Role of microglia cell activation in neurogenesis: focus on interleukin-1 beta (IL-1β ) signalling

Stephanie Koosje-Newman, Francisco Molina-Holgado. University of Roehampton, London, UK

A major problem limiting endogenous neurogenesis after brain insults is the poor survival of newly generated neurons as a result of a hostile environment involving proinflammatory microglia. However, under certain conditions following brain inflammation or injury, activated microglial cells are permissive to brain repair, providing neuroprotection or acting as a proneurogenic influence that supports the different steps of neurogenesis [1]. Exploring the relationship between the immune system and the central nervous system (CNS) offers great potential to generate a further, more in depth understanding of clinical presentations of neurodegeneration. Neural stem cells (NSC) have the potential to replace lost CNS cells during neurogenesis. However, the efficiency of neurogenesis is limited by various pro-inflammatory cytokines during neurodegeneration [2].

The aim of this study is to investigate if activation of microglial (BV-2) cells using lipopolisaccharide (LPS, serotype 026:B6, 1μμg/ml, t=24h-72h) and recombinant murine (rm) IFN-γγ (20ng/ml, t=24h-72h), can regulate NSC fate. Specifically, we focused on the cytokine IL-1β to determine whether or not, endogenous IL-1ββ is required for the microglia activation-induced modifications to NSC fate. Experiments were performed in NSC cultures prepared from the cortex of embryonic day 16 (ED16) C57BL6 mice (wild type, WT) and IL-1β knock-out (IL-1-/-) mice. Microglial cells, NSC from WT mice and/or NSC from IL-1 -/- mice were treated with LPS, IFN-γ and/or LPS/IFN-γ together and incubated for either 24n (acute activation) or 72h (subchronic activation). In addition, cell free conditioned medium (CM) from activated microglial cultures was added directly to the NSC (WT and IL-1-/-) to determine the participation of microglia activation and IL-1β on neurogenesis. MTT analyses were performed on microglial cultures after exposure to LPS, IFN-γ and LPS/IFN-γ to determine the cell viability of cells. Next we assessed, by BrdU labelling and serial dilution assays, if the exposure of NSC (WT and IL-1-/-) to CM regulates cell proliferation, differentiation or cell death. Exposure of microglia to IFN-γ induced a significant proliferative effect (10% increase cell viability) when compared to the control group (MTT analysis, n=4; P<0.001). Interestingly, the observed detrimental effects on cell viability in microglial cultures after exposure to LPS (40% decrease cell viability) were modulated by IFN-γ (MTT analysis, 20% increase cell viability, n=4; P<0.001). Similar results were shown in NSC from WT mice incubated with CM from with activated microglia (n=4). NSC from IL-1-/- mice were also treated with LPS and IFN-γ. Results differed from NSC from WT mice in terms of cell death and cell proliferation following treatments. In NSC from IL-1 -/- mice, exposure to LPS induced cell death in a significant manner (BrdU analysis, 40% decrease cell viability, t(6)=0.037; P=0.032). However, after exposure to IFN-γ,, no significant changes in the proliferative rate was observed (BrdU analysis, t(6)=0.134; P=0.055). Although, in NSC from IL-1-/- mice, exposure to both LPS and IFN-y increase proliferation in a significant manner (BrdU analysis, t(6)=0.391; P=0.002). The main findings of this study demonstrate that IFN-γ, despite its well-described pro-inflammatory actions, is not neurotoxic for microglial cells or NSC. These results suggest a new IFN-γ pathway that remains to be elucidated in the type or nature of the signalling.

[1] Molina-Holgado E & Molina-Holgado J. Neurochem. 114:1277, 2010

[2] García-Ovejero D et al. Biochem Soc Trans41:1577-82, 2013.