Armando J. Arroyo‐Mejias

Increased sensory nerve density has been described in type 2 inflammatory conditions and is linked to eosinophil and mast cell infiltration and neuropathic pain. To examine these relationships in eosinophilic gastrointestinal diseases, we developed a protocol using Ce3D (clearing-enhanced 3D) tissue clearing to evaluate mucosal gastrointestinal (GI) nerve remodeling and interactions with eosinophils and mast cells in whole-mount GI biopsies. Human gastric and esophageal biopsies were processed for 3-dimensional imaging. Tissues were fixed, permeabilized, and immunolabeled for mucosal nerves (Protein Gene Product 9.5), sensory neurons (substance P), eosinophils (eosinophil peroxidase), mast cells (tryptase), and epithelial cells (pan-cytokeratin). Vacuum-microwave–assisted staining was employed to enhance antibody penetration and protocol efficiency. Samples were cleared with Ce3D and imaged using confocal microscopy. Machine learning–enhanced quantitative analysis and modeling of nerve morphology and cellular interactions, including nerve length, branching, and spatial relationships between eosinophils, mast cells, and sensory neurons were performed. We established a 3-dimensional imaging method for whole-mount GI biopsies to characterize nerve architecture and eosinophil and mast cell interactions in the human esophagus and stomach. This approach enables high resolution and volumetric analyses and can be modified to assess other spatial neuroimmune interactions in human GI mucosal biopsies.

A central question in immunology is what features allow the immune system to respond in a timely manner to a variety of pathogens encountered at unanticipated times and diverse body sites. Two decades of advanced and static dynamic imaging methods have now revealed several major principles facilitating host defense. Suborgan spatial prepositioning of distinct cells promotes time‐efficient interactions upon pathogen sensing. Such pre‐organization also provides an effective barrier to movement of pathogens from parenchymal tissues into the blood circulation. Various molecular mechanisms maintain effective intercellular communication among otherwise rapidly moving cells. These and related discoveries have benefited from recent increases in the number of parameters that can be measured simultaneously in a single tissue section and the extension of such multiplex analyses to 3D tissue volumes. The application of new computational methods to such imaging data has provided a quantitative, in vivo context for cell trafficking and signaling pathways traditionally explored in vitro or with dissociated cell preparations. Here, we summarize our efforts to devise and employ diverse imaging tools to probe immune system organization and function, concluding with a commentary on future developments, which we believe will reveal even more about how the immune system operates in health and disease.