Spatial organization of immune cells during inflammation and effects of anti-inflammatory agents
Authors:   , Schäufele Tim Johann 
Published: November 2024

Background: Recognition of a pathogen by Toll-like receptors (TLRs) triggers acute inflammation, which can be broadly divided into proinflammatory and anti-inflammatory processes. The primary goal of an acute innate immunity is to eliminate the pathogen and restore tissue homeostasis, resulting in increased influx of vascular proteins and immune cells, including neutrophilic and eosinophilic granulocytes as well as dendritic cells (DCs), and monocytes. These immune cells respond to inflammatory mediators by phagocytosing, releasing cytotoxic compounds, and releasing additional mediators, such as prostaglandin E2 (PGE2). Currently, the inflammatory process is mainly understood from a time-dependent perspective, without considering the spatial organization and microenvironments of these immune cells. Furthermore, inflammation is often accompanied by pain, which is commonly treated with non-steroidal anti-inflammatory drugs (NSAIDs). However, accumulating evidence suggests that inhibiting cyclooxygenase (COX)-dependent prostanoid synthesis using NSAIDs can contribute to the chronification of inflammation and pain, but the underlying mechanisms responsible for this phenomenon remain elusive. Methods: The multiplex imaging technique “Multi-Epitope-Ligand-Cartography” (MELC) was used to examine the inflammatory response in a mouse model of zymosan-induced inflammation. This technique allows to visualize over 47 fluorescence-labeled antibodies on the same tissue slice. Zymosan particles were injected into the hind paw and localized via fluorescence labeling, which makes this model suitable for studying immune cell localization in regard to the position of the zymosan particles. To analyze high-dimensional image data obtained from MELC, a comprehensive bioinformatic analysis pipeline was established, including phenotype clustering and neighborhood analysis to reveal cell microenvironments. Results: The analysis of MELC data provided insights into the spatial organization of the acute innate inflammation, consisting of a core-region, a proinflammatory region (PI-region), and an anti-inflammatory region (AI-region), characterized by pathogen, proinflammatory M1-like macrophages, and anti-inflammatory M2-like macrophages, respectively. This architecture was established within 24 hours after zymosan injection and persisted over time. These data challenge the current time-dependent perspective of the inflammatory course, highlighting the importance of considering the spatial structure and the dynamic changes in the sizes of these regions over time. Next, the pharmacological impact of the NSAID meloxicam on the formation of the inflammatory architecture was investigated. Meloxicam treatment led to prolonged thermal hypersensitivity and induced specific alterations in the inflammatory architecture, including locally reduced polarization of M1-like macrophages, the disappearance of the PI-region, reduced neutrophil survival, and impaired phagocytic capacity of both neutrophils and macrophages, ultimately resulting in impaired pathogen clearance. Notably, the M2-like macrophages and the AI-region remained unaffected by meloxicam treatment. Interestingly, the number of eosinophils expressing the anti-inflammatory cytokine IL-4 increased in the core-region following meloxicam treatment. Conclusion: This thesis revealed the inflammatory architecture and demonstrated the ability of MELC analysis to assess the pharmacological impact of the NSAID meloxicam on this regional structure, providing a valuable perspective on the NSAID-associated chronification of inflammation and pain. Based on these findings, it is suggested that the administration of NSAIDs during pathogen-induced acute inflammation should be reevaluated, particularly in patients with compromised immune systems.