The aging lung: Physiology, disease, and immunity.

The population is aging at a rate never seen before in human history. As the number of elderly adults grows, it is imperative we expand our understanding of the underpinnings of aging biology. Human lungs are composed of a unique panoply of cell types that face ongoing chemical, mechanical, biological, immunological, and xenobiotic stress over a lifetime. Yet, we do not fully appreciate the mechanistic drivers of lung aging and why age increases the risk of parenchymal lung disease, fatal respiratory infection, and primary lung cancer. Here, we review the molecular and cellular aspects of lung aging, local stress response pathways, and how the aging process predisposes to the pathogenesis of pulmonary disease. We place these insights into context of the COVID-19 pandemic and discuss how innate and adaptive immunity within the lung is altered with age.

May the (Mechanical) Force Be with AT2.

Idiopathic pulmonary fibrosis is a fatal disease involving destruction of the lung alveolar structure. In this issue of Cell, Wu et al. (2020) show that impaired alveolar (AT2) stem cells produce mechanical tension that leads to spatially regulated fibrosis, initiating a new chapter in understanding what underlies the periphery to center progression of this lung disease.

Don't Stop Re-healin'! Cancer as an Ongoing Stem Cell Affair.

Tumors have long been suspected of hijacking stem cell mechanisms used for tissue maintenance and repair. Ge et al. now show that skin tumors exhibit merged chromatin profiles from distinct stem cell lineages. This "lineage infidelity" recreates a state akin to transient wound repair that persists to maintain uncontrolled growth.

Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis.

Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal 3D cocultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell cocultures. Gain- and loss-of-function experiments showed that BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1 null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide tools to understand the mechanisms of respiratory diseases at the single-cell level.

Early-stage lung cancer is driven by a transitional cell state dependent on a KRAS-ITGA3-SRC axis.

Glycine-12 mutations in the GTPase KRAS (KRASG12) are an initiating event for development of lung adenocarcinoma (LUAD). KRASG12 mutations promote cell-intrinsic rewiring of alveolar type-II progenitor (AT2) cells, but to what extent such changes interplay with lung homeostasis and cell fate pathways is unclear. Here, we generated single-cell RNA-seq (scRNA-seq) profiles from AT2-mesenchyme organoid co-cultures, mice, and stage-IA LUAD patients, identifying conserved regulators of AT2 transcriptional dynamics and defining the impact of KRASG12D mutation with temporal resolution. In AT2WT organoids, we found a transient injury/plasticity state preceding AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics, most notably retention of the injury/plasticity state. The injury state in AT2KRAS cells of patients, mice, and organoids was distinguishable from AT2WT states via altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors impaired AT2KRAS organoid growth. Together, our data show that an injury/plasticity state essential for lung repair is co-opted during AT2 self-renewal and LUAD initiation, suggesting that early-stage LUAD may be susceptible to interventions that target specifically the oncogenic nature of this cell state.

Author Correction: EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

We wish to correct two mutations in Supplementary Table 4 of this Letter. The NCI-H460 cell line was annotated as being mutant for TP53. NCI-H460 has been verified to be TP53 wild type by several sources1. The NCI-H2009 cell line was annotated as being mutant for PIK3CA. As annotated by COSMIC (ref. 24 of the original Letter) and CCLE (ref. 25 of the original Letter), the NCI-H2009 cell line has a mutation in PIK3C3, rather than PIK3CA. The cell line is wild type for PIK3CA. The Supplementary Information of this Amendment contains the corrected Supplementary Table 4. These errors do not affect our conclusions. The original Letter has not been corrected.

Threat of resurgence or hope for global eradication of poliovirus?

PURPOSE OF REVIEW: Recent outbreaks of poliomyelitis in countries that have been free of cases for decades highlight the challenges of eradicating polio in a globalized interconnected world beset with a novel viral pandemic. We provide an epidemiological update, advancements in vaccines, and amendments in public health strategy of poliomyelitis in this review. RECENT FINDINGS: Last year, new cases of wild poliovirus type 1 (WPV1) were documented in regions previously documented to have eradicated WPV1 and reports of circulating vaccine-derived poliovirus type 2 (cVDPV2) and 3 (cVDPV3) in New York and Jerusalem made international headlines. Sequencing of wastewater samples from environmental surveillance revealed that the WPV1 strains were related to WPV1 lineages from endemic countries and the cVDPV2 strains from New York and Jerusalem were not only related to each other but also to environmental isolates found in London. The evidence of importation of WPV1 cases from endemic countries, and global transmission of cVDPVs justifies renewed efforts in routine vaccination programs and outbreak control measures that were interrupted by the COVID-19 pandemic. After the novel oral poliovirus vaccine type 2 (nOPV2) received emergency authorization for containment of cVDPV2 outbreaks in 2021, subsequent reduced incidence, transmission rates, and vaccine adverse events, alongside increased genetic stability of viral isolates substantiates the safety and efficacy of nOPV2. The nOPV1 and nOPV3 vaccines, against type 1 and 3 cVDPVs, and measures to increase accessibility and efficacy of inactivated poliovirus vaccine (IPV) are in development. SUMMARY: A revised strategy utilizing more genetically stable vaccine formulations, with uninterrupted vaccination programs and continued active surveillance optimizes the prospect of global poliomyelitis eradication.

Application of VirCapSeq-VERT and BacCapSeq in the diagnosis of presumed and definitive neuroinfectious diseases.

Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and the complexity of bioinformatic analysis. Here we report the use of virus capture-based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Thirty-four samples were categorized: (1) patients with definitive CNS infection by routine testing; (2) patients meeting clinically the Brighton criteria (BC) for meningoencephalitis; (3) patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT, and DNA extracts were used for BacCapSeq analysis. Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3, 11/26 samples (42%) were positive for at least one pathogen; however, 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess the clinical value of this novel technique, clinicians should understand the benefits and limitations of using this modality.

COVID-19 neuropathology at Columbia University Irving Medical Center/New York Presbyterian Hospital.

Many patients with SARS-CoV-2 infection develop neurological signs and symptoms; although, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical centre. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit. Hospital-associated complications were common, including eight patients (20%) with deep vein thrombosis/pulmonary embolism, seven (17%) with acute kidney injury requiring dialysis and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 h of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischaemic changes in all brains, both global and focal; large and small infarcts, many of which appeared haemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, although none showed evidence of vasculitis. Eighteen patients (44%) exhibited pathologies of neurodegenerative diseases, which was not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR, RNAscope® and immunocytochemistry with primers, probes and antibodies directed against the spike and nucleocapsid regions. The PCR analysis revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in the nasal epithelia. RNAscope® and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in coronavirus disease 2019 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but more likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischaemia. Further studies are needed to define whether these pathologies, if present in patients who survive coronavirus disease 2019, might contribute to chronic neurological problems.

National Heart, Lung, and Blood Institute and Building Respiratory Epithelium and Tissue for Health (BREATH) Consortium Workshop Report: Moving Forward in Lung Regeneration.

The National Heart, Lung, and Blood Institute of the National Institutes of Health, together with the Longfonds BREATH consortium, convened a working group to review the field of lung regeneration and suggest avenues for future research. The meeting took place on May 22, 2019, at the American Thoracic Society 2019 conference in Dallas, Texas, United States, and brought together investigators studying lung development, adult stem-cell biology, induced pluripotent stem cells, biomaterials, and respiratory disease. The purpose of the working group was 1) to examine the present status of basic science approaches to tackling lung disease and promoting lung regeneration in patients and 2) to determine priorities for future research in the field.

Clinical use of steroids in viral central nervous system (CNS) infections: three challenging cases.

The role of adjunctive corticosteroids in reducing morbidity and mortality of viral CNS infections remains poorly defined. Clinicians are often left in a quagmire regarding steroid use in complex and rapidly evolving viral CNS infections. Limited studies have explored the underlying mechanisms behind the potential benefit of steroids. Here, we describe steroid use in three cases of viral CNS disease: varicella zoster virus (VZV), Powassan virus, and influenza A-associated acute necrotizing encephalopathy.

Localized semi-nonnegative matrix factorization (LocaNMF) of widefield calcium imaging data.

Widefield calcium imaging enables recording of large-scale neural activity across the mouse dorsal cortex. In order to examine the relationship of these neural signals to the resulting behavior, it is critical to demix the recordings into meaningful spatial and temporal components that can be mapped onto well-defined brain regions. However, no current tools satisfactorily extract the activity of the different brain regions in individual mice in a data-driven manner, while taking into account mouse-specific and preparation-specific differences. Here, we introduce Localized semi-Nonnegative Matrix Factorization (LocaNMF), a method that efficiently decomposes widefield video data and allows us to directly compare activity across multiple mice by outputting mouse-specific localized functional regions that are significantly more interpretable than more traditional decomposition techniques. Moreover, it provides a natural subspace to directly compare correlation maps and neural dynamics across different behaviors, mice, and experimental conditions, and enables identification of task- and movement-related brain regions.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

Non-small-cell lung cancer is the leading cause of cancer-related death worldwide. Chemotherapies such as the topoisomerase II (TopoII) inhibitor etoposide effectively reduce disease in a minority of patients with this cancer; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the polycomb repressive complex 2 (PRC2) is well known to tri-methylate histone H3 at lysine 27 (H3K27me3) and elicit gene silencing. Here we demonstrate that EZH2 inhibition has differential effects on the TopoII inhibitor response of non-small-cell lung cancers in vitro and in vivo. EGFR and BRG1 mutations are genetic biomarkers that predict enhanced sensitivity to TopoII inhibitor in response to EZH2 inhibition. BRG1 loss-of-function mutant tumours respond to EZH2 inhibition with increased S phase, anaphase bridging, apoptosis and TopoII inhibitor sensitivity. Conversely, EGFR and BRG1 wild-type tumours upregulate BRG1 in response to EZH2 inhibition and ultimately become more resistant to TopoII inhibitor. EGFR gain-of-function mutant tumours are also sensitive to dual EZH2 inhibition and TopoII inhibitor, because of genetic antagonism between EGFR and BRG1. These findings suggest an opportunity for precision medicine in the genetically complex disease of non-small-cell lung cancer.

Prematurity negatively affects regenerative properties of human amniotic epithelial cells in the context of lung repair.

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFß2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/ß-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFß transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFß and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFß deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

PP2A methylation controls sensitivity and resistance to ß-amyloid-induced cognitive and electrophysiological impairments.

Elevated levels of the ß-amyloid peptide (Aß) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer's disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aß exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aß-induced impairments. Neither transgene affected Aß production or the electrophysiological response to low concentrations of Aß, suggesting that these manipulations selectively affect the pathological response to elevated Aß levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aß levels in AD patients.

E-Cadherin Loss Accelerates Tumor Progression and Metastasis in a Mouse Model of Lung Adenocarcinoma.

Metastatic disease is the primary cause of death of patients with lung cancer, but the mouse models of lung adenocarcinoma do not accurately recapitulate the tumor microenvironment or metastatic disease observed in patients. In this study, we conditionally deleted E-cadherin in an autochthonous lung adenocarcinoma mouse model driven by activated oncogenic Kras and p53 loss. Loss of E-cadherin significantly accelerated lung adenocarcinoma progression and decreased survival of the mice. Kras;p53;E-cadherin mice had a 41% lung tumor burden, invasive grade 4 tumors, and a desmoplastic stroma just 8 weeks after tumor initiation. One hundred percent of the mice developed local metastases to the lymph nodes or chest wall, and 38% developed distant metastases to the liver or kidney. Lung adenocarcinoma cancer cell lines derived from these tumors also had high migratory rates. These studies demonstrate that the Kras;p53;E-cadherin mouse model better emulates the tumor microenvironment and metastases observed in patients with lung adenocarcinoma than previous models and may therefore be useful for studying metastasis and testing new lung cancer treatments in vivo.