Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Summary

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Abstract

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H⁺ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Introduction

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material^1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O₂^- and its derivative reactive oxygen species (ROS) ^2,4.

The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes ^5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na⁺/H⁺ exchanger or ClC family Cl^-/H⁺ antiporters, which promote phagocytic function, rely on H⁺ translocation ^9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils ^4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba ^13-16. The NADPH oxidase is an electrogenic enzyme that releases H⁺ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H⁺ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme ^17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH ^22-24.

In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing ^25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism ²⁸. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)¹¹, and the pH remains neutral or alkaline during the first 30 min acidifying only later ^29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH ^31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions ³³.

Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH ^34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies ³⁶, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses.

Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH ¹⁹. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm ³⁷. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

Protocol

Ethics Statement: All animal manipulations were performed in strict accordance with the guidelines of the Animal Research Committee of the University of Geneva.

1. Preparation of Phagocytic Targets

1. Add 20 mg of dried zymosan to 10 ml sterile phosphate buffered saline (PBS). Vortex and heat in a boiling water bath for 10 min. Cool and centrifuge at 2,000 x g for 5 min.
2. Remove the supernatant, resuspend in 1 ml PBS and sonicate for 10 min in a water bath sonicator. Transfer 500 µl to two 1.5 ml tubes. Centrifuge at 11,000 x g for 5 min.
3. Repeat the wash with 500 µl PBS. The boiled zymosan may be aliquoted, frozen and stored at -20 °C.
4. Wash zymosan twice in 500 µl freshly prepared 0.1 M Na₂CO₃, pH 9.3 as above (step 1.2).
5. Resuspend in 490 µl after the final wash. Add 10 µl of 10 mg/ml FITC dissolved in dimethyl sulfoxide (DMSO), vortex, cover with foil, and incubate with agitation for 4 hr at RT.
6. To remove unbound dye, wash as above (step 1.2), first with 0.5 ml DMSO + 150 µl PBS, then 0.5 ml DMSO + 300 µl PBS, then 0.5 ml DMSO + 450 µl PBS, then PBS alone.
7. Count zymosan using a haemocytometer. Add 1 µl of zymosan to 500 µl PBS, vortex, then add 10 µl to the edge of the haemocytometer chamber, where it meets the coverslip placed over the central grid. Mount the haemocytometer on a bright-field microscope equipped with a 20X objective.
8. Focus on the upper left-hand corner containing 16 squares and count all zymosan particles in this area using a hand tally counter. Repeat with the lower right-hand corner. Calculate the zymosan concentration using the following equation: Zymosan concentration = Count/2 x 500 x 10⁴ zymosan/ml. Labeled zymosan may be aliquoted, frozen and stored at -20 °C.
9. Opsonize the labeled zymosan with a rabbit anti-zymosan antibody at 1:100 dilution. Vortex and incubate for 1 hr in a 37 °C water bath. Wash with 500 µl PBS as above (step 1.2).
   Note: Sonication is highly recommended, particularly after opsonization, as zymosan tends to form clumps that can make analysis more difficult later on. Opsonization with 65% v/v rat or human serum may be used as an alternative.
10. Count zymosan using a haemocytometer as described in steps 1.7-1.8, and dilute to 100 x 10⁶ particles/ml. Opsonization with other phagocytic stimulators, such as complement or no opsonization, may be alternatively performed.

2. Live Video Microscopy

1. Isolate primary mouse neutrophils as described in detail elsewhere³⁸.
   1. Alternatively, use any phagocytic cell type. Keep neutrophils on ice in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal calf serum (v/v), 200 U penicillin/streptomycin, at a concentration of 10 x 10⁶ cells/ml until ready for use. Other cell types may be seeded 1-4 days before.
2. Add 2 x 10⁵ (20 µl) of neutrophils to the center of a 35 mm glass-bottom dish, spread the drop by adding 50 µl of medium and incubate at 37 °C for 5 min to allow cells to adhere.
3. Add 1 ml of Hank’s balanced salt solution (HBSS) warmed to 37 °C containing the specific MPO inhibitor 4-aminobenzoic hydrazide (4-ABH, 10 µM). Non-specific MPO inhibitors, such as sodium azide (5 mM), may also be used but have been recently suggested to exert additional non-specific effects on pH ³⁹.
4. Mount the dish on a widefield fluorescence microscope equipped with a 37 °C heating system and a 40X oil objective. Incubate cells without imaging for 10 min to allow cells and equipment to equilibrate.
5. Adjust the microscope settings to acquire a bright-field transmission image [phase or differential interference contrast (DIC) if available] with 440 nm or 490 nm excitation and 535 nm emission every 30 sec.
6. Adjust exposure time and acquisition frequency according to the microscope system to avoid too much photobleaching and phototoxicity and depending on the experimental questions being asked. Begin image acquisition.
7. After 2 min, add 10 x 10⁶ (50 µl) opsonized FITC-zymosan to the center of the dish. Adjust target:cell ratios depending on the type of phagocyte and target used.
8. Continue imaging for 30 min. After 30 min stop the time-lapse acquisition and start a timer. Move the stage and capture 10 or more snapshots in different fields of view to image other phagosomes, all within 5 min.

3. Calibration and Control Experiments

1. Prepare the pH calibration solutions (recipes are described in Table 1) prior to the day of experiment and freeze in 10 ml aliquots. Thaw the calibration solutions and warm to 37 °C in a water bath. Check the pH with a pH meter and record the actual pH of solutions.
2. Mount a peristaltic pump on the glass bottom dish prior to image acquisition and perform live video microscopy as described above (section 2).
3. At the end of the 30 min acquisition period turn the pump on to remove the solution within the dish, add 1 ml of the first calibration solution and stop the pump.
4. Wait until the signal stabilizes, usually 5 min. Repeat with each calibration solution.
   1. Perform at least one calibration per experimental day, and calibrate starting with the highest pH and working sequentially to the lowest, as well as starting from the lowest pH and working sequentially towards the highest.
5. As a control, add 100 µl of 100 µM NADPH oxidase inhibitor diphenyl propionium iodide (DPI) diluted in HBSS to cells in 900 µl HBSS in the glass bottom dish, and incubate for 10 min.
6. Start acquisition and add zymosan as above (step 2.7). After 15 min of phagocytosis, add 10 µl of 10 µM V-ATPase inhibitor concanamycin A (ConcA) diluted in HBSS and continue imaging for an additional 15 min.

4. Analysis

1. Analysis of time-lapse movies and phagosome snapshots.
   Note: The following instructions are specific for ImageJ. Step-by-step instructions may vary widely depending of the software used, but the functions described in this section are available in most professional image analysis software.
   1. Open the images with professional image analysis software. Subtract the background in the 490 channel by drawing a small square ROI with the square polygon selection tool in an area where there are no cells, and clicking Plugins> BG Subtraction from ROI. Then upload the same ROI on the 440 channel by clicking Edit> Selection> Restore Selection and repeat.
   2. Make a 32-bit ratio image of the 490 channel divided by the 440 channel by clicking Process>Image Calculator… , selecting the appropriate images in the Image1 and Image2 drop-down menus, then selecting ‘Divide’ in the Operation drop-down menu, and checking the 32-bit (float) result check-box.
   3. Change the color-coding look-up table to a ratio-compatible color-coding by clicking Image> Lookup Tables> Rainbow RGB. Threshold the ratio image to eliminate 0 and infinity pixels by clicking Image> Adjust> Threshold…. Adjust the scroll bars so that zymosan appears in red and click Apply, making sure the Set background pixels to NaN check box is selected.
   4. Combine this ratio stack with the bright-field channel into a multi-channel composite by clicking Image> Color> Merge Channels, and selecting the bright-field image in the gray channel dropdown menu and the ratio image in the green channel dropdown menu, and selecting the Keep source images check-box. Scan the time-lapse movies or snapshots to determine which phagosomes are to be analyzed. The bright-field channel is useful for this.
   5. Draw regions of interest (ROI) using the oval polygon selection tool around phagosomes, and add them to the ROI manager by clicking Analyze> Tools > ROI Manager> Add. Measure their zymosan intensity ratio by clicking More> Multi-Measure and de-selecting the One row per slice check-box.
   6. For time-lapse movies, track phagosomes one at a time by drawing an ROI, typing Ctrl+M to measure each time point, and moving the ROI when necessary to track intensity values over time. Copy the measurements from the Results window into a spreadsheet software.
      Note: Analysis of 15 (5 time courses per experiment x 3) independent experiments is ideal, but more or less may be required depending on the variability observed. Saving the ROIs is always recommended. Some software packages allow image registration and dynamic update of ROIs, facilitating this part of the analysis.
2. Conversion from fluorescence to pH values
   1. Measure the 490/440 ratio for phagosomes in the calibration time-lapse movies as described in section 4.1.
   2. In a spreadsheet, plot the ratio values over time, and calculate the average ratio values from all phagosomes within the field of view (usually, between 5-20) from 5 time-points within the middle of the time period corresponding to each pH calibration solution. (See also the red boxes in Figure 5A.)
   3. Plot these average 490/440 ratio values against the actual measured pH. Combine at least 3 independent calibrations into a single graph. Use a statistical or numerical software package to perform a nonlinear regression (best fit curve), usually to a sigmoidal equation.
      Note: For example, in Figure 5B the equation used was a Boltzmann sigmoidal [Y = Bottom + ((Top-Bottom)/(1+EXP((V50-X)/Slope))], where Y values are the 490/440 ratios, X are the pH values and the Top, Bottom, V50 and slope parameters are calculated by the software.
   4. Use the obtained equation to transform ratio values to pH.
      Note: For example, in Figure 5B the equation obtained for the calibration curve in the presence of sodium azide is 490/440 Ratio = 1.103 + [(2.89-1.103)/(1+EXP((6.993-pH)/0.9291)]. Solving for pH gives the following equation: pH = 6.993 - 0.9291*[LN((1.178/(Ratio-1.103))-1))].

Results

The following are representative results for an experiment where the phagosomal pH of primary mouse neutrophils isolated from the bone-marrow of wild-type or Hvcn1^-/- mice were compared. For a successful experiment, it is important to obtain enough phagosomes within the field of view during the whole duration of the time-lapse movie, while avoiding too many phagosomes, which will later be more difficult to segment during the image analysis. Figure 1 shows examples of...

Discussion

Although more time consuming than alternative methods, such as spectroscopy and FACS, which employ a similar strategy of using a pH sensitive dye coupled to targets but measure the average pH of a population of phagosomes, microscopy offers several advantages. First is that internal and external bound, but not internalized, particles can easily be distinguished without having to add other chemicals, such as trypan blue or antibodies, to quench or label external particles, respectively. Second is that following the cells ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zymosan A powder	Sigma-Aldrich	Z4250	Various providers exist
Fluorescein isothiocyanate	Sigma-Aldrich	F7250	Various providers exist
Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)	Life Technologies	Z2850	Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.
4-Aminobenzoic hydrazide (4-ABH)	Santa Cruz	sc-204107	Toxic, use gloves, various providers exist
Diphenyleneiodonium chloride (DPI)	Sigma-Aldrich	D2926	Toxic, use gloves, various providers exist
Concanamycin A (ConcA)	Sigma-Aldrich	27689	Toxic, use gloves, various providers exist
Nigericin	Sigma-Aldrich	N7143	Toxic, use gloves, various providers exist
Monensin	Enzo	ALX-380-026-G001	Toxic, use gloves, various providers exist
Phosphate buffered saline (PBS)	Life Technologies	14200-075	Toxic, use gloves, various providers exist
Hank's balance salt solution	Life Technologies	14025092	Ringer's balanced salt solution or other clear physiological buffers may be substituted.
Sodium carbonate (Na2CO3)	Sigma-Aldrich	S7795	Various providers exist
2-(N-Morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	M3671	Various providers exist
4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375	Various providers exist
N-Methyl-D-glucamine (NMDG)	Sigma-Aldrich	M2004	Various providers exist
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)	Sigma-Aldrich	3777	Various providers exist
Tris(hydroxymethyl)aminomethane (Tris)	Sigma-Aldrich	T1503	Various providers exist
Potassium chloride (KCl)	Sigma-Aldrich	P9333	Various providers exist
Sodium chloride (NaCl)	Sigma-Aldrich	S7653	Various providers exist
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266	Various providers exist
Absolute Ethanol (EtOH)	Sigma-Aldrich	2860	Various providers exist
Glass-bottom 35 mm petri dishes (Fluorodish)	World Precision Instruments	FD35-100	Ibidi µ-clear dishes or coverslips with appropriate imaging chambers may be sustituted
Sonicating water bath	O. Kleiner AG		A sonicator may be used instead, various instrument providers exist
Heamocytometer	Marienfeld GmbH		Various instrument providers exist
Widefield live imaging microscope	Carl Zeiss AG		Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.
Peristaltic pump (Dynamax RP-1)	Rainin		Various instrument providers exist
pH meter	Schott Gerate GmbH		Various instrument providers exist
Manual Counter	Milian SA		Various instrument providers exist