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Published on: September 19, 2010
Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system
Yoelvis Garcia-Mesa1, Taylor R Jay2, Mary Ann Checkley1
1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, 44106, USA.
Abstract:
The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.
Insights
Researchers developed a method to immortalize human and animal microglial cells for studying HIV in the central nervous system (CNS). These robust cell lines are crucial for understanding HIV molecular regulation in microglia.
Area of Science:
- Neuroscience
- Virology
- Cell Biology
Background:
- Microglia are primary reservoirs for Human Immunodeficiency Virus (HIV) in the central nervous system (CNS).
- Studying molecular events controlling HIV expression in microglia is essential for understanding CNS pathogenesis.
- Existing methods for primary microglial culture present limitations for long-term studies.
Purpose of the Study:
- To develop a reliable and robust method for immortalizing microglial cells from primary glia.
- To create characterized cell lines for investigating HIV molecular regulation in microglia.
- To establish a resource for studying microglial cell function in the context of HIV infection.
Main Methods:
- Primary human and animal glial cells were transformed using lentiviral vectors expressing SV40 T antigen (with or without hTERT).
- Immortalized cells were characterized for morphology, surface markers (CD11b, TGFβR, P2RY12), human origin, RNA expression (RNA-seq), migratory, phagocytic, and inflammatory activities.
- Cells were superinfected with HIV reporter viruses, and latently infected clones were selected for HIV reactivation studies.
Main Results:
- Successfully developed immortalized microglial cell lines with microglia-like morphology and characteristic surface markers.
- Confirmed human origin, RNA expression profiles, and functional activities (migration, phagocytosis, inflammatory response) of the immortalized cells.
- Demonstrated successful application of the method across multiple species (human, macaque, rat, mouse) and established HIV-reactive clones.
Conclusions:
- The developed immortalization method provides a robust and versatile tool for generating microglial cell lines.
- These characterized cell lines serve as an invaluable resource for studying HIV infection and molecular regulation in microglia.
- The cell lines will advance research into microglial cell biology and HIV pathogenesis within the CNS.
