Speaker
Description
In this study, fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence is applied as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages were obtained from human blood-circulating monocytes, polarised using established treatments, and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Fluorescence lifetime imaging microscopy (FLIM) quantified variations in NAD(P)H-derived fluorescent lifetimes in large field-of-view images of individual polarised macrophages also challenged, in real-time with small molecule perturbations of metabolism during imaging. We uncover FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) which strongly stratifies the phenotype of polarised human macrophages. This stratification and parameters emanating from a FLIM approach, served as the basis for machine learning models. Applying a random forest model, identified three strongly governing FLIM parameters, achieving a ROC AUC value of 0.944 when classifying human macrophages. Visualisation of the data show a clear classification of IFNγ-M1 and IL-4-M2 macrophages in response to real-time imaging when treated with FCCP. The excellent performance of machine learning models, applied on the data extracted from the non-invasive technique, underlines further the efficiency of this workflow. This workflow can be easily adopted to non-invasively characterise macrophage polarisation in in vivo models and in vitro multicellular organoid models to study foreign body interactions, biomaterial assessment, pharmaceutical research and screening and clinical applications such as disease diagnosis. Precise regulation of macrophage activation state is key to understanding disease control, tissue homeostasis and implant response, with this regulation shown to be directly related with macrophage intracellular metabolism. Therefore, impaired macrophage metabolism results in impaired function such as the case of diabetes, the foreign body response to biomaterials, obesity or cancer.
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