A cell's phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT-Spatially PhotoActivatable Color ...Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.
Purpose
More than 50 different monogenic disorders causing inflammatory bowel disease (IBD) have been identified. Our goal was to characterize the clinical phenotype, genetic workup, and immunologic ...alterations in an Ashkenazi Jewish patient that presented during infancy with ulcerative colitis and unique clinical manifestations.
Methods
Immune workup and whole-exome sequencing were performed, along with Sanger sequencing for confirmation. Next-generation sequencing of the TCRB and IgH was conducted for immune repertoire analysis. Telomere length was evaluated by in-gel hybridization assay. Mass cytometry was performed on patient’s peripheral blood mononuclear cells, and compared with control subjects and patients with UC.
Results
The patient presented in infancy with failure to thrive and dysmorphic features, consistent with a diagnosis of dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Severe ulcerative colitis manifested in the first year of life and proceeded to the development of a primary immunodeficiency, presenting as
Pneumocystis jiroveci
pneumonia and hypogammaglobulinemia. Genetic studies identified a deleterious homozygous C.3791G>A missense mutation in the helicase regulator of telomere elongation 1 (
RTEL1
), leading to short telomeres in the index patient. Immune repertoire studies showed polyclonal T and B cell receptor distribution, while mass cytometry analysis demonstrated marked immunological alterations, including a predominance of naïve T cells, paucity of B cells, and a decrease in various innate immune subsets.
Conclusions
RTEL1
mutations are associated with significant alterations in immune landscape and can manifest with infantile-onset IBD. A high index of suspicion is required in Ashkenazi Jewish families where the carriage rate of the C.3791G>A variant is high.
Crohn’s disease is an inflammatory bowel disorder that can affect any portion of the gastrointestinal tract, most commonly the terminal ileum near the ileocecal valve. Crohn’s disease can be ...characterized by transmural inflammation and deep fissuring ulcers that predispose to fistula formation and “skip” lesions separated by normal segments of bowel. While often affecting the terminal ileum near the ileocecal valve, Crohn’s disease presenting primarily in the appendix is a rare entity. In part due to its low prevalence, cases of appendiceal Crohn’s disease can be confused for acute, non-Crohn’s-related appendicitis on initial presentation. Although there are published cases of primary appendiceal Crohn’s disease in the medical literature, in most cases the diagnosis is made retrospectively following appendectomy for presumed appendicitis. We report on a case of Crohn’s disease that was diagnosed pre-operatively, primarily involved the appendix, and which progressed radiographically despite medical therapy and resolution of clinical symptoms. Unique management issues related to this case include the appropriateness of systemic therapy for disease isolated to the appendix, an inability to endoscopically obtain tissue for a definitive diagnosis, and the decision to proceed with surgery in an asymptomatic patient with progressive disease on imaging. Intraoperatively, the appendix was severely inflamed and densely adherent to the left pelvic side wall and adjacent to the left ovary and fallopian tube. A laparoscopic appendectomy was performed. Pathology demonstrated acute appendicitis as well as marked mural chronic inflammation and epithelioid granulomas, consistent with Crohn’s disease. Surgical resection may be the most appropriate treatment for Crohn’s disease primarily involving the appendix, obviating the need for systemic therapy and minimizing the risk for appendiceal perforation and fistula formation.
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein ...binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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•Meta-analysis of human, non-human primate, and mouse single-cell RNA-seq datasets for putative SARS-CoV-2 targets•Type II pneumocytes, nasal secretory cells, and absorptive enterocytes are ACE2+TMPRSS2+•Interferon and influenza increase ACE2 in human nasal epithelia and lung tissue•Mouse Ace2 is not upregulated by interferon, raising implications for disease modeling
Analysis of single-cell RNA-seq datasets from human, non-human primate, and mouse barrier tissues identifies putative cellular targets of SARS-CoV-2 on the basis of ACE2 and TMPRSS2 expression. ACE2 represents a previously unappreciated interferon-stimulated gene in human, but not mouse, epithelial tissues, identifying anti-viral induction of a host tissue-protective mechanism, but also a potential means for viral exploitation of the host response.
Studies are needed to determine the mechanisms of mucosal dysregulation in patients with inflammatory bowel diseases (IBDs) and differences in inflammatory responses of patients with ulcerative ...colitis (UC) vs Crohn’s disease (CD). We used mass cytometry (CyTOF) to characterize and compare immune cell populations in the mucosa and blood from patients with IBD and without IBD (controls) at single-cell resolution.
We performed CyTOF analysis of colonic mucosa samples (n = 87) and peripheral blood mononuclear cells (n = 85) from patients with active or inactive UC or CD and controls. We also performed single-cell RNA sequencing, flow cytometry, and RNA in situ hybridization analyses to validate key findings. We used random forest modeling to identify differences in signatures across subject groups.
Compared with controls, colonic mucosa samples from patients with IBD had increased abundances of HLA-DR+CD38+ T cells, including T-regulatory cells that produce inflammatory cytokines; CXCR3+ plasmablasts; and IL1B+ macrophages and monocytes. Colonic mucosa samples from patients with UC were characterized by expansion of IL17A+ CD161+ effector memory T cells and IL17A+ T-regulatory cells; expansion of HLA-DR+CD56+ granulocytes; and reductions in type 3 innate lymphoid cells. Mucosal samples from patients with active CD were characterized by IL1B+HLA-DR+CD38+ T cells, IL1B+TNF+IFNG+ naïve B cells, IL1B+ dendritic cells (DCs), and IL1B+ plasmacytoid DCs. Peripheral blood mononuclear cells from patients with active CD differed from those of active UC in that the peripheral blood mononuclear cells from patients with CD had increased IL1B+ T-regulatory cells, IL1B+ DCs and IL1B+ plasmacytoid DCs, IL1B+ monocytes, and fewer group 1 innate lymphoid cells. Random forest modeling differentiated active UC from active CD in colonic mucosa and blood samples; top discriminating features included many of the cellular populations identified above.
We used single-cell technologies to identify immune cell populations specific to mucosa and blood samples from patients with active or inactive CD and UC and controls. This information might be used to develop therapies that target specific cell populations in patients with different types of IBD.
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