Douglas Baumann and Dr. Brian Poole, Department of Microbiology and Molecular Biology
Systemic Lupus Erythematosus (Lupus) is an autoimmune disease that afflicts over 250,000 citizens of the United States. Although much remains to be learned about Lupus, the information gathered so far has proven to be extremely valuable in increasing survival rates and improving the quality of life of Lupus patients. As we continue to put time and energy into Lupus research, the result will be that we will be able to combat the disease more effectively. The first step in combating Lupus is to gain knowledge about the pathogenesis and mechanisms of the disease. By further developing our knowledge, we will be able to develop new ways to prevent and treat, or lessen the symptoms of, Lupus.
Advances in biology have enabled a more perfect understanding of some of the important cell signaling pathways related to Lupus. In the last two decades, Toll-like receptors (TLRs) have become key suspects for contributing to the development of autoimmunity. They are proteins that play a key role in the innate immune system. TLRs detect particular components or characteristics of invading microbes or pathogens and initiate signaling pathways that activate immune cell responses. TLR-7 recognizes single-stranded RNA; single-stranded RNA is a characteristic of many viral genomes. TLR-7 has been shown to play an important, yet somewhat poorly understood role in the development of Lupus. Numerous recent experiments give substantial weight to the argument that TLR-7 is a good therapeutic target for treatment of Lupus, yet still more needs to be understood about how TLR-7 contributes to Lupus.
The gene Interferon regulatory factor 5 (IRF-5) plays a vital role in the expression of type I interferon genes. IRF-5 acts as a direct transducer of virus-mediated signaling. IRF-5 is activated by TLR-7 signaling and is a key mediator in the TLR-7 signaling pathway. IRF-5 is of particular interest to experts studying Lupus because some particular single nucleotide polymorphisms (SNPs) of IRF-5 have been linked to Lupus(1). The particular IRF-5 rs2004640 T SNP has been linked to an increased likelihood of Lupus development(2) and is the primary focus of this research.
The purpose of this research was to determine whether the TLR-7 signaling pathway is overactive in individuals who are at genetic risk of developing Lupus. We hypothesized that TLR-7 signaling activity would be overactive in individuals at genetic risk compared to individuals who were not at increased risk. For the purpose of this study, a person at genetic risk of developing Lupus is one who is homozygous for the aforementioned single nucleotide polymorphism, or mutation, in the IRF-5 gene.
To test the hypothesis we obtained blood samples from volunteers and infected the B cells with Epstein – Barr virus (EBV). This process results in the formation of immortal cell lines known as Lymphoblastoid cell lines. Cell lines from risk individuals were paired up with cell lines from protective individuals. The paired cell lines were treated and incubated for 24 hours with varying concentrations of imiquimod, a TLR-7 ligand or activator. After the 24 hour incubation period, the supernatants from the cells were pulled off and the IL-6 levels in the supernatants were measured by an ELISA. IL-6 levels were measured because IL-6 is a cytokine that is produced as a result of TLR-7 signaling, thus IL-6 levels in the supernatant should reflect the signaling activity of TLR-7.
Figure 1 represents the IL-6 levels in risk cell lines compared to protective lines. Although the data didn’t produce a consistent trend, we did see that IL-6 levels in protective lines are almost always higher than IL-6 levels in risk lines at the 1:400 imiquimod dilution ratio. The data at the 1:400 dilution ratio even approached statistical significance. Figure 2 shows the average concentration of IL-6 in risk cell lines vs. protective cell lines. The data shows that IL-6 levels do increase sharply when treated with imiquimod but decrease slowly as the amount of imiquimod used decreases. Contrary to what we expected, we saw that protective lines generally produced more IL-6 than risk lines. This unexpected data will force us to rethink and reformulate our hypothesis.
I ran into a few difficulties during the course of this research project. The greatest difficulty was in working with the immortalized cell lines. Cell line care and maintenance is always a difficult task that requires skill, diligence, and some luck. Although most of the cell lines I had planned on treating were not a problem, there were a few that proved to be extremely difficult to prepare for treatment. Overall the project went as planned without any major obstacles.
Although I am somewhat disappointed that the research has not yet yielded any statistically significant data, I am pleased with the fact that in doing this research we have gained some knowledge about the mechanisms of Lupus development. I learned more fully to appreciate that science is a process of making good assumptions and testing those assumptions. The experience of performing this research and giving an accounting of my efforts has been immensely rewarding and has helped me gain greater appreciation for the value of diligent work and continuous improvement. I feel fortunate to have been able to take part in the ORCA grant program.
References
- Sigurdsson, S., Nordmark, G., Goring, H. H. H., Lindroos, K., Wiman, A. C., Sturfelt, G., Jonsen, A., Rantapaa-Dahlqvist, S., Moller, B., Kere, J., Koskenmies, S., Widen, E., Eloranta, M. L., Julkunen, H., Kristjansdottir, H., Steinsson, K., Alm, G., Ronnblom, L., and Syvanen, A. C. (2005) Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus, Am. J. Hum. Genet. 76, 528-537.
- Graham, R. R., Kozyrev, S. V., Baechler, E. C., Reddy, M. P. L., Plenge, R. M., Bauer, J. W., Ortmann, W. A., Koeuth, T., Escribano, M. F., Pons-Estel, B., Petri, M., Daly, M., Gregersen, P. K., Martin, J., Altshuler, D., Behrens, T. W., Alarcon-Riquelme, M. E., and Argentine Collaborative Grp; Spanish Collaborative, G. (2006) A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus, Nature Genet. 38, 550-555.