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.
