Macrophage efferocytosis is the phagocytic clearance of apoptotic cells. This fundamental process governs tissue homeostasis, immune tolerance, and the resolution of inflammation. Immune and metabolic reprogramming are essential to sustain efferocytic capacity. Maladaptive efferocytosis is mostly considered “incomplete or nonfunctional” in chronic diseases, but can also be characterized by misinterpreted, mistimed, or mischaracterized efferocytic events. A central barrier to progress has been the inability to define efferocytic macrophages as functional entities, rather than transient, poorly resolved states obscured by technical and conceptual limitations. Here, we review efferocytosis across health and disease by linking apoptotic cargo identity, macrophage processing capacity, and inflammatory and metabolic constraints to efferocytic throughput. We further highlight the technical challenges that cause the bona fide efferocytic states to be misclassified or excluded in high‑dimensional datasets. Applying an updated framework acknowledging the complexity of macrophage efferocytosis will ultimately enable the development of novel therapeutic strategies.

Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, type 2 immune-mediated disease of the esophagus characterized by eosinophil-predominant mucosal inflammation, epithelial remodeling, and subepithelial fibrosis. Although patient biopsies have established the EoE transcriptome and identified key cellular and molecular mediators, biopsy-based research is inherently correlative and cannot resolve causal disease mechanisms, temporal disease progression or the functional hierarchy of immune cell interactions. Thus, animal models are indispensable tools for addressing these limitations. In this Review, we examine the landscape of experimental EoE models. We assess the capacity of each model to recapitulate key disease features including lamina propria and intraepithelial eosinophilia, subepithelial fibrosis, basal cell hyperplasia, epithelial barrier dysfunction, and angiogenesis. We further map these models to human EoE transcriptomic overlap and disease endotype relevance. Across this analysis, we highlight mechanistic insights that were obtained from these models including the eosinophil-independence of IL-13-driven esophageal remodeling, the respective roles of thymic stromal lymphopoietin and IL-33, and the critical role of epithelial-expressed IL-13Rα1. We further highlight the profibrotic functions of amphiregulin-producing T helper 2 cells and colony-stimulating factor 1-dependent macrophages. We discuss the anatomical, genetic and functional limitations of current models and outline directions for the next generation of EoE preclinical systems.

Aims: Conventional two-dimensional (2D) culture systems fail to recapitulate the structural and functional complexity of the brain parenchyma, driving microglia toward an activated, non-homeostatic state. Here, we therefore developed a simple, robust, and scalable three-dimensional (3D) culture system using a Matrigel-collagen composite matrix to promote a more in vivo-like microglial phenotype.

Methods: Primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) from multiple donor lines were cultured either in conventional 2D conditions or embedded in a Matrigel-collagen 3D matrix. Phenotypes were characterized using confocal microscopy and morphometric analysis, live-cell imaging, transcriptomic profiling, electrophysiology, cytokine secretion assays upon inflammatory stimulation, and flow cytometry.

Results: In 3D culture, mouse microglia developed a markedly more ramified and structurally complex morphology, supported by filamentous actin-rich terminal processes. Live-cell imaging demonstrated enhanced dynamics of cell processes and a more sessile cell body, consistent with physiological microglia surveillance behavior. Transcriptomic profiling further revealed that microglia cultured in 3D downregulated genes associated with activation and proliferation while upregulating homeostatic markers, yet retained a robust cytokine response to inflammatory stimulation. Electrophysiological profiling similarly indicated a shift toward a more resting, in vivo-like state. Human iMGLs showed comparable morphological adaptations in 3D, with protein expression and functional readouts reflecting both shared homeostatic features and cell line- and context-dependent characteristics.

Conclusion: Together, our results demonstrate that a simple and accessible 3D culture system shapes microglial morphology, behavior, and molecular identity, establishing a versatile platform for investigating microglial physiology in a controlled yet physiologically more relevant environment.

Monocytes consist of several subsets, which differ in their phenotype and functional capacity. This heterogeneity was first shown in man, and later evidenced in other species. Here, we review the current knowledge on the phenotype and functionality of monocyte populations in species of veterinary interest, in comparison to man. The increasing availability of reagents for leukocyte phenotypic analyses, along with the growing application of -omic technologies, are enabling a better characterization of these subsets, and facilitating comparisons across species. The review demonstrates that classical, intermediate, and non-classical monocytes can be defined in pig, cattle, sheep, camel, buffalo, horse, and dog. However, more phenotypic, functional and transcriptomic studies are needed in some species to establish an accurate correspondence. A better understanding of shared and species-specific features of these monocyte subsets will pave the way for translational research on strategies selectively targeting these cells to treat different pathological conditions, including infectious, inflammatory and malignant diseases.

Macrophage efferocytosis is the phagocytic clearance of apoptotic cells. This fundamental process governs tissue homeostasis, immune tolerance, and the resolution of inflammation. Immune and metabolic reprogramming are essential to sustain efferocytic capacity. Maladaptive efferocytosis is mostly considered “incomplete or nonfunctional” in chronic diseases, but can also be characterized by misinterpreted, mistimed, or mischaracterized efferocytic events. A central barrier to progress has been the inability to define efferocytic macrophages as functional entities, rather than transient, poorly resolved states obscured by technical and conceptual limitations. Here, we review efferocytosis across health and disease by linking apoptotic cargo identity, macrophage processing capacity, and inflammatory and metabolic constraints to efferocytic throughput. We further highlight the technical challenges that cause the bona fide efferocytic states to be misclassified or excluded in high‑dimensional datasets. Applying an updated framework acknowledging the complexity of macrophage efferocytosis will ultimately enable the development of novel therapeutic strategies.

Monocytes consist of several subsets, which differ in their phenotype and functional capacity. This heterogeneity was first shown in man, and later evidenced in other species. Here, we review the current knowledge on the phenotype and functionality of monocyte populations in species of veterinary interest, in comparison to man. The increasing availability of reagents for leukocyte phenotypic analyses, along with the growing application of -omic technologies, are enabling a better characterization of these subsets, and facilitating comparisons across species. The review demonstrates that classical, intermediate, and non-classical monocytes can be defined in pig, cattle, sheep, camel, buffalo, horse, and dog. However, more phenotypic, functional and transcriptomic studies are needed in some species to establish an accurate correspondence. A better understanding of shared and species-specific features of these monocyte subsets will pave the way for translational research on strategies selectively targeting these cells to treat different pathological conditions, including infectious, inflammatory and malignant diseases.

Eosinophilic esophagitis (EoE) is a chronic, food antigen-driven, type 2 immune-mediated disease of the esophagus characterized by eosinophil-predominant mucosal inflammation, epithelial remodeling, and subepithelial fibrosis. Although patient biopsies have established the EoE transcriptome and identified key cellular and molecular mediators, biopsy-based research is inherently correlative and cannot resolve causal disease mechanisms, temporal disease progression or the functional hierarchy of immune cell interactions. Thus, animal models are indispensable tools for addressing these limitations. In this Review, we examine the landscape of experimental EoE models. We assess the capacity of each model to recapitulate key disease features including lamina propria and intraepithelial eosinophilia, subepithelial fibrosis, basal cell hyperplasia, epithelial barrier dysfunction, and angiogenesis. We further map these models to human EoE transcriptomic overlap and disease endotype relevance. Across this analysis, we highlight mechanistic insights that were obtained from these models including the eosinophil-independence of IL-13-driven esophageal remodeling, the respective roles of thymic stromal lymphopoietin and IL-33, and the critical role of epithelial-expressed IL-13Rα1. We further highlight the profibrotic functions of amphiregulin-producing T helper 2 cells and colony-stimulating factor 1-dependent macrophages. We discuss the anatomical, genetic and functional limitations of current models and outline directions for the next generation of EoE preclinical systems.

Aims: Conventional two-dimensional (2D) culture systems fail to recapitulate the structural and functional complexity of the brain parenchyma, driving microglia toward an activated, non-homeostatic state. Here, we therefore developed a simple, robust, and scalable three-dimensional (3D) culture system using a Matrigel-collagen composite matrix to promote a more in vivo-like microglial phenotype.

Methods: Primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) from multiple donor lines were cultured either in conventional 2D conditions or embedded in a Matrigel-collagen 3D matrix. Phenotypes were characterized using confocal microscopy and morphometric analysis, live-cell imaging, transcriptomic profiling, electrophysiology, cytokine secretion assays upon inflammatory stimulation, and flow cytometry.

Results: In 3D culture, mouse microglia developed a markedly more ramified and structurally complex morphology, supported by filamentous actin-rich terminal processes. Live-cell imaging demonstrated enhanced dynamics of cell processes and a more sessile cell body, consistent with physiological microglia surveillance behavior. Transcriptomic profiling further revealed that microglia cultured in 3D downregulated genes associated with activation and proliferation while upregulating homeostatic markers, yet retained a robust cytokine response to inflammatory stimulation. Electrophysiological profiling similarly indicated a shift toward a more resting, in vivo-like state. Human iMGLs showed comparable morphological adaptations in 3D, with protein expression and functional readouts reflecting both shared homeostatic features and cell line- and context-dependent characteristics.

Conclusion: Together, our results demonstrate that a simple and accessible 3D culture system shapes microglial morphology, behavior, and molecular identity, establishing a versatile platform for investigating microglial physiology in a controlled yet physiologically more relevant environment.