Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis.

Mice

C57BL/6 mice were obtained from the Animal Facility of the Faculty of Health Sciences at the University of Macau. B6 (Cg)-Tyr c-2J /J transgenic mice and B6.129P2-Lyz2 tm1(cre)Ifo /J transgenic mice were purchased from The Jackson Laboratory. STOCK Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J transgenic mice were kind gifts from Dr. Kai MIAO (University of Macau). The Animal Research Ethics Committee of the University of Macau approved all experiments.

Generation of macrophage-reporter mice

B6.129P2-Lyz2 tm1(cre)Ifo /J mice express Cre from the myeloid-specific LysM promoter. STOCK Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J mice are dual-reporter (membrane-targeted Tomato [mTOM]/ membrane-targeted enhanced green fluorescent protein [mEGFP]-floxed reporter) mouse strain. This strain expresses reporter gene mTOM (red) in the absence of Cre, whereas expresses reporter gene mEGFP (green) upon Cre activation. B6.129P2-Lyz2 tm1(cre)Ifo /J mice were crossed with STOCK Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J mice to get LysM-CremTmG mice in which LysM-Cre positive myeloid cells would express EGFP.

Mononuclear cell isolation

Whole blood of C57BL/6 mice was collected from retro-orbital venous plexus using heparinized (Sigma) microtubes. The heparinized blood was diluted with the four-times volume of PBS containing 0.5% bovine serum albumin (BSA), then layered on 4 mL Histopaque (1.077g/mL, Sigma) gradient layer. Mononuclear cells were collected from the 1.077-PBS interface. After the elimination of red blood cells (RBCs) via hypotonic lysis, mononuclear cells were resuspended in the completed RPMI-1640 medium for mitochondria staining and the next examination. PE anti-mouse CD115 (CSF-1R) antibody (AFS98, 1:80, Biolegend) was used to gate monocytes from mononuclear cells.

Macrophage preparation and drug treatments

Bone marrow-derived macrophages (BMDMs) were differentiated from mouse bone marrow cells 23 . Briefly, bone marrow cells were isolated from the femur and tibia of C57BL/6 mice (or LysM-Cre mTmG mice) and cultured in the completed RPMI- 1640 medium containing 20 ng/mL recombinant murine M-CSF (rmM-CSF, 315-02, PeproTech) or 15% L929 cells (ATCC) conditioned medium. On day 6, cells were harvested from Petri dishes by 20 mM EDTA treatment for further examination. For macrophage activation, BMDMs were stimulated for 24 hr with certain cytokines after removal of rmM-CSF. 20 ng/mL IFN-γ (315-05, PeproTech) plus 10 ng/ml LPS (O111:B4, Sigma-Aldrich) was used to induce M1 macrophages, 20 ng/mL IL-4 (214-14, Peprotech) to induce M2a, 10 µg/mL immune complex (150 μg/ml monoclonal anti-chicken egg albumin antibody [C6534] preincubated with 15 μg/ml ovalbumin [A5503] at 37°C for 30 min; Sigma Aldrich) plus 50 ng/ml LPS (O111:B4, Sigma-Aldrich) to induce M2b, and 50 ng/mL IL-10 (210-10, Peprotech) to induce M2c, respectively. For repolarization of M2a macrophages, BMDMs were primed with IL-4 for 24 hr to induce M2a macrophages; then cells were washed and treated with IFN-γ plus LPS for an additional 24 hr. To investigate the blocking of macrophages toward M1 polarization, the BMDMs were stimulated for 24 hr with IFN-γ plus LPS either in the presence or absence of 10 µM Mdivi-1 (Sigma-Aldrich) plus 20 µM fusion promoter M1 (Sigma-Aldrich), 100 µM 1400W (Beyotime), or 5 mM NAC (Sigma-Aldrich).

Flow Cytometry

Nonspecific binding was blocked by Fc-block (Mouse TruStain FcX™ PLUS, S17011E, 1:2000, Biolegend) before surface staining. Fluorescent-dye conjugated monoclonal antibodies were used to identify macrophage maturation and to differentiate their activation status, including Alexa Fluor 647 anti-mouse F4/80 antibody (BM8, 1:200, Biolegend), PE anti-mouse/human CD11b antibody (M1/70, 1:80, Biolegend), PE anti-mouse CD86 antibody (GL-1, 1:20, Biolegend), PE anti-mouse CD206 antibody (C068C2, 1:40, Biolegend), and PE anti-mouse CD163 antibody (TNKUPJ, 1:80, eBioscience). Mitochondria staining was performed according to the manufacturer's instructions (M7514, 100 nM, Invitrogen). Briefly, cells were incubated in the completed medium containing 100 nM MitoTracker Green FM for 30 min; then cells were washed twice with the completed medium. Cell viability was assessed by 7-AAD staining (1:100, Biolegend). JC-1 staining was performed according to the manufacturer's instructions (ab113850, Abcam). Briefly, cells were incubated in completed medium containing 5 μM JC-1 for 20 min; then cells were washed twice with the completed medium. 100 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP)-treated cells were used as a positive control. For the staining of intracellular arginase, cells were fixed and permeabilized with 100% methanol (chilled at -20°C) at room temperature for 5 min and were stained with anti-mouse arginase-1 antibody (24HCLC, 1:100, Invitrogen), followed by incubation with Alexa Fluor 488 Donkey anti-Rabbit Secondary Antibody (A-21206, 1:200, Invitrogen). For the staining on macrophages of mice ear, a single-cell suspension was prepared as described before 24 . Briefly, mice ear was chopped with scissors into small pieces and post immersed in the digestion cocktail (2.5 mg/ml Collagenase Type 2 [Worthington-Biochem], 2.5 mg/ml Collagenase Type IV [Gibco] and 0.5 mg/ml DNAse I [Roche]) at 37 °C for 90 min. Following incubation, digested skin samples were squeezed and filtered to get the suspension of cells. Data were acquired with a BD Accuri™ C6 Cytometer (BD Biosciences, USA) and analyzed using BD Accuri C6 Software (BD Biosciences) or FlowJo software (Tree Star). Transmission electron microscopy (TEM) BMDMs were stimulated as described above. Cells were first fixed with 2.5% glutaraldehyde at 4 °C overnight. Following fixation, cells were washed three times with PBS and postfixed in 1% osmic acid at 4 °C for 1.5 hours. Then cells were dehydrated in a graded ethanol series and embedded in acrylic resin. Cut sections were stained with uranyl acetate and lead citrate, and then imaged using a Hitachi H-7500 transmission electron microscope (Hitachi, Japan) equipped with a digital camera. Metabolism Assays XF-96 Extracellular Flux Analyzer (Seahorse Bioscience, USA) was used to analyze the metabolic profiles of activated macrophages 4 . 25,000 BMDMs were seeded in a 96 well culture plate and activated as indicated. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured after the sequential addition of 1 µM oligomycin; 1.5 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP); and 100 nM rotenone plus 1 µM antimycin A. Bradford assay (500-0205, Bio-rad) was conducted to normalize cell number after metabolic analysis 4 . Basal respiration can be calculated as the difference between OCR before drug injection and lower OCR after rotenone plus antimycin A injection. ATP production can be calculated as the difference between basal OCR and lower OCR after oligomycin injection. Spare respiratory capacity can be established by the difference between basal OCR and the highest OCR after FCCP injection. Basal glycolysis can be calculated from the measurement of ECAR before drug injection. Glycolytic capability can be calculated from the measurement of ECAR after oligomycin injection. The glycolytic reserve can be calculated as the difference between higher ECAR after oligomycin injection and highest ECAR after FCCP injection.

Glucose uptake assay

For glucose uptake capacity, BMDMs were starved in the glucose-free medium for 1 hr, then incubated in glucose-free medium containing 100 µM 2-NBDG (Invitrogen) for 30 min in 37 °C incubator. Cell viability was assessed by 7-AAD staining (1:100, Biolegend). Data were acquired with a BD Accuri™ C6 Cytometer (BD Biosciences, USA) and analyzed using BD Accuri C6 Software (BD Biosciences) or FlowJo software (Tree Star).

Macrophage function assay

BMDMs seeded in a 96 well culture plate were stimulated as described above. Intracellular reactive oxygen species (ROS) production and nitric oxide (NO) production were measured using DCFH-DA and DAF-FM DA respectively, according to the manufacturer's instructions (Beyotime). After incubation, fluorescence was detected by Microplate Reader (Molecular Devices, USA) immediately.

Confocal microscopy imaging and mitochondrial morphology analysis

Mitochondria staining was performed according to the manufacturer's instructions (M7512, Invitrogen). Briefly, BMDMs were incubated in the completed medium containing 100 nM MitoTracker Red CMXROS for 30 min and then 2 μg/mL Hoechst 33342 (Sigma Aldrich) for an additional 2 min. After incubation, cells were washed twice and replaced with the completed medium for live-cell imaging. For live-cell imaging, BMDMs on confocal dishes were placed in a micro-incubator system, which maintained 37 °C and 5% CO 2 within a humidified environment. Images were acquired using a Nikon A1MP+ fluorescence confocal microscope (Nikon, Japan) with a water-immersed 40× and 1.15 NA objective. We acquired MitoTracker images at 561 nm excitation, and Hoechst images at 405 nm excitation. Images were subsequently imported into ImageJ software for analysis. Analysis of mitochondrial morphology ( Figure S1 ) was performed using the macro MiNA in Fiji software (ImageJ) 25 . Briefly, the macro MiNA creates a skeleton of the mitochondrial network by several preprocessing steps ( Figure S1 A), then calculates several parameters reflecting mitochondrial morphology ( Figure S1 B), such as mean length of branches, mean network size (the mean number of branches per network), and mitochondrial footprints (mitochondrial coverage area). Three dimensional (3D) or two dimensional (2D) scatter plots were generated via Origin software (version 8.6). Structured illumination microscopy (SIM) For the super-resolution imaging of mitochondria, BMDMs were incubated in the completed medium containing 100 nM MitoTracker Red CMXROS for 30 min. After incubation, cells were washed twice and replaced with the completed medium for live-cell imaging. Super-resolution images were acquired using a Nikon N-SIM system (Nikon, Japan) with an oil-immersed 100× and 1.49 NA objective. Images for live-cell imaging (live N-SIM) were taken at a single Z-plane. Images were captured using Nikon NIS-Elements and reconstructed using slice reconstruction in NIS-elements. Cells used for live-cell imaging were maintained in a 37 °C, 5% CO 2, and humidified environment. For super-resolution imaging of single liposome, MitoTracker-loaded liposome and Matrigel were mixed and smeared on the confocal dish to immobilize the liposome. Liposomes were imaged with the same imaging conditions used in live-cell experiments. In vivo Matrigel plug assay Matrigel plug implanted in C57BL/6 mice was used for macrophage in vivo activation. To induce activated macrophages in mice, 0.25 mL Matrigel (BD Biosciences) containing rmM-CSF (100 ng/mL)+PBS was used for naive macrophage differentiation, Matrigel containing rmM-CSF+LPS (10ng/mL) + IFN-γ (20ng/mL) for M1, Matrigel containing rmM-CSF+IL-4 (20 ng/mL) for M2a, Matrigel containing rmM-CSF+immune-complexes (10 μg/mL)+LPS (50 ng/mL) for M2b, Matrigel containing rmM-CSF+IL-10 (50ng/mL) for M2c, and Matrigel containing PBS only for control group. All procedures were completed on ice. C57BL/6 mice were anesthetized with isoflurane, then Matrigel mixture was subcutaneously injected into the inguinal areas of mice. Five days after plug implantation, mice were euthanized by CO 2 asphyxiation for Matrigel plug excision. For ex vivo live-cell imaging, Matrigel plugs were labeled by MitoTracker Red CMXRos and Hoechst 33342. Then they were placed in the micro-incubator system on the confocal microscope for further observation. MitoTracker images were collected at 561 nm excitation, and Hoechst images were collected at 405 nm excitation. For immunofluorescence staining, Matrigel plugs were OCT-embedded and sectioned for further examination.

Mitotracker-loaded liposome preparation

Liposomes were prepared by the ethanol injection method as described before 26 . Briefly, 10 mg phospholipids and 5 mg cholesterol were dissolved in 500 µL ethanol, then MitoTracker Red CMXRos stocking solution was added to the above ethanolic solution. The resulting organic phase was dropped in 4 mL PBS under 100 rpm/min stirring. Unloaded MitoTracker Red CMXRos and ethanol solvent were removed by ultracentrifugation of liposome suspension at 14,000 rpm for 1 hour. The obtained precipitates were dispersed in PBS and stored at 4°C. After dilution with water, the particle size and zeta potential of MitoTracker-loaded liposomes were measured by a Zetasizer Nano-ZS (Malvern Instruments, UK). For in vitro testing, cells were incubated in a completed medium containing MitoTracker Red CMXRos-loaded liposome for 60 min. Then, cells were washed twice and replaced with the completed medium for live-cell imaging. To evaluate the MitoTracker release from liposome during the cell endocytosis process, we constructed the fluorescent green/red dyes double-loaded liposome by labeling the membrane of Mitotracker-loaded liposomes with the Green Fluorescent Cell Linker Kit, according to the manufacturer's instructions (Sigma). Briefly, precipitates of Mitotracker-loaded liposome was dispersed in Diluent C from the Kit and subsequently mixed with equal volumes of Diluent C containing PKH67 dye for 3 min at room temperature. Then, stop the staining by adding PBS containing 1% FBS and remove unloaded PKH67 by ultracentrifugation at 14,000 rpm for 1 hour. After re-suspension, cells were incubated in a completed medium containing MitoTracker and PKH67 double-loaded liposome for 60 min. For the control group, MitoTracker-loaded liposome and free PKH67 were sequentially stained for 60 min and for 3 min. Then, cells were washed twice and replaced with the completed medium for live-cell imaging. In vivo skin inflammation experiment and two-photon excited microscopy imaging The same experimental protocol was used and performed on c2J mice and LysM-CremTmG mice. To observe the mitochondrial structure of resident macrophages in vivo , mice were anesthetized by intraperitoneal injection of Avertin (25 µL of injection volume/g of body weight), then a single subcutaneous injection of MitoTracker-loaded liposomes (in 30 µL PBS) was administrated into the dorsal side of ear pinna with the hair removed. One day after liposome injection, mice were anesthetized by Avertin for mitochondria intravital imaging. To track the dynamics of mitochondria morphology after inflammatory stimulation, the LPS (30 µg in 30 µL PBS) was subcutaneously injected into the dorsal side of ear pinna after mice anesthetization. On the 3 rd -day post LPS injection, MitoTracker-loaded liposomes (in 30 µL PBS) were subcutaneously injected into the ear of mice to label mitochondria in vivo . On the 4 th -day post LPS injection, mice were anesthetized by Avertin for intravital imaging. Briefly, anesthetized mice ear pinnae were mounted on slides, immersed by glycerol and nipped by a cover glass. Images were acquired using a Nikon A1MP+ microscope (Nikon, Japan) with a water-immersed 40× and 1.15 NA objective. Two-photon laser excitation was provided by an 800-1300 nm tunable femtosecond laser (Insight X3, Spectra-Physics). For c2J mice, mitochondrial images were acquired at 1120 nm excitation. The fluorescence of MitoTracker Red CMXRos and second harmonic generation (SHG) images in the ear of c2J mice were collected in the wavelength range of 604-679 nm and 506-593 nm, respectively. For LysM-CremTmG mice, EGFP cell images were acquired at 970 nm excitation, and the fluorescence of EGFP and SHG was collected in the wavelength range of 506-593 nm and 415-485 nm, respectively. Mitochondrial images of LysM-Cre mTmG mice were acquired at 1180 nm excitation, and the fluorescence of MitoTracker Red CMXRos was collected in the wavelength range of 604-679 nm. The corresponding spectra were acquired with an Andor iDus 401 series Spectroscopy CCD (Andor, EU) coupled on the back-side port of the Nikon A1MP+ microscope and analyzed using Graphpad Prism software (version 6.0). For immunofluorescence staining, mice's ear was OCT-embedded and sectioned for further examination. For the flow cytometer, mice's ear was harvested and digested to get the cell suspension.

Immunofluorescence staining

BMDMs or tissue sections were fixed with 4% paraformaldehyde in PBS or permeabilized with 100% methanol (chilled at -20°C) at room temperature for 5 min, blocked with 1% BSA in PBS for 30 min. Primary antibodies were incubated overnight at 4°C, followed by the secondary antibody for 1 hr at room temperature if needed or Hoechst 33342 for 5 min at room temperature. Antibodies used were FITC anti-mouse F4/80 antibody (BM8, 1:100, Biolegend), Alexa Fluor 647 anti-mouse CD86 antibody (GL-1, 1:100, Biolegend), Alexa Fluor 647 anti-mouse CD206 antibody (C068C2, 1:200, Biolegend), PE anti-mouse CD163 antibody (TNKUPJ, 1:40, eBioscience), anti-mouse arginase-1 antibody (24HCLC, 1:100, Invitrogen), Alexa Fluor 488 donkey anti-rabbit secondary antibody (A-21206, 1:1000, Invitrogen). Images were acquired using a Nikon A1MP+ fluorescence confocal microscope (Nikon, Japan) with a water-immersed 40× and 1.15 NA objective.

Statistical Analysis

Data were evaluated with Graphpad Prism software (version 6.0) using unpaired two-tailed Student's t-tests. Data are expressed as means ± the standard deviation (SD). Differences were considered significant when p values were below 0.05.

Supplementary Material Supplementary figures. Click here for additional data file.