Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Protein cysteine thiols undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates protein-membrane and protein-protein interactions critical to an array of biological processes, from homeostatic lung surfactant function to cellular transformation. The most widely used S-acylation assays, including acyl-biotin exchange (ABE) and acyl resin-assisted capture (Acyl-RAC), utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. ABE and Acyl-RAC have enabled both the proteome-wide identification of S-palmitoylation sites and basic biochemical studies. Yet, these assays generally utilize hundreds of micrograms to milligrams of input material and require numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome this, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment from 20-50 micrograms of input protein. The method is compatible with protein-level detection of Sacylated proteins as well as S-acyl site-based identification and quantification using on-quartz isobaric (tandem mass tag) labeling and LC-MS/MS. Also described are conditions for long-term hydroxylamine storage, which further expedites the assay and minimizes waste. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional intact protein detection and chemoproteomic workflows.

Stem cell migration drives lung repair in living mice.

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

Transitional cell states sculpt tissue topology during lung regeneration.

Organ regeneration requires dynamic cell interactions to reestablish cell numbers and tissue architecture. While we know the identity of progenitor cells that replace lost tissue, the transient states they give rise to and their role in repair remain elusive. Here, using multiple injury models, we find that alveolar fibroblasts acquire distinct states marked by Sfrp1 and Runx1 that influence tissue remodeling and reorganization. Unexpectedly, ablation of alveolar epithelial type-1 (AT1) cells alone is sufficient to induce tissue remodeling and transitional states. Integrated scRNA-seq followed by genetic interrogation reveals RUNX1 is a key driver of fibroblast states. Importantly, the ectopic induction or accumulation of epithelial transitional states induce rapid formation of transient alveolar fibroblasts, leading to organ-wide fibrosis. Conversely, the elimination of epithelial or fibroblast transitional states or RUNX1 loss, leads to tissue simplification resembling emphysema. This work uncovered a key role for transitional states in orchestrating tissue topologies during regeneration.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Meta-Analysis of COVID-19 BAL Single-Cell RNA Sequencing Reveals Alveolar Epithelial Transitions and Unique Alveolar Epithelial Cell Fates.

Single-cell RNA sequencing (scRNA-seq) of BAL cells has provided insights into coronavirus disease (COVID-19). However, reports have been limited by small patient cohorts. We performed a meta-analysis of BAL scRNA-seq data from healthy control subjects (n = 13) and patients with COVID-19 (n = 20), sourced from six independent studies (167,280 high-quality cells in total). Consistent with the source reports, increases in infiltrating leukocyte subtypes were noted, several with type I IFN signatures and unique gene expression signatures associated with transcellular chemokine signaling. Noting dramatic reductions of inferred NKX2-1 and NR4A1 activity in alveolar epithelial type II (AT-II) cells, we modeled pseudotemporal AT-II-to-AT-I progression. This revealed changes in inferred AT-II cell metabolic activity, increased transitional cells, and a previously undescribed AT-I state. This cell state was conspicuously marked by the induction of genes of the epidermal differentiation complex, including the cornified envelope protein SPRR3 (small proline-rich protein 3), upregulation of multiple KRT (keratin) genes, inferred mitochondrial dysfunction, and cell death signatures including apoptosis and ferroptosis. Immunohistochemistry of lungs from patients with COVID-19 confirmed upregulation and colocalization of KRT13 and SPRR3 in the distal airspaces. Forced overexpression of SPRR3 in human alveolar epithelial cells ex vivo did not activate caspase-3 or upregulate KRT13, suggesting that SPRR3 marks an AT-I cornification program in COVID-19 but is not sufficient for phenotypic changes.

Efficient Adeno-associated Virus-mediated Transgenesis in Alveolar Stem Cells and Associated Niches.

Targeted delivery of transgenes to tissue-resident stem cells and related niches offers avenues for interrogating pathways and editing endogenous alleles for therapeutic interventions. Here, we survey multiple adeno-associated virus (AAV) serotypes, administered via intranasal and retroorbital routes in mice, to target lung alveolar stem cell niches. We found that AAV5, AAV4, and AAV8 efficiently and preferentially transduce alveolar type-2 stem cells (AT2s), endothelial cells, and PDGFRA+ fibroblasts, respectively. Notably, some AAVs show different cell tropisms depending on the route of administration. Proof-of-concept experiments reveal the versatility of AAV5-mediated transgenesis for AT2-lineage labeling, clonal cell tracing after cell ablation, and conditional gene inactivation in both postnatal and adult mouse lungs in vivo. AAV6, but not AAV5, efficiently transduces both mouse and human AT2s in alveolar organoid cultures. Furthermore, AAV5 and AAV6 can be used to deliver guide RNAs and transgene cassettes for homologous recombination in vivo and ex vivo, respectively. Using this system coupled with clonal derivation of AT2 organoids, we demonstrate efficient and simultaneous editing of multiple loci, including targeted insertion of a payload cassette in AT2s. Taken together, our studies highlight the powerful utility of AAVs for interrogating alveolar stem cells and other specific cell types both in vivo and ex vivo.

Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.

In both humans and mice, repair of acute kidney injury is worse in males than in females. Here, we provide evidence that this sexual dimorphism results from sex differences in ferroptosis, an iron-dependent, lipid-peroxidation-driven regulated cell death. Using genetic and single-cell transcriptomic approaches in mice, we report that female sex confers striking protection against ferroptosis, which was experimentally induced in proximal tubular (PT) cells by deleting glutathione peroxidase 4 (Gpx4). Single-cell transcriptomic analyses further identify the NFE2-related factor 2 (NRF2) antioxidant protective pathway as a female resilience mechanism against ferroptosis. Genetic inhibition and pharmacological activation studies show that NRF2 controls PT cell fate and plasticity by regulating ferroptosis. Importantly, pharmacological NRF2 activation protects male PT cells from ferroptosis and improves cellular plasticity as in females. Our data highlight NRF2 as a potential therapeutic target to prevent failed renal repair after acute kidney injury in both sexes by modulating cellular plasticity.

Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

Multi-apical polarity of alveolar stem cells and their dynamics during lung development and regeneration.

Epithelial cells of diverse tissues are characterized by the presence of a single apical domain. In the lung, electron microscopy studies have suggested that alveolar type-2 epithelial cells (AT2s) en face multiple alveolar sacs. However, apical and basolateral organization of the AT2s and their establishment during development and remodeling after injury repair remain unknown. Thick tissue imaging and electron microscopy revealed that a single AT2 can have multiple apical domains that enface multiple alveoli. AT2s gradually establish multi-apical domains post-natally, and they are maintained throughout life. Lineage tracing, live imaging, and selective cell ablation revealed that AT2s dynamically reorganize multi-apical domains during injury repair. Single-cell transcriptome signatures of residual AT2s revealed changes in cytoskeleton and cell migration. Significantly, cigarette smoke and oncogene activation lead to dysregulation of multi-apical domains. We propose that the multi-apical domains of AT2s enable them to be poised to support the regeneration of a large array of alveolar sacs.

Human lung organoids as a model for respiratory virus replication and countermeasure performance in human hosts.

Human respiratory viruses induce a wide breadth of disease phenotypes and outcomes of varying severity. Innovative models that recapitulate the human respiratory tract are needed to study such viruses, understand the virus-host interactions underlying replication and pathogenesis, and to develop effective countermeasures for prevention and treatment. Human organoid models provide a platform to study virus-host interactions in the proximal to distal lung in the absence of a human in vivo model. These cultures fill the niche of a suitable ex vivo model that represents the in vivo lung environment and encapsulates the structure and function of the native human lung.

Defined conditions for long-term expansion of murine and human alveolar epithelial stem cells in three-dimensional cultures.

Alveolar type 2 cells (AT2s) serve as stem cells of the alveoli and restore cell numbers after injury. Here, we describe a detailed protocol for the isolation, purification, and culture of murine and human AT2s. We have developed chemically defined and stroma-free culture conditions that enable expansion and maintenance of AT2s. The culture conditions are scalable and compatible with high-throughput chemical and genetic screenings and can potentially be used to generate large AT2 numbers for cell-based therapies. For complete details on the use and execution of this protocol, please refer to Katsura et al. (2020).

An In Vitro Microfluidic Alveolus Model to Study Lung Biomechanics.

The gas exchange units of the lung, the alveoli, are mechanically active and undergo cyclic deformation during breathing. The epithelial cells that line the alveoli contribute to lung function by reducing surface tension via surfactant secretion, which is highly influenced by the breathing-associated mechanical cues. These spatially heterogeneous mechanical cues have been linked to several physiological and pathophysiological states. Here, we describe the development of a microfluidically assisted lung cell culture model that incorporates heterogeneous cyclic stretching to mimic alveolar respiratory motions. Employing this device, we have examined the effects of respiratory biomechanics (associated with breathing-like movements) and strain heterogeneity on alveolar epithelial cell functions. Furthermore, we have assessed the potential application of this platform to model altered matrix compliance associated with lung pathogenesis and ventilator-induced lung injury. Lung microphysiological platforms incorporating human cells and dynamic biomechanics could serve as an important tool to delineate the role of alveolar micromechanics in physiological and pathological outcomes in the lung.

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.

Lung Regeneration: Cells, Models, and Mechanisms.

Lung epithelium, the lining that covers the inner surface of the respiratory tract, is directly exposed to the environment and thus susceptible to airborne toxins, irritants, and pathogen-induced damages. In adult mammalian lungs, epithelial cells are generally quiescent but can respond rapidly to repair of damaged tissues. Evidence from experimental injury models in rodents and human clinical samples has led to the identification of these regenerative cells, as well as pathological metaplastic states specifically associated with different forms of damages. Here, we provide a compendium of cells and cell states that exist during homeostasis in normal lungs and the lineage relationships between them. Additionally, we discuss various experimental injury models currently being used to probe the cellular sources-both resident and recruited-that contribute to repair, regeneration, and remodeling following acute and chronic injuries. Finally, we discuss certain maladaptive regeneration-associated cell states and their role in disease pathogenesis.

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair.

Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, after severe injury they accumulate and contribute to persistent inflammation. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making the cells vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.

Epithelial cell plasticity: breaking boundaries and changing landscapes.

Epithelial tissues respond to a wide variety of environmental and genotoxic stresses. As an adaptive mechanism, cells can deviate from their natural paths to acquire new identities, both within and across lineages. Under extreme conditions, epithelial tissues can utilize "shape-shifting" mechanisms whereby they alter their form and function at a tissue-wide scale. Mounting evidence suggests that in order to acquire these alternate tissue identities, cells follow a core set of "tissue logic" principles based on developmental paradigms. Here, we review the terminology and the concepts that have been put forward to describe cell plasticity. We also provide insights into various cell intrinsic and extrinsic factors, including genetic mutations, inflammation, microbiota, and therapeutic agents that contribute to cell plasticity. Additionally, we discuss recent studies that have sought to decode the "syntax" of plasticity-i.e., the cellular and molecular principles through which cells acquire new identities in both homeostatic and malignant epithelial tissues-and how these processes can be manipulated for developing novel cancer therapeutics.

Identification of a Repair-Supportive Mesenchymal Cell Population during Airway Epithelial Regeneration.

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα+ cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.

Human Lung Stem Cell-Based Alveolospheres Provide Insights into SARS-CoV-2-Mediated Interferon Responses and Pneumocyte Dysfunction.

Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here, we report a feeder-free, scalable, chemically defined, and modular alveolosphere culture system for the propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor angiotensin-converting enzyme receptor type-2 (ACE2) and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirror features of COVID-19 lungs, including emergence of interferon (IFN)-mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with IFNs recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low-dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres, thus, provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases.

The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro.

While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3a/b and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis.

Stem cells undergo dynamic changes in response to injury to regenerate lost cells. However, the identity of transitional states and the mechanisms that drive their trajectories remain understudied. Using lung organoids, multiple in vivo repair models, single-cell transcriptomics and lineage tracing, we find that alveolar type-2 epithelial cells undergoing differentiation into type-1 cells acquire pre-alveolar type-1 transitional cell state (PATS) en route to terminal maturation. Transitional cells undergo extensive stretching during differentiation, making them vulnerable to DNA damage. Cells in the PATS show an enrichment of TP53, TGFß, DNA-damage-response signalling and cellular senescence. Gain and loss of function as well as genomic binding assays revealed a direct transcriptional control of PATS by TP53 signalling. Notably, accumulation of PATS-like cells in human fibrotic lungs was observed, suggesting persistence of the transitional state in fibrosis. Our study thus implicates a transient state associated with senescence in normal epithelial tissue repair and its abnormal persistence in disease conditions.