We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.
Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.
Here the method for the use of live-cell video microscopy to study macrophage phagocytosis is described. Video microscopy offers multiple additional layers of information for analysis. One basic advantage is that uptake data can be generated (from a single experiment) for any time point throughout the 6 hr observation period. More importantly, the method described enables differential analysis of the individual stages of phagocytosis. We have, for example, shown that changes in overall uptake of C. albicans glycosylation and morphogenesis mutants by macrophage cell lines and primary macrophages can be either a consequence of changes in macrophage migration towards target cells or rate of engulfment once cell-cell contact is established 7.
There are a number of pitfalls to avoid when conducting these experiments. First, it is very important to ensure stable environmental conditions throughout the experimental procedure. This is best achieved in an environmental chamber that is set to the experimental conditions several hours before commencing the experiment. Video quality is highly dependent on sophisticated modules that use infrared lasers to automatically maintain the sample z-position regardless of mechanical or thermal changes thus eliminating the need for manual focus corrections.
Given the extended nature of the experiments, it is important to limit laser light exposure and associated photobleaching and photoconversion effects, which can be minimized by employing highly fluorescent markers, which facilitate low exposure times. The FITC staining protocol described here is quick and reliable. As FITC is a very bright and stable stain, low exposure times are required and this makes FITC ideal for extended time-lapse movies. However, we routinely use a variety of different target stains, including Calcofluor White and PKH dyes as well as tagged organisms.
Most of our published work is conducted using a wide-field microscope, but exposure can be further minimized by using a spinning disc confocal microscope and in our hands this is essential for time-lapse 3D video microscopy.
During this study, images were captured at 1 min intervals over a 6 hr phagocytosis assay. The interval between images can be adjusted depending on the process under investigation. For example, the interval can be decreased when investigating rapid processes such as vesicular trafficking. The minimum time interval will be limited by microscope and camera specifications. There are some considerations to bear in mind when adjusting timings. First, decreasing the interval between snapshots will mean fewer points can be imaged and file size will be considerably increased. On the contrary, increasing the image interval too much will make the movie lose continuity.
This approach can be applied in principle to study other pathogens and uptake of dying host cells. For example, we have recently shown that bone-marrow derived macrophages from sialoadhesin-deficient mice exhibit greatly reduced binding and phagocytosis of sialylated Campylobacter jejuni 8. However, target size is an important determinant for feasibility, as image analysis becomes increasingly difficult with a reduction in target cell size. Sophisticated image analysis software is essential for rapid video microscopy analysis, and this should be coupled with appropriate bioinformatics support to facilitate the generation of algorithms to study migration of individual cells and entire cell populations 7. Live-cell video microscopy in combination with sophisticated image analysis software for the minute-by-minute analysis of migration and individual macrophage-C.albicans interactions, provides unique insight into the complexity of C. albicans phagocytosis by macrophages. There is enormous potential to expand these methods to study other pathogens and phagocytes (dendritic cells, neutrophils) and to develop 3D video microscopy to image cell-cell interactions in greater detail or on more physiological surfaces such as epithelial and endothelial cell layers. This technique is part of the next generation of tools in the study of host-pathogen interactions and will help generate detailed spatial and temporal information on a broad range of dynamic processes.
The authors have nothing to disclose.
LPE is a Scottish Senior Clinical Fellow and acknowledges the support of the Chief Scientist Office (SCD/03). This work was funded by Wellcome Trust Project Grant to LPE (089930). NARG was funded by a Wellcome Trust Programme Grant (080088) and an equipment Grant (075470) (for DeltaVision), and by a FP7-2007-2013 Grant (HEALTH-F2-2010-260338-ALLFUN). We would like to thank the University of Aberdeen imaging facility, in particular Kevin MacKenzie, for helpful support and advice.
Name of the reagent | Company | Catalogue number |
Yeast nitrogen base without amino acids | Formedium | CYN0402 |
NaOH | Sigma | S5881-500G |
Adenine hemisulphate salt | Sigma | A3159-25G |
Agar technical (no. 3) | Oxoid | LP0013 |
D-glucose | Sigma | G7021-1KG |
SC-Ura dropout | Formedium | DSCK102 |
FITC | Sigma | F7250-1G |
Sodium carbonate | BDH | 301215M |
Sodium bicarbonate | BDH | 102475W |
PBS | Gibco | 18912-014 |
DMEM | Lonza | BE12-614F |
FCS | Life Technologies | 10106169 |
Penicillin/streptomycin | PAA | P11-010 |
L-glutamine | Lonza | BE17-605E |
LysoTracker Red DND-99 | Invitrogen | L7528 |
35 mm glass-dishes | PAA | PAA12305160X |