BACKGROUND:Tandem use of cigarettes and cannabis is increasing, particularly where recreational marijuana is legalized, posing a serious risk to fetal development. This study investigates the impact of cannabinoids (THC and CBD) and nicotine on fetal lung development using human lung explants from 10-16 weeks of gestation. Our results show that THC promotes epithelial cysting RESULTS: Our data revealed decreased proliferation (Ki-67) following treatment with CBD and CBD + THC+nicotine, whereas smooth muscle cell differentiation decreased across all conditions. Furthermore, the combination treatments induced a senescence-like phenotype as demonstrated by increased CDKN1A and CDKN2A expression and a strong reduction in LAMIN B1 staining. Additionally, DNA damage response markers ɣH2A.X and 53BP1 were significantly upregulated in the combination treatments. This was accompanied by an activation of the interferon pathway with increased expression of the downstream targets MX1 and IFI2. The addition of senolytics, Dasatinib and Quercetin (D + Q), reduced the senescence-associated secretory phenotype (SASP), such as IL-6, IL-8, M-CSF, MCP-3, MIP-1β, TNFα, MIP-3β, and TSLP, the senescence gene expressions, and reversed interferon pathway activation CONCLUSION: Our results demonstrate that co-exposure to nicotine and cannabis alters cellular proliferation and differentiation, induces a senescence-like phenotype and DNA damage response in human fetal lung cultures, which may be partially rescued using senolytics. IMPACT:This study aims to assess the effect of cannabis and nicotine use during pregnancy on fetal human lung development. The combinatorial exposure to CBD, THC, and nicotine during pregnancy increases senescence-like phenotype, DNA damage response, and senescence-associated secretory phenotypes (SASP) secretion. The use of senolytics (D + Q) may prove to be a promising therapeutic option, alleviating several of the adverse effects induced by the different substances.

Background: Children with Trisomy 21 (T21) experience high rates of respiratory disease, but the developmental origins of airway epithelial defects remain poorly defined. Airway integrity relies on balanced differentiation of basal, secretory, and ciliated cells, as well as intact tight junctions, which can result in mucociliary clearance issues, a phenomenon observed in T21. Since T21 is characterized by chronic type I interferon (IFN-I) hyperactivation, we investigated whether airway abnormalities arise in utero and whether IFN signaling contributes to these defects. Methods: Human prenatal and postnatal non-T21 and T21 lungs were analyzed by FISH, immunofluorescence (IF), and RT-qPCR to quantify epithelial cell populations and IFN-I pathway activation. Basal cells were isolated from prenatal large airways from matched samples and were cultured in air-liquid interface (ALI) to assess differentiation, ciliary function (ciliary beat frequency (CBF)), and barrier integrity (TEER, ZO-1 staining, and TEM). Non-T21 ALI cultures were treated with IFN-β (400 pg/mL) for 21 days to model T21-associated IFN-I signaling. Membranes were collected for gene and protein analysis. Results: In prenatal and postnatal T21 lungs, RT-qPCR and FISH demonstrated elevated IFN-I pathway activity, with increased MX1, IFI27, IFNAR1/IFNAR2, and IFNB1 expression compared to non-T21 lungs (p< 0.01). In prenatal T21 lungs, IF and RT-qPCR showed a trend toward reduced basal cells (TP63) and a significant expansion of secretory cells (SCGB1A1/SCGB3A2; p< 0.05), while ciliated cell numbers (FOXJ1) were unchanged. Postnatally, secretory cell expansion persisted (p< 0.05), but ciliated cells were significantly decreased, with fewer FOXJ1+/ARL13B+ cells and reduced FOXJ1 transcript levels (p< 0.05). Moreover, tight junctions were compromised in T21 prenatal airways, with disorganized ZO-1, abnormal tight junction ultrastructure, and a trend toward decreased OCLN expression (p< 0.05). In vitro, T21 ALI cultures from prenatal basal cells recapitulated the postnatal tissue phenotype, with increased secretory cells, reduced ciliated cells and CBF, and disrupted ZO-1 organization compared to non-T21 ALI (p< 0.05). IFN-β treated non-T21 ALI cultures showed an increase in secretory cell differentiation (SCGB1A1), impaired ciliated cell formation (FOXJ1+/ARL13B+), and ciliary dysfunction comparable to T21 ALI, with significantly reduced CBF relative to untreated controls as well as disrupted tight junction (p< 0.05). Conclusion: Airway epithelial dysregulation in T21 begins in utero, with a possible shift from basal toward secretory fates, disrupted tight junctions, and postnatal loss of ciliated cells. These defects are reproduced in T21 ALI cultures and can be recapitulated by chronic IFN-β exposure. This indicates that IFN-I hyperactivation is a central driver of abnormal airway epithelial differentiation and impaired ciliary function in T21, identifying IFN-I signaling as a potential therapeutic target for early-onset airway disease in Trisomy 21. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Objective: Trisomy 21 (T21), resulting in Down Syndrome, is the most common chromosomal abnormality worldwide. This multisystem disorder is associated with several developmental defects, including numerous lung anomalies. We have observed that prenatal T21 lungs have abnormal dilatation of the distal airways and acini accompanied by what appears to be a defect in cell adhesion, likely from compromised intercellular junctions. This study aimed to interrogate transcriptional changes across epithelial lineages in the human prenatal T21 lung and assess their potential impact on epithelial structure and development. Hypothesis: We hypothesized that signaling alterations in T21 lungs disrupt epithelial cell adhesion programs in a stage- and lineage-specific manner. Methods: We analyzed single nucleus (sn)RNA-seq data from 23 prenatal lung samples (14 Trisomy 21 and 9 Disomic 21; 13-20 post-conception weeks). After SoupX decontamination, stringent QC, and consensus doublet removal, data were normalized with SCTransform, integrated with RPCA, and clustered in Seurat. Epithelial nuclei were then subsetted, reprocessed, and subclustered (resolution 0.5), and annotated using ToppGene-derived markers. Differential expression was performed using NEBULA, modeling raw counts with karyotype, age, and sex as fixed effects and subject as a random effect. Pathway analyses included over-representation tests (GO/KEGG/Reactome) and GSEA on full ranked gene lists. Results: T21 exhibited developmentally dynamic and lineage-specific disruptions in epithelial adhesion pathways. In the early developmental window (< 15 weeks), T21 epithelium showed disruption of the cell-cell junction organization pathway, consistent with reduced expression of the tight-junction component, OCLN. These disruptions were confirmed by electron microscopy. By mid-gestation (16-20 weeks), T21 displayed a coordinated upregulation of junctional and integrin-ECM adhesion programs. Across epithelial subpopulations, we observed increased expression of adhesion-related genes, including: TJP1, VMP1, CDH2, CDH19, TIAM1, VCL, TLN2, ITGA6, ITGAV, ITGB1, FN1, COL3A1, and NPNT, with individual genes enriched in distinct clusters such as AT1, AT2, airway progenitor, and neuroendocrine. These gene sets enriched for “cell junction assembly”, “cell junction organization”, and “cell-matrix adhesion”, indicating continued disruption and a possible compensatory effect of cell-cell and cell-matrix adhesion programs in mid-gestation T21 lungs. The pulmonary neuroendocrine cluster displayed a distinct increase in neuronal Ig-domain adhesion molecules (CADM1, IGSF11, TENM3, NLGN4X, LRRC4C), suggesting aberrant stabilization of neuroepithelial bodies in T21. Conclusions: Our findings suggest that adhesion dysregulation is an important feature of prenatal T21 lung epithelium. A pattern of early tight-junction loss followed by later enhancement of junctional, integrin, and ECM pathways supports a model in which altered adhesion programs contribute to disrupted epithelial maturation, altered epithelial-matrix interactions, and aberrant neuroepithelial body stabilization in T21 lung development. Funding: NIH/NHLBI Office of The Director, National Institutes of Health (OD) R01HL155104; NIH/NHLBI R01HL141856; and NIH/NICHD R24HD000836. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rapid advances in single-cell technologies now allow measurement of thousands of transcripts and other molecular features of individual cells offering unprecedented insight into lung biology in homeostasis and in disease. The accelerated generation of multimodal data has, however, been accompanied by the reporting of putatively "novel" cell types described without consensus regarding their ontogeny, identity, function, or defining markers. To fully realize the value of the technological advances and to enable rigorous comparison across studies, respiratory research will benefit from standardized, quantitative, and biologically grounded cell classifications and nomenclature. Achieving the transformative potential of the multimodal data will depend on common, machine- and human-readable nomenclature, structured and expandable dictionary and atlas resources, and clear methodological standards that ensure consistency as technologies evolve. The American Thoracic Society (ATS) recognized the importance of promoting a common nomenclature to enhance equitable access and utility of the vast amounts of multimodal data generated by the lung research community. The Collaborative Cross-Consortium and Country Lung Cell Nomenclature Project (C3LCN) was adopted as an ATS Assembly Project in 2024. This is the consensus report outlining the goals and framework of the Project to foster coordinated progressive lung cell research to include: 1) providing best practices for analysis, publication and reporting of lung single-cell transcriptomic datasets; 2) establishing a contemporary lexicon for healthy adult human cells of the lower respiratory tract with structured, persistent, and resolvable identifiers; 3) defining a scalable taxonomy to organize a common lung cell nomenclature; 4) offering tools to support collaboration, knowledge dissemination, and translational advances rooted in modern lung biology augmenting, not replacing, pre-genomic biological knowledge; and 5) describing an infrastructure capable of incorporating new ontological refinements as higher-resolution, multimodal single-cell and spatial datasets emerge, cellular heterogeneity is better defined, and disease-associated abnormal cell types and reactive cell states are increasingly recognized and mechanistically interrogated. Together, this coordinated effort aims to provide the foundation necessary for a robust, harmonized, and expandable nomenclature for lung science.

Alveolar growth and repair are central processes in development and chronic lung disease, such as bronchopulmonary dysplasia (BPD), a neonatal lung disease without curative therapy. Alveolar epithelial type 2 (AT2) cells are the endogenous progenitor pool giving rise to alveolar epithelial type 1 cells and promoting alveolar repair. Since netrin-1, a regulator of cell homeostasis and stemness, has been linked to lung diseases, we now investigated its signaling and function in AT2 cells in a hyperoxia-based model of BPD and in lungs of infants with BPD. First, we demonstrated that prolonged hyperoxia reduced both netrin-1 and its receptor Unc5b in neonatal mouse lungs and in primary AT2 cells. Second, ex vivo studies using precision-cut lung slices (PCLS) and primary murine AT2 cell culture showed that netrin-1 regulates AT2 cell survival and the expression of Krüppel-like factor 4 (Klf4) through Unc5b, a transcription factor regulating cell survival. Third, single-cell and bulk transcriptomic analysis, as well as proximity-dependent biotin identification assay, showed Klf4 to be upregulated in AT2 cells during alveolarization, downstream of netrin-1, and to regulate AT2 cell survival. In vivo, Klf4 gene expression and protein abundance was significantly reduced in total lung homogenates and in AT2 cells of neonatal mice exposed to hyperoxia. Finally, KLF4+ cells, KLF4+ epithelial, and specifically KLF4+ AT2 cells were reduced in clinical BPD. In summary, our data identify a novel netrin-1-Unc5b-Klf4 axis in AT2 cells that is disrupted in BPD and could offer a novel target for endogenous alveolar repair.

Background: The Hedgehog (HH) signaling pathway is essential for lung branching morphogenesis, yet its role in human alveologenesis remains unclear. Bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants, is characterized by arrested alveolarization and elevated expression of HH pathway components. We hypothesized that HH pathway regulation is crucial for alveolar type II (AT2) cell differentiation during late-stage lung development. To further investigate mechanisms contributing to BPD we modeled hyperoxic injury in prenatal human lung explants. Methods Fresh human prenatal lung explants (18-22 weeks gestation) were cultured at air-liquid interface in CK+DCI medium to promote alveolar-like differentiation. Explants were treated with either a HH pathway activator (SAG) or inhibitor (5E1) and/or exposed to hyperoxic (40% O 2 ) or normoxic conditions for 7 days. RT-qPCR, immunofluorescence (IF), and fluorescent in situ hybridization (FISH) were used to assess target gene expression, cell fate, primary cilia (PC) dynamics, and tissue morphology. Bronchopulmonary dysplasia (BPD) lung tissues were collected, and the HH pathway, proliferation, PC, and progenitor markers were further evaluated by RT-qPCR and combined FISH-IF. Publicly available BPD RNA sequencing datasets were used to compare and correlate with in vitro results. Results: Under normoxia, SAG treated explants exhibited large distal cysts, increased proliferation, and higher PC frequency in mesenchymal and epithelial compartments (p< 0.05). We also observed decreased expression of AT2/AT1 markers as compared to non-treated explants (ABCA3, LAMP3, AGER and HOPX) (p< 0.05). Results were confirmed by FISH and IF analyses with a reduction in SFTPC alongside SOX9 (p< 0.05). Although SAG-treated explants contained fewer intermediate cells (KRT8 + /SFTPC + /CLDN4 + ), this population exhibited a 1.5-fold increase in PC compared with untreated controls. HH activation induced senescence signatures (CDKN1A, CDKN2A, TP53, P21, γH2AX; p< 0.01). Similarly, hyperoxia lead to HH activation, cystic morphology, enhanced proliferation, expansion of KRT8 + /SFTPC + /CLDN4 + intermediate cells with higher PC, and reduced distal/AT2/AT1 programs, including SOX9 (p< 0.05). Under hyperoxia, 5E1 partially rescued hyperoxia-induced defects such as reduced cysts and senescence, restored AT2/AT1 markers and intermediate-state balance, decreased proliferation (p< 0.05), and significantly lowered PC prevalence (p< 0.01). Public BPD datasets confirm HH, senescence, and PC-related gene upregulation with reduced alveolar markers. Conclusions: In our CK+DCI hyperoxia model of late human alveologenesis, we demonstrated that pharmacologic HH activation recapitulates hyperoxia by blocking AT2 maturation and differentiation, and inducing cystic remodeling, PC accumulation, proliferation, and senescence. 5E1 partially rescues these defects, and BPD tissues/public datasets corroborate the signature, nominating the HH-PC axis as a key focus to deepen mechanistic understanding of BPD. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Introduction:Single-cell RNA-sequencing analyses have revealed the existence of two distinct capillary cell populations in the human lung: general capillary cells (CAP1) and alveolar capillary cells (CAP2). Studies in mouse have shown that the splicing of Vegf-a evolves during embryonic development, creating a temporal pattern of expression for different isoforms, which contributes to the formation of pulmonary capillaries. Moreover, it was demonstrated that murine Vegf-a188 isoform promotes the emergence of CAP2 in vitro. Human homologs of these VEGF-A isoforms exist; however, their role in this process remains elusive. This study investigates the role of VEGF-A and its isoforms in the differentiation of lung capillaries during human prenatal development. Methods:A cohort of human prenatal tissues, aged from the late pseudoglandular to early canalicular stages of development (10-20 weeks of gestation), was used to study the emergence of CAP2 markers (TBX2, SOSTDC1, EDNRB, HPGD, APLN) in correlation with the expression of the different VEGF-A isoforms (VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189). Results:RT-qPCR analyses revealed a simultaneous expression of certain VEGF-A isoforms with several CAP2 markers, which peaked at around 18-20 weeks of gestation. Human prenatal lung explants were then treated with recombinant proteins of the different VEGF-A isoforms to study their impact on EC proliferation, as well as on the expression of CAP2 markers. While most of the isoforms did not impact EC proliferation, except for VEGF-A189 which downregulated it, almost all of them upregulated the expression of APLN, a major CAP2 marker. By using fluorescence in situ hybridization, we showed that this increase of expression was specific to the ECs. However, most of the isoforms induced a downregulation of EDNRB and HPGD. They also did not impact the expression of SOSTDC1 and TBX2. Discussion:Our study shows that the different VEGF-A isoforms do not have the same effect on human lung capillary differentiation as those observed with their homologs in mice, highlighting the importance of studying this process in the human model. Moreover, while it demonstrated that VEGF-A isoforms can induce APLN expression in ECs, it also revealed that CAP2 differentiation is most likely a multifactorial process, not only involving VEGF-A.

Despite significant advances in understanding lung development, the intricate cellular interactions and spatial organization of the developing human lung remain incompletely defined. Spatial transcriptomics enables gene expression profiling within the native tissue context, providing unprecedented insights into complex developmental processes. In this study, we applied the 10X Genomics Visium platform to characterize spatially resolved transcriptional profiles of prenatal human lungs during the pseudoglandular and canalicular stages.Spatial transcriptomic analysis of 12 prenatal lung samples (13–20 weeks gestation) identified 10 distinct transcriptional niches corresponding to unique combinations of epithelial, mesenchymal, endothelial, and immune cell populations. Unsupervised clustering revealed developmental shifts in spot/niche composition from the pseudoglandular to canalicular stage, with a progressive increase in alveolar epithelial spots and a concomitant decline in mesenchymal regions, particularly in peripheral lung areas. Differential gene expression analysis demonstrated stage-specific transcriptional transitions in individual spot types, including downregulation of cell cycle and structural pathways and upregulation of secretory pathways as the lung matures. Spatial organization analysis revealed increasing compartmentalization of pulmonary cell types, highlighting the progressive structuring of the distal lung microenvironment. In summary, this study provides a spatial map of the developing human lung, offering novel insights into pulmonary lineage dynamics and cellular interactions during early organogenesis.

RATIONALE:Trisomy 21 (T21), resulting in Down syndrome (DS), is the most prevalent chromosomal abnormality worldwide. While pulmonary disease is a major cause of morbidity and mortality in DS, the ontogeny of pulmonary complications remains poorly understood. We recently demonstrated that T21 lung anomalies, including airway branching and vascular lymphatic -abnormalities, are initiated in utero. Here, we aimed to describe molecular changes at the single-cell level in prenatal T21 lungs. METHODS:Single-cell RNA sequencing was used to generate transcriptomic profiles of individual human lung cells in tissue obtained from T21 (n = 5) and non-T21 (n = 4) prenatal lungs. Clustering of cells, marker identification, uniform manifold approximation and projection representation, and differential expression analysis were performed in Seurat. Cell type annotation and pathway analysis were annotated using ToppFun and a human fetal lung cell atlas. Spatial differences in cellular phenotypes were validated using immunofluorescence staining and fluorescent in situ hybridization. RESULTS:Our results detail changes in gene expression at the time of initiation of histopathological abnormalities in T21 prenatal lungs. Notably, we identify precocious differentiation of epithelial cells, widespread induction of key extracellular matrix molecules in mesenchymal cells, and hyperactivation of IFN signaling in endothelial cells. CONCLUSIONS:This single-cell dataset of T21 lungs greatly expands our understanding of antecedents to pulmonary complications and should facilitate efforts to mitigate respiratory disease in DS.

Trisomy 21 (T21) is the most common chromosomal disorder worldwide and the leading cause of intellectual disability. Individuals with T21 present with unique facial features, developmental challenges, and multiorgan system defects including cardiovascular, pulmonary, gastrointestinal, endocrine, neurologic, hematological, immunologic diseases, and other systems. We reviewed the latest changes and advances in T21 management over the last 10 years, focusing on new findings and improvements in diagnosing, preventing, and treating complications across different organ systems, which have led to a longer lifespan and better quality of life for children with T21.

Rationale: Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods: We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca 2 + dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results: Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p< 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p< 0.05; n=5–6), with increased γ-H2AX/p21 IF (p< 0.01; n=6), SA-β-gal (p< 0.05; n=5–6), and ER/mitochondrial stress (p< 0.05; n=5–6). Fura-2 AM showed greater Ca 2 + release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p< 0.01; n=5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p< 0.05; n=6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p< 0.01; n=6). In postnatal HLF, PQQ had the stronger effect (p< 0.05; n=5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p< 0.05; n=5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p< 0.05; n=5-6). Conclusion: These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background Cystic Fibrosis-related Bone Disease is an emerging challenge faced by 50 % of adult people with cystic fibrosis (CF). The multifactorial causes of this comorbidity remain elusive. However, congenital bone defects have been observed in animal models with CFTR mutations, suggesting its importance. The role of CFTR in bone cells development is unknown. Studies from human cells remain somewhat controversial depending on the cells used and the disease state of the patients from which the cells derived. Methods Therefore, we investigated the role of CFTR in osteoblast development using induced pluripotent stem cells generated from homozygous CF donors for F508del and non-CF controls. This approach allows for a clear understanding towards how the CFTR mutation may influence osteoblast differentiation independently from other confounding factors. Results We observed a lower capacity of differentiation in CF cells as compared to control, already from mesenchymal stem cells (MSC) stage, whereby they retained expression of the pluripotency marker OCT4. Furthermore, our results demonstrated a delayed osteoblast commitment and altered expression of specific markers, such as an increased RANKL/OPG ratio and decreased BMP2, suggesting a potentially perturbed bone homeostasis associated with CFTR mutation. Conclusions This is the first study of its kind, clearly demonstrating a role for CFTR mutation in delaying osteoblast differentiation and/or regeneration.

Human chromosomal anomalies, notably trisomies, disrupt gene expression, leading to diverse cellular and organ phenotypes. Increased cellular senescence (SEN) and oxidative stress in trisomies have gained recent attention. We assessed SEN, SEN-associated secretory phenotype (SASP), and oxidative stress on trisomy 13 (T13), T18, and T21 human fetal lung tissues and isolated primary human fetal lung fibroblasts. Telomerase-associated foci staining showed DNA damage primarily within T21 and T18 lungs. These results were confirmed by real-time quantitative PCR showing an increase of the SEN marker CDKN2B and SASP markers IL-6 and CXCL8. In contrast, lung tissues from T13 showed an upregulation of CDKN2A, whereas no significant changes in SASP marker genes were observed. γ-H2AX (H2A histone family member X) was upregulated in each genotype, particularly in T21. Isolated fibroblasts demonstrated a strong association between T21 and several SEN markers. An increase of γ-H2AX-positive cells were observed in fibroblasts from T21, T18, and T13, but only T21 exhibited an increase in P21 expression. Only T21 fibroblasts displayed a significant increase in reactive oxygen species levels, as indicated by MitoSOX and CellROX. This study provides the first evidence of a link between SEN and trisomy anomalies during prenatal human lung development, particularly in T21.

RATIONALE: Lung matrix remodeling by activated fibroblasts is a key pathomechanism of bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF). Epigenetic modifiers could contribute to the onset and progression of lung fibrosis. We recently identified Krüppel-like factor 4 (Klf4) as a key transcription factor in fibroblast cellular activity through an EP300-related interactome. Hence, we now studied if the Klf4-Ep300 axis is a regulator of the acetylome and of chromatin remodeling in fibroblasts in BPD and IPF, favoring thereby lung fibrosis.METHODS: (i) a neonatal murine exposed to hyperoxia (HYX) or normoxia (NOX) until postnatal day (P)14 or P28, (ii) transgenic mice with an ACTA2+ cell-specific deletion of Klf4, (iii) primary neonatal murine and human lung fibroblasts from lungs with IPF and control, and (iv) lungs from patients with BPD or IPF vs control. RESULTS: HYX caused fibrotic lung remodeling in neonatal hyperoxia-exposed mice that was associated with an active TGFβ signaling and reduced Klf4 abundance in proliferative ACTA2+ cells. These findings were related to changes in global and posttranslational protein acetylation as well as to an activation of EP300 in ACTA2+ cells in vivo and in cultured lung fibroblasts, that results in an inactivation of FoxO1. Transgenic mice with ACTA2+ cell-specific ablation of Klf4 exhibited higher matrix remodeling with increased number ACTA2+ cells as well as activation of EP300 and altered histone acetylation. Next, we found Klf4 to regulate proliferation, migration and differentiation in primary neonatal murine and in human lung fibroblasts. Chip-Seq, biotinylation identication assay (BioID), and EP300 inhibition supported a functional role of Klf4 as a key regulator of the global acetylome and FoxO1 acetylation. In addition, we performed ATAC-seq of murine lung fibroblasts from transgenic mice with ACTA2+ cell-specific ablation of Klf4 and from human donor fibroblasts with Klf4 knockdown. Deep epigenetic profiling uncovered a shifted transcription factor footprint favoring FoxO signaling and fibrosis following the loss of Klf4, along with a marked converging epigenetic profile with human IPF-fibroblasts. Finally, human lungs with BPD exhibited significantly reduced abundance of Klf4+ ACTA2+ and an increase in ACTA2+ cells. Similarly, fibrotic foci of IPF lungs showed loss of Klf4. Conversely, Klf4 overexpression exhibited an anti-proliferative and pro-apoptotic effect in IPF lung fibroblasts.CONCLUSION: Our study reveals a Klf4-Ep300 axis as a novel epigenetic modifier of chromatin architecture in lung fibroblasts through modulation of the acetylome, favouring thereby fibrosis and offering a possible therapeutic target for severe BPD and IPF.

Introduction: Lung development is a highly intricate process that encompasses a series of regulated signaling pathways, including the Hedgehog pathway (HH), ultimately leading to the establishment of mature and functional pulmonary structures. Throughout development, components of the HH pathway such as SHH, PTCH1 and HHIP are found to be spatially and temporally regulated. Primary cilia play a vital role in this process, as the HH pathway relies entirely on them. They act as a sensory organelle that transduce signals essential for epithelial and mesenchymal interactions. This study aims to characterize the role of primary cilia during lung development in humans. Methods: Immunofluorescence (IF) stainings were performed on formalin-fixed paraffin-embedded (FFPE) human fetal lung tissues between 10 and 23 weeks of gestation (n=31). Co-staining of ARL13B (cilia) and ACTA2 (smooth muscle cell marker) was performed to quantify the percentage of ACTA2+ cells harboring primary cilia (ACTA2+ ARL13B+). Additionally, to characterize the role of primary cilia in lung organogenesis, human fetal lung explants from 10 to 15 weeks of gestation were treated with 0.5 mM or 1 mM of chloral hydrate (CH) for 48 hours to inhibit primary ciliogenesis. RT-qPCR was used to assess the expression of different genes ( ACTA2, SHH, SOX2, SOX9, PTCH1 and HHIP ) following CH treatment. Results: Our results showed a significant decrease in ACTA2+ ARL13B + cells as pulmonary development progressed, from 15% at 10 weeks to 3% at 20 weeks or older (r 2 = 0.7130; p < 0.001). Next, we assessed the effect of primary cilia inhibition in these ACTA2+ cells harboring primary cilia. Our data showed that ACTA2+ ARL13B+ cell percentage in fetal lung explants is reduced following treatment with CH, confirming the inhibition of primary ciliogenesis. Furthermore, we observed decreased expression levels of Hedgehog pathway genes ( SHH, PTCH1 and HHIP), as well as ACTA2 and SOX9, while SOX2 remained unaffected in the explants after treatment. Conclusion: These findings suggest that primary cilia are more prevalent during early stages of lung development and diminish as the lung matures, reflecting a transition from a proliferative state to a more differentiated, specialized state. During development, primary cilia appear to play a critical role in regulating key components of the HH pathway. this work is funded by Region Grand-Est. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rationale: Trisomy 21 (T21), resulting in Down Syndrome (DS), is the most prevalent chromosomal disorder worldwide. Respiratory tract infections (RTIs) are one of the leading causes for which children with DS are hospitalized. The elevated susceptibility of children with DS to RTIs likely stems from structural differences in the airways and immune system abnormalities. Our studies previously demonstrated that the structural anomalies observed in T21 lungs are congenital. The aim of this study is to characterize immune cells in prenatal T21 lungs, potentially explaining vulnerability to RTIs. Methods: Single cell RNA sequencing was used to generate transcriptomic profiles of individual human lung cells in tissue obtained from T21 (n=5) and non-T21 (n=4) prenatal lungs. Immune cell clustering, cluster marker identification, and differential expression analysis was performed in Seurat, while functional classification was performed by Gene Set Enrichment Analysis (GSEA). Spatial differences in immune cell phenotypes were examined using immunofluorescent staining (IF) on paraffin-embedded prenatal lung tissue sections. Additionally, immune cells (CD45+) were isolated from prenatal T21 and non-T21 lung single cell suspensions using immunomagnetic columns, followed by RT-qPCR for different immune cell-type specific markers (i.e CD19 for B cells). Results: We identified 14 different immune cell clusters (Figure 1) from 2,818 immune cells (1408 Non-T21 and 1410 T21) which comprised ∼8% of the total cells sequenced. All major immune cell populations were identified, including natural killer (NK) cells, B cells, T cells, macrophages and dendritic cells. We identified a total of 83 genes as differentially expressed in at least one of the 14 different clusters (FDR<0.05). When assessing proportionality of the different cell types, a significant decrease in the percentage of B cells (p=0.0003) as well as a trend towards more CD16 NK cells (p=0.0585) was observed in T21 lungs as compared to non-T21. RT-qPCR further demonstrated B cell markers CD19 (p=0.0362), CD38 (p=0.0251), and CD22 (p=0.0200) were all significantly decreased in T21. RAG1, a marker associated with B cell maturation, also displayed a trend towards decreased expression in T21 immune cells (p=0.0902). CD38 IF staining was significantly decreased in prenatal T21 lungs as compared to non-T21 age and sex matched controls (p=0.0042). Summary: Our data demonstrate changes in the T21 pulmonary immune system in utero, primarily within the B cell population, which may be contributing to the increased susceptibility of RTIs observed in children with DS.

Rationale: Pulmonary diseases are a primary cause of morbidity and mortality in children with Down Syndrome, or Trisomy 21(T21). We previously reported the presence of structural and molecular changes in T21 human fetal lungs accompanied with increased type I interferon pathway (IFN-I), which regulates differentiation, proliferation, and ECM. We hypothesize that IFN-I alters T21 lung development in a tissue dependent manner. Methods: Immunofluorescent (IF) staining and fluorescent in situ hybridization (FISH) were performed on prenatal T21 and non-T21 age and sex matched control lungs to assess components of IFN-I. Gene expression was assessed by qRT-PCR. Non-T21 human prenatal lung explants were cultured for 72 hours in the presence or absence of 200ng IFNβ. Different combinations of epithelial (E) buds and fibroblasts (F) of T21 and non-T21 prenatal lungs were co-cultured in Matrigel (i.e. T21 E+ Non-T21 F; T21 E+ T21 F; etc.) for 5 days. Monocultures of epithelial buds (T21 and non-T21) were also treated with IFNβ. IF and gene expression of cultures were performed. Results: Expression of IFNAR1 (p=0.0271), IFNAR2 (p=0.0098) and downstream IFN-I targets (MX1: p=0.0062; IFI27: p=0.0077) were upregulated in T21 prenatal lungs as compared to non-T21 controls. Additionally, FISH for IFNAR1 and IFNAR2 demonstrated highest expression within the mesenchymal compartment of T21 lungs. Furthermore, severity of T21 histopathological scoring positively correlated with IFI27, suggesting a link between IFN-I and T21 anomalies (p=0.0081; R=0.649). To test the effect of IFN-I upregulation, non-T21 human fetal lung explants were treated with IFNβ. A significant increase in epithelial airway dilatation was observed following IFNβ treatment (p=0.0144). Ki67 IF demonstrated significantly decreased proliferation in IFN-treated explants (p=0.0426). The expression of SFTPC (gene and protein, p=0.0293 and p=0.0108) and SFTPB (p=0.0575) was higher in IFN-treated explants. Additionally, TP63 was significantly decreased in IFN-treated explants at the gene (p=0.0099) and protein (p=0.0168) level. Increased expression of COL1A1 (p=0.0034) and COL6A1 (p=0.0001) was also noted in IFN-treated explants. These observations are consistent with our findings in T21 lungs. Mono- and co-cultures were performed to better understand the role each tissue compartment (epithelial, mesenchymal) is contributing to the anomalies observed in developing T21 lungs. Regardless of epithelial genotype, co-culturing with T21 fibroblasts caused decreased TP63 and increased SFTPC expression, comparable to what was observed in epithelial buds treated with IFNβ. Conclusions: Our studies suggest that IFN-I signaling from the mesenchyme in T21 lungs is influencing the structural and cellular anomalies observed in prenatal T21 lungs.

Respiratory tract infections (RTIs) are amongst the leading causes of hospitalizations in children with Down syndrome (DS). Their elevated susceptibility likely stems from structural differences in the airways and immune system abnormalities. The aim of this study was to characterize immune cells in prenatal Trisomy 21 (T21) lungs, potentially explaining vulnerability to RTIs. Single-cell RNA sequencing was used to profile immune cells in prenatal T21 (n = 5) and non-T21 (n = 4) prenatal lungs. Spatial phenotypes were assessed via fluorescent in situ hybridization and immunofluorescent staining on prenatal lung tissue sections. Gene expression analysis was also performed on isolated immune cells from lung single-cell suspensions. Several major immune cell populations were identified. A total of 84 DEGs were identified in at least 1 of the 14 different clusters. A significant decrease in the percentage of B cells was observed in T21 lungs (FDR = 0.0037, * p < 0.05). Furthermore, qRT-PCR demonstrated B cell markers were significantly decreased in T21, including those associated with B cell maturation (* p < 0.05 and ** p < 0.01). Several of these markers were also decreased at the protein level (i.e., CD20 and CD38; * p < 0.05 and ** p < 0.01). Our data demonstrate changes in the T21 pulmonary immune system in utero, primarily within the B cell population, which may contribute to the increased susceptibility to RTIs observed in children with DS.