Author Archives: Yaneli Guerra Hernandez

Yaneli Guerra 

Mentor: Derek Peters, M.D., Ph.D

PI: Yarui Diao

Cellular response mediates the production of macrophages as early infiltrates following muscle injury. M1 inflammatory type macrophages react initially, followed by regenerative M2 macrophages which reduce inflammation, breakdown cell debris, and promote healing. The change in macrophage phenotype appears to be mediated by the TREM 2 gene which induces the M2 macrophage. A delay in the transition impairs myogenesis, demonstrating the importance of this precisely timed phenotypic switch. My project attempts to analyze the effect of the TREM 2 gene on macrophage polarization and the role it plays in regeneration. After knocking out the TREM2 gene in mice models and collecting muscle tissue samples, we perform a qrt-pcr using known marker genes of the M1/M2 macrophages. Levels of gene expression in the samples allow us to better understand the turnover (up/down regulation) of the macrophages and whether or not that is a consequence of the TREM2 knockout. Additionally, we perform H&E and immunostaining on muscle tissue to better visualize regenerating fibers as well as detect embryonic myosin, a known marker of regeneration. By better understanding the process of macrophage polarization, we can potentially induce gene expression of TREM2 to drive regeneration and develop new therapeutic strategies. 

My project in the lab is to analyze the effect of the TREM 2 gene on macrophage polarization; the hypothesis being that TREM2 upregulates macrophage polarization and positively drives regeneration. The procedures I perform to accomplish the further analysis of TREM2 varies day by day. The entire process begins with the knockout of the TREM2 gene in mice, which we will contrast to wild type (normal) mice. To knockout the gene successfully involves a series of specific breeding amongst the mice which is carefully orchestrated through conscientious genotyping. After that step is accomplished, we induce an injury to the two mice to test the different levels of regeneration. We perform what is known as a Hind Limb Ischemia (HLI) surgery on the mice which involves ligating the femoral artery in the hind limb of the mice to prevent adequate blood flow. Subsequently, we collect the muscle tissue from this damaged area and dissociate the tissue into single cells. Those cells are then further analyzed and sorted by being run through a FACs sorting machine. FACs allow us to categorize specific cell populations based on phenotypes and to better understand the characteristics of a single cell population. By this method, we separate and collect the macrophages present in the sample. After collecting, we then isolate the RNA in order to better gauge which genes are being expressed and their relative abundance through qrt-PCR. We look for specific M1 and M2 markers in order to better understand the turnover of the macrophages and whether or not one is being expressed more or less as a consequence of the TREM2 knockout.

As an alternative method of analysis, we perform staining on the muscle tissue samples themselves. One particular stain, H&E, involves performing a cross section of the tissue and fixing the sample in solution in order to get a clear visual. Through this, we can clearly see which fibers in the samples are effectively regenerating and which are not. To further verify, we perform immunostaining, which involves the staining of a specific marker of regeneration (if there is increased staining, that means there is increased regeneration in the respective sample). 

A smaller project we are currently working on involves culturing the cells we obtain from the muscle tissue samples into Bone Marrow Derived Macrophages (BMDM). We take BMDMs from each muscle tissue, the wildtype and the TREM2 KO, and induce the polarization of specific macrophages. We accomplish this induction by subjecting the cells to specific mediums such as IL-4/LPS that are known to cause polarization of specific macrophages. We then hope to perform a similar experiment as before in which we isolate the RNA and perform a qrt-PCR to see, once again, if the turnover of macrophages is affected in the different samples. 

My experience thus far has been a blast! Thanks for reading!

In undergrad, Diao studied at the University of Nanjing, China majoring in biotechnology. Cultural values drove him to pursue a career path as a scientist, rather than attending medical school (a contrasting priority to that of the US). Funnily enough, Diao stated his drive for biology in particular as a source of his struggles with physics and mathematics. His passion was then further fueled by a research program he entered in his junior year, working in the lab with PhD students. Later on when considering grad school as his next career step, he asked his PI about the process and considered programs close to home and thus he began his PhD in Biochemistry and Molecular Biology at Hong Kong University of Science and Technology. He focused his thesis on attempting to identify the proteins that impact the PAX7 gene. As background, proteins can function as an adapter protein to connect PAX7 with histone modification, which subsequently changes transcription and expression.  He discovered previously unstudied proteins and played a role in identifying the correct sequence mechanism. He was immediately blown away by his discovery and the implications behind it and became interested in gene regulation and epigenetics. When wrapping up his PhD, Diao applied to the Human Frontier Science Program (HFSP) fellowship, which centered around helping international students continue their studies in the US. As part of the program, scholars did have to enter a new lab and explore an alternative project to the research they had done thus far. He then began his post doc at the University of California, San Diego entering Dr. Bing Ren’s lab.  There he developed the first genetic screening method to study enhancer functions and was involved in a project to profile gene promoters and 3D genome organization. 

In his personal life, moving to a new country was a stressful transition. He arrived in San Diego with one luggage case and spent 2 months away from his family, particularly from his 4 year old daughter, who at the time, was still in Hong Kong. While the project was exciting, being in a new setting and in a new lab was incredibly difficult, especially given the language barrier. Communication with mentors and lab mates proved challenging, however the environment in the lab was quite welcoming; a factor which proved precedent when deciding on a faculty job here at Duke. Having faced some discrimination in the California as a person of color, he felt motivated to explore other states that would bring new experiences. Additionally, he felt the staff at Duke enjoyed their respective research projects and valued science on a similar wavelength. He continues to enjoy his research and his current lab centers on regeneration and genomics. 

While Diao has certainly progressed his career and now holds a high faculty position, he noted the many mistakes he made to get to where he is now. As a grad student that often included being lost in scientific conversations while still training and funnily enough, falling asleep during lab meetings and loudly snoring.

The Diao Lab primarily focuses on regeneration and genomics. My specific project revolves around the polarization of macrophages and induction on behalf of the TREM2 gene. 

Background: As a response to muscle injury, the immediate cellular response is the production of immune cells, particularly macrophages as early infiltrates. M1 type macrophages react initially, serving as sources of inflammation through the presence of pro-inflammatory cytokines, such as TNFα, IL-1, IL-6, IL-12, etc. Throughout healing, M1 type macrophages are replaced with M2 phenotype macrophages typically around the Day 3 mark. The M2 phenotype macrophages possess a more regenerative aspect and reduce levels of inflammation through production of anti-inflammatory cytokines such as IL-4 and IL-10. Additionally, they stimulate the breakdown of debris (damaged tissue) to promote healing. However, the change in phenotype of the macrophages appears to be mediated by the TREM 2 gene which induces the M2 macrophage. “If this transition is delayed and the M1 phenotype is prolonged, inflammatory cytokines persist and myogenesis is impaired, demonstrating the importance of this precisely timed phenotypic switch. Once M2 macrophages are present, they mark the beginning of the regenerative phase” (1).

The purpose of our project is to analyze the effect of the TREM 2 gene on macrophage polarization. By knocking out the TREM2 gene and measuring levels of respective macrophages, correlations can be stipulated as to whether or not the TREM2 increases levels of efferocytosis and phagocytosis or plays a role in inflammation. Based on the articles I have read thus far, there is an indication that the TREM 2 gene drives macrophage polarization which allows the M2 macrophage to begin its process of healing. In the lab, we have knocked out the TREM2 gene in mice and are analyzing the cells collected from animal muscle tissue. Once the macrophages are separated, they are run through FACs sorting, which allows us to categorize specific cell populations based on phenotypes and to better understand the characteristics of a single cell population. We then isolate the RNA in order to better gauge which genes are being expressed and their relative abundance through qrt-PCR. We look for specific M1 and M2 markers in order to better understand the turnover of the macrophages and whether or not one is being expressed more or less as a consequence of the TREM2 knockout. We have already thus far seen induced ischemia on the mice in which TREM2 is knocked out, which is an indication of the progression of peripheral arterial disease (revealing the regenerative aspects of TREM2). Further analysis needs to be completed, yet on a larger scale, exploring the process of macrophage polarization not only better aids our understanding of the mechanism, but can provide insights into new strategies for approaching and amplifying treatment targeting various diseases. 

1. TA;, Wosczyna MN;Rando. “A Muscle Stem Cell Support Group: Coordinated Cellular Responses in Muscle Regeneration.” Developmental Cell, U.S. National Library of Medicine, https://pubmed.ncbi.nlm.nih.gov/30016618/. 

This summer, I hope to explore alternative career paths both by diving deeper into the world of research through hands-on experience and by connecting and listening to faculty members speak of their own career timelines. I hope to gain a general sense of the responsibilities, protocols, and expectations corresponding to lab research and hope that I can apply the practices I learn later in my career. I expect to come out of the 8 week program with a better understanding of my own interests in the scientific field, an expanded knowledge of lab based methodologies, and to contribute meaningfully to my selected project! Aside from research, I hope to make valuable connections with both my mentors and my peers. I am excited to engage in the group activities set up for us and most importantly, to have fun this summer!