A vagal reflex evoked by airway closure.

Airway integrity must be continuously maintained throughout life. Sensory neurons guard against airway obstruction and, on a moment-by-moment basis, enact vital reflexes to maintain respiratory function1,2. Decreased lung capacity is common and life-threatening across many respiratory diseases, and lung collapse can be acutely evoked by chest wall trauma, pneumothorax or airway compression. Here we characterize a neuronal reflex of the vagus nerve evoked by airway closure that leads to gasping. In vivo vagal ganglion imaging revealed dedicated sensory neurons that detect airway compression but not airway stretch. Vagal neurons expressing PVALB mediate airway closure responses and innervate clusters of lung epithelial cells called neuroepithelial bodies (NEBs). Stimulating NEBs or vagal PVALB neurons evoked gasping in the absence of airway threats, whereas ablating NEBs or vagal PVALB neurons eliminated gasping in response to airway closure. Single-cell RNA sequencing revealed that NEBs uniformly express the mechanoreceptor PIEZO2, and targeted knockout of Piezo2 in NEBs eliminated responses to airway closure. NEBs were dispensable for the Hering-Breuer inspiratory reflex, which indicated that discrete terminal structures detect airway closure and inflation. Similar to the involvement of Merkel cells in touch sensation3,4, NEBs are PIEZO2-expressing epithelial cells and, moreover, are crucial for an aspect of lung mechanosensation. These findings expand our understanding of neuronal diversity in the airways and reveal a dedicated vagal pathway that detects airway closure to help preserve respiratory function.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Astragaloside IV restores Th17/Treg balance via inhibiting CXCR4 to improve chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has a high fatality rate and poses a great threat to human health. Astragaloside IV (AS-IV) is proven to attenuate cigarette smoke (CS)-induced pulmonary inflammation, based on which this research focuses on the mechanism of AS-IV in COPD. METHODS: To evaluate the effects of AS-IV, CD4+ T cells received different concentrations of AS-IV. CD4+ T cell viability, T helper 17 (Th17)/regulatory T (Treg) markers and CXCR4 expressions in CD4+ T cells or spleen/lung tissues were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, quantitative real-time polymerase chain reaction and Western blot. The proportions of Treg and Th17 cells were assessed by flow cytometry. Enzyme-linked immune sorbent assay was employed to determine cytokine contents in serum and lung tissues. RESULTS: AS-IV with concentration exceeding 40 µM inhibited CD4+ T cell viability. In vitro, AS-IV suppressed the expressions of CXCR4, retinoid-related orphan receptor γt (RORγt), and interleukin (IL)-17A as well as Th17 cells but promoted the expressions of forkhead box p3 (Foxp3) and IL-10 as well as Treg cells, while CXCR4 overexpression reversed the effects of AS-IV. In vivo, AS-IV alleviated COPD, and CS-induced Th17/Treg imbalance in mice and reduced CS-induced down-regulation of IL-10 in serum and lung tissues and Foxp3 and up-regulation of IL-1ß, tumor necrosis factor alpha (TNF-α), IL-6, and IL-17A in serum and lung tissues and RORγt. AS-IV mitigated CS-induced CXCR4 up-regulation. Above effects of AS-IV on mice were offset by CXCR4 overexpression. CONCLUSIONS: AS-IV restores Th17/Treg balance via impeding CXCR4 to ameliorate COPD.

p53 governs an AT1 differentiation programme in lung cancer suppression.

Lung cancer is the leading cause of cancer deaths worldwide1. Mutations in the tumour suppressor gene TP53 occur in 50% of lung adenocarcinomas (LUADs) and are linked to poor prognosis1-4, but how p53 suppresses LUAD development remains enigmatic. We show here that p53 suppresses LUAD by governing cell state, specifically by promoting alveolar type 1 (AT1) differentiation. Using mice that express oncogenic Kras and null, wild-type or hypermorphic Trp53 alleles in alveolar type 2 (AT2) cells, we observed graded effects of p53 on LUAD initiation and progression. RNA sequencing and ATAC sequencing of LUAD cells uncovered a p53-induced AT1 differentiation programme during tumour suppression in vivo through direct DNA binding, chromatin remodelling and induction of genes characteristic of AT1 cells. Single-cell transcriptomics analyses revealed that during LUAD evolution, p53 promotes AT1 differentiation through action in a transitional cell state analogous to a transient intermediary seen during AT2-to-AT1 cell differentiation in alveolar injury repair. Notably, p53 inactivation results in the inappropriate persistence of these transitional cancer cells accompanied by upregulated growth signalling and divergence from lung lineage identity, characteristics associated with LUAD progression. Analysis of Trp53 wild-type and Trp53-null mice showed that p53 also directs alveolar regeneration after injury by regulating AT2 cell self-renewal and promoting transitional cell differentiation into AT1 cells. Collectively, these findings illuminate mechanisms of p53-mediated LUAD suppression, in which p53 governs alveolar differentiation, and suggest that tumour suppression reflects a fundamental role of p53 in orchestrating tissue repair after injury.

Curcumin Induces Ferroptosis in A549 CD133+ Cells through the GSH-GPX4 and FSP1-CoQ10-NAPH Pathways.

BACKGROUND: Cancer stem cells (CSCs) are characterized by an ability for unlimited proliferation and efficiency of self-renewal. The targeting of lung CSCs (LCSCs)-related signaling pathways represent a promising therapeutic strategy for treatment of lung cancer. Ferroptosis a potential strategy for LCSCs treatment, and curcumin cloud induce ferroptosis. In this study, we aimed to observe the effects of curcumin on LCSCs via ferroptosis-related pathways. METHODS: In this study, A549 cluster of differentiation (CD)133+ and A549 CD133- cells were isolated using magnetic bead-based separation. Colony formation and sphere formation assays, as well as cells injection in non-obese diabetes/severe combined immune deficiency (NOD/SCID) mice, were used to analyze the tumorigenic ability of cells differentially expressing CD133. A549 CD133+ cells were treated with different doses of curcumin (0, 10, 20, 40, 80 µM). Cell viability, glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) expressions were measured. The 50% inhibitory concentration (IC50) of curcumin, two ferroptosis inducers, inhibitor of GPX4 (RSL3) and inhibitor of FSP1 (iFSP1), and a ferroptosis inhibitor, ferrostatin-1 (Fer-1), were used to investigate the mechanism underlying the effect of curcumin on ferroptosis in A549 CD133+ cells. RESULTS: A549 CD133+ cells had greater tumorigenic ability than A549 cells. Curcumin treatment suppressed the expressions of GPX4 (glutathione peroxidase 4) and FSP1 in A549 CD133+ cells, thereby inducing ferroptosis. RSL3 and iFSP1 respectively suppressed the GSH (glutathione)-GPX4 and FSP1 (ferroptosis suppressor protein 1)-CoQ10 (coenzyme Q10)-nicotinamide adenine dinucleotide (NADH) pathways in A549 CD133+ cells. However, the roles of curcumin were blocked by Fer-1 treatment. CONCLUSIONS: In this study, curcumin induced ferroptosis through inhibiting the GSH-GPX4 and FSP1-CoQ10-NADH pathways in A549 CD133+ cells, resulting in the inhibition of their self-renewal potential.

Carbon nanotubes: Structural defects as stressors inducing lung cell toxicity.

Lung toxicity of carbon nanotubes (CNTs) is matter of concern since very long time. However, their mechanism of toxicity is still not yet well defined. In this work, the role of structural defects as organic stressors of CNTs able to trigger their potential toxicity is investigated. Four commercial CNTs, with different carbon purity grade, are morphologically characterized by transmission electron microscopy (TEM) and the relative amount of structural defects are estimated through Raman spectroscopy, by measuring the intensity ratio D/G (ID/IG). The oxidative potential of CNTs is evaluated with cytochrome-C assay and reactive oxygen species (ROS) detection. Data show that CNTs with larger amounts of structural defects (higher ID/IG ratio) induce an increased ROS generation and consequent cytotoxicity and cellular damage, shown by TEM images of CNTs-cells interaction. Raman analyses of cells exposed to CNTs point out that the spectra of the CNTs inside the cells show no differences with respect of the signal recorded for cell-free CNTs, evidencing their biopersistence in lung cells. Raman spectra cannot provide direct indication of the existence of metals as impurity. It follows that the intensity ratio ID/IG can be taken as a predictive marker of the toxicity of a given CNT.

MMP-3-mediated cleavage of OPN is involved in copper oxide nanoparticle-induced activation of fibroblasts.

BACKGROUND: Copper oxide nanoparticles (Nano-CuO) are one of the most produced and used nanomaterials. Previous studies have shown that exposure to Nano-CuO caused acute lung injury, inflammation, and fibrosis. However, the mechanisms underlying Nano-CuO-induced lung fibrosis are still unclear. Here, we hypothesized that exposure of human lung epithelial cells and macrophages to Nano-CuO would upregulate MMP-3, which cleaved osteopontin (OPN), resulting in fibroblast activation and lung fibrosis. METHODS: A triple co-culture model was established to explore the mechanisms underlying Nano-CuO-induced fibroblast activation. Cytotoxicity of Nano-CuO on BEAS-2B, U937* macrophages, and MRC-5 fibroblasts were determined by alamarBlue and MTS assays. The expression or activity of MMP-3, OPN, and fibrosis-associated proteins was determined by Western blot or zymography assay. Migration of MRC-5 fibroblasts was evaluated by wound healing assay. MMP-3 siRNA and an RGD-containing peptide, GRGDSP, were used to explore the role of MMP-3 and cleaved OPN in fibroblast activation. RESULTS: Exposure to non-cytotoxic doses of Nano-CuO (0.5 and 1 µg/mL) caused increased expression and activity of MMP-3 in the conditioned media of BEAS-2B and U937* cells, but not MRC-5 fibroblasts. Nano-CuO exposure also caused increased production of cleaved OPN fragments, which was abolished by MMP-3 siRNA transfection. Conditioned media from Nano-CuO-exposed BEAS-2B, U937*, or the co-culture of BEAS-2B and U937* caused activation of unexposed MRC-5 fibroblasts. However, direct exposure of MRC-5 fibroblasts to Nano-CuO did not induce their activation. In a triple co-culture system, exposure of BEAS-2B and U937* cells to Nano-CuO caused activation of unexposed MRC-5 fibroblasts, while transfection of MMP-3 siRNA in BEAS-2B and U937* cells significantly inhibited the activation and migration of MRC-5 fibroblasts. In addition, pretreatment with GRGDSP peptide inhibited Nano-CuO-induced activation and migration of MRC-5 fibroblasts in the triple co-culture system. CONCLUSIONS: Our results demonstrated that Nano-CuO exposure caused increased production of MMP-3 from lung epithelial BEAS-2B cells and U937* macrophages, which cleaved OPN, resulting in the activation of lung fibroblasts MRC-5. These results suggest that MMP-3-cleaved OPN may play a key role in Nano-CuO-induced activation of lung fibroblasts. More investigations are needed to confirm whether these effects are due to the nanoparticles themselves and/or Cu ions.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates.

We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models.

CREB1 Transcriptionally Activates LTBR to Promote the NF-κB Pathway and Apoptosis in Lung Epithelial Cells.

Bronchopulmonary dysplasia (BPD) is a prevalent chronic pediatric lung disease. Aberrant proliferation and apoptosis of lung epithelial cells are important in the pathogenesis of BPD. Lymphotoxin beta receptor (LTBR) is expressed in lung epithelial cells. Blocking LTBR induces regeneration of lung tissue and reverts airway fibrosis in young and aged mice. This study is aimed at revealing the role of LTBR in BPD. A mouse model of BPD and two in vitro models of BPD using A549 cells and type II alveolar epithelial (ATII) cells were established by exposure to hyperoxia. We found that LTBR and CREB1 exhibited a significant upregulation in lungs of mouse model of BPD. LTBR and CREB1 expression were also increased by hyperoxia in A549 and ATII cells. According to results of cell counting kit-8 assay and flow cytometry analysis, silencing of LTBR rescued the suppressive effect of hyperoxia on cell viability and its promotive effect on cell apoptosis of A549 and ATII cells. Bioinformatics revealed CREB1 as a transcriptional factor for LTBR, and the luciferase reporter assay and ChIP assay subsequently confirmed it. The NF-κB pathway was regulated by LTBR. CREB1 induced LTBR expression at the transcriptional level to regulate NF-κB pathway and further modulate A549 and ATII cells viability and apoptosis. In conclusion, this study revealed the CREB1/LTBR/NF-κB pathway in BPD and supported the beneficial role of LTBR silence in BPD by promoting viability and decreasing apoptosis of lung epithelial cells.

Cofilin-1 and profilin-1 expression in lung microvascular endothelial cells exposed to titanium dioxide nanoparticles.

BACKGROUND: Air pollutants exacerbate chronic airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). However, the underlying mechanisms are yet to be determined. While a number of studies have reported adverse effects of nanoparticles on humans, little is known about their effects on the respiratory system. OBJECTIVES: To examine the protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to titanium dioxide (TiO2) nanoparticles, a common air pollutant. MATERIAL AND METHODS: A proteomics approach using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to determine the differences in protein expression at 8 h and 24 h, following the treatment of HMVEC-L with 20-µM or 40-µM TiO2 nanoparticles. RESULTS: Human lung microvascular endothelial cells treated with 20-µM TiO2 nanoparticles showed alterations of 7 protein spots, including molecules related to calcium regulation, transport, cytoskeleton, and muscle contraction. The treatment of HMVEC-L with 40-µM TiO2 nanoparticles resulted in alterations of 4 protein spots, with molecular functions related to the cytoskeleton, myosin regulation, actin modulation, as well as guanosine diphosphate (GDP) and guanosine triphosphate (GTP) regulation. To validate these results, immunohistochemical staining and western blotting analyses were performed on lung tissues collected from mice exposed to TiO2 nanoparticles. Cofilin-1 and profilin-1 were expressed in the endothelium, epithelium and inflammatory cells, and decreased in lung tissues of TiO2 nanoparticle-exposed mice compared to sham-treated controls. CONCLUSIONS: These results suggest that some of the differentially expressed proteins may play important roles in airway diseases caused by TiO2 nanoparticle exposure.

Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts.

The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.

Tumor-polarized GPX3+ AT2 lung epithelial cells promote premetastatic niche formation.

The cellular and molecular components required for the formation of premetastatic niche (PMN) to promote lung metastasis need to be further investigated. Lung epithelial cells have been reported to exhibit immunomodulatory roles in lung homeostasis and also to mediate immunosuppressive PMN formation in lung metastasis. Here, by single-cell sequencing, we identified a tumor-polarized subpopulation of alveolar type 2 (AT2) epithelial cells with increased expression of glutathione peroxidase 3 (GPX3) and high production of interleukin (IL)-10 in the PMN. IL-10-producing GPX3+ AT2 cells inhibited CD4+ T cell proliferation but enhanced regulatory T cell generation. Mechanistically, tumor exosome-inducing GPX3 expression is required for GPX3+ AT2 cells to preferentially produce IL-10 by stabilizing hypoxia-inducible factor 1 (HIF-1α) and promoting HIF-1α-induced IL-10 production. Accordingly, conditional knockout of GPX3 in AT2 cells suppressed lung metastasis in spontaneous metastatic models. Together, our findings reveal a role of tumor-polarized GPX3+ AT2 cells in promoting lung PMN formation, adding insights into immune evasion in lung metastasis and providing potential targets for the intervention of tumor metastasis.

In Vivo Profiling of Individual Multiciliated Cells during Acute Influenza A Virus Infection.

Influenza virus infections are thought to be initiated in a small number of cells; however, the heterogeneity across the cellular responses of the epithelial cells during establishment of disease is incompletely understood. Here, we used an H1N1 influenza virus encoding a fluorescent reporter gene, a cell lineage-labeling transgenic mouse line, and single-cell RNA sequencing to explore the range of responses in a susceptible epithelial cell population during an acute influenza A virus (IAV) infection. Focusing on multiciliated cells, we identified a subpopulation that basally expresses interferon-stimulated genes (ISGs), which we hypothesize may be important for the early response to infection. We subsequently found that a population of infected ciliated cells produce most of the ciliated cell-derived inflammatory cytokines, and nearly all bystander ciliated cells induce a broadly antiviral state. From these data together, we propose that variable preexisting gene expression patterns in the initial cells targeted by the virus may ultimately affect the establishment of viral disease. IMPORTANCE Influenza A virus poses a significant threat to public health, and each year, millions of people in the United States alone are exposed to the virus. We do not currently, however, fully understand why some individuals clear the infection asymptomatically and others become severely ill. Understanding how these divergent phenotypes arise could eventually be leveraged to design therapeutics that prevent severe disease. As a first step toward understanding these different infection states, we used a technology that allowed us to determine how thousands of individual murine lung epithelial cells behaved before and during IAV infection. We found that small subsets of epithelial cells exhibited an antiviral state prior to infection, and similarly, some cells made high levels of inflammatory cytokines during infection. We propose that different ratios of these individual cellular responses may contribute to the broader antiviral state of the lung and may ultimately affect disease severity.

SIRT1-mediated p53 deacetylation inhibits ferroptosis and alleviates heat stress-induced lung epithelial cells injury.

OBJECTIVE: Acute lung injury (ALI) is a common complication of heat stroke (HS) and a direct cause of death. However, the mechanism underlying ALI following HS remains unclear. METHOD: To investigate whether ferroptosis is involved in HS-ALI. We established a HS model of mice and mouse lung epithelial-2 cells (MLE-2). The severity of lung injury was measured by H&E staining, the wet-to-dry lung weight ratio, and Transmission electron microscopy. Potential markers of ferroptosis Fe2+, malondialdehyde (MDA), hydroxynonenal (4-HNE) and lipid peroxidation were detected. The percentages of cell death and viability induced by HS were assessed by LDH and CCK8 assays. SLC7A11, ACSL4, GPX4, SIRT1, p53, and p53 K382 acetylation levels were measured by Western blot. RESULTS: The administration of ferroptosis inhibitor ferrostatin-1(Fer-1) could significantly ameliorate lung injury, inhibiting levels of MDA and 4-HNE, and ameliorating HS-induced increased ACSL4, decreased SLC7A11 and GPX4, suggesting ferroptosis was involved in HS-induced ALI in vivo and in vitro. Moreover, SIRT1 expression decreased, and p53 K382 acetylation levels increased in MLE-2 cells. Activation of SIRT1 could improve lung epithelial ferroptosis caused by HS in vivo ang in vitro. Besides, the activation of SIRT1 could significantly reduce the p53 K382 acetylation levels, suggesting that activation of SIRT1 could prevent ferroptosis via inhibiting p53 acetylation. CONCLUSION: These findings substantiate the vital role of the SIRT1/p53 axis in mediating ferroptosis in HS-ALI, suggesting that targeting SIRT1 may represent a novel therapeutic strategy to ameliorate ALI during HS.

Human distal lung maps and lineage hierarchies reveal a bipotent progenitor.

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

Effect of α-tocopherol in alleviating the lipopolysaccharide-induced acute lung injury via inhibiting nuclear factor kappa-B signaling pathways.

Acute respiratory distress syndrome (ARDS) leads to the acute lung injury (ALI), a form of diffused alveolars injury, accompanied by severe inflammation and oxidative damage of alveolar epithelial cells. α-Tocopherol (α-TOH), one of the eight isoforms of vitamin E, is a natural antioxidant-free radical. We aimed to understand the effect of α-TOH and mechanism involved in inducing the ALI. Lipopolysaccharide (LPS) is injected into the trachea of mice to generate ALI mouse models. α-TOH was used to administrate the mice intragastrically to detect the expression of inflammatory factors and antioxidant molecules by enzyme linked immunosorbent assay, hematoxylin-eosin staining and immunohistochemical staining. Mouse alveolar epithelial cell line (MLE-12 cells) was used to determine the effect of α-TOH on alveolar epithelial cells. Inflammatory factors such as, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α shows significant increase in the lung tissues of the mice induced by LPS and reduction in the expressions of superoxide dismutase (SOD)1/2 and glutathione peroxidase (GSH-Px). After treatment with α-TOH, the inflammation and oxidative stress levels shows substantial reduction in the lung tissues of the mice. Moreover, α-TOH also increases the proliferation ability of MLE-12 cells in vitro and reduces apoptosis level. In addition, α-TOH reduces p65 phosphorylation and nuclear translocation in alveolar epithelial cells in vivo and in vitro, thus, inhibiting the activity of the nuclear factor kappa-B (NF-κB) signaling pathway. α-TOH reduces the inflammation and oxidative stress of lung tissue by inhibiting the NF-κB signaling pathway, thereby alleviating the LPS-induced ALI.

Knockdown of receptor interacting protein 140 (RIP140) alleviated lipopolysaccharide-induced inflammation, apoptosis and permeability in pulmonary microvascular endothelial cells by regulating C-terminal binding protein 2 (CTBP2).

The main pathological feature of acute lung injury (ALI) is pulmonary edema caused by increased permeability of pulmonary microvascular endothelial cells (PMVECs). LPS was has been confirmed to lead to cell damage and barrier dysfunction in PMVECs. Furthermore, receptor interacting protein 140 (RIP140) was discovered to be increased in LPS-induced human pulmonary microvascular endothelial cells (HPMECs), but the mechanism of RIP140 on LPS-induced HPMECs has not been investigated. In this study, an acute lung injury model was constructed in LPS-induced HPMECs. After RIP140 was downregulated, inflammation, apoptosis and cell permeability levels were detected by RT-qPCR, TUNEL staining and FITC-Dextran, respectively. Western blotting was used to detect the protein levels of related factors. The binding of RIP140 and C-terminal binding protein 2 (CTBP2) was predicted by database and verified by Co-IP. Subsequently, CTBP2 overexpression was transfected into cells and the above experiments were performed again. The results showed that inflammation, apoptosis and permeability levels of LPS-induced HPMECs were remarkably increased compared to the untreated control group. However, these levels were suppressed after RIP140 was silenced compared to the LPS-induced HPMECs group. Notably, the Co-IP study demonstrated that RIP140 and CTBP2 interacted with each other. Moreover, CTBP2 overexpression reversed the inhibitory effects of RIP140 silencing on LPS-induced inflammation, apoptosis and permeability levels in HPMECs. Together, the study found that interference of RIP140 could alleviate LPS-induced inflammation, apoptosis and permeability in HPMECs by regulating CTBP2.