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Dr. Claude Perreault and a team of Canadian and Finnish scientists has identified a protein able to stimulate the production of T-cells, the white blood cells involved in the recognition and the elimination of infectious agents. The discovery by researchers of the Institute for Research in Immunology and Cancer (IRIC) at the Université de Montréal in Canada and the University of Oulu in Finland - published in the latest edition of the journal Immunity - could help to combat age-related declines in immune response.
Scientist Live spoke with Dr. Perreault about the study, the role of Wnt4 in the immune system, and how Wnt4 could hold the key to prolonging life.
How did you first become involved in researching thymic atrophy?
I was interested in understanding two factors. First, why are T-Lymphocytes produced in the thymus and nowhere else. SSEDIT5 CMS - editing Scientist LiveSecondly, why does the thymus involute early in life so that it leaves older people immunodeficient. For example, thymic atrophy begins as early as one year of age. Progressive thymic involution is responsible for the fact that elderly individuals have very poor thymic function. They produce very little T-Lymphocytes and because of that they are more susceptible to infections, cancer, and autoimmune disease. We also found that one major characteristic of the thymus found nowhere else in lymphoid organs is the expression of a protein called Wnt4. We hypothesised that Wnt4 had a role in T-Lymphocyte development and that by providing high levels of Wnt4 to hematopoietic progenitor cells we would enhance thymopoiesis. That is how it began.
We did two series of experiments. In the first set, we induced over-expression of Wnt4 in hematopoietic stem cells and found that compared to mice that received standard cells those that received cells producing high levels of Wnt4 had a bigger thymus and produced 3-4 times more T-Lymphocytes. On the contrary we found that when we knocked out Wnt4 there was thymic atrophy. Overall, these studies suggest that Wnt4 is necessary for normal T-cell production and that over-expression of Wnt4 is sufficient to improve thymopoiesis. In the future, we hope to evaluate the best way to give Wnt4 to animals or humans in order to find whether this molecule can be used to treat thymic involution.
How does thymic atrophy occur?
Beginning at 1 year of age the size and function of the thymus decrease progressively so that we have barely any thymic function after the age of 50. Because of that, people older than fifty produce very little T-Lymphocytes and all their T-Lymphocytes are old because they were produced earlier in life. Because older people do not produce T-Lymphocytes the diversity of their T-Lymphocytes is considerably limited and they have problems when they encounter new pathogens. Each time they encounter a new virus, for example avian flu or SARS, older people are susceptible because they cannot produce the T-Lymphocytes to recognize the virus.
Just to provide a number, it has been estimated that Respiratory Syncitial Virus is responsible for 175,000 hospitalisations of elderly patients and 15,000 deaths in the United States per year. The same is true for cancer. One reason why cancer incidents increase in elderly people is because they cannot generate T-Lymphocytes and therefore cannot recognise and attack tumours. I would say thymic involution is probably one of the two most important barriers in increasing life expectancy, the other being neurodegeneration.
Why choose a Wnt protein considering its alternative roles in other pathways?
We chose it because we found that in previous studies, to our surprise, some T-Lymphocytes progenitors were present in lymph nodes that do not produce T-Lymphocytes. We found that the T-cell progenitors that were present normally did not expand in the lymph and underwent apoptosis. We concluded that there must be a factor that enhances the survival of T-cell progenitors that is present in the thymus but absent in the lymph node. And that this factor would be responsible for the fact that T-cell progenitors do generate T-Lymphocytes in thymus and not in lymph nodes.
Listen to the audio clip the end of the article to hear Dr. Perreault explain how gene expression profiling was used in his research.
What were your findings and can you place them in a broader immunologic context?
In the first experiment we injected a foetal liver cell transduced with Wnt4 into irradiated recipients and we waited for sixteen weeks. Then studied the hematopoietic organs and the lymphoid organs of recipients. When we examined the secondary lymphoid organs and blood cell subsets, there were minor differences. There was only one organ and lineage that was affected by Wnt4 over-expression. This was the thymus. Thymic cellularity was increased four-fold relative to controls. Thymopoesis depends on feeding of the thymus by bone marrow cells called multipotent progenitors (MP). We found in the bone marrow of the mice that although the overall solidarity was similar in the two groups, the small subset of bone marrow MPs was increased five fold in the recipients of Wnt4-transduced cells. To summarise, we had two major findings: an increase in MPs in the bone marrow and an increase in thymic cellularity.
Next, we studied the bone marrow MPs in greater detail. We found that in recipients of Wnt4 positive cells the multipotent progenitors had lower rates of apoptosis. Normally these multipotent progenitors have high apoptosis rates. This was prevented by high levels of Wnt4. We concluded that Wnt4 was a survival factor for bone marrow MPs, the cells responsible for feeding the thymus.
We also did gene profiling in bone marrow progenitor cells. We found that Wnt4 increased the expression of several genes that improve cell survival and decrease apoptosis. After that, we tried to understand how Wnt4 may affect the MPs and their progeny in the thymus. Wnt proteins can signal through two major pathways - canonical and non-canonical. The best known is the canonical pathway dependent on a protein called beta-catenin. Non-canonical is beta-catenin independent. We found that Wnt4 did not stabilize beta-catenin, so it did not signal through the canonical Wnt pathway. It worked through the non-canonical pathway and this pathway involved JNK, so Wnt 4 upregulated activation of JNK. After those Gain of Function studies (where we explored the consequences of the over-expression of Wnt 4), we then performed Loss of Function experiments.
We asked what would happen in mice that are Wnt 4 deficient? For that we had the help of a colleague in Finland [Seppo Vainio] who has generated Wnt4 knockout mice. Part of this phase of the study was complicated by the fact that Wnt4 is also important for kidney formation. So Wnt4 knockout mice lack kidneys and die within 24 hours. As a result, we could only study neonates that were Wnt4 knockout. We found that these neonates have thymic atrophy and had a decrease in MP in their bone marrow. We found that they had the mirror image of what we found in our Gain of Function experiment showing that not only Wnt4 is sufficient to increase the amount of bone marrow MPs and thymic cellularity, but it is also necessary in maintaining normal levels of T cells.
Listen to the audio clip at the end of the article to hear about how Dr. Perreault's research can be applied in potential therapies.
What is next for your laboratory in terms of research?
We are trying to understand where exactly Wnt4 has its effect. Is it in the bone marrow or in the thymus? As of today, our research indicates the thymus. But we want to understand exactly how Wnt4 does that.
(Reporting by Marc Landas )