Natural NADH and FAD Autofluorescence as Label-Free Biomarkers for Discriminating Subtypes and Functional States of Immune Cells

Immune cell activity is a major factor for disease progression in inflammatory bowel diseases (IBD). Classifying the type and functional state of immune cells is therefore crucial in clinical diagnostics of IBD. Label-free optical technologies exploiting NADH and FAD autofluorescence, such as multiphoton microscopy, have been used to describe tissue morphology in healthy and inflamed colon samples. Nevertheless, a strategy for the identification of single immune cell subtypes within the tissue is yet to be developed. This work aims to initiate an understanding of autofluorescence changes depending on immune cell type and activation state. For this, NADH and FAD autofluorescence signals of different murine immune cell subtypes under native conditions, as well as upon in vitro stimulation and cell death, have been evaluated. Autofluorescence was assessed using flow cytometry and multiphoton microscopy. Our results reveal significantly increased NADH and FAD signals in innate immune cells compared to adaptive immune cells. This allowed identification of relative amounts of neutrophils and CD4+ T cells in mixed cell suspensions, by using NADH signals as a differentiation marker. Furthermore, in vitro stimulation significantly increased NADH and FAD autofluorescence in adaptive immune cells and macrophages. Cell death induced a significant drop in NADH autofluorescence, while FAD signals were hardly affected. Taken together, these results demonstrate the value of autofluorescence as a tool to characterize immune cells in different functional states, paving the way to the label-free clinical classification of IBD in the future.

Automated summary

The activity of immune cells is a major factor in the progression of inflammatory bowel diseases. This study aims to understand the autofluorescence changes depending on the type and activation state of immune cells. The results show that innate immune cells have significantly increased autofluorescence signals compared to adaptive immune cells. In vitro stimulation also increases autofluorescence signals in adaptive immune cells and macrophages. Cell death leads to a significant decrease in NADH autofluorescence, while FAD signals remain unaffected. These findings demonstrate the value of autofluorescence as a tool to characterize immune cells in different functional states.