Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.

Single-cell transcriptomic analyses reveal cellular and molecular patterns of rubber tree response to early powdery mildew infection.

As a perennial woody plant, the rubber tree (Hevea brasiliensis) must adapt to various environmental challenges through gene expression in multiple cell types. It is still unclear how genes in this species are expressed at the cellular level and the precise mechanisms by which cells respond transcriptionally to environmental stimuli, especially in the case of pathogen infection. Here, we characterized the transcriptomes in Hevea leaves during early powdery mildew infection using single-cell RNA sequencing. We identified 10 cell types and constructed the first single-cell atlas of Hevea leaves. Distinct gene expression patterns of the cell clusters were observed under powdery mildew infection, which was especially significant in the epidermal cells. Most of the genes involved in host-pathogen interactions in epidermal cells exhibited a pattern of dramatically increased expression with increasing pseudotime. Interestingly, we found that the HbCNL2 gene, encoding a nucleotide-binding leucine-rich repeat protein, positively modulated the defence of rubber leaves against powdery mildew. Overexpression of the HbCNL2 gene triggered a typical cell death phenotype in tobacco leaves and a higher level of reactive oxygen species in the protoplasts of Hevea leaves. The HbCNL2 protein was located in the cytomembrane and nucleus, and its leucine-rich repeat domain interacted with the histidine kinase-like ATPase domain of the molecular chaperone HbHSP90 in the nucleus. Collectively, our results provide the first observation of the cellular and molecular responses of Hevea leaves to biotrophic pathogen infection and can guide the identification of disease-resistance genes in this important tree species.

LungMAP Portal Ecosystem: Systems-Level Exploration of the Lung.

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular, imaging, and pathological datasets. To centralize and standardize information across broad lung research efforts we expanded the LungMAP.net website into a new gateway portal. This portal connects a broad spectrum of research networks, bulk and single-cell multi-omics data and a diverse collection of image data that span mammalian lung development, and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, mouse), to enable consistent queries across technologies, cohorts, age, disease, and drug treatment. These atlases are provided as independent and integrated queryable datasets, with an emphasis on dynamic visualization, figure generation, re-analysis, cell-type curation, and automated reference-based classification of user-provided single-cell genomics datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, supported data types, data portals and datasets from LungMAP and external research efforts.

A single-cell atlas of the miracidium larva of Schistosoma mansoni reveals cell types, developmental pathways, and tissue architecture.

Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.

Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Single Cell Atlas: a single-cell multi-omics human cell encyclopedia.

Single-cell sequencing datasets are key in biology and medicine for unraveling insights into heterogeneous cell populations with unprecedented resolution. Here, we construct a single-cell multi-omics map of human tissues through in-depth characterizations of datasets from five single-cell omics, spatial transcriptomics, and two bulk omics across 125 healthy adult and fetal tissues. We construct its complement web-based platform, the Single Cell Atlas (SCA, www.singlecellatlas.org ), to enable vast interactive data exploration of deep multi-omics signatures across human fetal and adult tissues. The atlas resources and database queries aspire to serve as a one-stop, comprehensive, and time-effective resource for various omics studies.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell-specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single-cell RNA sequencing (scRNAseq) cell atlas. STUDY DESIGN: Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. METHODS: An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. RESULTS: The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. CONCLUSIONS: scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone-dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. LEVEL OF EVIDENCE: NA; Basic science Laryngoscope, 133:3506-3511, 2023.

Establishing a human bone marrow single cell reference atlas to study ageing and diseases.

Introduction: Ageing in the human bone marrow is associated with immune function decline that results in the elderly being vulnerable to illnesses. A comprehensive healthy bone marrow consensus atlas can serve as a reference to study the immunological changes associated with ageing, and to identify and study abnormal cell states. Methods: We collected publicly available single cell transcriptomic data of 145 healthy samples encompassing a wide spectrum of ages ranging from 2 to 84 years old to construct our human bone marrow atlas. The final atlas has 673,750 cells and 54 annotated cell types. Results: We first characterised the changes in cell population sizes with respect to age and the corresponding changes in gene expression and pathways. Overall, we found significant age-associated changes in the lymphoid lineage cells. The naïve CD8+ T cell population showed significant shrinkage with ageing while the effector/memory CD4+ T cells increased in proportion. We also found an age-correlated decline in the common lymphoid progenitor population, in line with the commonly observed myeloid skew in haematopoiesis among the elderly. We then employed our cell type-specific ageing gene signatures to develop a machine learning model that predicts the biological age of bone marrow samples, which we then applied to healthy individuals and those with blood diseases. Finally, we demonstrated how to identify abnormal cell states by mapping disease samples onto the atlas. We accurately identified abnormal plasma cells and erythroblasts in multiple myeloma samples, and abnormal cells in acute myeloid leukaemia samples. Discussion: The bone marrow is the site of haematopoiesis, a highly important bodily process. We believe that our healthy bone marrow atlas is a valuable reference for studying bone marrow processes and bone marrow-related diseases. It can be mined for novel discoveries, as well as serve as a reference scaffold for mapping samples to identify and investigate abnormal cells.

Single-cell atlas reveals a high selection of IgA1- or IgG1-expressing plasma cells in patients with psoriasis.

Psoriasis has been considered as a T-cell driven common inflammatory skin disease. However, thus far, the pathogenetic role of B cells in psoriasis have largely been neglected. To explore the effect of psoriasis on B-cell mediated humoral immune response, we combined single-cell RNA-sequencing (scRNA-seq) and single-cell antigen receptor lineage (BCR)-sequencing (scBCR-seq) analysis to characterize human PBMC (Peripheral blood mononuclear cells). Merged PBMC from three healthy donors and three patients with psoriasis revealed 27 major cellular clusters in the UMAP plots. Interestingly, we found that follicular helper T cells (TFH) and plasma cells (PCs) obviously increased in patients with psoriasis. Further, we demonstrated that BCL6-expressing TFH cells and their helping B cell-derived IGHA1- or IGHG1-expressing PCs, were increased in patients with psoriasis. By analyzing scBCR-seq data, we found that BCR had a low diversity of IGHA and IGHG but not IGHM and IGHD in patients with psoriasis. In line with a low diversity, IGHA and IGHG frequently used IGHV3 in patients with psoriasis. In addition, we found that CDR silent mutations in IGHA and CDR replacement mutations in IGHG increased from patients with psoriasis. Finally, we showed that patients with psoriasis had high BCR selection pressure in CDR and framework regions (FWR) on IGHG and IGHA but not IGHM and IGHD. Altogether, our results suggest that patients with psoriasis had high BCR selection pressure, partly from helping of BCL6-expressing TFH cells, may result in increased IGHG1- or IGHA1-expressing PCs. Further exploration of high BCR selection pressure will provide valuable clues for the treatment of psoriasis.

Cell-type-specific responses to the microbiota across all tissues of the larval zebrafish.

Animal development proceeds in the presence of intimate microbial associations, but the extent to which different host cells across the body respond to resident microbes remains to be fully explored. Using the vertebrate model organism, the larval zebrafish, we assessed transcriptional responses to the microbiota across the entire body at single-cell resolution. We find that cell types across the body, not limited to tissues at host-microbe interfaces, respond to the microbiota. Responses are cell-type-specific, but across many tissues the microbiota enhances cell proliferation, increases metabolism, and stimulates a diversity of cellular activities, revealing roles for the microbiota in promoting developmental plasticity. This work provides a resource for exploring transcriptional responses to the microbiota across all cell types of the vertebrate body and generating new hypotheses about the interactions between vertebrate hosts and their microbiota.

Decoding aging-dependent regenerative decline across tissues at single-cell resolution.

Regeneration across tissues and organs exhibits significant variation throughout the body and undergoes a progressive decline with age. To decode the relationships between aging and regenerative capacity, we conducted a comprehensive single-cell transcriptome analysis of regeneration in eight tissues from young and aged mice. We employed diverse analytical models to study tissue regeneration and unveiled the intricate cellular and molecular mechanisms underlying the attenuated regenerative processes observed in aged tissues. Specifically, we identified compromised stem cell mobility and inadequate angiogenesis as prominent contributors to this age-associated decline in regenerative capacity. Moreover, we discovered a unique subset of Arg1+ macrophages that were activated in young tissues but suppressed in aged regenerating tissues, suggesting their important role in age-related immune response disparities during regeneration. This study provides a comprehensive single-cell resource for identifying potential targets for interventions aimed at enhancing regenerative outcomes in the aging population.

Multicellular factor analysis of single-cell data for a tissue-centric understanding of disease.

Biomedical single-cell atlases describe disease at the cellular level. However, analysis of this data commonly focuses on cell-type-centric pairwise cross-condition comparisons, disregarding the multicellular nature of disease processes. Here, we propose multicellular factor analysis for the unsupervised analysis of samples from cross-condition single-cell atlases and the identification of multicellular programs associated with disease. Our strategy, which repurposes group factor analysis as implemented in multi-omics factor analysis, incorporates the variation of patient samples across cell-types or other tissue-centric features, such as cell compositions or spatial relationships, and enables the joint analysis of multiple patient cohorts, facilitating the integration of atlases. We applied our framework to a collection of acute and chronic human heart failure atlases and described multicellular processes of cardiac remodeling, independent to cellular compositions and their local organization, that were conserved in independent spatial and bulk transcriptomics datasets. In sum, our framework serves as an exploratory tool for unsupervised analysis of cross-condition single-cell atlases and allows for the integration of the measurements of patient cohorts across distinct data modalities.

Multi-omic analysis characterizes molecular susceptibility of receptors to SARS-CoV-2 spike protein.

In the post COVID-19 era, new SARS-CoV-2 variant strains may continue emerging and long COVID is poised to be another public health challenge. Deciphering the molecular susceptibility of receptors to SARS-CoV-2 spike protein is critical for understanding the immune responses in COVID-19 and the rationale of multi-organ injuries. Currently, such systematic exploration remains limited. Here, we conduct multi-omic analysis of protein binding affinities, transcriptomic expressions, and single-cell atlases to characterize the molecular susceptibility of receptors to SARS-CoV-2 spike protein. Initial affinity analysis explains the domination of delta and omicron variants and demonstrates the strongest affinities between BSG (CD147) receptor and most variants. Further transcriptomic data analysis on 4100 experimental samples and single-cell atlases of 1.4 million cells suggest the potential involvement of BSG in multi-organ injuries and long COVID, and explain the high prevalence of COVID-19 in elders as well as the different risks for patients with underlying diseases. Correlation analysis validated moderate associations between BSG and viral RNA abundance in multiple cell types. Moreover, similar patterns were observed in primates and validated in proteomic expressions. Overall, our findings implicate important therapeutic targets for the development of receptor-specific vaccines and drugs for COVID-19.

Comprehensive analysis to identify the influences of SARS-CoV-2 infections to inflammatory bowel disease.

Background: Coronavirus disease 2019 (COVID-19) and inflammatory bowel disease (IBD) are both caused by a disordered immune response and have direct and profound impacts on health care services. In this study, we implemented transcriptomic and single-cell analysis to detect common molecular and cellular intersections between COVID-19 and IBD that help understand the linkage of COVID-19 to the IBD patients. Methods: Four RNA-sequencing datasets (GSE147507, GSE126124, GSE9686 and GSE36807) from Gene Expression Omnibus (GEO) database are extracted to detect mutual differentially expressed genes (DEGs) for IBD patients with the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to find shared pathways, candidate drugs, hub genes and regulatory networks. Two single-cell RNA sequencing (scRNA-eq) datasets (GSE150728, PRJCA003980) are used to analyze the immune characteristics of hub genes and the proportion of immune cell types, so as to find common immune responses between COVID-19 and IBD. Results: A total of 121 common DEGs were identified among four RNA-seq datasets, and were all involved in the functional enrichment analysis related to inflammation and immune response. Transcription factors-DEGs interactions, miRNAs-DEGs coregulatory networks, and protein-drug interactions were identified based on these datasets. Protein-protein interactions (PPIs) was built and 59 hub genes were identified. Moreover, scRNA-seq of peripheral blood monocyte cells (PBMCs) from COVID-19 patients revealed a significant increase in the proportion of CD14+ monocytes, in which 38 of 59 hub genes were highly enriched. These genes, encoding inflammatory cytokines, were also highly expressed in inflammatory macrophages (IMacrophage) of intestinal tissues of IBD patients. Conclusions: We conclude that COVID-19 may promote the progression of IBD through cytokine storms. The candidate drugs and DEGs-regulated networks may suggest effective therapeutic methods for both COVID-19 and IBD.

Single-cell views of the Plasmodium life cycle.

Malaria-causing Plasmodium parasites undergo multiple phenotypic transitions as they cycle between diverse niches in the mammalian and mosquito hosts. Recent applications of single-cell technologies to Plasmodium have enabled the systematic investigation of the distinct stages across the life cycle. Most single-cell data have focused on the parasite exclusively, but a few studies have started to profile both parasite and host cells to shed light on the heterogeneity of cell states that underpin host-parasite interactions. In this opinion article, we highlight how atlasing initiatives are starting to be used to infer functional interactions between parasite and host and could be a powerful tool in drug discovery and vaccine development.

Single-cell atlas of splenocytes reveals a critical role of a novel plasma cell‒specific marker Hspa13 in antibody class-switching recombination and somatic hypermutation.

Our previous study had shown that member 13 (Hspa13) of heat shock protein family A (Hsp70) promotes plasma cell (PC) production and antibody secretion. To further explore Hspa13 expression and function, we combined single-cell RNA-sequencing and antigen receptor lineage (BCR) analysis to characterize sheep red cell‒primed splenocytes. The single-cell transcriptional profiles revealed that Hspa13 is specifically and highly expressed in PCs. These results suggest that Hspa13 is a novel PC-specific marker. In terms of its function, we found that the CD19cre-mediated conditional knock-out (cKO) of Hspa13 reduced the expression of Ebi3 and IL-10 in PCs. Ebi3 and IL-10 are important factors in IL-4‒secreting type 2 helper T cell (Th2) activation and differentiation. As expected, we found that the Hspa13 cKO reduced IL‒4-expressing follicular helper T (Tfh2) cells. Finally, the single-cell antigen receptor analysis demonstrated that the Hspa13 cKO reduced the Aicda-mediated antibody class-switching recombination (CSR) and somatic hypermutation (SHM) in germinal centers (GCs) B cells. Altogether, the single-cell atlas of splenocytes revealed a critical indirect role for the novel PC-specific marker Hspa13 in CSR and SHM in GC B cells by promoting Ebi3 and IL-10 expression in PCs to induce IL-4-expressing Tfh2 cells. Further exploration of Hspa13 expression and function will provide valuable clues for how to use Hspa13 in the treatment of autoimmune diseases.