AH&MRC profile: Mike McGuckin

Human inflammatory bowel diseases (IBD) such as Crohn’s Disease and ulcerative colitis (UC) are believed to involve inappropriate immune responses to the complex mix of microbes in the gut. A mucosal barrier made up of cells and secreted goodies normally keeps this plethora of pathogenic and normal bugs separated from the intestinal tissues.

It has long been known that this mucosal barrier is disrupted in IBD. However, the extent to which the mucin proteins that characterise this layer are involved in the chronic inflammatory cycle of IBD beyond simple physical protection is only just starting to become apparent.

Last year, Associate Professor Michael McGuckin and his team at the Mater Medical Research Institute (MMRI) in Brisbane published the first real in vivo evidence that mucins on the surface of the gut play an active and critical role in mucosal defence against infection and inflammation.

Now they have again led the field in establishing a functional link between mucin abnormalities and a novel mechanism underlying intestinal inflammation, not only in mice but also in human patients with a particular form of IBD.

At the Australian Health and Medical Research Congress in this week, McGuckin discussed his two mice strains, called Winnie and Eeyore, and explained just what they are teaching him about the gut.

McGuckin heads the Mucosal Diseases Program at the MMRI, focusing on mucosal barriers to infection and inflammation. He has close ties with several respiratory physicians and gastroenterologists around town, although he concentrates on epithelial mucins in the gastrointestinal tract, in collaboration with the director of gastroenterology at the adjacent Mater Hospital, Professor Tim Florin.

McGuckin has a long-standing interest in mucin glycoproteins, which are made by specialised cells found in all mucosal epithelial linings in the body.

These large molecules are about 70 per cent carbohydrate on a protein backbone and include mucins secreted into the extracellular matrix, as well as others that remain embedded in the apical membrane of mucosal epithelial cells and poke out into the lumen.

These cell surface-mucins form large disulphide-bonded polymers by virtue of characteristic cysteine-rich regions at either end of the protein structure. The polymers get together to form gel-like mucous layers covering the cell surface.

The most well know mucosal layer is of course in the gut, where the mucin proteins are made and secreted by the distinctive-looking goblet cells – so-called because of their flask-like appearance – dispersed throughout the gastrointestinal epithelia.

Exactly how these complex proteins actively participate in protecting the intestinal lining from infection, and how they contribute to the pathology associated with chronic infection and inflammation, are of prime interest to McGuckin’s team.

According to McGuckin, the field of mucin research is not all that popular, mainly because the genes are huge and the proteins are difficult to work with. “Howard Florey and Frank Macfarlane Burnet both dabbled in mucins and there have not been too many Aussies since,” he says.

“Macfarlane Burnet was the one who got mucins written up in the textbooks as an important part of primary defence, although this was based really on very little in terms of empirical data.”

---PB--- Mucins and the gut – a perfect couple

The human gut is constantly brimming with pathogenic and inflammatory signals, all on an epithelial surface area the size of a tennis court. “That is a big area to defend, and in fact, the overwhelming initial response of the immune system is not to over-respond to all these local insults,” McGuckin says.

Pathogenic microbes have evolved many ways to broach this mucosal barrier: that is their job after all and it probably happens on a microscale all of the time. Pathogens may break through the layer by evolved signalling mechanisms to trick the host cells, break it down using enzymes or go around the barrier via the goblet cell-free Peyer Patches.

“What we showed recently was that if pathogens, or normal flora for that matter, make it to the surface of the epithelial cells, the mucins come into play in extra ways – they try to block the bacteria adhering to the cell surface and also signal into the cell about the presence of the pathogen on the surface.”

If the pathogen threat increases above a certain level, adaptive immunity is engaged and inflammation kicks in, McGuckin says. “This in itself can damage the physical barrier to some extent, which is why the body does not really want to use it unless it really needs to.

“So, a lot of the signals coming from the mucins are probably designed to moderate the responses and help the cells decide what to do on a local level.” The affected epithelial cells may destroy themselves by apoptosis, or be ‘popped out’ from the cell layer and shed via the gut lumen.

At the AHMRC, McGuckin described work published in PLoS Medicine earlier this year on two mouse models of intestinal inflammation that were generated by random mutagenesis.

---PB--- A tale of two mice

The story started a few years ago when Professor Chris Goodnow at the Australian Phenomics Facility in Canberra contacted McGuckin to see if he was interested in a transgenic mouse strain the facility had produced. The major phenotype of the mouse strain was constant diarrhoea from a young age and chronic colitis as an adult.

McGuckin was indeed interested. Genotyping of the mice with the unfortunate bowel habits pinpointed a mutation in the gene that encodes MUC2, one of the main mucin components of the intestinal mucous barrier.

“Soon after, another mouse came out of the facility’s mutagenesis program with the same phenotype, and it also showed a single, although different, mis-sense mutation in the same gene,” McGuckin says.

“Our initial idea was that these mutations probably inhibited the mucous gel complex from forming properly, leading to a poor quality mucous and the observed bowel symptoms. It turned out of course to be far more complex.

“The mutations caused the mucin molecules to form an inappropriate complex during biosynthesis, which then put the cell into something called endoplasmic reticulum (ER) stress.”

ER stress is an evolutionarily conserved phenomenon that occurs when proteins misfold during biosynthesis. It is caused by a variety of cell disturbances – a genetic alteration, metabolic imbalance, toxin, viral insult – and has recently been associated with diseases such as neurodegeneration and diabetes.

“Normal cells always have a small amount of misfolded protein, simply because protein biosynthesis is a complex process and when it goes wrong, the cell has mechanisms in place to deal with it.

“The cell sees the misfolded protein as garbage, and the mistake is disposed of accordingly via proteasomal degradation.

“The trouble comes when these misfolded proteins are being made at a higher rate than normal, and you get an inappropriate accumulation of the misfolded molecule - this sends the cell into ER stress.”

The cell will then engage the unfolded protein response (UPR), which involves a whole bunch of cellular events designed to alleviate the problem. However, if the ER stress is too severe or goes on for too long, the cell has to pull the plug and the most likely outcome at that stage is apoptosis or programmed cell suicide.

Coming back to the mice with diarrhoea, the major findings from the initial characterisation were animals with a genetic predisposition to misfolding of this major protein, MUC2.

This abnormality induced substantial ER stress with associated pathology in the goblet cells and mucous layer structure, and finally the development of chronic intestinal inflammation.

---PB--- Depleted goblet cells

These observations prompted McGuckin’s team to go back and look more carefully at the early IBD literature. “One of the hallmarks of UC is depletion of, and smaller, goblet cells, which is exactly what we saw in these mice. Everyone has naturally assumed that this depletion is a response to the inflammation and not a primary cause of it, but it could be either.”

The team found several electron microscopy studies from the 1970s describing significant ER vacuolisation in gut tissue from patients with UC. “This morphology is exactly what we get in extreme forms of our mice. Interestingly, the feature was observed even in unaffected, non-inflamed areas of the intestine.”

Examination of a collection of the Mater group’s own UC patient samples also showed similar morphological features to those in the mouse models, as well as biochemical and histochemical markers of ER stress.

This strong similarity between the human and mouse disease was an important finding that strongly argues for ER stress-related mucin depletion being actively involved in the development of IBD.

“We now have a prospective trial going on where we collect tissue samples from patients and assess them biochemically and morphologically for ER stress,” he says.

“What our studies and another very recent one in another model (published in Cell recently) show, is that a cellular event related to protein misfolding can push these intestinal secretory cells into ER stress.”

This results in a range of inflammatory triggers, and even with completely normal underlying immunity, the result might be chronic inflammation of the intestine.

The other group, based in Boston, has now also shown some human genetic data that supports the Australian findings by linking a polymorphism in one of the known ER stress-related genes to intestinal inflammation in IBD, although it took a huge population size to find it.

McGuckin’s group is now trying to cement the idea that ER stress is a player in human disease. They think it is, but there still is the question of whether it is primary or secondary.

“How much of the inflammation is coming from the ER stress and how much is coming from the depleted mucosal barrier?”

McGuckin predicts that both scenarios come into play, but that the sequence will be impossible to determine as one cannot be dissected away from the other – they are simultaneous as well as interdependent – “and in the end the distinction is probably academic anyway,” he says.

“My guess is that environmental factors will be key to this whole thing, and the genetics will be very difficult to tease out as they are likely to be extremely complex in multi-factorial diseases such as IBD.”

Having said that, how one identifies potential environmental culprits also seems challenging, particularly in the gut.

“Is it history of infection, is it gut flora-related, is it what people eat or don’t eat, is it toxins, drugs – there are so many things that could induce the ER stress response.” Also, how the individual system responds to those factors could be part of the whole deal and might also come back to genetic factors.

Even if the ER stress-related effect is secondary in the pathogenesis of IBD, it is still a potentially novel druggable target. “If you can alleviate the ER stress with a drug then you can perhaps get a new angle at treatment of these diseases and, at worst, maintain them in remission.”

There are new drugs available and more on the way that work on the ER stress pathway and these will be tested for efficacy in the two mouse models. In addition, McGuckin will be looking at the current drugs for IBD. Early results seem to indicate that even low-dose steroids dampen the ER stress response quite considerably and decrease inflammatory signalling in the gut.

“I think what people are discovering over the last five years or so using these animal models is that you can create cell-specific defects that drive this IBD kind of inflammation pattern in the presence of completely normal immune function.”

Having to cope with the extra stress and cellular damage could be enough to kick a seemingly normal immune system into overdrive. “It is all clearly even more complex that we all thought and our studies on the ER stress mice opens up a whole new idea for looking at it.”