Running head: AN INVESTIGATION INTO THE ORAL TOLERANCE 1 ! ! ! ! An Investigation Into the Oral Tolerance In a Type 1 Diabetes Mouse Model Kristen M. Cuadra University of Florida
AN INVESTIGATION INTO THE ORAL TOLERANCE 2 Abstract Type 1 Diabetes (T1D) affects millions of Americans and around 16% of those affected are under the age of 20. It is an autoimmune disorder in which the individual's own immune system attacks the insulin producing beta cells of the body. This study focuses on preventing the onset of T1D by modifying certain strain s of Lactococcus lactis to secrete either pro insulin, IL 10, or both pro insulin and IL 10. There are three different strains of bacteria that will be use d throughout the study and we are measuring the levels at which these strains secrete either pro insulin, IL 10, or both pro insulin and IL 10. It was hypothesized that the levels of secretion would increase oral tolerance and the results support the hypothesis. However, the levels of the L.lactis secreting either pro insulin or IL 10 were higher than the L.lactis engineered to secrete both pro insulin and IL 10. The levels of secretion in the gut by L.lactis can help prevent the onset of T1D by inducing tolerogenic dendritic cells, which play a role in helping create an immune tolerance.
AN INVESTIGATION INTO THE ORAL TOLERANCE 3 Introduction Type 1 diabetes (T1D) is an autoimmune disease in which an individual's immune system attacks its own beta cells in the islets of Langerhans a specific cluster of cells in the pancreas. These beta cells create insulin to be released by the pancreas so wit hout these cells, the body does not have a mechanism for producing insulin. Researchers have been experimenting with a modified bacterium known as L.lactis for a possible cure for T1D L.lactis can be modified to secrete pro insulin as well as IL 10 and wh en present in the gut, this bacterium increases the rate at which these two proteins are secre ted. In s tudies relevant to my research experimenters have proven that the engineered L. lactis is successful in NOD mice. IL 10 and pro insulin are both particularly important in the bo dy's regulation of glucose and vital in preventing the pathogenesis of T1D. One study found that "the presence of exogenous IL 10 in the gut induced IL 10 secretion by certain cells. There was also an increase in the measure ment of IL 10 titers in the plasma" (Frossard, Steidler, & Eigenmann, 2007). This conclusion supported the idea that modified L.lactis can effectively begin the process of immune tolerance. Aim and Hypothesis My research will be expanding on the effects of the treatment of modified L. lactis I have one main study aim. It is focusing on L.lactis secretion of proteins. As of right now, it is known that the bacterium can be modifie d to secrete insulin and IL 10. H owever, the levels at which the proteins of interest are secreted are still unknown so I will attempt to answer this question. We predict that the L lactis strains will successfully secrete levels of pro insulin and IL 10 that will initiate tolerance to prevent the onset of T1D. Overall, the goal of the therapy is to induce tolerogenic dendritic cells in the gastrointestinal tract because they play an important role in stimulating and sustaining immune tolerance (Li & Shi, 2014) This immune tolerance will help prevent and reverse type 1 diabetes. Methods
AN INVESTIGATION INTO THE ORAL TOLERANCE 4 Our delivery vector is a GRAS organism, Lactococcus lactis, that has been genetically modified to secrete pro insulin, IL 10, or pro insulin and IL 10 NOD mice were orally gavaged 5x/week with the various strains of L.lactis. To confirm that the strains were secreting the proper pr oteins, we performed ELISA for IL 10 and/or proinsulin for each strain. The bacteria was diluted 1/1000 in GM17T(E) media overnight and allowed to reach stationary phase (16 hours later). From that ov ernight culture, we diluted an aliquot of bacteria 1/25 in BM9T media and allowed them to grow and secrete for 3 hours at 30 Â¡C The bacteria were spun down and supernatants were collected for ELISA. 1. Mercodia Proinsulin (PINS) ELISA This ELISA required a blank of zero, a coated plate, enzyme conjugate made of mouse monoclonal antibodies, enzyme conjugate buffer, assay buffer, substrate TBM, wa sh buffer, and stop solution in order to be run. A plate map was created for the ELISA that c onsisted of a blank, 4 standard measurements, and 2 different strains of L.lactis that included one with pro insulin alone and one with both pro insulin and IL 10 Each strain had three dilutions that were included in the plate: neat, diluted 1:100, and diluted 1:1000. Also, every well within the plate map is duplicated to ensure accuracy. When beginning the ELISA, we first prepared the enzyme conjugate and wash buffer solution. Then 50 L of the appropriate controls and samples were pipetted into the appropriate place, according to the plate map. Assay buffer is added to each well and then the plate was incubate d on a plate shaker (700 900 rpm) for one hour at room temperature (18 25 Â¡C ). After the incubation period, the plate was washed 6 times with 700 L of wash buffer using an automatic plate washer. Then 100 L of the enzyme conjugate that was prepared earlier in the process was added to each well and the plate was incubate d on the plate s haker for one hour at the same rpm and temperature as before. The plate was washed using the automatic plate washer as described before, 200 L of substrate TMB
AN INVESTIGATION INTO THE ORAL TOLERANCE 5 was added to each well and then incubate d for 15 minutes on the bench at room temperature. Lastly, 50 L of stop solution was added to each well. a. Data Collection : The optical density was read at 450 nm and it was read using the spectrophotometer called SpectraMax M5. b. Data Analysis : The average absorbance for the duplicated wells was plotted on the standard curve and the diluted samples (1:100 and 1:1000) were multiplied by their respective dilution factors. The SoftMax Pro 7.0 was used to analyze the results. 2. Mouse IL 10 ELISA Th is ELISA required the same starting materials as the pro insulin ELISA, however, the steps and measurements are different. The plate map was created to have 8 standard measurements, including a blank of 0 and 2 different strains of L.lactis (one with IL 10 alone and one with both pro insulin and IL 10). These 2 st rains were placed into wells in three dilutions : neat, diluted 1:100, and diluted 1:1000. The wells were also duplicated in order to check for accuracy. In order to begin the proced ure, the wells need ed to be coated with the capture antibody (100 L per well) and left to incubate overnight at a temperature of 4 Â¡C The next day, t he plate was washed 3 times with the wash buffer, 200 L of assay diluent was placed in each well, and the plate was incubated for an hour. After the hour, the plate was washed 3 times again with the wash buffer and 100 L of each standard and sample was placed into the appropriate wells according to the plate map. The plate was s ealed and incubated for 2 hours at room temperature. After the 2 hours, it was washed again, but 5 washes instead of 3, and 100 L of working detector was added to each well before inc ubating again for 1 hour. The plate was wash ed again ( 7 washes ) 100 L of substrate solution was added to each well, and then incubate d for 30 minutes in the dark. Finally, 50 L of stop solution was added to each well.
AN INVESTIGATION INTO THE ORAL TOLERANCE 6 a. Data Collection : The optical density was read at 450 nm and it was read using the spectrophotometer called SpectraMax M5. b. Data Analysis : The average absorbance for the duplicated wells was plotted on the standard curve and the diluted samples (1:100 and 1:1000) were multiplied by their respective dilution factors. The SoftMax Pro 7.0 was used to analyze the results. Results We hypothesized that the strains of L.lactis bacteria will secrete IL 10 and pro insulin at the levels needed to induce tolerogenic dendritic cells and stimulate oral tolerance of type 1 diabetes. The hypothesis was proven to be correct and the results of the study are shown below In both graph 1 a nd graph 2, it was proven that solo secretions work more effectively than when both pro insulin and IL 10 are made to secrete together. In graph 1, L.lactis was modified to secrete pro insulin alone and secreted about .20 ng/mL more pro insulin than the L. lactis that was modified to secrete both pro insulin and IL 10. In graph 2, the L.lactis that was engineered to secrete IL 10 alone secreted about 25 ng/mL more IL 10 than the L.lactis that was engineered to secrete both pro insulin and IL 10. It is clear that L.lactis secreting IL 10 is more efficient than both the L.lactis secreting pro insulin and the L.lactis secreting both pro insulin and IL 10. Graph 1 : Pro insulin Secretio n Graph 2: IL 10 Secretion
AN INVESTIGATION INTO THE ORAL TOLERANCE 7 Discussion and Conclu sions Type 1 diabetes is an autoimmune disorder so the main goal of this study was to find a way to induce tolerogenic dendritic cells because these are the cells that help maintain immune tolerance. Overall, the results have proven that using L.lactis to secret e pro insulin and IL 10 can induce oral tolerance to dia b etic auto antigens in order to prevent people from developing type 1 diabetes. The L.lactis engineered to secrete either pro insulin or IL 10 was more effective than the L.lactis that secreted both pro insulin and IL 10. If this treatment were transferred to humans, an immunosuppressant would need to be considered in order to make sure that the individual's immune system would not negatively react to the treatments used to prevent the onset of type 1 diabetes. Future research should focus on mechanistic studies in order to determine the exact mechanisms of prevention because this knowledge will allow researchers to create a more specific target treatment.
AN INVESTIGATION INTO THE ORAL TOLERANCE 8 References Frossard, C. P., Steidler, L., & Eigenmann, P. A. (2007). Oral administration of an IL 10 secreting Lactococcus lactis strain prevents food induced IgE sensitization. Journal of Allergy and Clinical Immunology,119 (4), 952 959. doi:10.1016/j.jaci.2006.12.615 Robert, S., & Steidler, L. (2014). Recombinant Lactococcus lactis can make the difference in antigen specific immune tolerance induction, the Type 1 Diabetes case. Microbial Cell Factories, 13 (Suppl 1). doi:10.1186/1475 2859 13 s1 s11 Takiishi, T., Cook, D. P., Korf, H., Sebastiani, G., Mancarella, F., Cunha, J., . Mathieu, C. (2016). Reversal of Diabetes in NOD Mice by Clinical Grade Proinsulin and IL 10 Secreting Lactococcus lactis in Combination With Low Dose Anti CD3 D epends on the Induction of Foxp3 Positive T Cells. Diabetes, 66 (2), 448 459. doi:10.2337/db15 1625