Inhalable textile microplastic fibers impair lung repair

Microplastics are a pressing global concern. Inhalation of microplastic fibers has been associated with interstitial lung disease related to alveolar epithelial damage in nylon flock workers. However, the means by which fibers affect lung tissue and epithelial growth remains unknown. Our aim was to assess the effects of nylon and polyester textile microplastic fibers on epithelial growth and differentiation using airway and alveolar lung organoids cultured from epithelial cell progenitors, isolated both from murine lungs and human lung tissue obtained from COPD patients. Exposure to nylon (11x30 µm) or polyester (15x53 µm) microfibers resulted in significantly fewer and smaller human and murine airway organoids after 14 days of culture, the effect being most profound with nylon. Alveolar organoids were not affected by these fibers. Incubation with nylon- or polyester-conditioned medium also resulted in fewer airway organoids. Effects were mainly observed in developing airway organoids; exposure of developed organoids from day 14 to day 21 to fibers or fiber-conditioned medium had no significant effect on organoid number or size. In conclusion, airway organoid formation is negatively impacted by the presence of textile microplastic fibers and this effect appears to be mediated by leaching additives. Our results suggest that microplastic fibers may especially harm the developing airways or airways undergoing repair. Further studies will focus on identifying these additives and the mechanism behind their effect. Importantly, wider investigations into the presence of microplastic fibers in human lung tissue are urgently needed to determine the actual risk of these fibers to human health.

The airways and alveoli of the human respiratory tract are lined by two distinct types of epithelium, which are the primary targets of respiratory viruses. We previously established long-term expanding human lung epithelial organoids from lung tissues and developed a ‘proximal’ differentiation protocol to generate mucociliary airway organoids. However, a respiratory organoid system with bipotential of the airway and alveolar differentiation remains elusive. Here we defined a ‘distal’ differentiation approach to generate alveolar organoids from the same source for the derivation of airway organoids. The alveolar organoids consisting of type I and type II alveolar epithelial cells (AT1 and AT2, respectively) functionally simulate the alveolar epithelium. AT2 cells maintained in lung organoids serve as progenitor cells from which alveolar organoids derive. Moreover, alveolar organoids sustain a productive SARS-CoV-2 infection, albeit a lower replicative fitness was observed compared to that in airway organoids. We further optimized 2-dimensional (2D) airway organoids. Upon differentiation under a slightly acidic pH, the 2D airway organoids exhibit enhanced viral replication, representing an optimal in vitro correlate of respiratory epithelium for modeling the high infectivity of SARS-CoV-2. Notably, the higher infectivity and replicative fitness of the Omicron variant than an ancestral strain were accurately recapitulated in these optimized airway organoids. In conclusion, we have established a bipotential organoid culture system able to reproducibly expand the entire human respiratory epithelium in vitro for modeling respiratory diseases, including COVID-19.

Background: Air pollution has been linked to COPD development; however, the precise mechanisms involved are unclear. We hypothesized diesel exhaust particles (DEP) may reduce the functionality of lung epithelial progenitors, whereas anti-oxidants may protect against the influence from DEP. Methods: Murine lung organoids were set up by co-culturing epithelial cells (EpCAM+/CD45-/CD31-) with CCL206 fibroblasts in Matrigel. Organoids were exposed to DEP (50-, 100-, 200 µg/ml) and/or NAC (1mM)/MitoQ (1μM). Results: The total organoid number was decreased by 200 μg/mL DEP by day7, yet increased by 50 and 100 μg/mL DEP. Subclassifying the organoids into airway/alveolar organoids revealed that airway organoid number was increased by 50 and 100 μg/mL DEP, and decreased by 200 μg/mL DEP by day14. Alveolar organoid number was decreased by 200 μg/mL DEP. Immunofluorescence studies confirmed that 200 μg/mL DEP decreased the number of acetylated-α tubulin+ and pro-SPC+ organoids. The size of alveolar organoids was also reduced by 200 μg/mL DEP. In line with these findings, the gene expression of FGF2, FGF7, FGF10 and HGF was decreased in fibroblasts by 200 μg/mL DEP. Interestingly, NAC (1mM) or MitoQ (1μM) rescued the number of alveolar organoids after exposure to 200 μg/mL DEP, but had no influence on airway organoids. NAC and MitoQ had no influence on organoid size. Conclusion: DEP functionally inhibit lung organoid formation by epithelial progenitors, which may contribute to defective alveolar repair in COPD. NAC and MitoQ protect against the damage from DEP in alveolar epithelial progenitors.

ABSTRACTHuman parainfluenza virus type 3 (HPIV-3) is a significant cause of lower respiratory tract infections, with the most severe disease in young infants, immunocompromised individuals, and the elderly. HPIV-3 infections are currently untreatable with licensed therapeutics, and prophylactic and therapeutic options are needed for patients at risk. To complement existing human airway models of HPIV-3 infection and develop an animal model to assess novel intervention strategies, we evaluated infection and transmission of HPIV-3 in ferrets. A well-characterized human clinical isolate (CI) of HPIV-3 engineered to express enhanced green fluorescent protein (rHPIV-3 CI-1-EGFP) was passaged on primary human airway epithelial cells (HAE) or airway organoids (AO) to avoid tissue culture adaptations. rHPIV3 CI-1-EGFP infection was assessed in vitro in ferret AO and in ferrets in vivo. Undifferentiated and differentiated ferret AO cultures supported rHPIV-3 CI-1-EGFP replication, but the ferret primary airway cells from AO were less susceptible and permissive than HAE. In vivo rHPIV-3 CI-1-EGFP replicated in the upper and lower airways of ferrets and targeted respiratory epithelial cells, olfactory epithelial cells, type I pneumocytes, and type II pneumocytes. The infection efficiently induced specific antibody responses. Taken together, ferrets are naturally susceptible to HPIV-3 infection; however, limited replication was observed that led to neither overt clinical signs nor ferret-to-ferret transmission. However, in combination with ferret AO, the ferret model of HPIV-3 infection, tissue tropism, and neutralizing antibodies complements human ex vivo lung models and can be used as a platform for prevention and treatment studies for this important respiratory pathogen.

Microplastics (<5 mm) are an increasing concern for environmental and human health, continuously detected in ecosystems worldwide and a variety of human tissues. While health effects remain unclear, experimental studies on microplastic particles have suggested adverse outcomes. Microplastic fibers, which shed from everyday items, are more toxic than particles and twice as prevalent, yet remain understudied. Microplastic studies vary widely and use various extraction techniques, with few validating recovery accuracy. These limited recovery studies primarily examine particles, raising concerns about the true abundance of microfibers. This study establishes baseline recovery rates of polyethylene terephthalate (PET) and polypropylene (PP) microfibers of varying lengths from formalin-fixed human cadaveric lung tissue. Following enzymatic and oxidative digestion, PET microfibers showed a recovery rate of 47%, while 87% of PP microfibers was recovered. Chemical alterations were assessed using laser direct infrared (LDIR) spectroscopy; optical microscopy and scanning electron microscopy (SEM) evaluated physical changes post-digestion. These findings provide insights into microfiber recovery, highlight potential over- and underestimations, and characterize the chemical and physical behavior of fibers within human tissue studies. Establishing accurate recovery methods is essential for advancing microfiber toxicology research and assessing potential health risks.

Recent studies have focused on understand lung repair mechanisms, which are critical for treating respiratory diseases. In this study, we develop alveolar organoids to investigate the complex interactions between alveolar epithelial cells, niche fibroblasts and macrophages, which are essential for lung development, maintenance and repair, especially under physiological injury. Our results suggest that alveolar organoids may be a model for epithelial cell regeneration and the inflammatory response in lung tissue. Alveolar organoid studies can also serve as models for various lung injuries and demonstrate mechanisms in the injured human lung.

IntroductionIdiopathic pulmonary fibrosis (IPF) is a fatal disease of lung parenchymal scarring triggered by alveolar epithelial cell dysfunction. Inherited forms of pulmonary fibrosis, including those caused by mutant forms of...

<b>Introduction:</b> Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide that is characterized by progressive loss of alveolar tissue. Endogenous lung stem cells and tissue repair pathways are thought be dysfunctional and their reactivation is a promising therapeutic approach for COPD. Wnt/β-catenin signaling is critical for stem cell function and is reduced in human COPD lungs. We previously demonstrated that therapeutic Wnt/β-catenin initiated tissue repair in COPD patient-derived lung tissue <i>ex vivo</i>. Recently, we identified an epithelial progenitor population enriched with club cells, which were activated by Wnt/β-catenin signaling to form alveolar organoids. Thus, we hypothesize that club cells represent a novel Wnt-responsive epithelial progenitor for tissue repair in COPD. <b>Methods:</b> Single cell RNA sequencing (scRNA seq), organoid assay, fate tracing studies, flow cytometry, immunofluorescence, FACS sorting, porcine pancreatic elastase (PPE) induced mouse COPD model. <b>Results:</b> 1) Using scRNA seq, we identified club cells as the major progenitor cells in the Wnt-responsive epithelial progenitor cell population. 2) We perform fate tracing studies using a <i>Scgb1a1Cre-ERT;Rosa-Tomato;TCF/Lef:H2B-GFP</i> mouse line combined with lung organoid assays to demonstrate that activation of the Wnt/β-catenin signaling using the GSK3 inhibitor CHIR9920 increased the number of alveolar organoids formed by club cells. 3) In a PPE-induced mouse COPD model, we identified an impaired organoid forming capacity of club cells, which could be restored by activation of the Wnt/β-catenin signaling. Importantly, the alveolar organoid formation was largely increased. <b>Conclusion:</b> Our study reveals a novel mechanism of club cell activation by Wnt/β-catenin signaling in COPD.

Mutations in the FOXF1 (forkhead box F1) gene, encoding the mesenchymal FOX (forkhead box) transcription factor, are linked to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. Although proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we used murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in patients with ACDMPV) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into type 1 alveolar epithelial cells (AEC1s). Alveolar organoids containing Foxf1WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1WT/S52F mice was associated with decreased expression of noncanonical WNT5A (Wnt family member 5A) ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in patients with ACDMPV.

The development of physiologically relevant in vitro 3D models is crucial for studying lung biology and disease mechanisms. While airway organoids have significantly improved our ability to mimic lung tissue, they lack key nonepithelial components that are essential for tissue homeostasis. Here, we describe the generation of human airway assembloids, combining airway organoids, stromal fibroblasts, and endothelial cells to better replicate the native lung environment. The model was generated from healthy lung tissue donors by using a scaffold-free culture system to promote cell self-organization. Assembloids exhibited long-term viability, maintained typical airway epithelial markers, and demonstrated functional characteristics, such as mucus production and ciliary beating. This technology provides a powerful platform for studying airway physiology, disease mechanisms, and therapeutic approaches, with potential applications in regenerative and personalized medicine. Our study established a novel, reproducible 3D assembloid model of the human airways, bridging the gap between traditional organoid cultures and complex tissue engineering strategies.

Aldehyde dehydrogenase ALDH3A1 rescues cigarette smoke-induced emphysema by conferring alveolar type 2 to type 1 cell transition.

Rationale: There is increasing evidence that Alveolar Type II (ATII) progenitor-cell dysfunction and senescence plays a role in emphysema pathogenesis. The WNT pathway plays a key role in ATII stem-cell niche homeostasis/activation and is dysregulated in chronic lung disease (including COPD). We therefore sought to identify potential culprit mediators of this pathway in the failed alveolar regeneration seen in emphysematous tissue of patients with COPD. Methods: We interrogated a large human ATII-enriched scRNA sequencing dataset (Hu, 2024) for WNT pathway proteins differentially expressed in COPD patient vs. healthy donor ATIIs. The WNT antagonist, WNT Inhibitory Factor-1 (WIF1) was significantly upregulated in COPD ATIIs. We confirmed this difference and co-localization with HTII-280 (ATII marker) in situ with IF staining of human COPD and healthy tissue (n=5 each group). We tested activity of recombinant WIF1 by WNT reporter cell line. We isolated ATII cells (HTII-280+ enriched) and fibroblasts from explanted lungs of COPD and healthy donors for individual testing and study in an alveolar organoid model (as previously described Katsura, 2020). Results: We confirmed significantly upregulated WIF1 expression in COPD vs. healthy donors via qPCR in isolated primary cells enriched for ATIIs. Given WIF1 overexpression is known to induce senescence in non-epithelial cell lines, we also quantified expression of p16 and p21 and found these are also significantly upregulated in COPD. We did find a moderate association by linear regression between fold-increase in senescence marker and WIF1 expression within patient samples with an R2 of.498 and.435 for p16 and p21, respectively. To study the effect on primary human ATIIs, we treated alveolar organoids with 100ng/mL WIF1 (dosing per WNT-reporter testing) for 14D. We found a small decrease in organoid forming capacity (progenitor cell capacity marker) after 14D treatment WIF1 (pilot data n=3 donors, Figure1a). We stimulated primary human fibroblasts isolated from COPD patients and healthy donors (n=2) with Wnt3a (canonical Wnt activator) and treated with WIF1 for 72hrs and found a significant increase in p21 expression in WIF1 treated fibroblasts (Figure1b). Conclusions: We demonstrate a novel potential mediator of ATII progenitor cell dysfunction in COPD associated emphysema, the WNT pathway antagonist – WIF1. We demonstrate this ATII cell derived antagonist is associated with increased markers of senescence in these ATIIs of patients with COPD. Furthermore, we show that this association may be driven by a paracrine signaling mechanism between source ATIIs and neighboring fibroblasts in which senescence is induced.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Targeted Deletion of Jun/AP-1 in Alveolar Epithelial Cells Causes Progressive Emphysema and Worsens Cigarette Smoke–Induced Lung Inflammation

Emphysema is an important phenotype of chronic obstructive pulmonary disease (COPD) characterized by alveolar destruction. Macrophage migration inhibitory factor (MIF)-deficient mice develop spontaneous emphysema and MIF, through one of its receptors CD74, was shown to induce type II alveolar epithelial cell proliferation in vitro. However, how MIF and through which of its receptors, is involved in alveolar repair in COPD and healthy conditions has not been elucidated yet. Therefore, we aimed to investigate the effects of MIF on lung alveolar repair using a model of lung organoids established by co-culturing fibroblasts with primary EpCAM+ cells, isolated from lung tissue of healthy mice or patients with or without COPD. MIF treatment of EpCAM+ cells from healthy mice resulted in significantly fewer alveolar organoids compared to untreated controls. A similar trend was found for human organoids derived from patients without COPD or COPD GOLD I. However, MIF treatment of EpCAM+ cells from severe COPD patients (GOLD III/IV) induced more and larger alveolar organoids compared to controls. This divergence may suggest differential expression of MIF receptors in severe COPD and we therefore investigated expression of CD74, CXCR2, CXCR4, and CXCR7 in a lung tissue RNA-sequencing dataset of control vs COPD GOLD IV patients. CXCR4 gene expression was significantly higher in severe COPD, whereas other MIF receptors were not differentially expressed. In conclusion, MIF only promoted alveolar organoid formation and growth in cells isolated from severe COPD lungs. As CXCR4 was shown to regulate migration of lung alveolar epithelial cells before, its higher expression in severe COPD may explain this divergent effect of MIF on lung alveolar repair.