Shannon Percival-Smith 31199136 December 2, 2017 Biology 463 Annotated Bibliography Almada, A. E., & Wagers, A. J. (2016). Molecular Circuitry of Stem Cell Fate in Skeletal Muscle Regeneration, Ageing and Disease. Nature Reviews Molecular Cell Biology, 17(5), 267–279. https://doi.org/10.1038/nrm.2016.7. This review paper outlines our current understanding of satellite cells, which are the adult, muscle-resident stem cells responsible for the ability of skeletal muscle to heal itself. It lists local and systemic signals that satellite cells respond to in the context of ageing and in degenerative muscular diseases, like Duchenne muscular dystrophy (DMD). There is a table with all of these factors listed, which will be useful in the background section of my project. In DMD, what was interesting to me was that satellite cells go through symmetrical cell division, which is abnormal. In normal mice, satellite cells asymmetrically divide to produce daughter cells with differential p38 concentrations. Asymmetrical cell division is a characteristic of stem cells that allows them to produce one differentiating cell, and one cell that is retained as undifferentiated. High p38 concentrations make these cells competent to myogenic lineage commitment thus giving rise to myoblasts. Colombo, Mario P, and Silvia Piconese. 2007. “Regulatory T-Cell Inhibition versus Depletion: The Right Choice in Cancer Immunotherapy.” Nature Reviews Cancer 7 (November). Nature Publishing Group: 880. http://dx.doi.org/10.1038/nrc2250. This paper outlined different methods of depleting and inhibiting regulatory T cells in mice. Some methods were genetic, and others were injections of antibodies that attach to cell-surface markers and cause the death of the cells. There were many different markers that can be used for this, and multiple antibodies for each marker. This was useful to my project because it gave me a feel for the different methods I could approach my question with. Originally, I was going to do an experiment that decreased the number of regulatory T cells in mice, but decided instead to test a mechanism by which they aid in regeneration. So, this paper is not immediately helpful for the Shannon Percival-Smith 31199136 December 2, 2017 current iteration of my final project, but it was useful for designing my original experiment. It also was a good starting point for thinking about possible mechanisms. Castiglioni, A., Corna, G., Rigamonti, E., Basso, V., Vezzoli, M., Monno, A., … RovereQuerini, P. (2015). FOXP3+ T cells recruited to sites of sterile skeletal muscle injury regulate the fate of satellite cells and guide effective tissue regeneration. PLoS ONE, 10(6), 1–18. https://doi.org/10.1371/journal.pone.0128094 This paper showed that depleting regulatory T cells impairs regeneration by decreased satellite cell expansion. They showed by in vitro co-culture experiments of satellite cells and FOX3+ T cells that satellite cells became active and were still able to fuse with one another (an important step in myogenesis). This indicated that Tregs are positive regulators of regeneration. The authors did not test any mechanisms to see how they did this, and assumed that Tregs have a direct impact on satellite cells to guide effective regeneration. This paper was important because it helped me form my research question and design an in vivo experiment to rule out the direct impact of Tregs on satellite cells through IL-10 signalling. The paper states that satellite cells express low to no IL-10R, so knocking this gene out in satellite cells will ensure no IL-10 signalling can occur between Tregs and satellite cells in those mice. Burzyn, D., Kuswanto, W., Kolodin, D., Shadrach, J. L., Cerletti, M., Jang, Y., … Mathis, D. (2013). A Special Population of Regulatory T Cells Potentiates Muscle Repair. Cell, 155(6): 1282-1295. https://doi.org/10.1016/j.cell.2013.10.054 This paper (among other experiments) included a characterization of the differences in gene expression of regulatory T cells from spleen and from damaged muscle. This is where I got the idea for my project to test the IL-10 knockout in Tregs, because Il10 has about 8-fold higher expression in muscle Tregs relative to spleen Tregs. I thought that it would be highly unlikely that this massive difference was due solely to chance. That being said, I cannot know for sure if muscle Tregs upregulate Il10, or if spleen Tregs downregulate Il10 because it was observational Shannon Percival-Smith 31199136 December 2, 2017 data. For my proposal, I chose to knockout IL-10 in regulatory T cells because IL-10 has previously been implicated as an anti-inflammatory, pro-regenerative cytokine, and it is one of the major secretions of Tregs. Together, I took this as evidence that IL-10 is important during the regenerative phase of muscle damage (around 10-14 days after acute damage). I hypothesize that IL-10 knocked out from Tregs is sufficient to cause impaired regeneration, and that this is not due to IL-10 signalling between Tregs and satellite cells. This paper helped me form my hypothesis. Lemos, Dario R, Farshad Babaeijandaghi, Marcela Low, Chih-Kai Chang, Sunny T Lee, Daniela Fiore, Regan-Heng Zhang, Anuradha Natarajan, Sergei a Nedospasov, and Fabio M V Rossi. 2015. “Nilotinib Reduces Muscle Fibrosis in Chronic Muscle Injury by Promoting TNF-Mediated Apoptosis of Fibro/adipogenic Progenitors.” Nature Medicine, 21 (7): 786– 94. doi:10.1038/nm.3869. This paper was done by the lab I am doing my honours thesis in. They characterized the kinetics of fibro/adipogenic progenitors (FAPs) during muscle damage, and found that FAP numbers peak about 3 days after damage, and then return to predamaged levels over the following 5 days. They also showed that FAP numbers stay high when regeneration is impaired, and if left long enough, FAPs differentiate into fibroblasts and adipocytes and cause fibrosis. They were able to use this data in future work (unpublished) to make inferences about which signals likely came from certain cell populations, based on correlations between numbers of cell populations and amount of cell signals. This has been helpful because it has taught me that the way you frame or phrase a question in the context of your research can have a big effect on the data that you end up collecting and the way you can answer your question. Shannon Percival-Smith 31199136 December 2, 2017 This paper also had a detailed methods section. They outline the important steps in many of the same techniques I used in my proposal, so it was a great reference with respect to that. It helped me design my inducible Cre/lox Mdx mice for my experiments. They also found: • By creating a Ccr2-/- mouse (a required receptor for macrophage infiltration), they showed that blood macrophages are required for the apoptosis of fibro/adipogenic progenitors. They determined this by damaging muscle with tiger snake venom (notexin) and using flow cytometry to measure FAP numbers at various time points after damage in Ccr2-/- mice and C57BL/6 mice (wild type). • That culturing FAPs in vitro and treating them with common macrophage secretions, including TNF-alpha, TGF-B1, and IL-6, result in FAP apoptosis in only the TNF-alpha treatment group. • They determined that anti-TNF treatment resulted in FAP persistence and increased collagen deposition, which is a hallmark of fibrosis.
