Inflammatory protein C5aR (green) expressed on the surface of an epithelial cell signals via endosomal signaling protein Rab5 (red) inside cells. This live cell image was taken using high spatial and temporal resolution microscopy – Lattice Light Sheet Microscope (LLSM). Credit: Kai-Chen Wu.
“The CoE is identifying important membrane proteins on innate immune cells that respond to infection by activating key signalling proteins inside.”
In 2012, Imaging CoE Centre scientists made the startling find that an abundant T cell population, termed Mucosal-associated invariant T (MAIT) cells were activated by metabolites of vitamin B (namely riboflavin) (Nature 2012).
Using a combination of chemistry, cellular immunology and structural biology, they subsequently showed that the most potent MAIT cell antigen arose from the convergence of two distinct metabolic pathways, namely riboflavin metabolism and metabolites from glycolysis (Nature 2014).
Armed with this information, the field is now empowered to understand MAIT cells in homeostasis, health and disease. To further enable this, Imaging CoE scientists developed novel biochemical reagents (MR1 tetramers) that have enabled the phenotypic characterisation of MAIT cells.
PROF. DAVID FAIRLIE
We are developing and deploying chemical probes and imaging approaches to study innate and innate-like immune cells and their properties. These studies are helping us to understand the molecular basis of innate immune cell mediated defence, how tissues function and repair, and how threats can be combatted with new kinds of drugs that target proteins on innate immune cells and related cancer cells.
Innate immune cells include many diverse types including mast cells, neutrophils, macrophages, dendritic cells and natural killer cells. Their maturation, temporal engagement, activation and interplay are still not well understood. Even the way they recognise pathogens and cancer cells through protein-protein interactions is still poorly understood. We now know that they also respond to chemical imbalances that disrupt cellular and tissue homeostasis.
The Imaging CoE is studying the different responses of innate immune cells to infectious and non-infectious stimuli, as well as their roles in cancer, metabolic and inflammatory disease.
New information can better inform on how, when and where innate immune cells work in different settings to protect our tissues and maintain good health. This information can also help us design more effective drugs for treating disease.
Scientists in our Centre are developing new approaches and technologies to observe and track key ligands and proteins that activate innate immunity to learn more molecular details about how mammals defend themselves against infection, tumours and injury.
- Develop fluorescent ligands for imaging the activation or inhibition of inflammation-related proteins (GPCRs) involved in innate immune responses to microbes, tumours or chemicals.
- Image the motility and migration of innate immune cells and cancer cells in vitro and in vivo.
- Discover, map and selectively block signalling pathways that mediate innate immunity.
- Discover links between surveillance, metabolism and inflammation in innate immunity.
- Develop fluorescent proteins, peptides and drugs to target protein-protein interactions in innate immune cells and their interactions with cancer cells.
- Target specific proteins on specific innate immune cells that mediate immune responses.
- Image dendritic cells and receptors to study structure and function.
- Image NK cells and receptors in innate immunity.
Linking inflammation and cance
University of Queensland researchers in the CoE for Advanced Molecular Imaging discovered an important clue connecting inflammation to cancer. They found that a key inflammatory protein (protease activated receptor 2) substantially overexpressed on the surface of innate immune cells
(e.g. mast cells, macrophages) is also present on cancer cells and similarly promotes their movement along chemical gradients.
The team led by Imaging CoE Chief Investigator, Professor David Fairlie will use this information and new molecular tools to better understand the molecular basis of signalling responses that control detection and eradication of cancer cells and immune cells damaged by infectious and chemical stimuli that can cause diseases. These studies could lay the groundwork for developing new therapeutic agents.
The researchers, including cell biologists PhD student Yuhong Jiang, Dr James Lim, Dr Jacky Suen and Ms Kai-Chen Wu, have learned that activation of PAR2 leads to similar signalling in cancer cells as in macrophages. They have now mapped the intermediate PAR2-dependent signalling pathways in cells that lead to recruitment of beta arrestins 1/2, calcium release, ERK 1/2 phosphorylation, RhoA activation, and inhibition of forskolin-induced cAMP accumulation.
Functional consequences of this signalling include secretion of inflammatory cytokines, apoptosis and cell migration.
Of potential therapeutic interest, these researchers have also identified a potent inhibitor of PAR2 that blocked all of these signalling pathways in cancer cells and in innate immune cells. This new chemical can also block production of inflammatory cytokines and PAR2-induced motility of human macrophages and migration of breast and colon cancer cells. Cancer cell migration and invasion is relevant to metastasis, the spread of cancer cells to different sites.
The research has led to a better understanding of intracellular signalling in macrophages versus cancer cells, the role of PAR2 in the cell membrane, and how activating compounds outside cells can trigger molecular signalling inside cells and produce functional responses associated with protection, cell migration, dysfunction and cell death.
Surface expressed protein PAR2 (green) and plasma membrane stain (red) co-staining. PAR2 induces migration of innate immune and cancer cells.
Innate immune signalling in macrophages
Rab proteins are key signalling molecules in innate immune cells. Impairment of Rab signalling pathways can result in immunodeficiencies, inflammatory diseases and cancers. Imaging CoE researchers have discovered intracellular trafficking of the C5a receptor via Rab GTPase.
Activation of C5aR leads to its internalisation, recycling and degradation and these events have been found to be associated with Rab5a in early endosomes. Rab5 is highly expressed in macrophages and is required for these events. Rab5 dysfunction prevents C5aR internalisation. This research has linked C5aR with Rab5a signalling to inflammatory responses in macrophages. The C5a receptor was tracked initially in HEK293 cells expressed C5aR conjugated to green fluorescent protein. Rab5a S34N was monitored using the td-Tomato orange fluorescent protein. The C5aR-Rab5a association was then found to regulate proinflammatory chemokine/cytokine mediators in primary human macrophages and the signaling pathway was mapped.
This work has allowed us to track the fate of C5aR from the cell surface and Rab5a from endosomes to unravel molecular and signalling mechanisms that lead to inflammation, disruptions to cell homeostasis, and restoration of normality.
Scientists at Imaging CoE are deploying novel compounds to modulate different innate cell functions mediated by these same proteins on and near the cell surface.
Human macrophages (top row) stained to monitor membrane-bound protein C5aR (green), endosomal signalling protein Rab5 (red) and nucleus (blue). Activation with unstained proinflammatory protein C5a induces internalisation of C5aR into the cytoplasm and co-localisation with Rab5 (bottom row).