Progress 12/01/12 to 11/30/15
Outputs
Target Audience:In spring of 2015, these data were presented to approximately 200 fellowundergraduates and faculty at the annual Northeast Regional Undergraduate Research Conference hosted by UMass Amherst. Three posters were presented by the undergraduate researchers conducting experiments supported by this Hatch grant. Changes/Problems:The major problem associated with this year's work stemmed from the loss of the Ndufa1S55Atransgenic mouse colony. Becouse of this unexpected event, we were unable to verify our in vitro rotenone studies with mice expressing a mitochondrial defect in Complex I. What opportunities for training and professional development has the project provided?This project has provided two semesters of training for two undergraduate students in the form of independent study research projects, two semesters of training for one undergraduate student in the form of an undergraduate honors research thesis project, and one graduate student in the form of mentoring and supervision undergraduate researchers. The following basic research skills were mastered: mouse necropsy, single-cell spleen preparation, CD4+ T cell isolation using magnetic beads, cell culture and Th1/Th2/Th17/iTreg polarization assays using cytokine and antibody cocktails, surface protein staining using fluorescently-tagged antibodies, intracellular protein staining using fluorescently-tagged antibodies, flow cytometric analysis, introduction to imaging flow cytometry, graphing and presenting scientific data at an annual Undergraduate Research Symposium in April, 2015, hosted by UMass Amherst. How have the results been disseminated to communities of interest?In spring of 2015, these data were presented to approximately 200 fellowundergraduates and faculty at the annual Northeast Regional Undergraduate Research Symposium hosted by UMass Amherst. Two posters were presented by the undergraduate researchers conducting experiments supported by this Hatch grant. The Role of Mitochondrial Energy Metabolism in Regulatory T Cell Differentiation and Function Adam Lerman1, E. Ilker Ozay2, Ankita Mitra1, and Lisa M. Minter1,2 1Department of Veterinary and Animal Sciences, 2Program in Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA, 01003 The Role of Mitochondrial Function on T-Cell Activation and Differentiation Victoria Mello1, Emrah Ilker Ozay2, Grace Trombley1, Adam Lerman1, and Lisa M. Minter1,2 1Deparment of Veterinary and Animal Sciences, 2Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, MA, 01003 What do you plan to do during the next reporting period to accomplish the goals?Although the funding period for this project has concluded, we will complete data and statistical analysis, and write and submit a manuscript detailing our findings before the end of the 2016 calendar year.
Impacts
What was accomplished under these goals?
In YEAR 3, we have accomplished the following towards completing AIM II: We have assessed the effects of rotenone on the in vitro differentiation of T helper type 1 (Th1), T helper type 2 (Th2) cells, T helper type 17 (th17) and induced regulatory T cells (iTregs) from wild type C57BL/6 mice, by conducting replicate experiments of at least three independent experiments for each condition of polarized cell tested (Th1, Th2, Th17, and iTreg) . Using single treatment of purified T cells under polarizing conditions in culture, we find that incubating T cells for 15 minutes with 25microM Rotenone prior to polarization, does not affect the ability of T cells to differentiate toward Th1, Th2, or Th17 development. Under these conditions, there was no effect on the expression of Notch1IC, compared to control-treated cells. Expression levels of the master transcriptional regulators that control T cell differentiation, T-bet (for Th1 cells), GATA3 (for Th2 cells), and RORgammat (for Th17 cells), were also unaffected compared to control treated cells, as assessed by intracellular staining and flow cytometry. However, we found that treating with 25microM Rotenone prior to polarization towards an iTreg phenotype, greatly diminished their capacity to adopt an iTreg fate. This was reflected in the decreased expression of the master transcriptional regulator, FoxP3 (for iTreg cells), as assessed by intracellular staining and flow cytometry. Interestingly, although the expression of Notch1IC remained unchanged in iTreg cells pretreated with 25mciroM Rotenone prior to polarization, we found that the intracellular localization of Notch1IC was greatly impacted. We used imaging flow cytometry to determine that in control iTreg cells, Notch1IC localized to the mitochondria. In stark contrast, in iTreg cells pretreated with 25microM Rotenone prior to polarization, Notch1IC was not associated with the mitochondria, but rather found entirely in the nucleus. Ndufa1S55A mice were not available for evaluation due to loss of the colony.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2015
Citation:
The Role of Mitochondrial Function on T Cell Activation and Differentiation
Victoria Mello1, Emrah Ilker Ozay2, and Lisa M. Minter1,2
1Department of Veterinary and Animal Sciences, 2Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, MA, 01003
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: We have had continued difficulty establishing a sufficiently large colony of Ndufa1 S55A mutant mice for parallel analysis of CD4+T cells cultured in vitro under Th1 and Th2 polarizing conditions. We are continuing to enlarge the colony to provide sufficient numbers of animals for characterization. What opportunities for training and professional development has the project provided? This project has provided two semesters of training for one undergraduate student in the form of an honors thesis research project. The following basic research skills were mastered: mouse necropsy, single-cell spleen preparation, CD4+ T cell isolation using magnetic beads, cell culture and Th1/Th2 polarization assays using cytokine and antibody cocktails, surface protein staining using fluorescently-tagged antibodies, intracellular protein staining using fluorescently-tagged antibodies, flow cytometric analysis, graphing and presenting scientific data. How have the results been disseminated to communities of interest? Results are not ready for dissemination at this point in the project. Replicates of data need to be obtained and statistical analyses applied. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we will complete evaluation of the effects of Rotenone on the in vitro differentiation of Th1 and Th2 cells, including assessing the effects of Rotenone treatment on the production of Th1- and Th2-specific cytokines. We will also evaluate the effects of Rotenone on the differentiation of Th 17 cells and regulatory T cells. If Ndufa1 S55A mice are ready for evaluation, we will repeat these experiments using purified CD4+T cell from Ndufa1 S55A mutant mice.
Impacts
What was accomplished under these goals?
In YEAR 2, we have accomplished the following towards completing AIM II. We have assessed the effects of rotenone on the in vitro differentiation of T helper type 1 (Th1) and T helper type 2 (Th2) cells from wild type C57BL/6 mice. Using both single treatment, and repeated treatments of purified T cells in culture, we find that Rotenone inhibits the differentiation of T cells toward Th1 and Th2 development. This includes the inhibition of Notch1 expression in Rotenone-treated cells. Rotenone-treatment also prevented expression of the master regulators of Th1 and Th2 differentiation, T-bet and GATA3, respectively, as assessed by intracellular staining and flow cytometry. We will use these data as baselines against which to compare the results of Ndufa1S55A mutant mouse T cells culture under the same conditions.
Publications
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Progress 12/01/12 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems: We have been unable to establish a breeding colony of Ndufa1S55A knock-in mice at UMass, due to technical difficulties that have prevented us from sucessfully genotyping the mice. To over come this obstacle, we have arranged to obtain spleens and thymi from Ndufa1S55A knock-in mice from our collaborator, Dr. Nagendra Yadava, who maintains these transgenic mice and their littermate controls at his institution. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? We plan to collect more data (replicates), both in vitro and in vivo to validate our preliminary findings. We will also repeat the experiments outlined above under TH1 and TH2 polarizing conditions to further explore the effects of Roteneone on T cell differentiation. Finally, we aim to provide an immunological challenge to Ndufa1S55A knock-in mice and their littermate controls to understand how chronic impairment of mitochondrial Complex 1 affects the abiilty of these mice to respond to such a challenge. Completing this last goal will be contingent on the availability of the mice needed, as breeding the mice is slow and sometimes difficult.
Impacts
What was accomplished under these goals?
In Year 1 we acomplished the following under AIM2: We have collected preliminary data on the following: 1) We have performed in vitro stimulation assays on wild-type T cells treated with increasing concentrations of Rotenone.We found that this reduces the expression of markers of T cell activation, specifically the high affininty subunit of the IL-2 receptor, CD25, and the expression of the signaling-competent form of the transmembrane recptor, NOTCH1. 2) We have assessed the percentages of cellular subsets in the spleen and thymus of 5 week old Ndufa1S55A knock-in mice. We found differences in the spleens, but not the thymi. B cells were increased, T cells were decreased, CD4 & CD8 T cells were decreased. 3) We assessed the the proliferation of CD4 and CD8 T cells from Ndufa1S55A knock-in mice upon stimulation with anti-CD3 & anti-CD28. We found that the proliferative potential was decreased following stimulation. 4) We assessed the cytokine production profiles of splecocytes and thymocytes from Ndufa1S55A knock-in mice. We found that the cytokine profiles of splenocytes from Ndufa1S55A knock-in mice are altered compared to littermate controls. 5) We assessed the expression of the signaling-competent form of the transmembrane recptor, NOTCH1, in splenocytes from Ndufa1S55A knock-in mice upon stimulation with anti-CD3 & anti-CD28. We found reduced upregulation of NOTCH1 complared to littermate controls. Conclusions to date: Rotenone treatment alters the activation potential of wild-type immune cells, including upregulated expression of NOTCH1, and these results are consistent with what is observed in immune cells from Ndufa1S55A knock-in mice upon stimulation with anti-CD3 & anti-CD28.
Publications
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