Human Vascularized Macrophage-Islet Organoids to Model Immune-Mediated Pancreatic ß cell Pyroptosis upon Viral Infection.

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and ß cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated ß cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both ß cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced ß cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated ß cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of ß cell damage during viral exposure.

Adapting nanopore sequencing basecalling models for modification detection via incremental learning and anomaly detection.

We leverage machine learning approaches to adapt nanopore sequencing basecallers for nucleotide modification detection. We first apply the incremental learning (IL) technique to improve the basecalling of modification-rich sequences, which are usually of high biological interest. With sequence backbones resolved, we further run anomaly detection (AD) on individual nucleotides to determine their modification status. By this means, our pipeline promises the single-molecule, single-nucleotide, and sequence context-free detection of modifications. We benchmark the pipeline using control oligos, further apply it in the basecalling of densely-modified yeast tRNAs and E.coli genomic DNAs, the cross-species detection of N6-methyladenosine (m6A) in mammalian mRNAs, and the simultaneous detection of N1-methyladenosine (m1A) and m6A in human mRNAs. Our IL-AD workflow is available at: https://github.com/wangziyuan66/IL-AD .

Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion.

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

KLHL21 suppresses gastric tumourigenesis via maintaining STAT3 signalling equilibrium in stomach homoeostasis.

OBJECTIVE: Precancerous metaplasia transition to dysplasia poses a risk for subsequent intestinal-type gastric adenocarcinoma. However, the molecular basis underlying the transformation from metaplastic to cancerous cells remains poorly understood. DESIGN: An integrated analysis of genes associated with metaplasia, dysplasia was conducted, verified and characterised in the gastric tissues of patients by single-cell RNA sequencing and immunostaining. Multiple mouse models, including homozygous conditional knockout Klhl21-floxed mice, were generated to investigate the role of Klhl21 deletion in stemness, DNA damage and tumour formation. Mass-spectrometry-based proteomics and ribosome sequencing were used to elucidate the underlying molecular mechanisms. RESULTS: Kelch-like protein 21 (KLHL21) expression progressively decreased in metaplasia, dysplasia and cancer. Genetic deletion of Klhl21 enhances the rapid proliferation of Mist1+ cells and their descendant cells. Klhl21 loss during metaplasia facilitates the recruitment of damaged cells into the cell cycle via STAT3 signalling. Increased STAT3 activity was confirmed in cancer cells lacking KLHL21, boosting self-renewal and tumourigenicity. Mechanistically, the loss of KLHL21 promotes PIK3CB mRNA translation by stabilising the PABPC1-eIF4G complex, subsequently causing STAT3 activation. Pharmacological STAT3 inhibition by TTI-101 elicited anticancer effects, effectively impeding the transition from metaplasia to dysplasia. In patients with gastric cancer, low levels of KLHL21 had a shorter survival rate and a worse response to adjuvant chemotherapy. CONCLUSIONS: Our findings highlighted that KLHL21 loss triggers STAT3 reactivation through PABPC1-mediated PIK3CB translational activation, and targeting STAT3 can reverse preneoplastic metaplasia in KLHL21-deficient stomachs.

ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors.

Lymphocytes can circulate as well as take residence within tissues. While the mechanisms by which circulating populations are recruited to infection sites have been extensively characterized, the molecular basis for the recirculation of tissue-resident cells is less understood. Here, we show that helminth infection- or IL-25-induced redistribution of intestinal group 2 innate lymphoid cells (ILC2s) requires access to the lymphatic vessel network. Although the secondary lymphoid structure is an essential signal hub for adaptive lymphocyte differentiation and dispatch, it is redundant for ILC2 migration and effector function. Upon IL-25 stimulation, a dramatic change in epigenetic landscape occurs in intestinal ILC2s, leading to the expression of sphingosine-1-phosphate receptors (S1PRs). Among the various S1PRs, we found that S1PR5 is critical for ILC2 exit from intestinal tissue to lymph. By contrast, S1PR1 plays a dominant role in ILC2 egress from mesenteric lymph nodes to blood circulation and then to distal tissues including the lung where the redistributed ILC2s contribute to tissue repair. The requirement of two S1PRs for ILC2 migration is largely due to the dynamic expression of the tissue-retention marker CD69, which mediates S1PR1 internalization. Thus, our study demonstrates a stage-specific requirement of different S1P receptors for ILC2 redistribution during infection. We therefore propose a fundamental paradigm that innate and adaptive lymphocytes utilize a shared vascular network frame and specialized navigation cues for migration.

Small molecule conjugates with selective estrogen receptor ß agonism promote anti-aging benefits in metabolism and skin recovery.

Estrogen is imperative to mammalian reproductivity, metabolism, and aging. However, the hormone activating estrogen receptor (ERs) α can cause major safety concerns due to the enrichment of ERα in female tissues and certain malignancies. In contrast, ERß is more broadly expressed in metabolic tissues and the skin. Thus, it is desirable to generate selective ERß agonist conjugates for maximizing the therapeutic effects of ERs while minimizing the risks of ERα activation. Here, we report the design and production of small molecule conjugates containing selective non-steroid ERß agonists Gtx878 or genistein. Treatment of aged mice with our synthesized conjugates improved aging-associated declines in insulin sensitivity, visceral adipose integrity, skeletal muscle function, and skin health, with validation in vitro. We further uncovered the benefits of ERß conjugates in the skin using two inducible skin injury mouse models, showing increased skin basal cell proliferation, epidermal thickness, and wound healing. Therefore, our ERß-selective agonist conjugates offer novel therapeutic potential to improve aging-associated conditions and aid in rejuvenating skin health.

Single Cell Analysis of Lung Lymphatic Endothelial Cells and Lymphatic Responses during Influenza Infection.

Tissue lymphatic vessels network plays critical roles in immune surveillance and tissue homeostasis in response to pathogen invasion, but how lymphatic system per se is remolded during infection is less understood. Here, we observed that influenza infection induces a significant increase of lymphatic vessel numbers in the lung, accompanied with extensive proliferation of lymphatic endothelial cells (LECs). Single-cell RNA sequencing illustrated the heterogeneity of LECs, identifying a novel PD-L1+ subpopulation that is present during viral infection but not at steady state. Specific deletion of Pd-l1 in LECs elevated the expansion of lymphatic vessel numbers during viral infection. Together these findings elucidate a dramatic expansion of lung lymphatic network in response to viral infection, and reveal a PD-L1+ LEC subpopulation that potentially modulates lymphatic vessel remolding.

IgG is an aging factor that drives adipose tissue fibrosis and metabolic decline.

Aging is underpinned by pronounced metabolic decline; however, the drivers remain obscure. Here, we report that IgG accumulates during aging, particularly in white adipose tissue (WAT), to impair adipose tissue function and metabolic health. Caloric restriction (CR) decreases IgG accumulation in WAT, whereas replenishing IgG counteracts CR's metabolic benefits. IgG activates macrophages via Ras signaling and consequently induces fibrosis in WAT through the TGF-ß/SMAD pathway. Consistently, B cell null mice are protected from aging-associated WAT fibrosis, inflammation, and insulin resistance, unless exposed to IgG. Conditional ablation of the IgG recycling receptor, neonatal Fc receptor (FcRn), in macrophages prevents IgG accumulation in aging, resulting in prolonged healthspan and lifespan. Further, targeting FcRn by antisense oligonucleotide restores WAT integrity and metabolic health in aged mice. These findings pinpoint IgG as a hidden culprit in aging and enlighten a novel strategy to rejuvenate metabolic health.

Airway Basal Stem Cells in COVID-19 Exhibit a Proinflammatory Signature and Impaired Mucocililary Differentiation.

Airway basal stem cells (BSCs) play a critical role in epithelial regeneration. Whether coronavirus disease (COVID-19) affects BSC function is unknown. Here, we derived BSC lines from patients with COVID-19 using tracheal aspirates (TAs) to circumvent the biosafety concerns of live-cell derivation. We show that BSCs derived from the TAs of control patients are bona fide bronchial BSCs. TA BSCs from patients with COVID-19 tested negative for severe acute respiratory syndrome coronavirus 2 RNA; however, these so-termed COVID-19-exposed BSCs in vitro resemble a predominant BSC subpopulation uniquely present in patients with COVID-19, manifested by a proinflammatory gene signature and STAT3 hyperactivation. Furthermore, the sustained STAT3 hyperactivation drives goblet cell differentiation of COVID-19-exposed BSCs in an air-liquid interface. Last, these phenotypes of COVID-19-exposed BSCs can be induced in control BSCs by cytokine cocktail pretreatment. Taken together, acute inflammation in COVID-19 exerts a long-term impact on mucociliary differentiation of BSCs.

A Spatiotemporal and Machine-Learning Platform Accelerates the Manufacturing of hPSC-derived Esophageal Mucosa.

Human pluripotent stem cell-derived tissue engineering offers great promise in designer cell-based personalized therapeutics. To harness such potential, a broader approach requires a deeper understanding of tissue-level interactions. We previously developed a manufacturing system for the ectoderm-derived skin epithelium for cell replacement therapy. However, it remains challenging to manufacture the endoderm-derived esophageal epithelium, despite both possessing similar stratified structure. Here we employ single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for human esophageal development. We illuminate the cellular diversity, dynamics and signal communications for the developing esophageal epithelium and stroma. Using the machine-learning based Manatee, we prioritize the combinations of candidate human developmental signals for in vitro derivation of esophageal basal cells. Functional validation of the Manatee predictions leads to a clinically-compatible system for manufacturing human esophageal mucosa. Our approach creates a versatile platform to accelerate human tissue manufacturing for future cell replacement therapies to treat human genetic defects and wounds.

Age-related STAT3 signaling regulates severity of respiratory syncytial viral infection in human bronchial epithelial cells.

Respiratory syncytial virus (RSV) can cause severe disease especially in infants; however, mechanisms of age-associated disease severity remain elusive. Here, employing human bronchial epithelium models generated from tracheal aspirate-derived basal stem cells of neonates and adults, we investigated whether age regulates RSV-epithelium interaction to determine disease severity. We show that following RSV infection, only neonatal epithelium model exhibited cytopathy and mucus hyperplasia, and neonatal epithelium had more robust viral spread and inflammatory responses than adult epithelium. Mechanistically, RSV-infected neonatal ciliated cells displayed age-related impairment of STAT3 activation, rendering susceptibility to apoptosis, which facilitated viral spread. In contrast, SARS-CoV-2 infection of ciliated cells had no effect on STAT3 activation and was not affected by age. Taken together, our findings identify an age-related and RSV-specific interaction with neonatal bronchial epithelium that critically contributes to severity of infection, and STAT3 activation offers a potential strategy to battle severe RSV disease in infants.

Targeting the SOX2/CDP protein complex with a peptide suppresses the malignant progression of esophageal squamous cell carcinoma.

Emerging evidence indicates that SOX2 is an oncogene for esophageal squamous cell carcinoma (ESCC). However, direct targeting of SOX2 is not feasible given that this transcription factor plays important roles in the maintenance of tissues such as the brain. Here, we identified CDP (Homeobox protein cut-like 1 or CASP) as a unique SOX2 binding partner enriched in ESCC with Duolink proximity ligation assay, bimolecular fluorescence complementation (BiFc) and immunoprecipitation. We then screened a peptide aptamer library using BiFc and immunoprecipitation and identified several peptide aptamers, including P58, that blocked the CDP/SOX2 interaction, leading to the inhibition of ESCC progress in vitro and in vivo. Upon administration, synthetic peptide P58, containing the YGRKKRRQRRR cell-penetrating peptide and the fluorophore TAMRA, also blocked the growth and metastasis of ESCC in both mice and zebrafish. Therefore, targeting the SOX2 binding partner CDP with peptide P58 offers an alternative avenue to treat ESCC with increased SOX2 levels.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy.

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Upregulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

Systematic dissection of coordinated stromal remodeling identifies Sox10+ glial cells as a therapeutic target in myelofibrosis.

Remodeling of the tissue niche is often evident in diseases, yet, the stromal alterations and their contribution to pathogenesis are poorly characterized. Bone marrow fibrosis is a maladaptive feature of primary myelofibrosis (PMF). We performed lineage tracing and found that most collagen-expressing myofibroblasts were derived from leptin-receptor-positive (LepR+) mesenchymal cells, whereas a minority were from Gli1-lineage cells. Deletion of Gli1 did not impact PMF. Unbiased single-cell RNA sequencing (scRNA-seq) confirmed that virtually all myofibroblasts originated from LepR-lineage cells, with reduced expression of hematopoietic niche factors and increased expression of fibrogenic factors. Concurrently, endothelial cells upregulated arteriolar-signature genes. Pericytes and Sox10+ glial cells expanded drastically with heightened cell-cell signaling, suggesting important functional roles in PMF. Chemical or genetic ablation of bone marrow glial cells ameliorated fibrosis and improved other pathology in PMF. Thus, PMF involves complex remodeling of the bone marrow microenvironment, and glial cells represent a promising therapeutic target.

Human Airway Basal Cells Undergo Reversible Squamous Differentiation and Reshape Innate Immunity.

Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent "vulnerable spots" within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division.

BACKGROUND & AIMS: Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. METHODS: Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. RESULTS: Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. CONCLUSION: SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.