Collagen VII is an essential anchoring protein in the basement membrane zone, maintaining the attachment of stratified and pseudostratified epithelia to the underlying interstitial matrix. However, collagen VII is largely unexplored in normal lungs and idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive accumulation of extracellular matrix and aberrant re-epithelialization of fibrotic lung parenchyma. Analysis of collagen VII protein and mRNA encoded by COL7A1 gene in IPF distal lungs demonstrated elevated levels compared with those in normal lungs. To investigate its cellular source and spatial distribution in lung tissue, immunohistochemistry, RNAscope in situ hybridization, and cell culture experiments, in combination with analysis of public transcriptomic data sets were performed. In the IPF lungs, collagen VII was abundant in pathologically remodeled airways and honeycomb cysts, associated with increased basal cell populations. In contrast, in the control lungs, collagen VII was mainly localized in larger airways. RNA sequencing data revealed that epithelial basal cells and KRT5-/KRT17+ aberrant basaloid cells are the primary sources of COL7A1 mRNA expression. Furthermore, COL7A1 mRNA was observed in mesenchymal subsets, and both COL7A1 mRNA and the protein were observed in fibroblast foci, another histopathologic feature of IPF. In vitro, COL7A1 mRNA expression was increased in normal human lung fibroblasts treated with transforming growth factor-β1. These findings suggest that collagen VII could be involved in the process of abnormal re-epithelialization in lung fibrosis.

Rationale The human airway epithelium depends on a coordinated hierarchy of stem-and differentiated cells to maintain tissue integrity and respond to injury. Defining the transcriptional and translational programs that govern these processes is critical for understanding airway disease and advancing regenerative therapies. Objectives To map the transcriptional landscape of the human airway epithelium and identify regulatory factors controlling basal stem cell function and epithelial differentiation. Methods We performed single-cell RNA sequencing on bronchial biopsies from nine healthy never-smokers, categorized into young (<40 years) and aged (>60 years) cohorts. Unbiased cell type annotation and pseudotime trajectory analysis were used to define cell states and transcription factor dynamics. Measurements and Main Results All major airway epithelial cell types were identified, with conserved composition and transcriptional programs across age groups. Basal stem cells (BSCs) exhibited elevated ribosomal gene expression, indicating increased translational readiness. Pseudotime analysis revealed transitions from basal to differentiated states, with MYC , JUN , and FOS upregulated in proliferative suprabasal cells. HLF emerged as a BSC-enriched transcription factor downregulated upon differentiation. Functional assays showed that HLF overexpression suppresses proliferation in airway BSC and in lung squamous carcinoma cells, while Hlf -deficient mice display basal cell hyperplasia and deficient differentiation. In lung cancer datasets, low HLF expression correlated with worse patient survival. Conclusions This study defines conserved gene programs in the human airway epithelium and identifies HLF as a novel regulator of BSC proliferation and potential tumor suppressor. These findings may inform the development of regenerative therapies and contribute to improved understanding and treatment of lung disease. ### Competing Interest Statement The authors have declared no competing interest. Swedish Heart-Lung Foundation, 20230487, 20220339, 20210340 Swedish Cancer Society, 20 1326, 23 3117), Swedish Foundation for Strategic Research, SBE13-0130 Swedish Research Council, 2018-02631 Sjoberg Foundation

The aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples. Four NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning® Elplasia® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations). We successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected. The establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.

Summary The maternal body helps in providing nutrients and degrading toxic metabolites instead of the fetal body; disruptions in these mechanisms affect normal fetal development. Sterol 27-hydroxylase ( Cyp27a1 ) is involved in the alternative pathway of bile acid synthesis, which is enhanced during pregnancy. However, its role in fetal development remains unclear. Here, we demonstrate that maternal Cyp27a1 activity is essential for progression of normal pregnancy and fetal organ formation. Depletion of maternal Cyp27a1 reduced the pregnancy rate and litter size. Newborn mice died of respiratory distress syndrome resulting from the absence of mature alveolar epithelial cells. These phenotypes were caused by 7α-hydroxycholesterol (7α-HC) accumulating in Cyp27a1 -deficient mice. Mechanistically, 7α-HC destabilized the Fau protein, mediating ribosome assembly, the downregulation of which caused poor polysome formation, lower protein synthesis, and impaired lung maturation. Overall, this study revealed an essential mechanism of securing fetal development by degrading a toxic metabolite in the maternal body.

Cell-based therapies hold great promise in re-establishing organ function for many diseases, including untreatable lung diseases such as idiopathic pulmonary fibrosis (IPF). However, many hurdles still remain, in part due to our lack of knowledge about the disease-driving mechanisms that may affect the cellular niche and thereby possibly hinder the function of any transplanted cells by imposing the disease phenotype onto the newly generated progeny. Recent findings have demonstrated increased ciliation of lung cells from IPF patients, but how this affects ciliated cell function and the airway milieu is not well-known. Here, we performed single-cell RNA sequencing on primary ciliated (FOXJ1(+)) cells isolated from IPF patients and from healthy control donors. The sequencing identified multiple biological processes, such as cilium morphogenesis and cell signaling, that were significantly changed between IPF and healthy ciliated cells. Ferritin light chain (FTL) was downregulated in IPF, which suggests that iron metabolism may be affected in the IPF ciliated cells. The RNA expression was confirmed at the protein level with histological localization in lung tissue, prompting future functional assays to reveal the potential role of FTL. Taken together, our data demonstrate the importance of careful analyses in pure cell populations to better understand the IPF disease mechanism.

Background:The mast cell-specific metalloprotease CPA3 has been given important roles in lung tissue homeostasis and disease pathogenesis. However, the dynamics and spatial distribution of mast cell CPA3 expression in lung diseases remain unknown.Methods:Using a histology-based approach for quantitative spatial decoding of mRNA and protein single cell, this study investigates the dynamics of CPA3 expression across mast cells residing in lungs from control subjects and patients with severe chronic obstructive pulmonary disease (COPD) or idiopathic lung fibrosis (IPF).Results:Mast cells in COPD lungs had an anatomically widespread increase of CPA3 mRNA (bronchioles p < 0.001, pulmonary vessels p < 0.01, and alveolar parenchyma p < 0.01) compared to controls, while granule-stored CPA3 protein was unaltered. IPF lungs had a significant upregulation of both mast cell density, CPA3 mRNA (p < 0.001) and protein (p < 0.05), in the fibrotic alveolar tissue. Spatial expression maps revealed altered mast cell mRNA/protein quotients in lung areas subjected to disease-relevant histopathological alterations. Elevated CPA3 mRNA also correlated to lung tissue eosinophils, CD3 T cells, and declined lung function. Single-cell RNA sequencing of bronchial mast cells confirmed CPA3 as a top expressed gene with potential links to both inflammatory and protective markers.Conclusion:This study shows that lung tissue mast cell populations in COPD and IPF lungs have spatially complex and markedly upregulated CPA3 expression profiles that correlate with immunopathological alterations and lung function. Given the proposed roles of CPA3 in tissue homeostasis, remodeling, and inflammation, these alterations are likely to have clinical consequences.

Aging attenuates the ability of the lung to regenerate lost or damaged tissue and could be responsible for the decline in airway stem cell function. In addition, other naturally occurring aging phenomena such as cellular senescence and epigenetic alterations can impact the progression of many chronic lung diseases (such as COPD and Cancer). Still, how aging affects the human lung on a cellular and molecular level remains unknown. Therefore, it is important to delineate the molecular and cellular changes that occur within the “healthy” aging lung in order to understand and provide a context for the development of lung disease. To elucidate this, we have performed single cell RNA sequencing analysis along with psuedotime analysis on human airway cells from 4 aged healthy non-smoking subjects (65-74 years) and 5 younger healthy non-smoking subjects (20-39 years). The analysis showed that aged basal cells (KRT5+, p63+) expressed significantly higher levels of the AP-1 transcription factor family related genes, as well as an enrichment for genes involved in ROS production and oxidative phosphorylation. Furthermore, the pseudotime analysis identified AGR2 and TFF3, to have an increased expression pattern in ciliated (FOXJ1+) and secretory (SCGB1A1+) aged cells when compared to younger counterparts. In contrast, we found a significant downregulation of genes involved in antigen presentation on the aged ciliated and secretory cells indicating that these cells become less efficient in antigen presentation with age. Taken together, our data reveals that the aging process affects numerous gene and regulatory pathways that need to be further investigated for their involvement in health and disease

Chronic obstructive pulmonary disease (COPD) is characterized by aberrant inflammatory and repair responses, leading to bronchitis, small airway remodeling, and emphysema. It is the third leading cause of death worldwide and is caused by exposure to tobacco smoke or environmental pollutants (1). Importantly, the inflammatory processes in COPD are known to persist long after smoking cessation (2). However, the underlying mechanisms that initiate and drive inflammation are still not clear, and currently, no pharmacological treatments are available to alter the course of the disease. These mechanisms include defective epithelial repair in the airways and alveoli, suggesting that the endogenous lung stem cells may be the initial drivers of the disease. In line with that, research has demonstrated that basal (stem) cells in the airway are among the first cells to be affected by inhaled toxins, resulting in epithelial damage, defective repair, and subsequent inflammation (3, 4). The mechanisms triggering the initiation of this inflammation are not understood, but it is clear that the epithelial cells are in constant communication with the inflammatory cells. It was recently shown that a subpopulation of basal cells can drive the inflammatory processes observed in COPD, suggesting that these inflammatory signals represent a potential therapeutic target to inhibit tissue destruction and promote regeneration of the normal cells that still exist in the airways at end-stage COPD (5, 6). In the alveolar compartment, the alveolar type 2 (AT2) cells are the stem cells responsible for homeostasis and regeneration (Figure 1A). Even though AT2 cells have been evaluated in multiple single-cell OMICS studies (7–9), the mechanistic alterations in AT2 cells in COPD are unknown, and the question remains as to whether a diseasepropagating subpopulation of AT2 cells exists in COPD. Hence, a deeper understanding of the AT2 cells in patients with COPD is needed to understand their contribution to disease. In this issue of the Journal (pp. 708–719), Watanabe and colleagues (10) describe their approach to this problem, which included single-cell RNA sequencing of patients with COPD and yielded interesting findings with regard to the alterations in AT2 cells in COPD. By leveraging the power of single-cell RNA sequencing, they show a comprehensive molecular profile of the distal COPD lung, including the identification of a potential new AT2 cell subpopulation, labeled AT2i (inflammatory) cells, that the authors present as the potential subpopulation responsible for driving distal lung inflammation in COPD. The first question that arises in any article with a focus on single-cell RNA sequencing relates to quality control and sample distribution among all the sequencing-derived clusters and specifically in the population of interest. They produced an unsupervised uniform manifold approximation and projection of all their cells attained from COPD, healthy smokers, and never-smokers. In the uniform manifold approximation and projection, they show that they have a good distribution of their cells with respect to the known markers for distal lung cells in the three sample cohorts. This verification is important, as they proceeded to explore the COPD epithelial cell heterogeneity in greater depth. They explored epithelial cell heterogeneity using a predictive “closeness centrality” analysis that determines how “similar” the cells are to each other. Strikingly, they show that the epithelial cells are more heterogenous in subjects with COPD when compared with smokers without COPD or the never-smoker populations. An immediate question that arises is “what are the cells that are different in COPD when compared with the other populations?” Thus, the authors next looked more closely at the AT2 cells, because of their role in maintaining alveolar homeostasis. They noted that three AT2 cell clusters could be identified in patients with COPD. In these three clusters, they focused on the AT2 cell fraction that increased in correlation to COPD severity and in relation to the expression of inflammatory chemokine-related genes, such as CXCL1, CXCL1, CXCL2, CXCL3, CXCL8, CCL2, and CCL20. These genes were verified at the protein level through coimmunostaining with surfactant protein C. They labeled this population as AT2i cells (Figure 1B). To further validate their results, they integrated their data with publicly available datasets and found cells in one cluster that expressed similar chemokine markers to those associated with AT2i cells, thereby confirming their findings. However, it remains unclear which factors trigger activation of quiescent AT2 cells to AT2i cells and which differentiation trajectory they follow during lung regeneration. To answer that, in part, they used a pseudotime analysis to study gene expression as a function of an artificial time vector, which indicated that the AT2i population undergoes dysfunctional differentiation, with an enrichment of genes driving inflammatory pathways. In addition, to further scrutinize the role of the AT2i cells, Watanbe and colleagues looked into potential immune–response network interactions that could exist between epithelial and immune cells. They found that the strongest immune–epithelial interaction in the AT2i cell population was in COPD when compared with smokers without COPD and neversmokers. Although the work fromWatanabe and colleagues provides intriguing results, several drawbacks remain. One drawback, and this could be said of other single-cell–based analyses, is the small sample size (number of donors and cells per donor). Another drawback is the representation of COPD classifications within this analysis (Global Initiative for Chronic Obstructive Lung Disease I–IV). This would provide further subclassification and context for their findings indicating that the percentage of AT2i population increases with

Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here, as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AML, while FLT3-ITD/mutations conferred resistance. The samples responding to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of the protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. These findings illustrate the value of an unbiased, multiplex screening platform for developing combinatorial therapeutic approaches for AML.

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.

Airway inflammation and remodeling are characteristic features of asthma, with both contributing to airway hyperresponsiveness (AHR) and lung function limitation. Airway smooth muscle (ASM) accumulation and extracellular matrix deposition are characteristic features of airway remodeling, which may contribute to persistent AHR. Laminins containing the α2-chain contribute to characteristics of ASM remodeling in vitro and AHR in animal models of asthma. The role of other laminin chains, including the laminin α4 and α5 chains, which contribute to leukocyte migration in other diseases, is currently unknown. The aim of the current study was to investigate the role of these laminin chains in ASM function and in AHR, remodeling, and inflammation in asthma. Expression of both laminin α4 and α5 was observed in the human and mouse ASM bundle. In vitro, laminin α4 was found to promote a pro-proliferative, pro-contractile, and pro-fibrotic ASM cell phenotype. In line with this, treatment with laminin α4 and α5 function-blocking antibodies reduced allergen-induced increases in ASM mass in a mouse model of allergen-induced asthma. Moreover, eosinophilic inflammation was reduced by the laminin α4 function-blocking antibody as well. Using airway biopsies from healthy subjects and asthmatic patients, we found inverse correlations between ASM α4-chain expression and lung function and AHR, whereas eosinophil numbers correlated positively with expression of laminin α4 in the ASM bundle. This study, for the first time, indicates a prominent role for laminin α4 in ASM function and in inflammation, AHR, and remodeling in asthma, whereas the role of laminin α5 is more subtle.

Mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a protein with anti-inflammatory properties and the archetypal member of the dual-specificity phosphatases (DUSPs) family that have emerged over the past decade as playing an instrumental role in the regulation of airway inflammation. Not only does MKP-1 serve a critical role as a negative feedback effector, controlling the extent and duration of pro-inflammatory MAPK signalling in airway cells, upregulation of this endogenous phosphatase has also emerged as being one of the key cellular mechanism responsible for the beneficial actions of clinically-used respiratory medicines, including β 2 -agonists, phosphodiesterase inhibitors and corticosteroids. Herein, we review the role and regulation of MKP-1 in the context of airway inflammation. We initially outline the structure and biochemistry of MKP-1 and summarise the multi-layered molecular mechanisms responsible for MKP-1 production more generally. We then focus in on some of the key in vitro studies in cell types relevant to airway disease that explain how MKP-1 can be regulated in airway inflammation at the transcriptional, post-translation and post-translational level. And finally, we address some of the potential challenges with MKP-1 upregulation that need to be explored further to fully exploit the potential of MKP-1 to repress airway inflammation in chronic respiratory disease.

Corticosteroids are effective anti-inflammatory therapies widely utilized in chronic respiratory diseases. But these medicines can lose their efficacy during respiratory infection resulting in disease exacerbation. Further in vitro research is required to understand how infection worsens lung function control in order to advance therapeutic options to treat infectious exacerbation in the future. In this study, we utilize a cellular model of bacterial exacerbation where we pretreat A549 lung epithelial cells with the synthetic bacterial lipoprotein Pam3CSK4 (a TLR2 ligand) to mimic bacterial infection and tumor necrosis factor α (TNFα) to simulate inflammation. Under these conditions, Pam3CSK4 induces corticosteroid insensitivity; demonstrated by substantially reduced ability of the corticosteroid dexamethasone to repress TNFα-induced interleukin 6 secretion. We then explored the molecular mechanism responsible and found that corticosteroid insensitivity induced by bacterial mimics was not due to altered translocation of the glucocorticoid receptor into the nucleus, nor an impact on the NF-κB pathway. Moreover, Pam3CSK4 did not affect corticosteroid-induced upregulation of anti-inflammatory MAPK deactivating phosphatase-MKP-1. However, Pam3CSK4 can induce oxidative stress and we show that a proportion of the MKP-1 produced in response to corticosteroid in the context of TLR2 ligation was rendered inactive by oxidation. Thus to combat inflammation in the context of bacterial exacerbation we sought to discover effective strategies that bypassed this road-block. We show for the first time that known (FTY720) and novel (theophylline) activators of the phosphatase PP2A can serve as non-steroidal anti-inflammatory alternatives and/or corticosteroid-sparing approaches in respiratory inflammation where corticosteroid insensitivity exists.

Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti‐inflammatory pharmacotherapies, including corticosteroids, are front‐line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid‐inducible gene, mitogen‐activated protein kinase (MAPK) phosphatase 1 (MKP‐1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP‐1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein—tristetraprolin (TTP). But whether corticosteroid‐induced MKP‐1 acts via p38 MAPK‐mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK‐dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro‐asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL‐6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP‐1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone‐induced MKP‐1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro‐inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153–2158, 2016. © 2016 Wiley Periodicals, Inc.

BACKGROUND:IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2 ) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known.METHODS AND RESULTS:We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells.CONCLUSION:As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.