Imagine a great dane standing next to a Chihuahua. With the Great Dane weighing almost twenty times as much as the Chihuahua, it’s impressive that these two animals are members of the same species, Canis familiaris. In addition to size differences, there is an inverse relationship between breed size and longevity.1 The genetic basis behind the wide variation in sizes has been a prominent subject of research, and in recent years, several specific regions in the canine genome have been found to influence size.2 In 2008, a genetic analysis of large and small breed dogs discovered a difference in the promoter region of the gene for a small protein, insulin-like growth factor 1 (IGF1), which is produced in the liver and circulates throughout the body. The promoter region of a gene helps regulate when the gene is transcribed into a protein. The change in the promoter region of IGF1 means less of the protein is produced and consequently, smaller breeds have lower levels of IGF1 in their blood.3 Cells with a receptor protein, insulin-like growth factor 1 receptor (IGF1R), in their membranes can respond to IGF1. When IGF1 binds to IGF1R, a cascade of events occurs within the cell leading to cell growth, proliferation, and differentiation. The interaction of IGF1 and IGF1R is especially important for the growth and development of an organism.4 As IGF1 has been studied more, it has become apparent that IGF1 plays a role beyond growth, specifically in anti-inflammatory immune pathways.5 The goal of my summer research is to investigate if the canine immune system responds to IGF1 with the hypothesis that IGF1 could play a role in health differences between breeds.
This summer, I have been working in the lab of Dr. Jennifer Punt and Dr. John Wagner at the University of Pennsylvania. Jenni is currently Dean of One Health at the University of Pennsylvania School of Veterinary Medicine, as well as Haverford’s Pre-Vet advisor. Additionally, Jenni was previously a biology professor at Haverford. As an aspiring student of veterinary medicine, I’ve been in contact with Jenni since I arrived on Haverford’s campus. Interested in gaining an experience with a veterinarian different from those I’ve had with general clinical practice, I asked Jenni if I could work in her lab for the summer, and she said yes! I then applied for, and received, the KINSC Summer Scholar’s Scholarship to fund my research. In the lab, I’m working with three Penn undergrads, a second year Penn Vet student, and two high school students. It’s been a great experience involving teamwork, communication, and research.
The majority of my work has been focused on using a technique called flow cytometry to investigate our hypothesis that there are canine immune cells that can respond to IGF1. The easiest way to tell if a cell can respond to IGF1 is to see if the cell has the receptor, IGF1R. Luckily, there is a well-established technique for identifying proteins, like IGF1R, on cells. It involves capitalizing on another type of protein that is naturally produced as part of the immune system – an antibody. Antibodies have two regions to them – an Fc region and a Fab region. The Fc region is constant between many different antibodies, whereas the Fab region is variable and binds to a specific target called an antigen. This is a property of antibodies taken advantage of by scientists. Essentially, we can manufacture antibodies that bind specifically to any protein of interest. Since cells and antibodies are miniscule and cannot be readily visualized, antibodies of interest can be made such that they are conjugated with a small molecule that fluoresces: a fluorophore.
This is where flow cytometry comes in. A flow cytometer uses fluidics and optics to analyze cells one-by-one. Through a system of lasers, the machine can determine the volume of the cell (forward scatter), the internal complexity of the cell (side scatter), and whatever fluorophores are attached to its surface. By incubating immune cells with various fluorophore-conjugated antibodies, we can begin to characterize the cells that can respond to IGF1.
So where do we find these immune cells? The immune system is partly composed of white blood cells, found, as expected, in the blood (among other places). The diagram below illustrates the basic pathway for how blood cells, including white blood cells, are made.
For our project, we received canine blood samples from Penn Vet’s Veterinary Clinical Investigations Center (VCIC). Since the blood contains more than just white blood cells, we had to get rid of some of the extra material. One way to do this is to destroy, or lyse, the red blood cells with ammonium chloride. We can also spin the blood through a density gradient. The hypodense layer (above the gradient) is called the peripheral blood mononuclear cell (PBMC) fraction and contains mostly monocytes and lymphocytes (T and B cells).
As I mentioned previously, existing research indicates a connection between IGF1 and anti-inflammatory pathways, specifically pathways that suppress the immune system. Within many types of immune cells, there have been subsets identified whose main functions appear to be suppressive. The ones my project has focused on are polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs), a subset of neutrophils; monocytic myeloid derived suppressor cells (M-MDSCs), a subset of monocytes; and T regulatory cells (T regs), a subset of T cells. Luckily, all three of these cell types are found in the PBMC fraction of blood.
To determine which, if any, of these might respond to IGF1, we stained them for characteristic surface markers in addition to IGF1R. The list of antibodies we used is below. Due to the limitations of our flow machine, we were only able to stain with four at a time, but we were innovative in the combinations we used.
Work done in the lab last summer yielded some promising results that suggested the vast majority of PMN-MDSCs might express IGF1R. PMN-MDSCs are generally defined as being low density (PBMC) fraction and CADO48A+ (an antigen present on canine neutrophils.)
This figure, made by Trevor Esilu ‘21 who worked in the lab last summer, looks at the CADO48A+ population, labeled as neutrophils, and highlights their expression of IGF1R (gray) versus the whole sample (black). In whole blood, only about 2-3% of neutrophils express IGF1R. In contrast, 87% of the CADO48A+ cells (which are PMN-MDSCs) in the low density fraction express IGF1R. This result was very exciting!
At the beginning of this summer, we conducted an experiment that is a basic control for any antibody staining. Many cell types, especially neutrophils, have receptors on their surface that can bind to the Fc region of an antibody. If this happens, the cell will still be “positive” according to the flow cytometry, even though the antibody has not actually bound to the correct target.
In order to prevent this from happening, the sample has to be flooded with generic antibodies that do not fluoresce. This can be done by adding serum to the sample. Serum is blood without any cells, but it contains a wide variety of particles including hormones, lipids, cholesterol, sugars, proteins, and most important, antibodies. Generally, whole blood samples already contain serum, but PBMCs, due to the nature of their isolation, do not. One of the first things I did was conduct an experiment to see if the addition of serum changes the expression profile of IGF1R. The results from that experiment are below.
In both samples, whole blood and PBMC, without serum there is significant expression of IGF1R. Once the serum is added, however, this positive reading almost disappears. This was a disappointing way to start out my project, but we recognized that there was still some expression of IGF1R, we just still had to determine exactly where.
We still had some more suppressor populations to investigate. We decided to move onto T regs. To investigate if T regs expressed IGF1R, we stained with for IGF1R along with three characteristics of T regs: CD4, CD5, and Foxp3. Some of those results are below.
These graphs show the IGF1R expression in CD5+ CD4+ cells that are either Foxp3+ (left) or Foxp3– (right). As you can see, there is a population of the T regs (CD5+ CD4+ Foxp3+) cells that do express IGF1R. Additionally, the proportion of IGF1R+ cells is higher in the Foxp3+ cells than the Foxp3– cells. While these results clearly show expression of IGF1R in some T regs, some of our other samples indicated no expression of IGF1R in T regs. While the lack of reproducibility is disappointing, these results are very interesting and provide direction for further investigation.
While my part in the project will likely end this summer, our work will be continued by a Superlab class at Penn in the fall. Hopefully they can use our results to continue investigating IGF1R expression in T regs by testing more samples and varying ages, sizes, and disease states. Other future directions of the project include investigating potential IGF1R expression in M-MDSCs and possible stimuli that can induce IGF1R expression. I look forward to seeing which directions the project takes!
– Johanna ’21
(1) Galis, F., Sluijs, I. V. D., Dooren, T. J. M. V., Metz, J. A. J., and Nussbaumer, M. (2007, March 15) Do large dogs die young? J. Exp. Zoolog. B Mol. Dev. Evol.
(2) Boyko, A. R., Quignon, P., Li, L., Schoenebeck, J. J., Degenhardt, J. D., Lohmueller, K. E., Zhao, K., Brisbin, A., Parker, H. G., vonHoldt, B. M., Cargill, M., Auton, A., Reynolds, A., Elkahloun, A. G., Castelhano, M., Mosher, D. S., Sutter, N. B., Johnson, G. S., Novembre, J., Hubisz, M. J., Siepel, A., Wayne, R. K., Bustamante, C. D., and Ostrander, E. A. (2010) A Simple Genetic Architecture Underlies Morphological Variation in Dogs. PLOS Biol. 8, e1000451.
(3) Sutter, N. B., Bustamante, C. D., Chase, K., Gray, M. M., Zhao, K., Zhu, L., Padhukasahasram, B., Karlins, E., Davis, S., Jones, P. G., Quignon, P., Johnson, G. S., Parker, H. G., Fretwell, N., Mosher, D. S., Lawler, D. F., Satyaraj, E., Nordborg, M., Lark, K. G., Wayne, R. K., and Ostrander, E. A. (2007) A Single IGF1 Allele Is a Major Determinant of Small Size in Dogs. Science 316, 112–115.
(4) Laron, Z. (2001) Insulin-like growth factor 1 (IGF-1): a growth hormone. Mol. Pathol. 54, 311–316.
(5) Smith, T. J. (2010) Insulin-Like Growth Factor-I Regulation of Immune Function: A Potential Therapeutic Target in Autoimmune Diseases? Pharmacol. Rev. 62, 199–236.