Publikationsdatenbank

Development and characterization of a new in vitro method to study the cellular immune response of microglia and astrocytes and the underlying crosstalk (2024)

Art

Hochschulschrift

Autor

Zelenka, Laura (WE 7)

Quelle

Berlin, 2024 — xi, 111 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/42258

Kontakt

Institut für Mikrobiologie und Tierseuchen

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51843 / 66949
mikrobiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Diseases of the central nervous system (CNS) are a global health threat to both humans and animals with high mortality rates and frequent occurrence of convalescence, occasionally also including long-term sequela. Thus, the role of the different CNS cell types involved is at the forefront of comprehending the development, function, and diseases of the brain. The CNS cell type glia includes microglia and astrocytes, both are crucial players in the brain and are involved in pathogen defense, innate immune activation, and signaling. Their intimate crosstalk maintains the delicate balance between homeostasis and rapid detection of invading pathogens and has progressively gathered research attention. Therefore, the improvement of methods for the isolation, cultivation, and purification of distinct CNS-derived cell types at a high viability and purity is indispensable in order to study the individual contributions of cellular populations to the proper function of the brain. The objective of this study was to develop an in vitro protocol for the isolation, cultivation, and purification of highly viable and pure microglia and astrocytes from neonatal mice and subsequently investigate their cell type-specific effects as well as their contribution to the CNS immune response and cellular crosstalk. In the first step, a glial isolation protocol was developed by optimizing pre-existing techniques for the in vitro isolation and cultivation of microglia and astrocytes from whole neonatal murine brains. Microglial cell yields were maximized by stimulating with macrophage colony-stimulating factor (M-CSF) and applying multiple microglial harvests derived from the same mixed glial culture. Then, a purification protocol for magnetic-activated cell sorting (MACS®) was improved by using cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspensions. Thereby, microglia and astrocytes were successfully isolated, cultivated, and MACS-purified at a purity of 99-100%, as confirmed by fluorescence-activated cell sorting (FACS) analysis. Furthermore, glial cells generated with our novel protocol are highly viable (~100%) and show a preserved integrity, as demonstrated by field emission scanning electron microscopy (FESEM). Subsequently, non-purified and MACS-purified microglial and astrocyte monocultures were stimulated with different TLR ligands. Their pro- and anti-inflammatory response was determined by using Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) enzyme-linked immunosorbent assays (ELISA). In fact, these experiments demonstrated a significantly weakened protein response in MACS-purified microglia and astrocytes in contrast to the non-purified glial monocultures, providing the first hint of a microglia-astrocyte crosstalk. In the next step, our hypothesis of an existing crosstalk was reinforced by stimulating TLR2-knockout (TLR2-KO) microglial and astrocyte monocultures with wild-type (WT) supernatants (SN) derived from the respective other glial cell type. Here, a significant TNF protein release by TLR2-KO astrocyte monocultures was observed, which were activated with microglial WT SN in response to Pam3CSK4, in comparison to the untreated control. Interestingly, transcriptome analysis using RNA sequencing (RNA-seq) unraveled a wide array of significantly up- and down-regulated genes in comparison to their untreated controls, which may be candidate mediators of the intimate molecular glial conversation. Finally, co-culture experiments with microglia and astrocytes coming from different genetic backgrounds were performed. Interestingly, a significant TNF protein release by Pam3CSK4-activated WT microglia co-cultured with TLR2-KO astrocytes was detected in comparison to the untreated control, confirming the results from the prior monoculture experiments. In this thesis, we describe a novel protocol to optimize in vitro isolation, cultivation, and purification of neonatal murine primary microglia and astrocytes. With our proposed method, we maximized microglial yields with the big advantage of sacrificing as few mice as possible following the principles of the 3Rs (replacement, reduction, and refinement). By using ~100% pure glial mono-/co-cultures derived from mice with different genotypes, we were able to analyze the microglia-astrocyte crosstalk on a cell-specific level. Moreover, unlike previous protocols, we showed that the immune response during the crosstalk underlies a TLR2/1-dependent mechanism. Consequently, we postulate our novel protocol to be a suitable and efficient method to investigate cell type-specific effects which contributes to immune modulation as well as cellular crosstalk in future approaches. Thereby, we further extended the possibilities to study glial cells in different experimental issues on brain function as well as CNS infections.