Stimulation protocol for BV2 Microglia-induced neurotoxicity In Vitro model by co-stimulating and priming with Lipopolysaccharide and interferon gamma

Microglia are the resident macrophages responsible for the immune surveillance in the central nervous system (CNS). They respond to noxious stimuli by releasing inflammatory mediators and initiating inflammatory responses. Chronic inflammation mediated by microglia may cause loss of neuronal structure or function, a condition known as microglia-induced neurotoxicity. To study microglia and their neurotoxic effect, an in vitro microglia-induced neurotoxicity model (MINT) can be developed. Consequently, a microglia stimulant is needed for the MINT model to stimulate microglia into an inflammatory and neurotoxic phenotype. Lipopolysaccharide (LPS) is a widely used microglia stimulant in neuroinflammatory studies and represents an exogenous response, specifically of Gram-negative bacteria infection. We were interested in a microglia stimulant for the MINT model that resembles generic neurotoxicity rather than a disease-specific stimulant. Interferon gamma (IFN-γ), a cytokine expressed in response to tissue damage, represents a more generic and endogenous response of CNS damage. However, we report here that single stimulation with IFN-γ alone did not induce considerable amounts of reactive oxygen species (ROS) production in BV2 microglia. Two other stimulating approaches were then explored, namely co-stimulation (LPS/IFN-γ) and priming (primedIFN-γ/LPS) to induce the inflammatory phenotype of BV2 microglia. Co-stimulation was performed by adding LPS and IFN-γ simultaneously to BV2 cells. Concentrations used were 10 ng/mL LPS and 10 ng/mL IFN-γ (LPS10IFN-γ10), 100 ng/mL LPS and 5 ng/mL IFN-γ (LPS100IFN- γ5), 200 ng/mL LPS and 2.5 ng/mL IFN-γ (LPS200IFN-γ2.5). Meanwhile, priming was performed by treating BV2 microglia with IFN-γ, followed by LPS. Cells were treated with 10 ng/mL IFN-γ (primedIFN-γ10) or 50 ng/mL IFN-γ (primedIFN-γ50), followed by subsequent stimulation with 10 ng/mL LPS (LPS10), 100 ng/mL LPS (LPS100), 200 ng/mL (LPS200), or 1000 ng/mL (LPS1000). Activation state of microglia was determined via intracellular ROS production, nitric oxide (NO) production, CD40 expression, and migration. Based on the highest ROS and NO production, two concentrations each from co-stimulation (LPS10IFN-γ10 and LPS200IFN-γ2.5) and priming (primedIFN-γ10LPS100 and primedIFN-γ50LPS100) were selected to be used for the MINT model. Importantly, we also demonstrated that the inflammatory phenotype of microglia stimulated using co-stimulation and priming can be distinct. Co-stimulation with LPS200IFN-γ2.5 induced a higher intracellular ROS production (9.2-fold increase) and CD40 expression (28031 ± 8810.2 MFI), compared to priming with primedIFN-γ50LPS100 (4.0-fold increase in ROS and 16764 ± 1210.8 MFI of CD40). Co-stimulation also induced cell migration. On the other hand, priming with primedIFN-γ50LPS100 resulted in a higher NO production (64 ± 1.4 µM) compared to co-stimulation with LPS200IFN-γ2.5 (44 ± 1.7 µM). Unexpectedly, priming inhibited cell migration towards LPS. Taken together, the findings from this project revealed the ability of co-stimulation and priming in stimulating microglia into an inflammatory phenotype, and the heterogeneity of microglia responses towards different stimulating approaches.

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