Visualizing the Immune Response to Epithelial Injury in Mouse Small Intestinal Mucosa Reveals a Two-Phase Respond of Polymorphonuclear Leucocytes in LysM-GFP Mice

Intact intestinal barrier function is crucial for the maintenance of dietary nutrients resorption and restricts uptake of luminal antigens and toxins. Our previous studies have shown that larger epithelial defects (> 7 µm in size), induced by UV-laser application, leads to immigration of polymorphonuclear leucocytes, identified by their typical shaped nuclei and high motility of 25 µm/min. The lamina propria, underlying the epithelium, accommodates numerous eosinophils in physiological state but no neutrophils, which are believed to be the first immune cells at a site of tissue injury. We applied intravital autofluorescence 2-photon microscopy in LysM-GFP mice, in which neutrophils are brightly labeled, but eosinophils are identified by autofluorescence, highly fluorescent granules and their typical bi-lobular shaped nuclei. After epithelium injury with UV-laser application, we visualized the first unlabeled eosinophils after 1-3 minutes in the basal part of the epithelium, where they moved vigorously. Labeled neutrophils were visible crawling within the blood vessels in the lamina propria a few minutes after tissue damage but arrived at the site of epithelial damage after 15 minutes at the earliest. We show that eosinophils are the first immune cells to arrive at an epithelial damage in mouse small intestine, coming directly from the lamina propria, whereas neutrophils have to be recruited from the blood vessels.