In vivofluorescence lifetime imaging captures metabolic changes in macrophages during wound responses in zebrafish

Abstract

39The effector functions of macrophages across the spectrum of activation states in vitro are linked to 40 profound metabolic rewiring. However, the metabolism of macrophages remains poorly characterized in 41 vivo. To assess changes in the intracellular metabolism of macrophages in their native inflammatory 42 microenvironment, we employed two-photon fluorescence lifetime imaging microscopy (FLIM) of 43 metabolic coenzymes NAD(P)H and FAD. We found that pro-inflammatory activation of macrophages in 44 vivo was associated with a decrease in the optical redox ratio [NAD(P)H/(NAD(P)H+FAD)] relative to a 45 pro-resolving population during both infected and sterile inflammation. FLIM also resolved temporal 46 changes in the optical redox ratio and lifetime variables of NAD(P)H in macrophages over the course of 47 sterile inflammation. Collectively, we show that non-invasive and label-free imaging of autofluorescent 48 metabolic coenzymes is sensitive to dynamic changes in macrophage activation in interstitial tissues. This 49 imaging-based approach has broad applications in immunometabolism by probing in real time the 50 temporal and spatial metabolic regulation of immune cell function in a live organism. 51 52 Significance 53 Metabolic regulation of macrophage effector functions has recently emerged as a key concept in immune 54 cell biology. Studies rely on in vitro and ex vivo approaches to study macrophage metabolism, however 55 the high plasticity of these cells suggest that removal from their native microenvironment may induce 56 changes in their intracellular metabolism. Here, we show that fluorescence lifetime imaging microscopy 57 of metabolic coenzymes captures dynamic changes in the metabolic activity of macrophages while 58 maintaining them in their endogenous microenvironment. This approach also resolves variations on a 59 single-cell level, in contrast to bulk measurements provided by traditional biochemical assays, making it a 60 potentially valuable tool in the field of immunometabolism. 61 62 63 64 65 66 67 68 69 70 71 72 Nicotinamide adenine dinucleotide (NADH/NAD+) and flavin adenine dinucleotide (FADH 2 /FAD) are 97 endogenous metabolic coenzymes, and serve as electron carriers in numerous metabolic pathways 98including glycolysis, the Krebs cycle, electron transport chain and oxidative phosphorylation (17, 18). 99These coenzymes are autofluorescent when reduced and oxidized, respectively, and allow for 100 fluorescence lifetime imaging microscopy (FLIM) to quantify intracellular metabolism using intensity 101 and lifetime measurements (17, 18). NADH and NADPH have overlapping spectral properties, and for 102 accuracy NAD(P)H is used to reflect their combined signals (19). Fluorescence intensity can be used to 103 determine the optical redox ratio that provides an assessment of the redox state of the cell (20). Multiple 104 definitions of the optical redox ratio exist, but here we use NAD(P)H/(NAD(P)H+FAD), since an 105 increase in the optical redox ratio intuitively corresponds with an increase i...

Intravital Metabolic Autofluorescence Imaging Captures Macrophage Heterogeneity Across Normal and Cancerous Tissue

“…The use of fluorescent reporter mice in the current study provided macrophage-specific labels while circumventing these issues of antibody delivery and non-specific binding. The mCherry reporter used in the current study also did not interfere with NAD(P)H and FAD autofluorescence, similarly observed in previous in vivo autofluorescence studies (Hoffmann and Ponik, 2020;Miskolci et al, 2020). Identifying macrophage-specific autofluorescence features without fluorescent reporters remains challenging to due to substantial macrophage heterogeneity in vivo.…”

Label-free optical imaging of cell function and collagen structure for cell-based therapies

“…M1-like macrophages have upregulated glycolysis while M2-like macrophages generally are more dependent on oxidative metabolism [ 34 ]. These metabolic differences between M1-like and M2-like macrophages are detected by autofluorescence imaging of NAD(P)H and FAD [ 41 - 45 ]. High resolution optical imaging, combined with single-cell segmentation, allows quantification of the changes in metabolism at the cell level, and successfully distinguishes subpopulations of macrophages [ 15 ].…”

“…Although the combination of autofluorescence lifetime imaging with SHG imaging is currently underexplored for the characterization of cell-seeded biomaterials and scaffolds, we expect increased use of autofluorescence lifetime imaging concurrent with SHG imaging for characterization of regenerative engineered devices. NAD(P)H and FAD fluorescence lifetime imaging allows differentiation of macrophage phenotypes, due to their different metabolic activities [ 41 - 45 ]. Since the pro-inflammatory macrophage (M1) and anti-inflammatory macrophage (M2) macrophages exhibit different immune functions, autofluorescence imaging can be used to study macrophage polarization states to monitor wound healing progression.…”