Stem cell migration drives lung repair in living mice.

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 cells (AT2s) act as stem cells to replenish both themselves and alveolar type 1 cells (AT1s); however, the complex orchestration of stem cell activity after injury is poorly understood. Here, we establish longitudinal imaging of AT2s in murine intact tissues ex vivo and in vivo in order to track their dynamic behavior over time. We discover that a large fraction of AT2s become motile following injury and provide direct evidence for their migration between alveolar units. High-resolution morphokinetic mapping of AT2s further uncovers the emergence of distinct motile phenotypes. Inhibition of AT2 migration via genetic depletion of ArpC3 leads to impaired regeneration of AT2s and AT1s in vivo. Together, our results establish a requirement for stem cell migration between alveolar units and identify properties of stem cell motility at high cellular resolution.

Ventilatory capacity in CLAD is driven by dysfunctional airway structure.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) encompasses three main phenotypes: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS) and a Mixed phenotype combining both pathologies. How the airway structure in its entirety is affected in these phenotypes is still poorly understood. METHODS: A detailed analysis of airway morphometry was applied to gain insights on the effects of airway remodelling on the distribution of alveolar ventilation in end-stage CLAD. Ex vivo whole lung µCT and tissue-core µCT scanning of six control, six BOS, three RAS and three Mixed explant lung grafts (9 male, 9 female, 2014-2021, Leuven, Belgium) were used for digital airway reconstruction and calculation of airway dimensions in relation to luminal obstructions. FINDINGS: BOS and Mixed explants demonstrated airway obstructions of proximal bronchioles (starting at generation five), while RAS explants particularly had airway obstructions in the most distal bronchioles (generation >12). In BOS and Mixed explants 76% and 84% of bronchioles were obstructed, respectively, while this was 22% in RAS. Bronchiolar obstructions were mainly caused by lymphocytic inflammation of the airway wall or fibrotic remodelling, i.e. constrictive bronchiolitis. Proximal bronchiolectasis and imbalance in distal lung ventilation were present in all CLAD phenotypes and explain poor lung function and deterioration of specific lung function parameters. INTERPRETATION: Alterations in the structure of conducting bronchioles revealed CLAD to affect alveolar ventilatory distribution in a regional fashion. The significance of various obstructions, particularly those associated with mucus, is highlighted. FUNDING: This research was funded with the National research fund Flanders (G060322N), received by R.V.

Mucosal immune alterations at the early onset of tissue destruction in chronic obstructive pulmonary disease.

Rationale: COPD is characterized by chronic airway inflammation, small airways changes, with disappearance and obstruction, and also distal/alveolar destruction (emphysema). The chronology by which these three features evolve with altered mucosal immunity remains elusive. This study assessed the mucosal immune defense in human control and end-stage COPD lungs, by detailed microCT and RNA transcriptomic analysis of diversely affected zones. Methods: In 11 control (non-used donors) and 11 COPD (end-stage) explant frozen lungs, 4 cylinders/cores were processed per lung for microCT and tissue transcriptomics. MicroCT was used to quantify tissue percentage and alveolar surface density to classify the COPD cores in mild, moderate and severe alveolar destruction groups, as well as to quantify terminal bronchioles in each group. Transcriptomics of each core assessed fold changes in innate and adaptive cells and pathway enrichment score between control and COPD cores. Immunostainings of immune cells were performed for validation. Results: In mildly affected zones, decreased defensins and increased mucus production were observed, along CD8+ T cell accumulation and activation of the IgA pathway. In more severely affected zones, CD68+ myeloid antigen-presenting cells, CD4+ T cells and B cells, as well as MHCII and IgA pathway genes were upregulated. In contrast, terminal bronchioles were decreased in all COPD cores. Conclusion: Spatial investigation of end-stage COPD lungs show that mucosal defense dysregulation with decreased defensins and increased mucus and IgA responses, start concomitantly with CD8+ T-cell accumulation in mild emphysema zones, where terminal bronchioles are already decreased. In contrast, adaptive Th and B cell activation is observed in areas with more advanced tissue destruction. This study suggests that in COPD innate immune alterations occur early in the tissue destruction process, which affects both the alveoli and the terminal bronchioles, before the onset of an adaptive immune response.

iDESC: identifying differential expression in single-cell RNA sequencing data with multiple subjects.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) technology has enabled assessment of transcriptome-wide changes at single-cell resolution. Due to the heterogeneity in environmental exposure and genetic background across subjects, subject effect contributes to the major source of variation in scRNA-seq data with multiple subjects, which severely confounds cell type specific differential expression (DE) analysis. Moreover, dropout events are prevalent in scRNA-seq data, leading to excessive number of zeroes in the data, which further aggravates the challenge in DE analysis. RESULTS: We developed iDESC to detect cell type specific DE genes between two groups of subjects in scRNA-seq data. iDESC uses a zero-inflated negative binomial mixed model to consider both subject effect and dropouts. The prevalence of dropout events (dropout rate) was demonstrated to be dependent on gene expression level, which is modeled by pooling information across genes. Subject effect is modeled as a random effect in the log-mean of the negative binomial component. We evaluated and compared the performance of iDESC with eleven existing DE analysis methods. Using simulated data, we demonstrated that iDESC had well-controlled type I error and higher power compared to the existing methods. Applications of those methods with well-controlled type I error to three real scRNA-seq datasets from the same tissue and disease showed that the results of iDESC achieved the best consistency between datasets and the best disease relevance. CONCLUSIONS: iDESC was able to achieve more accurate and robust DE analysis results by separating subject effect from disease effect with consideration of dropouts to identify DE genes, suggesting the importance of considering subject effect and dropouts in the DE analysis of scRNA-seq data with multiple subjects.

COVID-19 progression in hospitalized patients using follow-up in vivo CT and ex vivo microCT.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19) which can lead to acute respiratory distress syndrome (ARDS) and evolve to pulmonary fibrosis. Computed tomography (CT) is used to study disease progression and describe radiological patterns in COVID-19 patients. This study aimed to assess disease progression regarding lung volume and density over time on follow-up in vivo chest CT and give a unique look at parenchymal and morphological airway changes in "end-stage" COVID-19 lungs using ex vivo microCT. Methods: Volumes and densities of the lung/lobes of three COVID-19 patients were assessed using follow-up in vivo CT and ex vivo whole lung microCT scans. Airways were quantified by airway segmentations on whole lung microCT and small-partition microCT. As controls, three discarded healthy donor lungs were used. Histology was performed in differently affected regions in the COVID-19 lungs. Results: In vivo, COVID-19 lung volumes decreased while density increased over time, mainly in lower lobes as previously shown. Ex vivo COVID-19 lung volumes decreased by 60% and all lobes were smaller compared to controls. Airways were more visible on ex vivo microCT in COVID-19, probably due to fibrosis and increased airway diameter. In addition, small-partition microCT showed more deformation of (small) airway morphology and fibrotic organization in severely affected regions with heterogeneous distributions within the same lung which was confirmed by histology. Conclusions: COVID-19-ARDS and subsequent pulmonary fibrosis alters lung architecture and airway morphology which is described using in vivo CT, ex vivo microCT, and histology.

A Multi-omic Analysis of the Human Lung Reveals Distinct Cell Specific Aging and Senescence Molecular Programs.

Age is a major risk factor for lung disease. To understand the mechanisms underlying this association, we characterized the changing cellular, genomic, transcriptional, and epigenetic landscape of lung aging using bulk and single-cell RNAseq (scRNAseq) data. Our analysis revealed age-associated gene networks that reflected hallmarks of aging, including mitochondrial dysfunction, inflammation, and cellular senescence. Cell type deconvolution revealed age-associated changes in the cellular composition of the lung: decreased alveolar epithelial cells and increased fibroblasts and endothelial cells. In the alveolar microenvironment, aging is characterized by decreased AT2B cells and reduced surfactant production, a finding that was validated by scRNAseq and IHC. We showed that a previously reported senescence signature, SenMayo, captures cells expressing canonical senescence markers. SenMayo signature also identified cell-type specific senescence-associated co-expression modules that have distinct molecular functions, including ECM regulation, cell signaling, and damage response pathways. Analysis of somatic mutations showed that burden was highest in lymphocytes and endothelial cells and was associated with high expression of senescence signature. Finally, aging and senescence gene expression modules were associated with differentially methylated regions, with inflammatory markers such as IL1B, IL6R, and TNF being significantly regulated with age. Our findings provide new insights into the mechanisms underlying lung aging and may have implications for the development of interventions to prevent or treat age-related lung diseases.

microRNA-33 deficiency in macrophages enhances autophagy, improves mitochondrial homeostasis, and protects against lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein (SREBP) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.

Cluster analysis of transcriptomic datasets to identify endotypes of idiopathic pulmonary fibrosis.

BACKGROUND: Considerable clinical heterogeneity in idiopathic pulmonary fibrosis (IPF) suggests the existence of multiple disease endotypes. Identifying these endotypes would improve our understanding of the pathogenesis of IPF and could allow for a biomarker-driven personalised medicine approach. We aimed to identify clinically distinct groups of patients with IPF that could represent distinct disease endotypes. METHODS: We co-normalised, pooled and clustered three publicly available blood transcriptomic datasets (total 220 IPF cases). We compared clinical traits across clusters and used gene enrichment analysis to identify biological pathways and processes that were over-represented among the genes that were differentially expressed across clusters. A gene-based classifier was developed and validated using three additional independent datasets (total 194 IPF cases). FINDINGS: We identified three clusters of patients with IPF with statistically significant differences in lung function (p=0.009) and mortality (p=0.009) between groups. Gene enrichment analysis implicated mitochondrial homeostasis, apoptosis, cell cycle and innate and adaptive immunity in the pathogenesis underlying these groups. We developed and validated a 13-gene cluster classifier that predicted mortality in IPF (high-risk clusters vs low-risk cluster: HR 4.25, 95% CI 2.14 to 8.46, p=3.7×10-5). INTERPRETATION: We have identified blood gene expression signatures capable of discerning groups of patients with IPF with significant differences in survival. These clusters could be representative of distinct pathophysiological states, which would support the theory of multiple endotypes of IPF. Although more work must be done to confirm the existence of these endotypes, our classifier could be a useful tool in patient stratification and outcome prediction in IPF.

Unagi: Deep Generative Model for Deciphering Cellular Dynamics and In-Silico Drug Discovery in Complex Diseases.

Human diseases are characterized by intricate cellular dynamics. Single-cell sequencing provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in-silico drug interventions. Here, we introduce UNAGI, a deep generative neural network tailored to analyze time-series single-cell transcriptomic data. This tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modeling and discovery. When applied to a dataset from patients with Idiopathic Pulmonary Fibrosis (IPF), UNAGI learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation via proteomics reveals the accuracy of UNAGI's cellular dynamics analyses, and the use of the Fibrotic Cocktail treated human Precision-cut Lung Slices confirms UNAGI's predictions that Nifedipine, an antihypertensive drug, may have antifibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including a COVID dataset, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond IPF, amplifying its utility in the quest for therapeutic solutions across diverse pathological landscapes.

Characterization of the COPD alveolar niche using single-cell RNA sequencing.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

BAL Transcriptomes Characterize Idiopathic Pulmonary Fibrosis Endotypes With Prognostic Impact.

BACKGROUND: Given the plethora of pathophysiologic mechanisms described in idiopathic pulmonary fibrosis (IPF), we hypothesize that the mechanisms driving fibrosis in IPF may be different from one patient to another. RESEARCH QUESTION: Do IPF endotypes exist and are they associated with outcome? STUDY DESIGN AND METHODS: Using a publicly available gene expression dataset retrieved from BAL samples of patients with IPF and control participants (GSE70867), we clustered IPF samples based on a dimension reduction algorithm specifically designed for -omics data, called DDR Tree. After clustering, gene set enrichment analysis was performed for functional annotation, associations with clinical variables and prognosis were investigated, and differences in transcriptional regulation were determined using motif enrichment analysis. The findings were validated in three independent publicly available gene expression datasets retrieved from IPF blood samples. RESULTS: One hundred seventy-six IPF samples from three centers were clustered in six IPF clusters, with distinct functional enrichment. Although clinical characteristics did not differ between the clusters, one cluster conferred worse sex-age-physiology score-corrected survival, whereas another showed a numeric trend toward worse survival (P = .08). The first was enriched for increased epithelial and innate and adaptive immunity signatures, whereas the other showed important telomere and mitochondrial dysfunction, loss of proteostasis, and increased myofibroblast signatures. The existence of these two endotypes, including the impact on survival of the immune endotype, was validated in three independent validation cohorts. Finally, we identified transcription factors regulating the expression of endotype-specific survival-associated genes. INTERPRETATION: Gene expression-based endotyping in IPF is feasible and can inform clinical evolution. As endotype-specific pathways and survival-associated transcription factors are identified, endotyping may open up the possibility of endotype-tailored therapy.

Lung Microenvironments and Disease Progression in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis (fHP) is an interstitial lung disease caused by sensitization to an inhaled allergen. Objectives: To identify the molecular determinants associated with progression of fibrosis. Methods: Nine fHP explant lungs and six unused donor lungs (as controls) were systematically sampled (4 samples/lung). According to microcomputed tomography measures, fHP cores were clustered into mild, moderate, and severe fibrosis groups. Gene expression profiles were assessed using weighted gene co-expression network analysis, xCell, gene ontology, and structure enrichment analysis. Gene expression of the prevailing molecular traits was also compared with idiopathic pulmonary fibrosis (IPF). The explant lung findings were evaluated in separate clinical fHP cohorts using tissue, BAL samples, and computed tomography scans. Measurements and Main Results: We found six molecular traits that associated with differential lung involvement. In fHP, extracellular matrix and antigen presentation/sensitization transcriptomic signatures characterized lung zones with only mild structural and histological changes, whereas signatures involved in honeycombing and B cells dominated the transcriptome in the most severely affected lung zones. With increasing disease severity, endothelial function was progressively lost, and progressive disruption in normal cellular homeostatic processes emerged. All six were also found in IPF, with largely similar associations with disease microenvironments. The molecular traits correlated with in vivo disease behavior in a separate clinical fHP cohort. Conclusions: We identified six molecular traits that characterize the morphological progression of fHP and associate with in vivo clinical behavior. Comparing IPF with fHP, the transcriptome landscape was determined considerably by local disease extent rather than by diagnosis alone.

Oral Health Stakeholders: A Time for Alignment and Action.

Policy Points Since the Surgeon General's report in 2000, multiple stakeholder groups have engaged in advocacy to expand access to oral health coverage, integrate medicine and dentistry, and to improve the dental workforce. Using a stakeholder map across these three policy priorities, we describe how stakeholder groups are shaping the oral health policy landscape in this century. While the stakeholders are numerous, policy has changed little despite invested efforts and resources. To achieve change, multiple movements must coalesce around common goals and messages and a champion must emerge to lead the way. The ongoing COVID-19 pandemic and political changes due to the 2020 elections can open a window of opportunity to unite stakeholders to achieve comprehensive policy change.

The transition from normal lung anatomy to minimal and established fibrosis in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: The transition from normal lung anatomy to minimal and established fibrosis is an important feature of the pathology of idiopathic pulmonary fibrosis (IPF). The purpose of this report is to examine the molecular and cellular mechanisms associated with this transition. METHODS: Pre-operative thoracic Multidetector Computed Tomography (MDCT) scans of patients with severe IPF (n = 9) were used to identify regions of minimal(n = 27) and established fibrosis(n = 27). MDCT, Micro-CT, quantitative histology, and next-generation sequencing were used to compare 24 samples from donor controls (n = 4) to minimal and established fibrosis samples. FINDINGS: The present results extended earlier reports about the transition from normal lung anatomy to minimal and established fibrosis by showing that there are activations of TGFBI, T cell co-stimulatory genes, and the down-regulation of inhibitory immune-checkpoint genes compared to controls. The expression patterns of these genes indicated activation of a field immune response, which is further supported by the increased infiltration of inflammatory immune cells dominated by lymphocytes that are capable of forming lymphoid follicles. Moreover, fibrosis pathways, mucin secretion, surfactant, TLRs, and cytokine storm-related genes also participate in the transitions from normal lung anatomy to minimal and established fibrosis. INTERPRETATION: The transition from normal lung anatomy to minimal and established fibrosis is associated with genes that are involved in the tissue repair processes, the activation of immune responses as well as the increased infiltration of CD4, CD8, B cell lymphocytes, and macrophages. These molecular and cellular events correlate with the development of structural abnormality of IPF and probably contribute to its pathogenesis.

Small airway loss in the physiologically ageing lung: a cross-sectional study in unused donor lungs.

BACKGROUND: Physiological lung ageing is associated with a gradual decline in dynamic lung volumes and a progressive increase in residual volume due to diminished elastic recoil of the lung, loss of alveolar tissue, and lower chest wall compliance. However, the effects of ageing on the small airways (ie, airways <2·0 mm in diameter) remain largely unknown. By using a combination of ex-vivo conventional CT (resolution 1 mm), whole lung micro-CT (resolution 150 µm), and micro-CT of extracted cores (resolution 10 µm), we aimed to provide a multiresolution assessment of the small airways in lung ageing in a large cohort of never smokers. METHODS: For this cross-sectional study, we included donor lungs collected from 32 deceased never-smoking donors (age range 16-83 years). Ex-vivo CT and whole lung high-resolution CT (micro-CT) were used to determine total airway numbers, stratified by airway diameter. Micro-CT was used to assess the number, length, and diameter of terminal bronchioles (ie, the last generation of conducting airways); mean linear intercept; and surface density in four lung tissue cores from each lung, extracted using a uniform sampling approach. Regression ß coefficients are calculated using linear regression and polynomial models. FINDINGS: Ex-vivo CT analysis showed an age-dependent decrease in the number of airways of diameter 2·0 mm to less than 2·5 mm (ß coefficient per decade -0·119, 95% CI -0·193 to -0·045; R2=0·29) and especially in airways smaller than 2·0 mm in diameter (-0·158, -0·233 to -0·084; R2=0·47), between 30 and 80 years of age, but not of the larger (≥2·5 mm) diameter airways (-0·00781, -0·04409 to 0·02848; R2=0·0007). In micro-CT analysis of small airways, the total number of terminal bronchioles per lung increased until the age of 30 years, after which an almost linear decline in the number of terminal bronchioles was observed (ß coefficient per decade -2035, 95% CI -2818 to -1252; R2=0·55), accompanied by a non-significant increase in alveolar airspace size (6·44, -0·57 to 13·45, R2=0·10). Moreover, this decrease in terminal bronchioles was associated with the age-related decline of pulmonary function predicted by healthy reference values. INTERPRETATION: Loss of terminal bronchioles is an important structural component of age-related decline in pulmonary function of healthy, non-smoking individuals. FUNDING: Research Foundation-Flanders, KU Leuven, Parker B Francis Foundation, UGent, Canadian Institutes for Health.

Profibrotic epithelial TGF-ß1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-ß (TGF-ß) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-ß signaling is not known. Here, we demonstrate that TGF-ß1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-ß1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-ß-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-ß1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.