Determination of microglial phenotypes by intracellular signaling
Extrinsic signals determine whether microglia are activated to become beneficial or detrimental phenotypes. The specificity of the stimuli largely reflects different signalling and receptors for inflammatory stimuli, which promotes activation of a distinct panel of transcription factors and, ultimately, a stimulus-specific gene expression program. Molecular mechanisms responsible for the shift between phenotypes under pathological conditions are poorly known. Studies by partner 9 on global gene profiling and signal transduction in primary microglial cells exposed to different stimuli revealed two distinct phenotypes: proinflammatory and antiinflammatory, cytoprotective phenotype characterized by selective induction of genes such as STATs, c-Myc, Jmdj3, and the negative regulator of transcription Id1 likely being crucial modulators of the two phenotypes. This was associated with specific histone modifications contributing to dynamic changes in chromatin structure.
To explore the physiological roles of signalling cascades and the interplay between transcription factors, partner 9 will silence the expression/activity of specific transcription factors or histone modifying enzymes with siRNA or pharmacological inhibitors in microglial cells and analyze resulting phenotypes (surface markers, gene expression and cytokine profiles). Characterization of phenotypes will involve the platform based on human iPSC-derived cells that will be set up by partner 7. Partner 9 will also test whether pharmacological or cytokine-mediated modulation of microglia phenotypes is possible. The most promising findings will be verified in animal models of stroke provided by partner 2 and spinal cord demyelination/remyelination provided by partner 4, in microglia/macrophages sorted by flow cytometry from animal brains or spinal cords. To differentiate the role of brain-derived macrophages and BMDMs, infiltrating peripheral monocytes will be selectively ablated using either antibody-mediated depletion or conditional ablation by diphtheria toxin.
Ellert-Miklaszewska A, Dabrowski M, Lipko M, Sliwa M, Maleszewska M, Kaminska B. Molecular definition of the pro-tumorigenic phenotype of glioma-activated microglia. Glia 61(7):1178-90, 2013.
Sielska M, Przanowski P, Wylot B, Gabrusiewicz K, Maleszewska M, Kijewska M, Zawadzka M, Kucharska J, Vinnakota K, Kettenmann H, Kotulska K, Grajkowska W, Kaminska B. Distinct roles of CSF family cytokines in macrophage infiltration and activation in glioma progression and injury response. J Pathol. 230(3):310-21, 2013
Tasks and methodology
- In vitro culture of microglia
- Silencing of transcription factors and histone modifying enzymes with siRNA and pharmacological tools
- Determination of microglial phenotypes by quantifying surface markers, gene expression, and cytokine profiles
- Sorting of microglia and profiling of signalling pathways in sorted cells
- In vivo ablation of infiltrating peripheral monocytes
Spanish National Research Council CSIC, Role of microglial signalling in vivo, 10 month