How the Dysfunction of Tregs is influencing the development of autoimmunity

Based on the study by Sumida et al. (2018): Activated β-catenin in Foxp3+ regulatory T cells links inflammatory environments to autoimmunity

            The immune system is a bodily system comprised of different cell types that respond to dangerous foreign material, called pathogens, that enter our body. The immune system is the essential way through which humans are able to survive in an environment with many pathogens. For a variety of reasons, the immune system can sometimes dysfunction and cause harm to the body instead of protecting it-this is called autoimmunity. One cell type that is part of the normal immune system is a regulatory T cell (Treg), which helps to suppress activity of other immune cells, thus preventing an overactivation of the immune system in the absence of danger signals. In a normally functioning body, Tregs secrete cytokines, which are molecules produced by a cell that travel throughout the body to cause other cell types to act or stop acting in certain ways. Different types of cytokines are produced by different cells in the immune system; Tregs normally produce large amounts of interleukin 10, (IL-10) a cytokine that suppresses the immune system. During autoimmunity, instead of secreting large amounts of IL-10, Tregs secrete higher amounts of interferon-gamma (IFNγ) which acts to increase the immune response through inflammation.^{1, 2} Multiple sclerosis (MS) is among the identified autoimmune diseases that displays this dysregulation of Tregs and the disease can be triggered by a combination of genetic and environmental factors. One identified environmental factor that increases risk and severity of MS, a high-salt diet (HSD), was explored further by Sumida et al. (2018) to determine its effects on Treg dysregulation.

           Sumida et al. (2018) were specifically observing the secretion of cytokines in memory

Treg cells (mTregs). Memory immune cells are cells that have been activated in the past and remain in different parts of the body to respond faster to the same type of pathogen. It was found that patients with MS express mTregs with a higher IFNγ to IL-10 secretion ratio than in healthy patients. This identification makes the results of this study important as a potential area of research for treating MS patients. The researchers went on to determine that mTregs cultured for 96 hours in a high salt environment differentially increased their expression of IFNγ and decreased the expression of IL-10. It is important to note here that the concentration of NaCl used in the cell culture is not as physiological levels. The research team added 40 mmol salt in the NaCl cell culture, which is not equivalent to the increase in salt in a high-salt diet of a human.^{3, 4} This means that conditions used in the experiment may not mirror conditions in the MS patients and may give false significance. In future experiments, the research team should re-evaluate the effects of HSD on the dysregulation of Tregs with more equivalent concentrations of NaCl. The researchers continued with these concentrations (40 mmol) for cell culture throughout the rest of the paper.

Figure 1: mRNA expression of IFNγ and IL-10 in a high salt environment after 96 hours. IFNγ increases and IL-10 decreases, leading to a pro-inflammatory environment.

         Moving forward, the team went on to identify what genes were dysregulated in Treg
cells that caused them to produce more IFNγ instead of IL-10. They found that CTNNB1, the gene for β-catenin, was one of the genes that was most highly increased in Tregs that produced more IFNγ and IL-10 than Tregs that were not producing either. The researchers continued manipulations with CTNNB1 in order to determine if this was the main factor that leads Tregs to become inflammatory rather than immunosuppressive. They conducted more tests with Tregs and found that expression of β-catenin was highest in IFNγ producing Tregs than in any other Treg population. When β-catenin was blocked using PKF (an inhibitor), significantly less IFNγ was produced by Tregs. Under the same PKF conditions, Tregs also produced less IL-10, but had a smaller decrease than was seen in IFNγ production. This is extremely important for future directions because, in the case of autoimmune diseases, it is essential to decrease the amount of IFNγ being produced by Tregs without decreasing the production of IL-10. The smaller impact that PKF had on IL-10 levels is important, but this is still not an ideal treatment because because it decreased IL-10 production. It would be better to develop a treatment that does not reduce IL-10 at all.

            Up until this point, all of the experiments that were conducted were in cell culture. Next, the researchers moved to conducting research in mice. The researchers genetically modified mice to continuously express β-catenin using a method called Cre/lox. Mice that continuously expressed β-catenin showed an increase in inflammation throughout all portions of their body. The researchers found that the physical changes in the genetically modified mice were similar to changes in mice with Treg-specific Foxo1 depletion, which have previously been used as models of autoimmunity.⁵ Taking this observation into consideration, the research team identified that there were higher levels of Foxo1 and Foxo3a phosphorylation (activation) in the modified mice. This led to an increase in immune cell types that are activated by IFNγ and a decrease in Tregs, which are activated by IL-10. Immune cells activated by IFNγ include CD8⁺ cells, which attack and destroy cells that might be infected with pathogens. This leads to inflammation and, without Tregs, CD8⁺ cells continue to destroy healthy cells in the body. This continued inflammation is a hallmark of autoimmunity.

Figure 2: Phenotypic changes in mice continually expressing β-catenin which resemble mice that are deficient in Foxo1, a common murine model of autoinflammatory diseases. 

            Finally, the research team identified the protein PTGER2 as being increased in high salt IFNγ producing Tregs, but not in IL-10 producing Tregs. They observed that deletion of PTGER2 resulted in reduced β-catenin activation and thus, decreased IFNγ production, but not decreased IL-10 production. Additionally, reduced β-catenin led to decreased expression of PTGER2. This suggests that PTGER2 and β-catenin act to reinforce each other in high salt environments which may prolong inflammation. This was the most important finding from the team’s research since PTGER2 can directly be targeted to decrease the amount of IFNγ without reducing IL-10.

            Sumida et al. (2018) developed a specific mechanism by which IFNγ and IL-10 become imbalanced in Tregs in a high salt environment. It is very important that they characterized a pathway

and β-catenin/PTGER2 feed-forward loop because this allows for multiple different parts of the pathway that can be therapeutically targeted to reduce inflammation in autoimmune diseases. In future experiments, Sumida et al. and other research teams can focus on inhibiting PTGER2 rather than generally targeting β-catenin, as β-catenin was proven to also be important in the creation of IL-10, an immunosuppressive cytokine. Additionally, future research needs to be conducted on the PTGER2/β-catenin pathway to determine if it is dysregulated by other environmental factors that are associated with increased risk or severity of autoimmune diseases. It is one accomplishment to identify and target this pathway in patients with high salt diets, but if PTGER2 can be artificially downregulated in other patient populations to produce the same effects, this would be monumental in the management of autoimmune diseases.

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Paper blog post is based upon

Sumida, T., Lincoln, M. R., Ukeje, C. M., Rodriguez, D. M., Akazawa, H., Noda, T., . . . Hafler,

D. A. (2018). Activated β-catenin in Foxp3 regulatory T cells links inflammatory

environments to autoimmunity. Nature Immunology,19(12), 1391-1402.

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