Biophysical forces mediated by respiration maintain lung alveolar epithelial cell fate.

Lungs undergo mechanical strain during breathing, but how these biophysical forces affect cell fate and tissue homeostasis are unclear. We show that biophysical forces through normal respiratory motion actively maintain alveolar type 1 (AT1) cell identity and restrict these cells from reprogramming into AT2 cells in the adult lung. AT1 cell fate is maintained at homeostasis by Cdc42- and Ptk2-mediated actin remodeling and cytoskeletal strain, and inactivation of these pathways causes a rapid reprogramming into the AT2 cell fate. This plasticity induces chromatin reorganization and changes in nuclear lamina-chromatin interactions, which can discriminate AT1 and AT2 cell identity. Unloading the biophysical forces of breathing movements leads to AT1-AT2 cell reprogramming, revealing that normal respiration is essential to maintain alveolar epithelial cell fate. These data demonstrate the integral function of mechanotransduction in maintaining lung cell fate and identifies the AT1 cell as an important mechanosensor in the alveolar niche.

Human distal airways contain a multipotent secretory cell that can regenerate alveoli.

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease.

The COPD GWAS gene ADGRG6 instructs function and injury response in human iPSC-derived type II alveolar epithelial cells.

Emphysema and chronic obstructive pulmonary disease (COPD) most commonly result from the effects of environmental exposures in genetically susceptible individuals. Genome-wide association studies have implicated ADGRG6 in COPD and reduced lung function, and a limited number of studies have examined the role of ADGRG6 in cells representative of the airway. However, the ADGRG6 locus is also associated with DLCO/VA, an indicator of gas exchange efficiency and alveolar function. Here, we sought to evaluate the mechanistic contributions of ADGRG6 to homeostatic function and disease in type 2 alveolar epithelial cells. We applied an inducible CRISPR interference (CRISPRi) human induced pluripotent stem cell (iPSC) platform to explore ADGRG6 function in iPSC-derived AT2s (iAT2s). We demonstrate that ADGRG6 exerts pleiotropic effects on iAT2s including regulation of focal adhesions, cytoskeleton, tight junctions, and proliferation. Moreover, we find that ADGRG6 knockdown in cigarette smoke-exposed iAT2s alters cellular responses to injury, downregulating apical complexes in favor of proliferation. Our work functionally characterizes the COPD GWAS gene ADGRG6 in human alveolar epithelium.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Airway derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD.

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

Lung regeneration: a tale of mice and men.

The respiratory system is the main site of gas exchange with the external environment in complex terrestrial animals. Within the trachea and lungs are multiple different tissue niches each consisting of a myriad of cells types with critical roles in air conduction, gas exchange, providing important niche specific cell-cell interactions, connection to the cardiovascular system, and immune surveillance. How the respiratory system responds to external insults and executes the appropriate regenerative response remains challenging to study given the plethora of cell and tissue interactions for this to occur properly. This review will examine the various cell types and tissue niches found within the respiratory system and provide a comparison between mouse and human lungs and trachea to highlight important similarities and differences. Defining the critical gaps in knowledge in human lung and tracheal regeneration is critical for future development of therapies directed towards respiratory diseases.

Activation of STING Signaling Pathway Effectively Blocks Human Coronavirus Infection.

The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.IMPORTANCE The highly infectious and lethal SARS-CoV-2 is posing an unprecedented threat to public health. Other coronaviruses are likely to jump from a nonhuman animal to humans in the future. Novel broad-spectrum antiviral therapeutics are therefore needed to control known pathogenic coronaviruses such as SARS-CoV-2 and its newly mutated variants, as well as future coronavirus outbreaks. STING signaling is a well-established host defense pathway, but its role in coronavirus infection remains unclear. In the present study, we found that activation of the STING signaling pathway robustly inhibits infection of HCoV-OC43 and SARS-CoV-2. These results identified the STING pathway as a novel target for controlling the spread of known pathogenic coronaviruses, as well as emerging coronavirus outbreaks.

Whole blood RNA sequencing reveals a unique transcriptomic profile in patients with ARDS following hematopoietic stem cell transplantation.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is characterized by the acute onset of hypoxemia and bilateral lung infiltrates in response to an inciting event, and is associated with high morbidity and mortality. Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for ARDS. We hypothesized that HSCT patients with ARDS would have a unique transcriptomic profile identifiable in peripheral blood compared to those that did not undergo HSCT. METHODS: We isolated RNA from banked peripheral blood samples from a biorepository of critically ill ICU patients. RNA-Seq was performed on 11 patients with ARDS (5 that had undergone HSCT and 6 that had not) and 12 patients with sepsis without ARDS (5 that that had undergone HCST and 7 that had not). RESULTS: We identified 687 differentially expressed genes between ARDS and ARDS-HSCT (adjusted p-value < 0.01), including IFI44L, OAS3, LY6E, and SPATS2L that had increased expression in ARDS vs. ARDS-HSCT; these genes were not differentially expressed in sepsis vs sepsis-HSCT. Gene ontology enrichment analysis revealed that many differentially expressed genes were related to response to type I interferon. CONCLUSIONS: Our findings reveal significant differences in whole blood transcriptomic profiles of patients with non-HSCT ARDS compared to ARDS-HSCT patients and point toward different immune responses underlying ARDS and ARDS-HSCT that contribute to lung injury.

Lung repair and regeneration: Advanced models and insights into human disease.

The respiratory system acts as both the primary site of gas exchange and an important sensor and barrier to the external environment. The increase in incidences of respiratory disease over the past decades has highlighted the importance of developing improved therapeutic approaches. This review will summarize recent research on the cellular complexity of the mammalian respiratory system with a focus on gas exchange and immunological defense functions of the lung. Different models of repair and regeneration will be discussed to help interpret human and animal data and spur the investigation of models and assays for future drug development.

Dysregulated alveolar epithelial cell progenitor function and identity in Hermansky-Pudlak syndrome pulmonary fibrosis.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of endosomal protein trafficking associated with pulmonary fibrosis in specific subtypes, including HPS-1 and HPS-2. Single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity while double mutant HPS1/2 mice exhibit spontaneous fibrosis with aging, which has been attributed to HPS mutations in alveolar epithelial type II (AT2) cells. We utilized HPS mouse models and human lung tissue to investigate mechanisms of AT2 cell dysfunction driving fibrotic remodeling in HPS. Starting at 8 weeks of age, HPS mice exhibited progressive loss of AT2 cell numbers. HPS AT2 cell was impaired ex vivo and in vivo. Incorporating AT2 cell lineage tracing in HPS mice, we observed aberrant differentiation with increased AT2-derived alveolar epithelial type I cells. Transcriptomic analysis of HPS AT2 cells revealed elevated expression of genes associated with aberrant differentiation and p53 activation. Lineage tracing and modeling studies demonstrated that HPS AT2 cells were primed to persist in a Krt8+ reprogrammed transitional state, mediated by p53 activity. Intrinsic AT2 progenitor cell dysfunction and p53 pathway dysregulation are novel mechanisms of disease in HPS-related pulmonary fibrosis, with the potential for early targeted intervention before the onset of fibrotic lung disease.

Airway epithelial cell identity and plasticity are constrained by Sox2 during lung homeostasis, tissue regeneration, and in human disease.

Maintenance of the cellular boundary between airway and alveolar compartments during homeostasis and after injury is essential to prohibit pathological plasticity which can reduce respiratory function. Lung injury and disease can induce either functional alveolar epithelial regeneration or dysplastic formation of keratinized epithelium which does not efficiently contribute to gas exchange. Here we show that Sox2 preserves airway cell identity and prevents fate changes into either functional alveolar tissue or pathological keratinization following lung injury. Loss of Sox2 in airway epithelium leads to a loss of airway epithelial identity with a commensurate gain in alveolar and basal cell identity, in part due to activation of Wnt signaling in secretory cells and increased Trp63 expression in intrapulmonary basal-like progenitors. In idiopathic pulmonary fibrosis, loss of SOX2 expression correlates with increased WNT signaling activity in dysplastic keratinized epithelium. SOX2-deficient dysplastic epithelial cells are also observed in COVID-19 damaged lungs. Thus, Sox2 provides a molecular barrier that suppresses airway epithelial plasticity to prevent acquisition of alveolar or basal cell identity after injury and help guide proper epithelial fate and regeneration.

An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung.

Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This "stem cell collision" is part of a multistep process that is now revealed to generate an injury-induced tissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.

TGF-ßR2 signaling coordinates pulmonary vascular repair after viral injury in mice and human tissue.

Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-ß signaling through TGF-ßR2 (transforming growth factor-ß receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelial Tgfbr2 exhibited prolonged injury and diminished vascular repair. Loss of endothelial Tgfbr2 prevented autocrine Vegfa (vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-ßR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-ß signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-ßR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-ßR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection.

Postinfectious Pulmonary Complications: Establishing Research Priorities to Advance the Field: An Official American Thoracic Society Workshop Report.

Continued improvements in the treatment of pulmonary infections have paradoxically resulted in a growing challenge of individuals with postinfectious pulmonary complications (PIPCs). PIPCs have been long recognized after tuberculosis, but recent experiences such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the importance of PIPCs following other lower respiratory tract infections. Independent of the causative pathogen, most available studies of pulmonary infections focus on short-term outcomes rather than long-term morbidity among survivors. In this document, we establish a conceptual scope for PIPCs with discussion of globally significant pulmonary pathogens and an examination of how these pathogens can damage different components of the lung, resulting in a spectrum of PIPCs. We also review potential mechanisms for the transition from acute infection to PIPC, including the interplay between pathogen-mediated injury and aberrant host responses, which together result in PIPCs. Finally, we identify cross-cutting research priorities for the field to facilitate future studies to establish the incidence of PIPCs, define common mechanisms, identify therapeutic strategies, and ultimately reduce the burden of morbidity in survivors of pulmonary infections.

Marginated Neutrophils in the Lungs Effectively Compete for Nanoparticles Targeted to the Endothelium, Serving as a Part of the Reticuloendothelial System.

Nanomedicine has long pursued the goal of targeted delivery to specific organs and cell types but has yet to achieve this goal with the vast majority of targets. One rare example of success in this pursuit has been the 25+ years of studies targeting the lung endothelium using nanoparticles conjugated to antibodies against endothelial surface molecules. However, here we show that such "endothelial-targeted" nanocarriers also effectively target the lungs' numerous marginated neutrophils, which reside in the pulmonary capillaries and patrol for pathogens. We show that marginated neutrophils' uptake of many of these "endothelial-targeted" nanocarriers is on par with endothelial uptake. This generalizes across diverse nanomaterials and targeting moieties and was even found with physicochemical lung tropism (i.e., without targeting moieties). Further, we observed this in ex vivo human lungs and in vivo healthy mice, with an increase in marginated neutrophil uptake of nanoparticles caused by local or distant inflammation. These findings have implications for nanomedicine development for lung diseases. These data also suggest that marginated neutrophils, especially in the lungs, should be considered a major part of the reticuloendothelial system (RES), with a special role in clearing nanoparticles that adhere to the lumenal surfaces of blood vessels.

DOT1L regulates lung developmental epithelial cell fate and adult alveolar stem cell differentiation after acute injury.

AT2 cells harbor alveolar stem cell activity in the lung and can self-renew and differentiate into AT1 cells during homeostasis and after injury. To identify epigenetic pathways that control the AT2-AT1 regenerative response in the lung, we performed an organoid screen using a library of pharmacological epigenetic inhibitors. This screen identified DOT1L as a regulator of AT2 cell growth and differentiation. In vivo inactivation of Dot1l leads to precocious activation of both AT1 and AT2 gene expression during lung development and accelerated AT1 cell differentiation after acute lung injury. Single-cell transcriptome analysis reveals the presence of a new AT2 cell state upon loss of Dot1l, characterized by increased expression of oxidative phosphorylation genes and changes in expression of critical transcription and epigenetic factors. Taken together, these data demonstrate that Dot1l controls the rate of alveolar epithelial cell fate acquisition during development and regeneration after acute injury.

Hyperactive mTORC1 in lung mesenchyme induces endothelial cell dysfunction and pulmonary vascular remodeling.

Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells, resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of patients with LAM develop pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified EC dysfunction characterized by increased EC proliferation and migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we used an mTORC1 gain-of-function mouse model with a Tsc2 KO (Tsc2KO) specific to lung mesenchyme (Tbx4LME-Cre Tsc2fl/fl), similar to the mesenchyme-specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from mice exhibited marked transcriptomic changes despite an absence of morphological changes to the distal lung microvasculature. In contrast, 1-year-old Tbx4LME-Cre Tsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single-cell RNA-Seq of 1-year-old mouse lung cells identified paracrine ligands originating from Tsc2KO mesenchyme, which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand-EC receptor crosstalk highlights the importance of an altered mesenchymal cell/EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in patients with LAM and in Tbx4LME-Cre Tsc2fl/fl mice did not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrated dysfunctional EC responses that contributed to pulmonary vascular remodeling.

Guided construction of single cell reference for human and mouse lung.

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.