediastinal lymph nodes. Interestingly, such challenge also induced production of IgG autoantibodies directed against lung tissue lysate, alveolar epithelial cell proteins and elastin fragment, while putrescine, one of metabolites generated by the bacterium, might play an important role in the autoantibody production. Furthermore, all of these effects were partly but significantly abrogated in mice with deletion of the IL-33 receptor ST2. Collectively, these data support the hypothesis that COPD is progressed at least partly by airways microbiota such as E. tarda initiating autoimmune attack of the airways epithelium mediated at least partly through the IL-33-ST2 axis.

Idiopathic pulmonary fibrosis (IPF) is a devastating condition characterized by progressive lung scarring and uncontrolled fibroblast proliferation, inevitably leading to organ dysfunction and mortality. Although elevated iron levels have been observed in patients and animal models of lung fibrosis, the mechanisms linking iron dysregulation to lung fibrosis pathogenesis, particularly the role of macrophages in orchestrating this process, remain poorly elucidated. Here we evaluate iron metabolism in macrophages during pulmonary fibrosis using both in vivo and in vitro approaches. In murine bleomycin- and amiodarone-induced pulmonary fibrosis models, we observed significant iron deposition and lipid peroxidation in pulmonary macrophages. Intriguingly, the ferroptosis regulator glutathione peroxidase 4 (GPX4) was upregulated in pulmonary macrophages following bleomycin instillation, a finding corroborated by single-cell RNA sequencing analysis. Moreover, macrophages isolated from fibrotic mouse lungs exhibited increased transforming growth factor (TGF)-β1 expression that correlated with lipid peroxidation. In vitro, iron overload in bone marrow-derived macrophages triggered lipid peroxidation and TGF-β1 upregulation, which was effectively suppressed by ferroptosis inhibitors. When cocultured with iron-overloaded macrophages, lung fibroblasts exhibited heightened activation, evidenced by increased α-smooth muscle actin and fibronectin expression. Importantly, this profibrotic effect was attenuated by treating macrophages with a ferroptosis inhibitor or blocking TGF-β receptor signaling in fibroblasts. Collectively, our study elucidates a novel mechanistic paradigm in which the accumulation of iron within macrophages initiates lipid peroxidation, thereby amplifying TGF-β1 production, subsequently instigating fibroblast activation through paracrine signaling. Thus, inhibiting iron overload and lipid peroxidation warrants further exploration as a strategy to suppress fibrotic stimulation by disease-associated macrophages. NEW & NOTEWORTHY This study investigates the role of iron in pulmonary fibrosis, specifically focusing on macrophage-mediated mechanisms. Iron accumulation in fibrotic lung macrophages triggers lipid peroxidation and an upregulation of transforming growth factor (TGF)-β1 expression. Coculturing iron-laden macrophages activates lung fibroblasts in a TGF-β1-dependent manner, which can be mitigated by ferroptosis inhibitors. These findings underscore the potential of targeting iron overload and lipid peroxidation as a promising strategy to alleviate fibrotic stimulation provoked by disease-associated macrophages.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory condition characterized by persistent inflammation and oxidative stress, which ultimately leads to progressive restriction of airflow. Extensive research findings have cogently suggested that the dysregulation of essential transition metal ions, notably iron, copper, and zinc, stands as a critical nexus in the perpetuation of inflammatory processes and oxidative damage within the lungs of COPD patients. Unraveling the intricate interplay between metal homeostasis, oxidative stress, and inflammatory signaling is of paramount importance in unraveling the intricacies of COPD pathogenesis. This comprehensive review aims to examine the current literature on the sources, regulation, and mechanisms by which metal dyshomeostasis contributes to COPD progression. We specifically focus on iron, copper, and zinc, given their well-characterized roles in orchestrating cytokine production, immune cell function, antioxidant depletion, and matrix remodeling. Despite the limited number of clinical trials investigating metal modulation in COPD, the advent of emerging methodologies tailored to monitor metal fluxes and gauge responses to chelation and supplementation hold great promise in unlocking the potential of metal-based interventions. We conclude that targeted restoration of metal homeostasis represents a promising frontier for ameliorating pathological processes driving COPD progression.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.