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

Proteins 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 critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 µg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, 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. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 µg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

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

Proteins 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 critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, 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. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Recent advances in lung organoid development and applications in disease modeling.

Over the last decade, several organoid models have evolved to acquire increasing cellular, structural, and functional complexity. Advanced lung organoid platforms derived from various sources, including adult, fetal, and induced pluripotent stem cells, have now been generated, which more closely mimic the cellular architecture found within the airways and alveoli. In this regard, the establishment of novel protocols with optimized stem cell isolation and culture conditions has given rise to an array of models able to study key cellular and molecular players involved in lung injury and repair. In addition, introduction of other nonepithelial cellular components, such as immune, mesenchymal, and endothelial cells, and employment of novel precision gene editing tools have further broadened the range of applications for these systems by providing a microenvironment and/or phenotype closer to the desired in vivo scenario. Thus, these developments in organoid technology have enhanced our ability to model various aspects of lung biology, including pathogenesis of diseases such as chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, and infectious disease and host-microbe interactions, in ways that are often difficult to undertake using only in vivo models. In this Review, we summarize the latest developments in lung organoid technology and their applicability for disease modeling and outline their strengths, drawbacks, and potential avenues for future development.

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.

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.

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.

Coupled myovascular expansion directs cardiac growth and regeneration.

Heart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here, we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled. We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth. Our work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.

Epigenetic basis of oncogenic-Kras-mediated epithelial-cellular proliferation and plasticity.

Oncogenic Kras induces a hyper-proliferative state that permits cells to progress to neoplasms in diverse epithelial tissues. Depending on the cell of origin, this also involves lineage transformation. Although a multitude of downstream factors have been implicated in these processes, the precise chronology of molecular events controlling them remains elusive. Using mouse models, primary human tissues, and cell lines, we show that, in Kras-mutant alveolar type II cells (AEC2), FOSL1-based AP-1 factor guides the mSWI/SNF complex to increase chromatin accessibility at genomic loci controlling the expression of genes necessary for neoplastic transformation. We identified two orthogonal processes in Kras-mutant distal airway club cells. The first promoted their transdifferentiation into an AEC2-like state through NKX2.1, and the second controlled oncogenic transformation through the AP-1 complex. Our results suggest that neoplasms retain an epigenetic memory of their cell of origin through cell-type-specific transcription factors. Our analysis showed that a cross-tissue-conserved AP-1-dependent chromatin remodeling program regulates carcinogenesis.

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.

Chromatin Remodeling of Colorectal Cancer Liver Metastasis is Mediated by an HGF-PU.1-DPP4 Axis.

Colorectal cancer (CRC) metastasizes mainly to the liver, which accounts for the majority of CRC-related deaths. Here it is shown that metastatic cells undergo specific chromatin remodeling in the liver. Hepatic growth factor (HGF) induces phosphorylation of PU.1, a pioneer factor, which in turn binds and opens chromatin regions of downstream effector genes. PU.1 increases histone acetylation at the DPP4 locus. Precise epigenetic silencing by CRISPR/dCas9KRAB or CRISPR/dCas9HDAC revealed that individual PU.1-remodeled regulatory elements collectively modulate DPP4 expression and liver metastasis growth. Genetic silencing or pharmacological inhibition of each factor along this chromatin remodeling axis strongly suppressed liver metastasis. Therefore, microenvironment-induced epimutation is an important mechanism for metastatic tumor cells to grow in their new niche. This study presents a potential strategy to target chromatin remodeling in metastatic cancer and the promise of repurposing drugs to treat metastasis.

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.

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia.

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

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.

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.

IL-1 and TNFα Contribute to the Inflammatory Niche to Enhance Alveolar Regeneration.

Inflammatory responses are known to facilitate tissue recovery following injury. However, the precise mechanisms that enhance lung alveolar regeneration remain unclear. Here, using an organoid-based screening assay, we find that interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) enhance the proliferation of AEC2s while maintaining their differentiation capacity. Furthermore, we find that expression of IL-1ß and TNFα are induced in the AEC2 niche following influenza-induced injury in vivo, and lineage tracing analysis revealed that surviving AEC2s around the damaged area contribute to alveolar regeneration. Through genetic and pharmacological modulation of multiple components of the IL-1-nuclear factor κB (NF-κB) signaling axis, we show that cell-intrinsic as well as stromal mediated IL-1 signaling are essential for AEC2 mediated lung regeneration. Taken together, we propose that the IL-1/TNFα-NF-κB signaling axis functions as a component of an inflammation-associated niche to regulate proliferation of surviving AEC2s and promote lung regeneration.

ABL kinase inhibition promotes lung regeneration through expansion of an SCGB1A1+ SPC+ cell population following bacterial pneumonia.

Current therapeutic interventions for the treatment of respiratory infections are hampered by the evolution of multidrug resistance in pathogens as well as the lack of effective cellular targets. Despite the identification of multiple region-specific lung progenitor cells, the identity of molecules that might be therapeutically targeted in response to infections to promote activation of progenitor cell types remains elusive. Here, we report that loss of Abl1 specifically in SCGB1A1-expressing cells leads to a significant increase in the proliferation and differentiation of bronchiolar epithelial cells, resulting in dramatic expansion of an SCGB1A1+ airway cell population that coexpresses SPC, a marker for type II alveolar cells that promotes alveolar regeneration following bacterial pneumonia. Furthermore, treatment with an Abl-specific allosteric inhibitor enhanced regeneration of the alveolar epithelium and promoted accelerated recovery of mice following pneumonia. These data reveal a potential actionable target that may be exploited for efficient recovery after pathogen-induced infections.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.