Abstract
Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. Dysfunction of mitochondrial reprogramming can occur during bacterial and viral infections compromising hosts’ immune signaling. Comparative evaluation of these alterations in response to bacterial and viral ligands can provide insights into a cell’s ability to mount pathogen-specific responses. In this study, we used two-photon excitation fluorescence (TPEF) imaging to quantify reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels in the cell and to calculate the optical redox ratio (ORR), an indicator of mitochondrial dysfunction. Analyses were performed on RAW264.7 cells and murine bone marrow derived macrophages (BMM) stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. Responses were cell type dependent, with primary cells having significantly higher levels of FAD and higher oxygen consumption rates suggesting BMM may be more dependent on mitochondrial metabolism. Our findings also suggest that FAD-TPEF intensity may be a better predictor of mitochondrial activity and localization since it demonstrates unique mitochondrial clustering patterns in LPS vs. Poly(I:C) stimulated macrophages. Collectively, we demonstrate that TPEF imaging is a powerful label-free approach for quantifying changes in mitochondrial function and organization in macrophages following bacterial and viral stimuli.
Highlights
Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses
To set a baseline of mitochondrial activity, we first evaluated changes in OXPHOS activity in RAW264.7 cells and bone marrow derived macrophages (BMM) stimulated with LPS and Poly(I:C) using the Seahorse XFp extracellular efflux analyzer
These results suggest that RAW264.7 cells, which are highly proliferative, may be less dependent on OXPHOS for energy production[16] and that cell lines vs. primary cells may differ in their prioritization of mitochondrial function
Summary
Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. In response to viruses or synthetic viral ligands such as Polyinosinic:polycytidylic acid (Poly(I:C)), sustained OXPHOS activity and increased ROS production are required to mount functional antiviral immune r esponses[13,14,15] These studies suggest that mitochondria might play a central role in modulating pathogen-specific responses. Recent studies have used NAD(P)H-TPEF images to evaluate structural changes in the mitochondrial organization in terms of the dynamic fusion or fission[34,35,36] These metrics, implemented independently or in combination, have been demonstrated to be powerful tools for quantifying the mitochondrial activity in cells and tissue for example, in cancer c ells[32,37] and their response to t reatment[33,38], and in tissue engineering and stem cell d ifferentiation[39,40]. We demonstrate a novel application of TPEF imaging in evaluating single cell mitochondrial reprogramming in macrophages stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. We show that the distance of the FAD-rich structures relative to the centre (nucleus) of the cell, as well as the spatial correlation between the FAD-TPEF and NAD(P)H-TPEF intensities in each cell, can provide valuable new insights into the potential mechanisms by which mitochondria contribute to pathogen-specific responses
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