AH&MRC profile: Helen Ball

Tryptophan is the least abundant of the 20 standard amino acids and a member of the essential acid group – we can’t synthesise it from other compounds so have to take it in through our diet.

It has three roles in the body: it is incorporated into proteins, a small amount is used as a precursor for serotonin and melatonin, and it is metabolised into kynurenine, which is used for a number of purposes including the production of niacin, the control of blood pressure and in reproduction.

The kynurenine pathway is of great interest as it plays a major role in immunomodulation and central nervous system disorders. The pathway is triggered by two enzymes: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). TDO is highly expressed in the liver and works to control levels of tryptophan in the bloodstream, and if inhibited can increase levels of serotonin.

IDO, on the other hand, is expressed in the intestines, the placenta and in endothelial cells. While it is not clear what it does in the intestines, in the placenta it works to suppress the immune system of the mother so she is able to tolerate the presence of the alien life form known as the fetus. In endothelial cells, IDO is switched on by the cytokine interferon gamma and is highly active in inflammation.

Dr Helen Ball began studying IDO and the kynurenine pathway to try to understand their implications in cerebral malaria. “We know that the disease is dependent on cytokines such as interferon gamma and lymphotoxin alpha, so that started our interest in the kynurenine pathway,” Ball says. “Interferon gamma obviously has many effects but one of them is as the major inducer of this pathway.”

Last year, Ball and her colleagues at the University of Sydney had a bit of a breakthrough. Working on a mouse model of cerebral malaria, they found a huge induction of IDO activity, but there was an imbalance in the levels of two downstream metabolites of the kynurenine pathway, the neuro-toxic quinolinic acid and the neuro-protective kynurenic acid. These metabolites can interact with an N-methyl-D-aspartate (NMDA) class of receptors through opposing actions.

“We thought this might contribute to pathology because it would contribute to the seizures you see in cerebral malaria,” Ball says. “We did a lot of work looking at the activity of IDO in the brain and measuring these metabolites.

“Then we got the idea of creating a knockout mouse model, but were rather disappointed as the phenotype was not at all different. There have also been some other results – for example, when people used an inhibitor of IDO in pregnancy it can result in the loss of pregnancy, and these IDO knockout mice breed normally. So there were indications that there were perhaps some redundancies there.”

Ball suspected there was something else playing a part so began to look for possible homologues of IDO. She came across what is now called IDO2, sitting right next to the IDO1 gene on chromosome 8. “It was really quite a surprise because it was sitting there next to the IDO1 gene all along and no one had noticed it in the past 20 years,” she says. “It seemed like there had been a gene duplication and you get these two IDO isoforms.”

She explained her work to the AH&MR Congress in Brisbane yesterday.

---PB--- IDOs and disease states

While cerebral malaria and bacterial meningitis are the main diseases looked at by Ball and her colleagues at the Molecular Immunopathology Unit at the university’s Bosch Institute, a greater understanding of the different roles of the IDOs and the kynurenine pathway have much larger implications.

Ball and her colleagues, including the head of the institute, Professor Nicholas Hunt, are delving deeper into the complexities of the pathway, which may lead to further understanding of disease states like hypertension, diabetes, disorders of the central nervous system and even cancer.

Tryptophan is essential for cell proliferation, so the theory is that the IDO-induced catabolism of tryptophan into kynurenines works to suppress this proliferation. “IDO1 is mostly induced in inflammatory situations, and it does this via two broad mechanisms,” Ball says.

“You can deplete tryptophan in the microenvironment and that will stop cell proliferation – it will stop proliferation of microbes and it can stop proliferation of T cells, so it can inhibit your immune response.

“And then some of the downstream metabolites have been found to have various direct effects on immune responses, particularly in metabolites that are implicated in central nervous system disorders. You find a higher activity of the pathway in the brain in disorders like AIDS-associated dementia and Alzheimer’s disease.”

What is intriguing about IDO2 is that it has quite different expression patterns to IDO1. Like IDO1 it is expressed in the placenta so may have a role in pregnancy, but unlike IDO1 it does not seem to be inducible by cytokines. It is also highly expressed in the kidney, although no one knows what its function is there; and it is expressed in spermatozoa, so it should have a role in male reproduction.

“From some of our work in the IDO1 gene knockout mice we believe there isn’t really any IDO2 or TDO activity in the brain, so we think that the metabolites are perhaps coming from peripheral sources and they can diffuse into the brain because the blood-brain barrier breaks down in cerebral malaria.”

The team is currently eagerly awaiting an IDO2 knockout mouse, created by Dr George Prendergast’s lab at the Lankenau Institute for Medical Research in Philadelphia. Prendergast’s lab discovered IDO2 at the same time as Ball but came to it from a different angle. His team concentrates on cancer modifier genes as targets for drug development, in particular the genes RhoB and Bin1.

IDO1 is of great interest to cancer geneticists because it modifies the reactions of T cells to cancer cells, and Bin1 regulates IDO1. This group is developing small molecule inhibitors of IDO in a breast cancer model.

“IDO has been an interesting target as an anti-cancer agent as IDO activity is associated with a poorer prognosis,” Ball says. “In the tumours, it is actually suppressing immune response or is somehow involved in the draining lymph nodes of the tumours. You get suppression of immune responses against the tumour, basically.

“So when they’ve used an inhibitor of IDO, the D racemer of 1-methyl-tryptophan, it has the best anti-tumour effect in mouse models. However, it has been a bit confusing because the best inhibitor for IDO1 is the L racemer. So this group actually set out to look for a protein that was inhibited by 1-D-MT and they found IDO2 as well. It is more selective for the D racemer.

“It hasn’t really been shown that IDO2 is the enzyme that is really involved in cancer so that still remains to be demonstrated.”

Ball was contacted by the Prendergast group when she published her discovery of IDO2 last year, and the teams are now happily collaborating. The US group has created a knockout model and is about to ship it over to Sydney, where further work will be done.

---PB--- Biosketch

Helen Ball did her PhD looking at the structure of a gene through neuropeptide Y receptors at the Garvan Institute in Sydney. She then travelled overseas and settled for a time in New York, where she worked on a transcription factor involved in a chromosomal translocation in leukaemia at Mt Sinai Medical Centre.

On her return to Australia, she became interested in biological techniques like microarrays and laser capture microscopy, and joined the University of Sydney’s Department of Pathology. The Bosch Institute, a virtual institute at the university headed by Professor Nick Hunt, has a long history of research into cerebral malaria.

Ball predominantly studies the pathogenesis of cerebral malaria through the dysregulation of cytokine networks and also lung pathology in malaria. When Ball published her discovery of IDO2 in Gene last year, she was contacted by both the Prendergast team and by Dr Hajime Yuasa from Kochi University in Japan, who recently spent a year in the Hunt laboratory researching the evolution of the IDO enzymes.

Ball and colleague Dr Lars Jermiin hypothesise that the IDO genes probably arose about 300 million years ago as a result of gene duplication. Yuasa began cloning them from Australian native marsupials and mammals, and suggested that IDO2 may be the original gene.