Autofluorescence signals of immune cells as a tool for describing their functional status

Inflammatory bowel diseases affect increasing numbers of patients around the world. To direct medical therapy, endoscopy and histological evaluation of the tissue are performed. New diagnostic tools, such as multiphoton microscopy (MPM), could improve microscopic tissue evaluation directly during endoscopy. With this method, autofluorescent signals can be detected in real-time without the need of additional fluorescent staining. MPM has been previously used to describe the morphology and architecture of murine and human mucosal inflammation. However, MPM could even allow the identification of various subtypes of immune cells and their functional states in the mucosa based on cell-type specific differences of autofluorescence. To evaluate immune cell autofluorescence, we analysed NADH and FAD signals of murine immune cells using MPM and flow cytometry. All cells were measured in their native form and after in-vitro stimulation. We also studied the effect of apoptosis on cell autofluorescence using flow cytometry, by staining dead cells with Sytox. Our results show an increase in the autofluorescence of NADH and FAD in innate immune cells compared to cells of the adaptive immune system, as measured by MPM (e.g. NADH: 577.2 ± 103.4 vs. 318.4 ± 39.3) and flow cytometry (e.g. NADH: 1379.0 ± 592.6 vs. 276.8 ± 33.6). Of all cell types that were analysed, we found that dendritic cells had the highest NADH and FAD signals. Furthermore, in-vitro stimulation increased autofluorescence signals of adaptive immune cells (e.g. NADH: 318.4 ± 39.3 vs. 637.7 ± 270.8 in MPM) and affected NADH and FAD values of stimulated innate immune cells as well. When comparing dead and alive cells by flow cytometry, we observed a decrease in the NADH signal of apoptotic cells whereas the FAD signal remained constant. Using cell area as an additional characteristic of the cell together with autofluorescence signals, we could clearly differentiate between dead and alive cells and between the different immune cell types. All in all, our results demonstrate the potential of using autofluorescent signals as a tool to characterize single cell types and functional states in a tissue and therefore may lead the path to successful clinical applications of MPM during endoscopic assessment of mucosal inflammation. Publication History Publication Date: 07 September 2021 (online) © 2021. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany