Crosstalk of Highly Purified Microglia and Astrocytes in the Frame of Toll-like Receptor (TLR)2/1 Activation.

The major immune cells of the central nervous systems (CNS) are microglia and astrocytes, subsets of the glial cell population. The crosstalk between glia via soluble signaling molecules plays an indispensable role for neuropathologies, brain development as well as homeostasis. However, the investigation of the microglia-astrocyte crosstalk has been hampered due to the lack of suitable glial isolation methods. In this study, we investigated for the first time the crosstalk between highly purified Toll-like receptor (TLR)2-knock out (TLR2-KO) and wild-type (WT) microglia and astrocytes. We examined the crosstalk of TLR2-KO microglia and astrocytes in the presence of WT supernatants of the respective other glial cell type. Interestingly, we observed a significant TNF release by TLR2-KO astrocytes, which were activated with Pam3CSK4-stimulated WT microglial supernatants, strongly indicating a crosstalk between microglia and astrocytes after TLR2/1 activation. Furthermore, transcriptome analysis using RNA-seq revealed a wide range of significant up- and down-regulated genes such as Cd300, Tnfrsf9 or Lcn2, which might be involved in the molecular conversation between microglia and astrocytes. Finally, co-culturing microglia and astrocytes confirmed the prior results by demonstrating a significant TNF release by WT microglia co-cultured with TLR2-KO astrocytes. Our findings suggest a molecular TLR2/1-dependent conversation between highly pure activated microglia and astrocytes via signaling molecules. Furthermore, we demonstrate the first crosstalk experiments using ∼100% pure microglia and astrocyte mono-/co-cultures derived from mice with different genotypes highlighting the urgent need of efficient glial isolation protocols, which particularly holds true for astrocytes.

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes.

BACKGROUND: The crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. NEW METHOD: We modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. RESULTS: We generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. COMPARISON WITH EXISTING METHOD(S): Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99-100%). CONCLUSIONS: We have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.