The puzzle of arthritis susceptibility, have we found the missing pieces?

 I was at a meeting of rheumatologists in Shanghai in April this year. This brought together scientists and clinicians from around the world to discuss the role of the genome and relevant biological pathways that influence susceptibility to arthritis.

Arthritis has many guises, it can result in painful hot and inflammed joints. If severe enough, the inflammation in patients with ankylosing spondylitis can result in a fused and deformed backbone and chronic disability.

There are a number of additional conditions that are associated with arthritis such as psoriasis and gut inflammation. This group of diseases are collectively known as the spondyloarthropthathies (SpA) since they exhibit shared but also distinct features.

Why does the clinician categorise and group arthritis conditions you might ask? The clinician’s desire to label and categorise is based on a need to understand the disease. Following a diagnosis, the patient can be treated in a consistent fashion and according to accepted guidelines. Originally a clinician grouped patients with similar features and symptoms and gave the condition a name. Giving a disease a name was a coup for a clinician, very much in the way biologists get excited about naming a new species of insect. As more technological advances were made, clinicians were able to examine the tissue microscopically and the biochemistry of the patients’ tissues and fluids. By comparison with healthy people, specific cells and molecules were found to be altered in patients. These features and biomarkers were helpful to monitor the disease course and test the effectiveness of different treatments. Different patient groups had shared and unique biomarkers, supporting a different mechanism of disease in the different patient groups. Things have moved on considerably over the last 10-15 years and scientists have delved even deeper, studying our genetic blueprint, the genome, a collective word for the DNA contained within all our chromosomes.

Why do some people develop an inflammatory arthritis or other autoimmune/inflammatory diseases and others do not? Is it a purely a chance event, influenced by lifestyle choices or by the environment that we live in? Maybe the events that predispose to arthritis aren’t so stochastic, scientists suggest that predisposition to disease may be more predictable. The critical finding in the 1990s was that diseases of SpA could be inherited and this was done by studying families and tracking the incidence of disease in twins and siblings. These studies discovered that if you have a twin who has ankylosing spondylitis, you are far more likely to develop the disease compared to a non identical brother or sister. This means that the more genes in common you have with an person who has SpA, the more likely it is that you will also develop the disease. Although I must mention that the presence of identical gene or genes only increases the risk of getting the disease, it is not a foregone conclusion.

Given the genetic link with disease, this makes the genome an exciting and 'fruitful' place to explore. New technologies have enabled us to study the genetic map, to find the genes that increase our risk of disease. Researchers have invested considerable time and money in characterising the genes that confer disease risk, namely the gene variants that are more prevalent in people with arthritis compared to the healthy population. These studies also identified the gene variants that were much more prevalent in the healthy population, and so revealed the gene variants that can provide protection from disease. So how are these variant genes identified? Variations in our genes between individuals or even on our pairs of chromosomes can be determined very quickly and easily using a technique called ‘SNP’ analysis. These single Gene Nucleotide Polymorphisms (SNPs) are essentially a tag to identify a region in the genome that has variability. These variable regions might directly identify the region that is conferring risk or track closely with regions of the genome that are associated with an individual having a greater or lower risk of developing disease. Analysing the prevalence of certain SNPs in healthy and patients groups is a really powerful way to find out whether certain ‘SNPs’ are associated with imparting increased risk to inflammatory disease and arthritis. A study that can detect association between variant regions in the genome and susceptibility to disease  is called a genome wide association study (GWAS).

It was the work of Matt Brown and his collaborators in the 2000s that pioneered the use of large cohorts of healthy and ankylosing spondylitis patients to identify ‘SNPs’ that were either under represented or over represented in people that had succumbed to arthritis. Once a disease SNP was identified scientists scrambled to identify which gene it might be tagging. Not all SNPs directly tag a gene and so there maybe a number of candidate genes close to the SNP that might be conferring disease protection or increased risk. The upshot of all this genetic anlysis and 'bean counting' ( ie statistics!) was that several genes were identified. One gene of particular interest was an immune hormone receptor, IL-23R. This provided important evidence that the IL-23 pathway influenced disease susceptibility in humans. This was an exciting finding as IL-23 was also shown to be a critical molecule in many animal models of arthritis

As we look for more and more genes, this is when GWAS gets tricky, when the effects are real but are subtle, so more individuals needed to be studied. Over the last 10 years, larger numbers of patients and healthy controls have been analysed by different research groups around the world and the data combined and analysed to achieve greater statistical power. These ‘super’ analyses essentially increased the chances of identifying many of the important genes that confer disease risk. It is important to bear in mind that this approach won't identify all genes that increase risk, there is a limit to the detection of associations of rare variants with disease and alternative approaches are required to identify these.

Identify the gene(s) and you identify the cause? Well yes maybe but it’s not as easy as you think. We have known since the 1980’s that people who expressed HLA-B27 (a specific subtype of an immune system molecule, see my previous blog) were much more likely to develop the debilitating rheumatological condition called ankylosing spondylitis. Having been aware that this gene imparts a 20 fold increased risk to developing arthritis, researchers have strived over the last 30 years to identify the nexus between this gene and the mechanism of the disease pathway, but this connection has remained elusive.

So has GWAS enabled us to get over this 30 year stumbling block with HLA-B27, I think it has and here is the reason why. It is really important that we pick up most of the genes that confer risk because they can then be mapped onto biological pathways. Following GWAS for ankylosing spondylitis, some very discrete pathways were identified, like a game of ‘join the dots’, several pathways started to light up with multiple hits. This provides important evidence for particular mechanisms of disease susceptibility and also identifies potential therapeutic routes for treatment.  The missing pieces of the puzzle are at last being found and a picture of the disease mechanism is starting to develop. With recent data from the GWAS, we can now be more confident about the  disease pathway that HLA-B27 is acting upon. Recent data has identified the genes that are working with HLA-B27 to increase that risk, and this information has guided the type of research that we do. Like a gold miner, if you are told where to dig for your gold you have a much better chance of finding it rather than digging around aimlessly hoping to 'strike it lucky'. Have arthritis researchers found their gold? This means for us to identify an approach to prevent, treat and cure arthitis. Well not yet, but we are digging in the right places, so watch this space.

Just before I finish, I just wanted to add that although it is becoming increasingly clear that differences in the human genome can contribute increased or decreased risk to susceptibility to arthritis, it is becoming apparent that disease risk may also be imparted by the presence of other genomes residing within our body. The presence of bacteria within our gastrointestinal tract provide us with trillions of genomes . These genomes contained within the micro-organisms that reside within our gut, are now being carefully analysed by DNA sequencing using the latest DNA sequencing technologies. Spurred on by a growing body of evidence that suggests that the diversity of bacteria (coined the microbiome), plays an important part in protection from or susceptibility to inflammatory diseases, arthritis researchers have begun to address whether arthritis patients exhibited similarities in their microbiome. This is a new and exciting area of research and hopefully I will be able to report back on this soon,

I haven't got time to write about the other features or imprints on our genome that exist, which may define the disease more closely or even provide more clues to the disease process, This is world of epigenetics which I will write about another time.

Self knowledge for the Immune system

Does my body need to know self?

The ability of the immune system to reject transplanted organs seems a bit unfortunate when the organs are required for a life saving function. This makes you think, why is it so important that our body is able to know what is self?

To understand the rejection process I need to give you some information on how self/ non self recognition occurs. You may have heard about tissue types and tissue matching, this is as an important process in organ donation to prevent avoid organ rejection. When someone does a 'tissue match' they analyse the properties of a family of molecules that are encoded by a region of the genome called The Major Histocompatibility Complex. These molecules play important functional roles on the surface of cells. A particular family of these surface molecules have a special groove in their structure which can be used to sample the internal and external protein environment of cells. Sampled bits of proteins called peptides (like small sections of a necklace) are held in this groove and offered up to be screened by survelliance cells of the the immune system. If we call these surface molecules that have this function,'presenting molecules', this will make things easier to explain.

Humans like many other animals have a lot of diversity in their presenting molecules, inheriting and expressing one full set from their mother and the other from their father. This is the diversity that causes issues with organ transplants because it is not common to find identical presenting molecules on both the donor and recipient's tissues.  Even a few mismatched molecules that are recognised  as 'foreign' can excite an immune response sufficient to cause damage to the donor tissue. The foreign presenting molecules are also chopped up and used as stimulatory peptides, adding more fuel to the rejection process. The difficulty in getting a perfect match with all the different presenting molecules is the main reason why organ recipients are given immune suppression to increase chances of a successful transplant.

This raises the critical question, why are the presenting molecules so diverse?

It has been the dogma that this diversity is important as it imparts individuals with greater 'fitness' due to protection from infection by pathogens. We need a variety of presenting molecules to give us a better likelihood of binding the peptides from chopped up bits of viruses and bacteria. If pathogen derived peptides are not presented up for surveillance the pathogen could evade a protective immune response. This has important implications since individuals with presenting molecules that lack the ability to present a large repertoire of bacterial/viral/fungal peptides may be more susceptible to infection. As a scientist in an immunology lab it is not uncommon to have your presenting molecules typed. I know that I have inherited a very good presenting molecule that enables protection from TB, a mycobacterial infection and it is pretty useful for certain flu peptides as well. Overall this diversity is good to give us a wide variety of presenting molecules to cover all possible bases so that even when a  rare opportunistic infection occurs the immune system can respond. This property of the immune system is not good for transplants but very good for survival against infection.

So does this answer the question, organ rejection is simply a co-incidental effect occurring as a consequence of diversity selected for protection against pathogens. It would seem fantastical to think that this diversity was selected due to threat of invasion of cells from another individual. Sitting on a sofa next to my husband am I risk of invasion from the cells of his body? Although most cells in our body lack the replicative qualities to infect another individual, transformed cells such as cancer cells may have a greater infective potential.  There are rare cases where tumours have been known to be passed to surgeons that are operating on an individual suffering from cancer. More commonly cancer cells are passed to recipients receiving an organ donation. The cancer cells piggy backing a ride into the donor and because the recipient is immuno-suppressed they are able to grow in the recipient because their immune system has been disabled.

Well until recently, the idea that tumours could be infectious in healthy individuals was thought to be rather far fetched, until some recent research involving the Tasmanian devil came to light. This Marsupial from Tasmania is a rather aggressive creature that has recently become endangered due to a facial tumour disease. What became evident is that the devils were catching the tumours from other affected individuals. This transfer of cells was possible due to the rather aggressive behaviour which includes face biting during disputes over food and mates. This infectious cancer known as devil facial tumour disease has spread virulently thought the Tasmanian devil population This phenomenon is threatening their survival as a species because the animal typically starves to death three months after infection because the tumors interfere with feeding.

Studies on the genetic makeup of the Tasmanain devil and analysis of their presenting molecules revealed a really interesting finding. These devils had been through an evolutionary 'bottleneck' and are highly related. This has the consequence that these creatures also have very similar presenting molecules. It was proposed the lack of presenting molecule diversity was a significant contributing factor that prevented the devils's immune system from ridding themselves from the 'infectious' tumour. The key reason for susceptibility was the tumour wasn't being recognised as foreign tissue. This isn't the only example of infectious tumours,there is also evidence from other species such as wolves and dogs which succumb to infectious tumours contracted during mating and coincidently also have limited presenting cell diversity. In this case, the infection is transient and the animals recover from the disease.

Maybe an infectious threat from foreign tissue was a selecting pressure that also contributed to diversity in presenting molecules over evolutionary time, Not such a way out idea after all?

One thing to note, not all animal of limited diversity succumb to tumour 'infection'. Cheetahs are highly related due to a similar 'bottle neck' effect but show no evidence of this phenomenon. There are obviously other factors to consider. Another caveat in is this theory is that more recent studies revealed that the Tasmian devil tumours do seem to have extra infectious characteristics, since skin grafts from other devils are rejected effectively by the host devil. Nethertheless, this phenomenon looks very interesting. Could these characteristics be acquired in human cells and a wave of infectious cancer become a threat to humans? Well the chances of this happening seem very low but the use of immuno-supressive agents and the spread of viruses that result in immunodeficiency, might provide a larger pool of individuals who could act as vectors from which a tumour with infectious potential might develop. Please don't lose any sleep over this though!

Just to finish off, it is worth mentioning that knowledge of self and the diversity of our presenting molecules may have had other evolutionary pressures. Recent studies shown that presenting molecules can influence mate choice, but that's another story.

Foot notes:
First, don't confuse this infectious tumour phenomenon with tumours that occur as a result of infection by pathogens, eg human HPV infection that causes cervical cancer, here the tumours arise from our own transformed tissue resulting from an infectious virus.
Secondly, there are additional surface molecules that play a  role in immune responses, that are called minor histocompability antigens which have an alternative way of orchestrating immune responses and also contribute significantly to organ rejection.

What is an individual?

Viewing the world as a human, I look out into the world and see myself as an individual, within a family and within a community and then as a member of the larger population of our world. A super organism of human and other life forms?

I would think of myself as a single organism. But where are the boundary lines? Biologically my body is made of millions of cells, living and dying thoughout my lifetime, I am full of bacteria and micro-organisms that are essential for my health and sometimes not so healthy existence. An organism may be either unicellular (a single cell) or, as in the case of humans, comprise many trillions of cells grouped into specialized tissues and organs. When we look further into the cell we see the nucleus, a specialised and contained region that contains the genetic material, the blueprint for cells and its multi cellular form.

This raises the question, Is an organism defined by the DNA contained within the nucleus?

Well not completely!

As a eucaryote organism, my cells also have DNA that resides outside the nucleus, the DNA is situated within small organelles called mitochondria that look spookily like little bacteria. The Mitochondria are the power generators of the cell, essential to our existence. Possibly the ancestors of the mitochondria were early cell invaders back in the mists of time, parasites, which then evolved a more symbiotic relationship with its host. Now millions of years later, the cell has become a permanent home for these 'bacteria' within our cells. Although still hosting some of its its unique coding DNA, evolution has occurred and now the nuclear DNA also codes for proteins that are required for the mitochondria's existence as well. The original parasitic mitochondrial ancestor would have been defined as an organism in its own right, but now the mitochondria has lost that identity. The distinction of an individual and an organism become harder to define when we look at some strange creatures that exist in our world.

Mixotrichia Paradoxia is one of these strange creatures that exists in symbiosis with four other organisms that provide critical structures or metabolic processes that Mixotrichia lacks. As a good analogy it is similar to thinking about your own body with the legs and arms provided by another creature. Looking down the electron microscope the Mixotrichia looked like may other singled organisms but the thing that give it away as being a little bit unusual was that it had BOTH cilia and flagella, apparently that is a no no in these kinds of cells. This encouraged scientists to look a bit harder, delving more thoroughly they realised that the flagella and cillia were actually separate organisms in their own right.

Mixotricha has four anterior flagella, that are used for steering, these are a paramecium. Movement is achieved by the beating movement of 250,000 hairlike stuctures which incredibly, were found to be helical bacteria, these are attached to the cell surface and provide the cell with cilia-like movements. Mixotricha also has rod-shaped bacteria arranged in an ordered pattern on the surface of the cell but no one knows what they do! Last but not least, Mixotrichia has spherical bacteria inside the cell which function like mitochondria.

So is Mixotrichia  and its symbionts, one individual, or many, one organism or many, a beautiful composite organism? This intriguing collection of creatures interacting symbiotically to define the the characteristics of Mixotricha paradoxia is a wonderful thing.

The definition of the boundaries of what is an organism becomes even more blurred, when you look at the larger scale and the perspective becomes even more interesting. Mixotricha paradoxia is an essential organism within the gut of the termite that allows it to obtain nutrients from wood and plant material, Mixotricha, allows the termite to exploit its barren environment, using poor food sources to sustain life. Most importantly, the termite relies on being a member of a larger community of termites in the termite mound, all working together as super organism to create the successful termite community.