Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis
Identifieur interne : 000830 ( Pmc/Curation ); précédent : 000829; suivant : 000831Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis
Auteurs : Yue Li [République populaire de Chine] ; Yuan He [République populaire de Chine] ; Kai Miao [République populaire de Chine] ; Ying Zheng [République populaire de Chine] ; Chuxia Deng [République populaire de Chine] ; Tzu-Ming Liu [République populaire de Chine]Source :
- Theranostics [ 1838-7640 ] ; 2020.
Abstract
Highly plastic macrophages are pivotal players in the body's homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of
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DOI: 10.7150/thno.40495
PubMed: 32194843
PubMed Central: 7053213
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Imaging of macrophage mitochondria dynamics <italic>in vivo</italic> reveals cellular activation phenotype for diagnosis</title><author><name sortKey="Li, Yue" sort="Li, Yue" uniqKey="Li Y" first="Yue" last="Li">Yue Li</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="He, Yuan" sort="He, Yuan" uniqKey="He Y" first="Yuan" last="He">Yuan He</name><affiliation wicri:level="1"><nlm:aff id="A2">Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Miao, Kai" sort="Miao, Kai" uniqKey="Miao K" first="Kai" last="Miao">Kai Miao</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Zheng, Ying" sort="Zheng, Ying" uniqKey="Zheng Y" first="Ying" last="Zheng">Ying Zheng</name><affiliation wicri:level="1"><nlm:aff id="A2">Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Deng, Chuxia" sort="Deng, Chuxia" uniqKey="Deng C" first="Chuxia" last="Deng">Chuxia Deng</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Liu, Tzu Ming" sort="Liu, Tzu Ming" uniqKey="Liu T" first="Tzu-Ming" last="Liu">Tzu-Ming Liu</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32194843</idno><idno type="pmc">7053213</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053213</idno><idno type="RBID">PMC:7053213</idno><idno type="doi">10.7150/thno.40495</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000830</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000830</idno><idno type="wicri:Area/Pmc/Curation">000830</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000830</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Imaging of macrophage mitochondria dynamics <italic>in vivo</italic> reveals cellular activation phenotype for diagnosis</title><author><name sortKey="Li, Yue" sort="Li, Yue" uniqKey="Li Y" first="Yue" last="Li">Yue Li</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="He, Yuan" sort="He, Yuan" uniqKey="He Y" first="Yuan" last="He">Yuan He</name><affiliation wicri:level="1"><nlm:aff id="A2">Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Miao, Kai" sort="Miao, Kai" uniqKey="Miao K" first="Kai" last="Miao">Kai Miao</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Zheng, Ying" sort="Zheng, Ying" uniqKey="Zheng Y" first="Ying" last="Zheng">Ying Zheng</name><affiliation wicri:level="1"><nlm:aff id="A2">Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Deng, Chuxia" sort="Deng, Chuxia" uniqKey="Deng C" first="Chuxia" last="Deng">Chuxia Deng</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author><author><name sortKey="Liu, Tzu Ming" sort="Liu, Tzu Ming" uniqKey="Liu T" first="Tzu-Ming" last="Liu">Tzu-Ming Liu</name><affiliation wicri:level="1"><nlm:aff id="A1">Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR</wicri:regionArea></affiliation></author></analytic><series><title level="j">Theranostics</title><idno type="eISSN">1838-7640</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Highly plastic macrophages are pivotal players in the body's homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of <italic>in vivo</italic> characterization methods to reveal the dynamic activation of macrophages impedes these studies in various disease contexts.</p><p><bold>Methods</bold>: Here,<italic> in vitro</italic> bone marrow-derived macrophages (BMDMs) and <italic>in vivo</italic> Matrigel plug were used to evaluate how mitochondria dynamics supports cellular activation and functions. We conducted macrophage repolarization <italic>in vitro</italic> to track mitochondria dynamics during the shift of activation status. For <italic>in vivo</italic> diagnosis, a novel MitoTracker-loaded liposome was first developed to label macrophage mitochondria in mice before/after inflammatory stimulation.</p><p><bold>Results</bold>: Based on the typical activation of <italic>in vitro</italic> BMDMs, we found glycolysis based macrophages have punctate and discrete mitochondria, while OXPHOS active macrophages have elongated and interconnected mitochondria. M1, M2a, M2b, and M2c activated BMDMs showed clustered and differentiable features in mitochondrial morphology. These features also hold for Matrigel plug-recruited macrophages in mice. Furthermore, with the interventions on M2a macrophages <italic>in vitro</italic>, we demonstrated that mitochondria morphology could be a metabolic index to evaluate macrophage activation status under drug manipulation. Using the MitoTracker-loaded liposomes, we further achieved subcellular imaging of macrophage mitochondria<italic> in vivo</italic>. Their organization dynamics revealed the dynamic change from anti-inflammatory macrophages to inflammatory ones<italic> in vivo</italic> under the lipopolysaccharide (LPS) challenge.</p><p><bold>Conclusion</bold>: These results reveal that subcellular imaging of mitochondria organization can characterize the activation status of macrophage <italic>in vitro</italic> and <italic>in vivo</italic> at a single-cell level, which is critical for the studies of noninvasive diagnosis and therapeutic drug monitoring.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Martinez, Fo" uniqKey="Martinez F">FO Martinez</name></author><author><name sortKey="Gordon, S" uniqKey="Gordon S">S Gordon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pollard, Jw" uniqKey="Pollard J">JW Pollard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="O Neill, La" uniqKey="O Neill L">LA 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Theranostics</journal-id><journal-id journal-id-type="iso-abbrev">Theranostics</journal-id><journal-id journal-id-type="publisher-id">thno</journal-id><journal-title-group><journal-title>Theranostics</journal-title></journal-title-group><issn pub-type="epub">1838-7640</issn><publisher><publisher-name>Ivyspring International Publisher</publisher-name><publisher-loc>Sydney</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32194843</article-id><article-id pub-id-type="pmc">7053213</article-id><article-id pub-id-type="doi">10.7150/thno.40495</article-id><article-id pub-id-type="publisher-id">thnov10p2897</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Paper</subject></subj-group></article-categories><title-group><article-title>Imaging of macrophage mitochondria dynamics <italic>in vivo</italic> reveals cellular activation phenotype for diagnosis</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Li</surname><given-names>Yue</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Yuan</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Miao</surname><given-names>Kai</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zheng</surname><given-names>Ying</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Deng</surname><given-names>Chuxia</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Tzu-Ming</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="corresp" rid="FNA_envelop">✉</xref></contrib></contrib-group><aff id="A1"><label>1</label>Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China</aff><aff id="A2"><label>2</label>Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao SAR, China</aff><author-notes><corresp id="FNA_envelop">✉ Corresponding author: Tzu-Ming Liu, Professor, Faculty of Health Sciences, University of Macau, Taipa, Macao SAR, China. Telephone: +0853 8822 4693; Fax numbers: +0853 8822 2314; E-mail address: <email>tmliu@um.edu.mo</email></corresp><fn fn-type="COI-statement"><p>Competing Interests: The authors have declared that no competing interest exists.</p></fn></author-notes><pub-date pub-type="collection"><year>2020</year></pub-date><pub-date pub-type="epub"><day>3</day><month>2</month><year>2020</year></pub-date><volume>10</volume><issue>7</issue><fpage>2897</fpage><lpage>2917</lpage><history><date date-type="received"><day>21</day><month>9</month><year>2019</year></date><date date-type="accepted"><day>14</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© The author(s)</copyright-statement><copyright-year>2020</copyright-year><license license-type="open-access"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">https://creativecommons.org/licenses/by/4.0/</ext-link>). See <ext-link ext-link-type="uri" xlink:href="http://ivyspring.com/terms">http://ivyspring.com/terms</ext-link> for full terms and conditions.</license-p></license></permissions><abstract><p>Highly plastic macrophages are pivotal players in the body's homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of <italic>in vivo</italic> characterization methods to reveal the dynamic activation of macrophages impedes these studies in various disease contexts.</p><p><bold>Methods</bold>: Here,<italic> in vitro</italic> bone marrow-derived macrophages (BMDMs) and <italic>in vivo</italic> Matrigel plug were used to evaluate how mitochondria dynamics supports cellular activation and functions. We conducted macrophage repolarization <italic>in vitro</italic> to track mitochondria dynamics during the shift of activation status. For <italic>in vivo</italic> diagnosis, a novel MitoTracker-loaded liposome was first developed to label macrophage mitochondria in mice before/after inflammatory stimulation.</p><p><bold>Results</bold>: Based on the typical activation of <italic>in vitro</italic> BMDMs, we found glycolysis based macrophages have punctate and discrete mitochondria, while OXPHOS active macrophages have elongated and interconnected mitochondria. M1, M2a, M2b, and M2c activated BMDMs showed clustered and differentiable features in mitochondrial morphology. These features also hold for Matrigel plug-recruited macrophages in mice. Furthermore, with the interventions on M2a macrophages <italic>in vitro</italic>, we demonstrated that mitochondria morphology could be a metabolic index to evaluate macrophage activation status under drug manipulation. Using the MitoTracker-loaded liposomes, we further achieved subcellular imaging of macrophage mitochondria<italic> in vivo</italic>. Their organization dynamics revealed the dynamic change from anti-inflammatory macrophages to inflammatory ones<italic> in vivo</italic> under the lipopolysaccharide (LPS) challenge.</p><p><bold>Conclusion</bold>: These results reveal that subcellular imaging of mitochondria organization can characterize the activation status of macrophage <italic>in vitro</italic> and <italic>in vivo</italic> at a single-cell level, which is critical for the studies of noninvasive diagnosis and therapeutic drug monitoring.</p></abstract><kwd-group><kwd>macrophage</kwd><kwd>mitochondria</kwd><kwd>liposome</kwd><kwd><italic>in vivo</italic> imaging</kwd><kwd>multiphoton microscopy</kwd></kwd-group></article-meta></front><floats-group><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold> Mitochondrial morphology varies from peripheral blood monocytes to bone marrow-derived macrophages (BMDMs). (A)</bold> Schematic diagram showing the isolation of mononuclear leukocytes from the peripheral blood of mice using Histopaque (1.077 g/mL) banding method. <bold>(B)</bold> Fluorescence confocal images of mitochondria (green color, MitoTracker Green FM), nuclei (blue color, Hoechst 33342), and surface marker (red color, CD115-PE) in monocytes (M) and lymphocytes (L). Scale bar, 10 µm. <bold>(C)</bold> Schematic diagram showing the preparation of naive bone marrow-derived macrophages (BMDMs) using macrophage colony-stimulating factor (M-CSF) stimulation. The macrophage lineage was validated by the F4/80-Alexa 647 vs CD11b-PE scatter plot. <bold>(D)</bold> Fluorescence confocal images of mitochondria (red color, MitoTracker Red CMXRos) and cellular membrane (green color, PKH67 Green) in BMDMs. Scale bar, 20 µm. <bold>(E)</bold> Mitochondrial network morphology of mononuclear cells (corresponds to Figure <xref ref-type="fig" rid="F1">1</xref>B) and BMDMs (corresponds to Figure <xref ref-type="fig" rid="F1">1</xref>D) produced by ImageJ macro tools. Scale bar, 10 µm. <bold>(F)</bold> Mitochondrial organization analysis of monocytes and BMDMs. Mitochondrial footprint reflects mitochondrial mass in a cell, mean network size is the mean number of branches per network, while mean branch/rod length is the average size of all rods/branches. Dots are data from individual cells (n=10). <bold>(G)</bold> Flow cytometer data of MitoTracker Green FM fluorescence in monocytes (red line) and BMDMs (gray area). All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g001"></graphic></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold> Mitochondria organization correlates with the activation status of BMDMs <italic>in vitro</italic>. (A)</bold> Schematic diagram showing the activation of bone marrow-derived macrophages (BMDMs) using different cytokines. Naive BMDMs were represented by gray color, M1 by red color, M2a by blue color, M2b by violet color, and M2c by green color. <bold>(B)</bold> After 24 hr of cytokine treatment, a flow cytometer was used to assess CD86, CD206, CD163 expression of BMDMs (naive, M1, M2a, M2b, and M2c). <bold>(C)</bold> After 24 hr of cytokine treatment, reactive oxygen species (ROS) and nitric oxide (NO) production of BMDMs were measured using DCFH-DA and DAF-FM DA fluorescent probes, respectively. <bold>(D)</bold> Flow cytometer was used to assess the intracellular arginase-1 expression of BMDMs. Fold changes of expression relative to naive were evaluated by mean fluorescence intensity (MFI). <bold>(E)</bold> Fluorescence confocal images of mitochondria (red color, MitoTracker Red CMXRos), and nuclei (blue color, Hoechst 33342) in BMDMs. Scale bar, 10 µm. <bold>(F)</bold> 2D scatter plots from mitochondrial network analysis (footprints, network size, and branch length) performed on BMDMs (naive, M1, M2a, M2b, and M2c). The quadrants of fission/fusion were added to enclose clustered macrophages in feature space based on their mitochondrial organization. The coordinates of each dot represent the feature values of individual cells (n≥10). (<bold>G</bold>) Representative structured illumination microscopy (SIM) images of mitochondria (red color, MitoTracker Red CMXRos) in BMDMs. Scale bar, 10 µm. All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g002"></graphic></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold> Mitochondria regulate the cellular respiration of activated BMDMs <italic>in vitro</italic>. (A)</bold> Representive transmission electron microscopy (TEM) images of mitochondria in BMDMs. Scale bar, 1 µm. <bold>(B)</bold> Mitochondrial membrane potential (ΔΨm) was measured using JC-1 probes. 100 μM FCCP-treated naive BMDMs as a positive control. A shifted population to lower fluorescence in the JC-1 aggregates channel shows the mitochondrial membrane potential is compromised. <bold>(C)</bold> The oxygen consumption rate (OCR) of BMDMs at baseline and in response to sequential treatment with oligomycin (OM), FCCP, and rotenone plus antimycin A (Rot/AA). <bold>(D)</bold> Basal respiration, ATP production, maximum respiration, and spare respiratory capacity of BMDMs at different activation status. <bold>(E)</bold> Glycolysis, glycolytic capability, and glycolytic reserve of BMDMs at different activation status. (<bold>F</bold>) Glucose uptake capability of BMDMs was measured using 2-NBDG fluorescent probes. Fold changes of 2-NBDG uptake were evaluated by MFI. (<bold>G</bold>) The mapping of metabolic phenotype based on the basal OCR and basal ECAR of BMDMs (naive, M1, M2a, M2b, and M2c). All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g003"></graphic></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold> Mitochondrial dynamics reflects the activation status of Matrigel plug-recruited macrophages <italic>in situ</italic>. (A)</bold> Schematic overview of experiment procedures, including Matrigel mixture preparation, subcutaneous injection and <italic>ex vivo</italic> imaging on Matrigel plugs. To generated activated macrophages in mice, M-CSF+PBS were used for naive macrophage differentiation, M-CSF+LPS+IFN-γ for M1, M-CSF+IL-4 for M2a, M-CSF+immune-complexes+LPS for M2b, M-CSF+IL-10 for M2c, and PBS only for the control group. <bold>(B)</bold> Immunofluorescence imaging of F4/80 expression (green) of fixed cells in implanted Matrigel plugs. The nuclei are stained with Hoechst 33342 (blue). Scale bar, 100 µm. <bold>(C)</bold><italic>Ex vivo</italic> live-cell imaging on mitochondria (red color, MitoTracker Red CMXRos), and nuclei (blue color, Hoechst 33342) in different types of macrophages recruited by Matrigel plugs (naive, M1, M2a, M2b, and M2c). Scale bar, 10 µm. <bold>(D)</bold> Quantitative analysis of mitochondrial organization on macrophages from Matrigel plugs (footprint, network size, and branch length). Dots are data from individual cells (n≥10). <bold>(E)</bold> Comparison of 2D scatter plots from mitochondrial organization parameters between BMDMs (Naive, M1, M2a, M2b, and M2c) <italic>in vitro</italic> and macrophages of Matrigel plugs (Naive <italic>in vivo</italic>, M1<italic> in vivo</italic>, M2a<italic> in vivo</italic>, M2b<italic> in vivo</italic>, M2c<italic> in vivo</italic>). The quadrants of fission/fusion were added to enclose clustered macrophages in feature space based on their mitochondrial organization. Dots are data of individual cells (n≥10). All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g004"></graphic></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold> Mitochondrial morphology changes along with the repolarization of M2a macrophages. (A)</bold> Schematic illustration of the M1-activation of anti-inflammatory M2a macrophages. BMDMs were primed with IL-4 for 24 hr to generate M2a macrophages; then cells were treated with IFN-γ plus LPS for an additional 24 hr to generate M2a→M1 macrophages. <bold>(B)</bold> Flow cytometer data of CD86-PE and CD206-PE expression in M1, M2a, and M2a→M1 macrophages. <bold>(C)</bold> The ROS and NO production levels in naive, M1, M2a, and M2a→M1macrophages, measured by DCFH-DA and DAF-FM DA fluorescent probes, respectively.<bold> (D)</bold> Changes of OCR in M1, M2a, and M2a→M1 macrophages with intervention. <bold>(E)</bold> Changes of glycolysis capability in M1, M2a, and M2a→M1 macrophages with intervention. <bold>(F)</bold> Glucose uptake capabilities of naive, M1, M2a, and M2a→M1 macrophages were measured by 2-NBDG fluorescent probes. <bold>(G)</bold> Fluorescence confocal images of mitochondria (red color, MitoTracker Red CMXRos), and nuclei (blue color, Hoechst 33342) in M2a and M2a→M1 macrophages. Scale bar, 20 µm. <bold>(H)</bold> Changes of the mitochondrial organization during the repolarization of M2a macrophages (M2a, M2a→M1). n=10. All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g005"></graphic></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold> NO blocks the mitochondrial fusion of M1 macrophages. (A)</bold> Schematic illustration of the intervention on M1 macrophage activation. The BMDMs were stimulated for 24 hr with IFN-γ plus LPS either in the presence or absence of Mdivi-1 (mitochondrial fission inhibitor) plus MfpM1 (mitochondrial fusion promoter), 1400W (the inhibitor of iNOS), or NAC (N-acetylcysteine, the ROS scavenger). <bold>(B)</bold> ROS production and NO production of macrophages were measured using DCFH-DA and DAF-FM DA fluorescent probes respectively. <bold>(C)</bold> Glucose uptake capability of macrophages (M1, M1[+Mdivi-1+MfpM1], M1[+1400w], and M1[+NAC]) were measured using 2-NBDG fluorescent probes. <bold>(D)</bold> Changes of OCR during M1 polarization with intervention (control, [+Md+MfpM1], [+1400w], and [+NAC]). <bold>(E)</bold> Changes of glycolytic capability during M1 polarization with intervention (control, [+Md+MfpM1], [+1400w], and [+NAC]). <bold>(F)</bold> Mitochondrial morphology of macrophages (M1, M1[+Mdivi-1+MfpM1], M1[+1400w], and M1[+NAC]). Mitochondria are red (MitoTracker Red CMXRos), and nuclei are cyan (Hoechst 33342). Scale bar, 20 µm. <bold>(G)</bold> Changes of mitochondrial organization during M1 polarization with intervention (control, [+Md+MfpM1], [+1400w], and [+NAC]). n=10. All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g006"></graphic></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p><bold> Characterization of MitoTracker-loaded liposomes <italic>in vitro</italic>. (A)</bold> Schematic structure of MitoTracker-loaded liposome and the representative fluorescent image of liposome (red color, MitoTracker Red CMXRos) captured by SIM. Scale bar, 1 µm. <bold>(B-C)</bold> The distribution of sizes and surface zeta potentials of MitoTracker-loaded liposomes diluted by water.<bold> (D)</bold> The fluorescence confocal images of labeled membrane and mitochondria in naive BMDMs from C57BL/6 mice. Two ways of labeling, MitoTracker-loaded liposomes subsequently with free PKH67 dye (upper panel) and MitoTracker\PKH67 double-loaded liposomes simultaneously (lower panel), were compared. Cell morphology (gray, bright field), mitochondria (red color, MitoTracker Red CMXRos) and membrane structures (green color, PKH67 Green). Scale bar, 20 µm. <bold>(E)</bold> The fluorescence confocal images of labeled mitochondria in BMDMs (M1 and M2a activation) from C57BL/6 mice. Two ways of labeling, free MitoTracker, and MitoTracker-loaded liposomes, were compared. Mitochondria (red color, MitoTracker Red CMXRos). Scale bar, 20 µm. All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g007"></graphic></fig><fig id="F8" position="float"><label>Figure 8</label><caption><p><bold> MitoTracker-loaded liposomes target mitochondria of macrophages in mice. (A)</bold> Experimental protocol. (1) To observe the mitochondria of resident macrophages, MitoTracker-loaded liposomes were subcutaneously injected into the dorsal side of mice ear pinna one day before <italic>in vivo</italic> imaging. (2) The LPS was subcutaneously injected into the dorsal side of mice ear pinna to prime inflammation. On the day 3 after LPS injection, MitoTracker-loaded liposomes were subcutaneously injected into the LPS-challenged mice ear. On the day 4 and day 7 after LPS injection, mice were anesthetized for two-photon <italic>in vivo</italic> imaging. <bold>(B)</bold> Second harmonic generation (SHG, green) images of collagens and two-photon fluorescence images of mitochondria (red) in c2J mice ear before/after LPS challenge. The excitation wavelength was 1120 nm. Scale bar, 10 µm. <bold>(C)</bold> 2D scatter plots of mitochondrial organization parameters in macrophages from Matrigel plugs (Naive <italic>in vivo</italic>, M1<italic> in vivo</italic>, M2a<italic> in vivo</italic>, M2b<italic> in vivo</italic>, and M2c<italic> in vivo</italic>) and c2J mice ear (before LPS, 4d after LPS, and 7d after LPS). The quadrants of fission/fusion were added to enclose clustered macrophages in feature space based on their mitochondrial organization. Dots are data of individual cells (n≥10). <bold>(D)</bold> Second harmonic generation (SHG) images of collagens (blue; excited at 970 nm) and two-photon fluorescence images of macrophage mitochondria (red; excited at 1180 nm) in LysM-Cre\mTmG mice ear before/after LPS challenge. Scale bar, 10 µm. All data shown were representative of two or three independent runs of experiments.</p></caption><graphic xlink:href="thnov10p2897g008"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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