THEME 8 IMAGING INNATE IMMUNE RESPONSES

Human macrophages (nucleus stained blue) treated with a novel fluorescent compound (red) that inhibits Mycobacterium tuberculosis. This class of compounds may mediate immune responses via an unknown intracellular target yet to be discovered.
Image by James Lim.

“The Imaging CoE is investigating how molecules enter innate immune cells and activate key signalling proteins inside to trigger immune responses to infection.”

Innate immune cells provide the first line of defence against infection and injury. Innate immune cells include mast cells, eosinophils, monocytes, macrophages, neutrophils, dendritic cells and NK cells. Some innate immune cells reside in our tissues and are exposed to the environment, others are recruited to tissues in response to infection or injury.

Imaging CoE researchers also study epithelial cells that form a physical, chemical and immunological barrier on tissue surfaces (skin, lungs, GI tract, organs, blood vessels) exposed to the environment. These cells are the first point of contact with infectious organisms and environmental stress.

Collectively, these cells monitor changes to tissues and maintain them in a healthy state, quickly identifying threats and eliminating them along with damaged cells.

 

PROF. DAVID FAIRLIE

AT A GLANCE

The group studies innate and innate-like immune cells and their responses to biological and chemical agents that cause inflammation. These studies are helping us to understand the molecular basis of innate immune-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.

The maturation, temporal engagement and activation of the various kinds of innate immune cells and the interplay between them are still not well understood. Even the ways in which they recognise pathogens, nutrients, chemicals and endogenous proteins through protein interactions are still poorly understood. We now know that innate immune cells can respond to chemical, metabolic and even mechanical disturbances to correct disrupted cellular and tissue homeostasis. However, the wide range of stimuli capable of disrupting homeostasis is only just beginning to be elucidated, requiring new studies on how our innate immune system responds to each newly identified environmental stimulus.

The Imaging CoE is imaging molecular events underlying the different responses of innate immune cells to infectious and non-infectious stimuli, as well as their roles in cancer, metabolic and inflammatory disease.

ACTIVITY PLAN

  1. Develop fluorescent ligands for imaging the activation or inhibition of inflammation-related proteins involved in innate immune responses to microbes, tumours or chemicals.
  2. Image the motility and migration of innate immune cells and cancer cells in vitro and in vivo.
  3. Discover, map and selectively block signalling pathways that mediate innate immunity.
  4. Discover links between surveillance, metabolism and inflammation in innate immunity.
  5. Develop fluorescent proteins, peptides and drugs to target protein-protein interactions in innate immune cells and their interactions with cancer cells.
  6. Target specific proteins on specific innate immune cells that mediate immune responses.
  7. Image dendritic cells, NK cells and receptors to study structure and function in innate immunity.

HIGHLIGHT

Linking inflammation to fibrosis

Imaging CoE researchers at the University of Queensland have discovered some important clues connecting inflammation in the liver to tissue scarring that can lead to liver cirrhosis and cancer. They found an important group of enzymes that contributes to inflammation in the liver and drives secretion of proteins that cause fibrosis and hepatic cell death.

Viral hepatitis infections, high fat diets or excessive alcohol consumption can result in persistent inflammation and tissue damage in the liver and reduce its capacity to regenerate and maintain functions. When damaged tissue is not repaired there is a build-up of tissue scarring or fibrosis that blocks blood flow in the liver and starves liver cells to death. Liver fibrosis can progress to cirrhosis and then to liver cancer.

The team of researchers, led by Dr Ken Loh, Dr Rebecca Fitzsimmons, Dr Abishek Iyer and Chief Investigator, Prof. David Fairlie, administered a chemical to mice to induce liver inflammation, fibrosis, and liver damage that are very similar to the clinical features of chronic human liver disease. They also delivered drug-like molecular probes to the mice to investigate the cellular basis of chronic hepatic inflammation leading to liver fibrosis. During the 12-week time period required to develop fibrosis, the researchers analysed histological (immunohistochemistry, TUNEL), biochemical (enzymes, proteins) and immunological (flow cytometry, qPCR, Legendplex, ELISA) changes in liver pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression.

By imaging cells, proteins and tissues during the development of fibrosis in mice, they were able to identify enzymes that were key promoters of inflammation in chronic liver disease. They found drug-like molecular probes that were able to block the enzymes and suppress chronic liver inflammation and fibrosis in mice. They discovered that the probes stopped the accumulation and activation of IL-33 dependent group 2 innate lymphoid cells (ILC2). This, in turn, reduced secretion of inflammatory cytokines that characterise type 2 inflammation and prevented liver cells from depositing collagen and other matrix proteins that cause thickening and scarring of connective tissue.

The Imaging CoE team, which includes collaborators in the Centre for Inflammation and Disease Research at the University of Queensland, is using this information to better understand the molecular basis of inflammatory signalling that regulates onset, detection and repair of damage to liver cells. These studies can provide new insights to molecular events that trigger liver inflammation, escalate liver damage or initiate repair, and the new knowledge can lay the foundation for new preventative and therapeutic approaches to managing the chronic inflammation that leads to liver fibrosis.

Imaging CoE researcher Dr Ken Loh (Institute for Molecular Bioscience, UQ) standing in front of a new flow cytometer and in vivo imaging platform (BD LSRFortessa X-20) that permits very sensitive and high-resolution multicolour analysis of different immune cell populations.

ACHIEVEMENT

Innate immune signalling

In 2019 we developed fluorescent small molecules to study inflammatory mechanisms associated with G protein-coupled receptors expressed on innate immune cells, including mast cells, macrophages, dendritic cells and epithelial cells of the kidney and colon. We also developed fluorescent compounds that inhibited growth of Mycobacterium tuberculosis in human macrophages, and they are now being used by external collaborators to investigate inflammatory mechanisms that combat bacteria.

The group developed novel compounds for modulating GPCR signalling that drives cell function. We imaged fluorescent receptors and extracellular ligands administered to human cells and discovered new intracellular signalling pathways and mechanisms that control key events such as receptor internalisation, transactivation of other receptors, cooperativity between signalling pathways and proteins, and we have linked receptor activation via specific pathways to some innate immune cell functions, including migration of innate immune cells and cancer cells.

The group continued studies on Rab proteins as key intracellular signalling molecules in macrophages. This is important as Rab signalling can cause immunodeficiency, inflammatory diseases and cancers. Imaging CoE researchers have studied intracellular trafficking of GPCRs via Rab GTPase, including receptor internalisation, recycling and degradation in endosomes. We profiled GPCR-Rab5a signalling pathways to inflammatory chemokines/cytokines in primary human macrophages.

Epithelial cells that line the gut and lung mucosa produce glycopeptides called mucins, which collectively form the mucus secretions that protect tissue from exposure to bacteria in the environment. Using molecular and cellular imaging techniques, we have learned the molecular basis of the cellular machinery responsible for this important protective process. We have also studied certain agents in the gut and lungs that compromise mucin production, switching off their cellular synthesis, and exposing mucosal tissue epithelia directly to bacteria. We have identified how to block these events and promote immune responses that protect the integrity of the intestinal and lung mucosa.

Innate immune cells such as macrophages, and certain kinds of epithelial cells, switch their metabolism of glucose to undergo glycolysis in response to different types of extracellular stimuli. This leads to inflammation and immune signatures that are glycolysis-dependent. We are investigating these mechanisms and imaging protein-protein interactions that prime certain types of cells for altered metabolic handling of glucose leading to immune responses that are tailored for restoring cellular homeostasis. The complex molecular events underpinning these phenomena are being unravelled using a combination of imaging, novel chemical probes, and immunological profiling.

Top: Proteases induce internalisation of a surface-expressed GPCR called PAR2 (green) from the cell membrane to endosomes in cancer cells (bottom left) leading to intracellular responses. Genetic knockout of specific signaling proteins that couple to PAR2 prevent its internalisation (bottom right)
Credit: Kai-Chen Wu.
Bottom: Imaging of mouse distal epithelial colon tissue sections stained by Alcian blue and PAS. Staining shows mucin-producing goblet cells in mice given 80 μL saline (left) versus a PAR2 agonist (100 μg/mouse) intracolonically. Mucin glycopeptides (blue) are depleted from colonic crypts 4h after PAR2 activation.
Credit: Eunice Poon.