Welcome! You have reached the homepage for the laboratory of Dr. Bryan Heit. Our lab is part of the Department of Microbiology and Immunology at Western University, and we are members of the Center for Human Immunology, the lead centre for the CIHR Human Immunology Network.
Our interests surround the function of phagocytes – white blood cells which ingest (phagocytose) pathogens, particles, and dead cells. We focus on the cellular and molecular processes which control the function of these cells during the maintenance of homeostasis, infection and chronic inflammatory disease. Central to most of our studies is the study of efferoctyosis – the phagocytic removal of apoptotic (dying) cells, and how failures in this process lead to inflammation, autoimmunity and infection.
Phagocytes are a class of white blood cells which have the capacity to engulf large particles such as bacterial and fungal pathogens, and subsequently destroy the engulfed material. The term phagocyte literally translates to “cell that eats”, which is an apt description of the primary function of these cells in our bodies. While there are many types of phagocytes, the Heit lab focuses primarily on macrophages, which play key roles in both maintaining our bodies and in fighting infections.
We use a combination of advanced microscopy techniques, gene expression analysis and functional assays to investigate the activity of macrophages. Some examples of the methods we employ can be found on our YouTube channel.
In this video the activity of a signaling pathway regulating efferocytosis – the engulfment and destruction of apoptotic (dying) cells by phagocytes such as macrophages – is monitored by fluorescent microscopy.
These macrophages are expressing synthetic genes which mark the plasma membrane of the cell in red (PM-RFP), and the signaling lipid PI(3)P in green (FYVE-GFP). These cells are engulfing small beads coated in a lipid mixture which mimics dying cells (*). Efferocytosed apoptotic cells are engulfed into a vacuole derived from the plasma membrane – visible in this video as the red ring around the efferocytosed beads. Activity of the signaling lipid on the efferosome can be observed as the appearance and disappearance of a green ring around the efferocytosed beads.
While this example tracks PI(3)P signaling, other signaling pathways and cellular responses can be investigated by expressing genes, or using dyes, which report the activity of these other pathways.
This video shows an example of how we visualize and quantify efferocytosis. During efferocytosis, apoptotic (dying) cells are taken up piecemeal by the engulfing macrophage. TO visualize this process we pre-label the cytosol of the apoptotic cell with a protein reactive fluorophore such as Cell Proliferation Dye e670, and coat the surface of the apoptotic cell with NHS-biotin. The apoptotic cells are then ‘fed’ to the macrophages, and after the desired period of time, the sample fixed and any non-internalized apoptotic cell material labelled with fluorescent streptavadin.
This video was captured 90 minutes after the apoptotic cell was fed to the macrophage. About half of the cell has been engulfed by the macrophage, and with the other half awaiting engulfment.
In this short Youtube video we demonstrate a simple method of measuring cholesterol uptake and intracellular transport in macrophages. In this experiment, macrophages are cultured in a media containing oxLDL (bad cholesterol) and a cholesterol/lipid-reactive dye (Nile red). Live cell time-lapse microscopy is then used to monitor the accumulation of cholesterol by the cell. The movement of cholesterol within the cell can also be observed; accumulating first in the peri-nuclear region (within the Endoplasmic Reticulum) and later trafficking to discrete punctuate structures – lipid droplets. This is the first step in foam cell formation, with the stress of cholesterol accumulation eventually leading to the death of the cell.
We have published a brief Youtube video showing a phagosomal pH measurement experiment. This method can be used to assess phagosome, efferosome or endosome acidification using any target that can be conjugated to FITC, and using any cell type. The method automatically compensates for photobleaching and incorporates an in situ pH calibration to convert FITC ratios into pH units.
In this video the pH of phagosomes in primary human macrophages following uptake of IgG-coated 5µm beads (pathogen mimics) is quantified. pH is measured by imaging FITC conjugated to the beads using two excitation wavelengths – a 440 nm excitation, which is pH-independent and allows for photobleaching correction, and a 490 nm excitation which is pH-dependent, with emission decreasing with decreasing pH.
After the time-lapse video is completed a calibration curve is calculated by profusing the imaging chamber with nigericin-containing buffer at known pH’s (4.0, 5.0, 6.0 and 7.0). The nigericin acts as a proton ionophore, equalizing the pH in the phagosome/efferosome lumen to the pH of the extracellular media. FITC images at 440 nm and 490 nm excitation are captured for each pH.
Post-imaging, the background is subtracted from the Ex440 and Ex490 channels and the 490/440 ratio calculated. A pH can be assigned to each bead based on the calibration curve generated using he nigericin-containing media.
A detailed protocol can be found at: Steinberg, B.E., and S. Grinstein. 2007. Assessment of phagosome formation and maturation by fluorescence microscopy. Methods Mol. Biol. 412: 289–300.