Human Lung Tissue Research Articles

Reduced Cathepsin L expression and secretion into the extracellular milieu contribute to lung fibrosis in systemic sclerosis.

Lung fibrosis is the leading cause of death in SSc, with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis. Fibrosis was induced experimentally using TGF-β in vitro, in primary human lung fibroblasts (pLFs), and ex vivo, in human lung tissues. ES and CTSL expression was quantified using ELISA, RT-qPCR, immunoblotting or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues. ES levels were significantly reduced in media conditioned by TGF-β-induced pLFs. TGF-β-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles, further reducing its availability in the ECM. Media conditioned by TGF-β-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL. Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy.

A Practical Valorization Approach for Mitigating Textile Fibrous Microplastics in the Environment: Collection of Textile-Processing Waste Microfibers and Direct Reuse in Green Thermal-Insulating and Mechanical-Performing Composite Construction Materials

Microplastic (MP) contamination is an urgent environmental issue to address. Fibrous microplastics (FMPs) are the principal MP type in the air and have already been found in human stool and lung tissues. FMPs are generated from the lifecycle of synthetic and blended textiles and are expected to increase due to fast fashion. Among textile processes, the finishing of fabrics is estimated to generate 5000 t/year of textile waste fibers in Italy, including FMPs. To limit FMPs spread, this paper suggests, for the first time, the direct collection of blended finishing textile waste microfibers and reuse in designing thermal-insulating and mechanical-performing fiber-reinforced cementitious composites (FRCs). The microfibers were thoroughly characterized (size, morphology, composition, and density), and their use in FRCs was additionally evaluated by considering water absorption and release capacity. Untreated, water-saturated, and NaOH-treated microfibers were considered in FRCs up to 4 wt%. Up to a +320% maximum bending load, +715% toughness, −80% linear shrinkage, and double-insulating power of Portland cement were observed by increasing microfiber contents. NaOH-treated and water-saturated microfibers better enhanced toughness and linear shrinkage reduction. Therefore, green and performant composite construction materials were obtained, allowing for the mitigation of more than 4 kg FMPs per ton of cement paste. This is a great result considering the FMP contamination (i.e., 2–8 kg/day fallout in Paris), and that FRCs are promising and shortly-widely used construction materials.

SARS-CoV-2 Omicron Induces Enhanced Mucosal Interferon Response Compared to other Variants of Concern, Associated with Restricted Replication in Human Lung Tissues.

SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC-a difference, which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Notably, blocking the innate immune signaling restored Omicron replication in the lung tissues. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.

Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission.

The first dominant SARS‐CoV‐2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS‐CoV‐2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co‐circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan‐Hu‐1 and Delta variant. However, severity is believed to be less owing to the reduced syncytia formation and lower multiplication in the human lung tissue. Perhaps most challenging is that several studies indicate that the efficacy of the available vaccines have been reduced against Omicron variant (8–127 times reduction) as compared to the Wuhan‐Hu‐1 variant. The administration of booster vaccine, however, compensates with the reduction and improves the efficacy by 12–35 fold. Concerningly though, the broadly neutralising monoclonal antibodies, including those approved by FDA for therapeutic use against previous SARS‐CoV‐2 variants, are mostly ineffective against Omicron with the exception of Sotrovimab and recent reports suggest that the Omicron BA.2 is also resistant to Sotrovimab. Currently two new Omicron variants BA.4 and BA.5 are emerging and are reported to be more transmissible and resistant to immunity generated by previous variants including Omicron BA.1 and most monoclonal antibodies. As new variants of SARS‐CoV‐2 will likely continue to emerge it is important that the evolution, and biological consequences of new mutations, in existing variants be well understood.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1β and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

A layer-level multi-scale architecture for lung cancer classification with fluorescence lifetime imaging endomicroscopy

In this paper, we introduce our unique dataset of fluorescence lifetime imaging endo/microscopy (FLIM), containing over 100,000 different FLIM images collected from 18 pairs of cancer/non-cancer human lung tissues of 18 patients by our custom fibre-based FLIM system. The aim of providing this dataset is that more researchers from relevant fields can push forward this particular area of research. Afterwards, we describe the best practice of image post-processing suitable per the dataset. In addition, we propose a novel hierarchically aggregated multi-scale architecture to improve the binary classification performance of classic CNNs. The proposed model integrates the advantages of multi-scale feature extraction at different levels, where layer-wise global information is aggregated with branch-wise local information. We integrate the proposal, namely ResNetZ, into ResNet, and appraise it on the FLIM dataset. Since ResNetZ can be configured with a shortcut connection and the aggregations by Addition or Concatenation, we first evaluate the impact of different configurations on the performance. We thoroughly examine various ResNetZ variants to demonstrate the superiority. We also compare our model with a feature-level multi-scale model to illustrate the advantages and disadvantages of multi-scale architectures at different levels.

Abstract 1586: An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma

Abstract Lung adenocarcinomas (LUAD) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1586.

Abstract 3865: Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues. We have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and identified T cell subsets within tumors using CD3 and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues. Citation Format: Anushka Dikshit, Sayantani Basak, Ching-Wei Chang, Michaeline Bunting, Kim Collins. Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3865.

Mesencephalic astrocyte-derived neurotrophic factor attenuates acute lung injury via inhibiting macrophages' activation.

Acute lung injury (ALI) is an urgent respiratory disease without effective treatment. Mesencephalic astrocyte-derived neurotrophic factor (MANF)has been demonstrated to play a suppressive role in some inflammatory conditions. However, the effect of MANF on ALI has not yet been reported. In this study, we collected bronchoalveolar lavage fluid (BALF) from the patients with or without pulmonary inflammation, and used lipopolysaccharide (LPS) to induce mice ALI model. Mono-macrophage-specific MANF knockout (MKO) mice were constructed and recombinant human MANF protein was used to ALI mice. We found that the endogenous MANF protein in both human BALF and mice lung tissues was increased in inflammatory conditions. MANF level in the macrophages of inflammatory lung was higher than that in normal controls in both human and mice. MANF deficiency in macrophages induced lung inflammation and aggravated LPS-induced lung injury. MANF lowered LPS-induced lung injury, inhibited macrophage polarization to M1 functional type. Meanwhile, MANF inhibited-LPS induced activation of NF-κB signal pathway by down regulating phosphorylated p65in lung tissue and macrophages. These results indicate that MANF acts as a suppressor in ALI via negatively regulating NF-κB activation and macrophages polarization, which may be a novel potential target and shed light on ALI therapy.

Getting a grip on microplastics’ risks

Tiny plastic particles less than 5 mm in diameter are popping up nearly everywhere scientists look. They are in water, food, and air, as well as in human blood, lung tissue, and stool. But it is unclear whether micro- and nanosized plastics pose any risks to human health or the environment. Researchers are starting to investigate sources of microplastics. They are also exploring how the particles affect human cells in test systems that mimic digestion and inhalation. But microplastics are complex mixtures with different shapes, sizes, and chemistries, making them difficult to study. Because of limited exposure data, regulators are struggling to understand the risks. The situation is reminiscent of the challenges faced by researchers studying the toxicology of engineered nanomaterials more than a decade ago. Lessons learned from nanotoxicology could help pave a better path to assess the risks of microplastics. When scientists at the US National Institute of

A local human Vδ1 T cell population is associated with survival in nonsmall-cell lung cancer

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA−CD27− effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

Human Lung Tissue Implanted on the Chick Chorioallantoic Membrane as a Novel In Vivo Model of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first invivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.

Abstract PR006: An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma

Abstract Lung adenocarcinomas (LUADs) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR006.

Hedgehog Signaling Pathway Orchestrates Human Lung Branching Morphogenesis.

The Hedgehog (HH) signaling pathway plays an essential role in mouse lung development. We hypothesize that the HH pathway is necessary for branching during human lung development and is impaired in pulmonary hypoplasia. Single-cell, bulk RNA-sequencing data, and human fetal lung tissues were analyzed to determine the spatiotemporal localization of HH pathway actors. Distal human lung segments were cultured in an air-liquid interface and treated with an SHH inhibitor (5E1) to determine the effect of HH inhibition on human lung branching, epithelial-mesenchymal markers, and associated signaling pathways in vitro. Our results showed an early and regulated expression of HH pathway components during human lung development. Inhibiting HH signaling caused a reduction in branching during development and dysregulated epithelial (SOX2, SOX9) and mesenchymal (ACTA2) progenitor markers. FGF and Wnt pathways were also disrupted upon HH inhibition. Finally, we demonstrated that HH signaling elements were downregulated in lung tissues of patients with a congenital diaphragmatic hernia (CDH). In this study, we show for the first time that HH signaling inhibition alters important genes and proteins required for proper branching of the human developing lung. Understanding the role of the HH pathway on human lung development could lead to the identification of novel therapeutic targets for childhood pulmonary diseases.

Identification of condition-specific regulatory mechanisms in normal and cancerous human lung tissue.

BackgroundLung cancer is the leading cause of cancer death in both men and women. The most common lung cancer subtype is non-small cell lung carcinoma (NSCLC) comprising about 85% of all cases. NSCLC can be further divided into three subtypes: adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and large cell lung carcinoma. Specific genetic mutations and epigenetic aberrations play an important role in the developmental transition to a specific tumor subtype. The elucidation of normal lung versus lung tumor gene expression patterns and regulatory targets yields biomarker systems that discriminate lung phenotypes (i.e., biomarkers) and provide a foundation for the discovery of normal and aberrant gene regulatory mechanisms.ResultsWe built condition-specific gene co-expression networks (csGCNs) for normal lung, LUAD, and LUSC conditions. Then, we integrated normal lung tissue-specific gene regulatory networks (tsGRNs) to elucidate control-target biomarker systems for normal and cancerous lung tissue. We characterized co-expressed gene edges, possibly under common regulatory control, for relevance in lung cancer.ConclusionsOur approach demonstrates the ability to elucidate csGCN:tsGRN merged biomarker systems based on gene expression correlation and regulation. The biomarker systems we describe can be used to classify and further describe lung specimens. Our approach is generalizable and can be used to discover and interpret complex gene expression patterns for any condition or species.Supplementary InformationThe online version contains available at 10.1186/s12864-022-08591-9.

Quantitative Expression of Drug, Phospholipid and Nucleoside Transporters in the Lung Tissues across Species

ObjectiveIt has been proven that transporters play a pivotal role in organ distribution of drugs, and subsequently in drug efficacy and toxicity. Active transport processes may affect the local pulmonary disposition of drugs administered directly to the lung by the inhaled route as well as the uptake of drugs to the lung from the systemic circulation. While drug transporter expressions and their roles in drug disposition and elimination in the major elimination organs including intestine, liver and kidney are well‐characterized, the expression of drug transporters and the comparison across species in the lung remain overall poorly understood. For example, azithromycin levels in pulmonary tissue remained to be high for up to 5 days after a single oral dose, in contrast to much lower plasma levels. However, the mechanisms associated with azithromycin lung loading and efficacy remain unknown. The aim of this study was to comprehensively determine the mRNA and protein expression levels of drug transporters in the lung in human and preclinical species using quantitative targeted proteomics approaches. The findings can be useful to further explore potential mechanisms in lung specific disposition.MethodsTotal cellular mRNA was isolated from human lower lung tissues using a Qiagen mRNA isolation kit. qRT‐PCR was performed, and β‐actin mRNA expression was used to normalize target gene expression. The mRNA levels were expressed as a fold change using the 2‐ΔΔCt method. The total membrane proteins of lung tissues from human, monkey, dog and rat were extracted by Native Membrane Protein Extraction Kit, and subsequently digested by trypsin followed by the treatment of DTT and iodoacetamide (IAA). The levels of transporter proteins were quantified using a Sciex 7500 triple‐quadruple mass spectrometer coupled to a Shimadzu LC (SLC‐30A) system with optimized ionization conditions. The Na K‐ATPase level in the lung was measured as the membrane protein marker.Results and conclusionIn the current investigation, thirteen ABC efflux transporters and twenty‐three solute carriers (SLC) were characterized. Among those, mRNAs of eleven ABC transporters and fifteen SLC transporters were detected by the RT‐PCR method. In addition, eight ABC transporter and eleven SLC transporter proteins were quantified using the targeted proteomics LC‐MS/MS method. The mRNA expression pattern agrees with the protein expression; however, no quantitative correlation can be drawn between mRNA and protein expression. While OATP2B1 was only detected in human lung tissues, OATP2A1 and 3A1 are expressed in the lungs of human, monkey dog and rat. The expressions of three equilibrative nucleoside transporters (ENT1/2/3) in human lung tissues were higher than those in monkey. The expression of ABCA1, ABCA3 and ABCG1, the ABC transporters that are the regulators of cellular cholesterol and phospholipid homeostasis, were detected in all tested species, and found to be high in rat. Collectively, the comparison of transporter expression profiles can contribute to a better understanding of drug distribution and transport in the lung and aid pulmonary drug discovery and development.

Using Droplet Digital PCR to Detect Cyanobacteria in Human Lung Tissue

Objective and HypothesisCyanobacteria produce a variety of secondary metabolites, including toxins, that are harmful to human and animal health. The objective of this study is to develop a quantitative method that can reliably detect cyanobacteria in human lung tissue. We propose that with cyanobacteria‐specific 16S rDNA primers, droplet digital PCR (ddPCR) can provide a reliable and sensitive method to detect Cyanobacteria in human tissue.MethodsTwo cyanobacteria strains were used in this validation: a toxin‐producing strain, Microcystis aeruginosa (LB 2385), and a non‐toxin‐producing, strain Synechococcus sp. (LB 2390). Escherichia coli served as a negative bacterial control. DNA was extracted from cultures and frozen, human lung tissue with DNeasy Blood & Tissue kit (QIAGEN) and quantified using the dsDNA High‐Sensitivity Assay (Invitrogen) on a Qubit 3.0 fluorometer. The ddPCR reactions consisted of FAM labelled cyanobacterial 16S rDNA target primer/probe and HEX labelled RPP30 reference primer/probe (Bio‐RAD). Oil droplets were generated using the Bio‐Rad Automated Droplet Generator and the emulsified PCRs were run in a 96‐well plate on a C1000 Touch™ Thermal Cycler. The plates were read on a Bio‐Rad QX200 droplet reader and the data analyzed using the QuantaSoft v1.7.4 software. The Limit of Detection (LOD) was found by initially diluting LB 2385 and LB 2390 to 4 ng/mcL in nuclease‐free water, followed by serial 2x dilutions until it was below the detection limit by ddPCR. This was repeated using normal human lung tissue instead of nuclease‐free water.ResultsThe ddPCR assay developed in this study showed high specificity and sensitivity in detecting both strains of cyanobacteria. Under both conditions tested (spiking in water vs 10 ng human DNA (hDNA)), LB 2385 DNA could be reliably detected at a concentration of 61 fg/mcL, whereas LB 2390 DNA could be reliably detected at a concentration of 31 fg/mcL. This corresponds to 1‐2 copies/mcL of cyanobacterial DNA in the original samples. In the presence of hDNA, the sensitivity for cyanobacterial DNA decreased as the concentration of hDNA increased (2‐30 ng/mcL), possibly due to a physical barrier or competition between the primer/probes. In the hDNA condition, RPP30 could be reliably detected at 2‐3 copies/mcL with a concentration of ~60 pg/mcL. RPP30 is valuable for normalizing sample input, reducing biases caused by DNA quantification variations and pipetting errors. A strong linear correlation between sample input and the number of positive droplets (i.e., the concentration detected by ddPCR) was observed for both cyanobacteria 16S rDNA and RPP30 DNA targets. This assay also demonstrated high intra‐ and inter‐assay reproducibility.ConclusionsA highly specific and sensitive ddPCR assay was developed to detect the presence of cyanobacteria in human lung tissue. The ability to detect cyanobacteria in human samples will allow further research into the role cyanobacteria plays in human health and disease.

Chlorine inhalation toxicology‐on‐a‐chip

Accidental or environmental exposures to chlorine (Cl2 ) gas represent a major health threat. In particular, inhalation toxicology of Cl2 is an area of active research that focuses on investigating the deleterious effects of Cl2 on the human respiratory system and developing effective countermeasures to mitigate the risk of Cl2 -induced lung injury. While traditional cell culture models have gained widespread use as a simple and convenient research tool for in vitro investigation of Cl2 toxicity, they have limited capacity to recapitulate the complexity of native human lung tissues and thus largely fail to generate accurate predictions on pulmonary responses to Cl2 . Here we present two distinct microengineered biomimetic models that reconstruct the small airway and alveolar regions of the human lung for experimental studies of respiratory toxicity of Cl2 in the distal lung, which remains an outstanding question in inhalation toxicology of Cl2 . Our microengineered small airway model is composed of two microfabricated 3D chambers separated by a semipermeable membrane. The design of this microdevice makes it possible to mimic tissue compartmentalization in native airways by permitting long-term co-culture of primary human small airway epithelial cells, primary human pulmonary microvascular endothelial cells, and lung fibroblasts in a physiological 3D microenvironment to engineer fully differentiated airway epithelium directly exposed to air and supported by vascularized, perfusable 3D stromal tissues. The alveolar model, which is constructed in the same device, uses primary human type II pneumocytes harvested from iPSC-derived human alveolar organoids, some of which are transdifferentiated into type I alveolar epithelial cells during the course of controlled microfluidic culture in our device. In this study, these models were capable of producing human lung tissues that exhibited differentiated phenotype and functional capacity that could be maintained for extended periods (over 1 month). Notably, our data revealed the potential of the underlying pulmonary vasculature to accelerate and promote differentiation and maturation of lung epithelial cells during air-liquid interface culture. Using the lung-on-a-chip systems in conjunction with an in-house Cl2 exposure system, we conducted a preliminary study in which the devices were exposed to various concentrations of Cl2 gas (10, 50, 100 and 300 ppm) for 15 min. Our preliminary data showed the deleterious potential of Cl2 to induce acute injury of both small airway and alveolar tissues in a dose-dependent manner. Studies are underway for more extensive screening of Cl2 toxicity and in-depth analysis of lung injury in our models at the molecular, cellular, and tissue levels. We believe that the microengineered systems developed in this work represent an important advance in our ability to model acute respiratory responses to toxic gases and may provide a potentially powerful in vitro platform to study biochemical threats and develop medical countermeasures against them.

Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation.

Extracellular vesicles (EVs) are single‐membrane vesicles that play an essential role in long‐range intercellular communications. EV investigation has been explored largely through cell‐culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single‐cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV‐DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA‐TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle‐mediated systematic communications.

HH pathway orchestrates the epithelial‐mesenchymal crosstalk during human lung branching morphogenesis

RationaleHedgehog (HH) pathway plays an essential role in lung branching morphogenesis in rodents. However, little is known about the role of this pathway in human lung development. We hypothesize that HH pathway is necessary for branching during human lung development and hence in pulmonary hypoplasia.MethodsExpression of HH pathway components was analyzed in single cell and bulk RNA‐sequencing data from publicly available lung datasets. Fresh human fetal lung tissues between gestational ages 10‐18 weeks were collected. RT‐qPCR, fluorescent in situ hybridization (FISH) and immunofluorescence staining (IF) were performed to determine spatiotemporal localization of HH pathway actors. In addition, distal lung segments were cultured in air‐liquid interface for up to 48 hours in the presence or absence of HH inhibitor (5E1). RT‐qPCR, western blot and IF were performed to investigate the effect of HH pathway inhibition on epithelial‐mesenchymal markers and associated signaling pathways.ResultsBulk and scRNA‐seq analysis showed an early expression of HH pathway components during human lung development. We noted an inverse expression between activators and inhibitors receptors (PTCH1/HHIP) over time. PTCH1 had a higher expression at 20 weeks compared to 14 weeks while the opposite was observed for HHIP. HHIP FISH staining was restricted to ACTA2+ airway smooth muscle cells while PTCH1 was primarily localized in mesenchymal populations adjacent to the tip of airway epithelial buds where SHH ligand is produced. Inhibiting HH signaling ex vivo reduced branching during development (2.5‐fold vs control, p<0.01, n=8). Consequently, SOX9expression, an epithelial progenitor marker, was increased whereas ACTA2 was decreased (respectively 1.5‐ and 2‐fold vs control, p<0.05, n=8). These results were confirmed by western blot and IF staining. No change was observed for SOX2 gene expression while a significant increase in protein expression was observed. FGF10 increased following HH inhibition (1.5‐fold vs control, p<0.01, n=8), but the expression of ETV4and 5 decreased (1.5‐fold vs control, p<0.05, n=8). Finally, we demonstrated that HH signaling elements (including SHH, PTCH1, GLI2) were downregulated in lung tissues from congenital diaphragmatic hernia patients.ConclusionWe have determined for the first‐time gene expression and localization of HH pathway during human lung morphogenesis. Moreover, we showed that HH signaling inhibition alters important genes and proteins expression, such as epithelial‐mesenchymal progenitors and associated pathways, required for proper branching processes. Understanding the role of HH pathway on human lung development could lead to the identification of novel therapeutic targets for childhood pulmonary diseases.