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Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands

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...

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Autores principales: Allen, Christian Harry, Ahmed, Duale, Raiche-Tanner, Olivia, Chauhan, Vinita, Mostaço-Guidolin, Leila, Cassol, Edana, Murugkar, Sangeeta
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8263786/
https://www.ncbi.nlm.nih.gov/pubmed/34234166
http://dx.doi.org/10.1038/s41598-021-93043-9
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author Allen, Christian Harry
Ahmed, Duale
Raiche-Tanner, Olivia
Chauhan, Vinita
Mostaço-Guidolin, Leila
Cassol, Edana
Murugkar, Sangeeta
author_facet Allen, Christian Harry
Ahmed, Duale
Raiche-Tanner, Olivia
Chauhan, Vinita
Mostaço-Guidolin, Leila
Cassol, Edana
Murugkar, Sangeeta
author_sort Allen, Christian Harry
collection PubMed
description 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.
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spelling pubmed-82637862021-07-09 Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands Allen, Christian Harry Ahmed, Duale Raiche-Tanner, Olivia Chauhan, Vinita Mostaço-Guidolin, Leila Cassol, Edana Murugkar, Sangeeta Sci Rep Article 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. Nature Publishing Group UK 2021-07-07 /pmc/articles/PMC8263786/ /pubmed/34234166 http://dx.doi.org/10.1038/s41598-021-93043-9 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Allen, Christian Harry
Ahmed, Duale
Raiche-Tanner, Olivia
Chauhan, Vinita
Mostaço-Guidolin, Leila
Cassol, Edana
Murugkar, Sangeeta
Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title_full Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title_fullStr Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title_full_unstemmed Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title_short Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
title_sort label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8263786/
https://www.ncbi.nlm.nih.gov/pubmed/34234166
http://dx.doi.org/10.1038/s41598-021-93043-9
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