A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease.

Single-cell-based methods such as flow cytometry or single-cell mRNA sequencing (scRNA-seq) allow deep molecular and cellular profiling of immunological processes. Despite their high throughput, however, these measurements represent only a snapshot in time. Here, we explore how longitudinal single-cell-based datasets can be used for deterministic ordinary differential equation (ODE)-based modelling to mechanistically describe immune dynamics. We derived longitudinal changes in cell numbers of colonic cell types during inflammatory bowel disease (IBD) from flow cytometry and scRNA-seq data of murine colitis using ODE-based models. Our mathematical model generalised well across different protocols and experimental techniques, and we hypothesised that the estimated model parameters reflect biological processes. We validated this prediction of cellular turnover rates with KI-67 staining and with gene expression information from the scRNA-seq data not used for model fitting. Finally, we tested the translational relevance of the mathematical model by deconvolution of longitudinal bulk mRNA-sequencing data from a cohort of human IBD patients treated with olamkicept. We found that neutrophil depletion may contribute to IBD patients entering remission. The predictive power of IBD deterministic modelling highlights its potential to advance our understanding of immune dynamics in health and disease.

Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Integrating single-cell multi-omics and prior biological knowledge for a functional characterization of the immune system.

The immune system comprises diverse specialized cell types that cooperate to defend the host against a wide range of pathogenic threats. Recent advancements in single-cell and spatial multi-omics technologies provide rich information about the molecular state of immune cells. Here, we review how the integration of single-cell and spatial multi-omics data with prior knowledge-gathered from decades of detailed biochemical studies-allows us to obtain functional insights, focusing on gene regulatory processes and cell-cell interactions. We present diverse applications in immunology and critically assess underlying assumptions and limitations. Finally, we offer a perspective on the ongoing technological and algorithmic developments that promise to get us closer to a systemic mechanistic understanding of the immune system.

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms.

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400-650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.